Efficient side-chain deacylation of polymyxin B1 in recombinant Streptomyces strains

Efficient side-chain deacylation of polymyxin B1 in recombinant Streptomyces strains | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Efficient side-chain deacylation of polymyxin B1 in recombinant Streptomyces strains Xiaojing Wang, Kai Wu, Hanzhi Zhang, Jing Liu, Zhijun Yang, Jing Bai, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1655594/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Objectives: Polymyxins are antibacterial polypeptides used as “last resort” therapy option for multidrug-resistant Gram-negative bacteria. The expansion of polymyxin-resistant infections has inspired development of novel polymyxin derivatives, and deacylation is one of the critical steps in generating those antibiotics. Deacylase from Actinoplanes utahensis hydrolyze the acyl moieties of echinocandins, and also efficiently deacylates daptomycin, ramoplanin and other important antibiotics. Here, deacylase was studied considering its potential usefulness in deacylating polymyxin B1. Results: All the six recombinant strains containing the deacylase gene catalysed hydrolysis of polymyxin B1, yielding cyclic heptapeptide. The efficiency of recombinant S. albus (SAL701) was higher than others, whereby deacylation was the most efficient at 40 °C in 0.2 M Tris buffer (pH 8.0) with 0.2 M Mg 2+ . The optimal substrate concentration of SAL701 was increased from 2.0 to 6.0 g/L. SAL701 was highly thermostable, showing no loss of activity at 50°C for 12 h, and the mycelia could be recycled at least three times without loss of catalytic activity. SAL701 could not deacylate β-lactam substrate such as penicillin G and cephalosporin C. Deacylase catalyzes the amide bond 1 closest to the nucleus of polymyxin B1 rather than the other bond, suggesting that it has high catalytic site specificity. Homology modeling and the docking results implied Thr190 in deacylase can facilitate hydrolysis with high regioselectivity. Conclusions: These results show that SAL701 is effective in increasing cyclic heptapeptide moiety of polymyxin B1. These properties of the biocatalyst may enable its development in the industrial production of polymyxins antibiotics. Deacylase polymyxin B1 Streptomyces host bioconversion antibiotic Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Polymyxins are antibacterial polypeptides first isolated from cultures of Bacillus polymyxa strains, and polymyxin B1 is one of the main components, which is shown in Fig. 1 . 1 The core scaffold contains a cyclic heptapeptide linked to the side chain of a linear tripeptide with an N-terminal fatty acyl. 2 They have been reused as “last resort” therapy option for multidrug-resistant Gram-negative bacteria. 1 The situation is worsen by the emergence of polymyxin-resistant infections. The development of novel polymyxin derivatives increase its activity and reduce toxicity. 2 The antibacterial activity of polymyxin B1 is mainly affected by changing the amino acid of the side chain. 3 Numerous novel compounds have been semi-synthesized by deacylating and replacing their side chains with a variety of different chains. 4 , 5 Cui replaced the amino acid l-Dab in position 3 of polymyxin B1 with d-Ser to synthesize polymyxin S2, which showed enhanced activity against Escherichia coli , Acinetobacter baumannii and Klebsiella pneumonia , and reduced toxicity. 6 , 7 Therefore, polymyxin B1 analogues with increased antibacterial activity can generate by direct modifications of the cyclic peptide. 6 Enzyme modification is an efficient strategy for deacylation. Deacylase from Actinoplanes utahensis hydrolyzes the amide bonds of cyclic lipopeptides, which has broad substrate specificity for both the acyl side chain and cyclic peptide analogs of echinocandins, ramoplanin, daptomycin and other important antibiotics. 8 – 10 The enzyme hydrolyze the acyl moieties of antibiotic echinocandins, and forms a cyclic hexapeptide moiety and long-chain fatty acid. 11 , 12 The resulting cyclic hexapeptide can be further reacylated to produce a series of analogs useful as therapeutic antibiotics in clinical practice, such as anidulafungin. 13 , 14 Ramoplanin is a lipoglycodepsipeptide with antibacterial activity against major Gram-positive bacteria. 15 , 16 Numerous novel ramoplanin derivatives have been semi-synthesized by deacylating and replacing their side chains with different kinds of carboxylic acids. 17 Among them, the 2-methylphenylacetic acyl group, displays increased tolerability and high activity, indicating its potential for overcoming the limitations of ramoplanin in vivo . 18 , 19 Deacylation is the key step in generating the aforementioned antibiotics, and deacylase may be useful in the antibiotic industry for catalyzing the hydrolysis of not only echinocandins and ramoplanin, but also teicoplanin, daptomycin and its derivatives, as well as other related antibiotics with corresponding acyl side chains. 20 , 21 In this study, we attempted to expand the substrate spectrum of recombinant Streptomyces with deacylase gene, and we found that the enzyme could hydrolyze polymyxin B1. In addition, we tried to predict the specificity of the catalytic site through structure analysis, and payed attention to its potential biotechnological application. Materials And Methods Strains and culture medium S. lividans TK24, S. coelicolor , S. albus , S. avermitilis K139, S. ambofaciens 2283, S. griseus , and A. utahensis NRRL 12052 were stored in our laboratory. Escherichia coli ET12567/PUZ8002, a donor strain for conjugation between E. coli and Streptomyc es , was purchased from Huayueyang Biological Technology Co., Ltd. (Beijing, China). Plasmids pDS701, pSET152 containing a deacylase gene under the control of PermE, were constructed in our laboratory as described earlier. 21 All chemicals, biochemicals, restriction enzymes, media and molecular biological reagents were of analytical grade and obtained from standard commercial sources. Gauserime synthetic agar plates (2% soluble starch, 0.05% NaCl, 0.05%K 2 HPO 4 ·3H 2 O,0.1% KNO 3 , 0.05% MgSO 4 ·7H 2 O, 0.001% FeSO 4 ·7H 2 O, and 1.5% agar powder [pH 7.4]) were used to culture strains for sporulation. Seed medium (2.5% sucrose, 2.0% oatmeal, 0.25% yeast powder, 0.1% K 2 HPO 4 , 0.05% KCl, 0.05%MgSO 4 ·7H 2 O, and 0.0002% FeSO 4 ·7H 2 O) and fermentation medium (2% sucrose, 1% peanut meal, 0.1% KH 2 PO 4 , and 0.025% MgSO 4 ·7H 2 O) were used to ferment A. utahensis. And seed medium (1.0% glucose, 0.5% yeast powder, and 1% peptone) and fermentation medium (2.5% glucose, 1% beanflour, 0.3% NaCl, and 0.3% CaCO 3 ) were used to ferment Streptomyces. 21 Heterologous over-expression of deacylase gene Plasmid pDS701 was introduced into different Streptomyces by intergeneric conjugation from E. coli ET12567 according to standard procedures. 22 Recombinant strains were cultured in medium supplemented with 50 μg/mL apramycin, and further confirmed using PCR amplification with universal primers M13F-47 and M13R-48. Wild-type and recombinant cultures were grown on Gauserime synthetic agar plates at 30°C for sporulation. An agar piece was inoculated into 50 mL seed medium for 30 h at 30°C on a shaker at 220 rpm, the resulting mycelial suspension was plused (2%) to fresh fermentation medium and further incubated for 48 h. S. albus without the plasmid was fermented as a control. Determination of bioconversion efficiency We used the whole-cell reaction system for bioconversion, because it is convenience, high efficiency, reusability and safety. The wet mycelia were sedimented by centrifugation and washed twice with 0.1 M Tris buffer (pH 8.0). The 10 g cell pellet (20% m/v) was resuspended in a 50 mL Tris buffer. The enzymatic reaction was initiated by adding polymyxin B1 (2 g/L) and allowed to continue for 5 h at 30°C with shaking at 50 rpm. The reaction mixture was immersed in an ice bath and stopped by adding an equivalent volume of methanol. The mixtures were centrifuged, and the supernatants were filtered through a 0.22 μm pore diameter membrane. An analytical HPLC system was used to measure the cyclic heptapeptide polymyxin B1 released during the hydrolysis of polymyxin B1. Chromatographic separation was carried out on a Waters 2695 HPLC System (Milford, MA, USA) consisting of analytical C18 column (250 × 4.6 mm; 5 μm; Agilent Technologies, Santa Clara, CA, USA) with a mixture of sodium sulfate solution (30 mM, pH was adjusted to 2.3 with phosphoric acid) and acetonitrile in a ratio of 77.5:22.5 V/V as the mobile phase. 7 HPLC was performed at a flow rate of 0.8 mL/min for 35 min with UV detection at 215 nm. The compounds were identified using ESI-MS analysis performed on an Agilent HPLC 1260 coupled with a 6550 quadrupole time-of-flight massspectrometry system. The method was linear at concentrations of 0.5-1500 μM with a correlation coefficient of 0.999. A standard curve was drawn using different amounts of cyclic heptapeptide polymyxin B1 and peak areas. The molar bioconversion rates were compared according to the following formula: bioconversion rate = ([product of daptomycin cyclic heptapeptide polymyxin B1]/substrate addition) × 100%. 23 All enzyme measurements were performed in triplicate, and the maximum error was less than 5%. Optimization of bioconversion conditions Enzymatic activity was determined using the standard assay conditions for each case but using different buffers. The effect of pH was examined at different pH values ranging from 3.0 to 8.0 in disodium hydrogen phosphate-citrate buffer, from pH 5.5 to 8.0 in phosphate buffer, and from pH 7.5 to 10.0 in Tris buffer at 0.1 M constant ionic strength and 30°C by 2 g/L polymyxin B1. The effect of metal ions was evaluated by adding 0.2 M KCl, NaCl, MgCl 2 , CuSO 4 or FeSO 4 to the Tris buffer. The effect of the ion concentration in the phosphate buffer and Tris buffer was also tested from 0.05 to 0.3 M. The effect of temperature of SAL701 was measured at temperatures ranging from 25°C to 70°C in 0.1 M Tris buffer pH 8.0 by 2 g/L polymyxin B1. The reaction time was prolonged to 96 h to determine the biotransformation efficiency. The optimum concentration of substrate was determined by adding different amounts of polymyxin B1 (1-12 g/L) under the optimized conditions in a total reaction volume of 200 mL using wild-type A. utahensis NRRL 12052, recombinant strains SAL701, SCO701 and SAM701. T hermostability and recycling of SAL701 The mycelia of Streptomyces strains carrying the deacylase gene were incubated at 4, 30 and 50°C under the optimized conditions. At different time points during incubation, mycelia samples were withdrawn and assayed to determine their deacylase activity using polymyxin B1 as a substrate. The mycelia were added to a 50 mL reaction buffer containing 2 g/L polymyxin B1 under the optimized conditions. After the enzymatic reaction for 5 h, the solution was centrifuged at 3000 × g for 30 min, and the supernatant was measured using HPLC as described above. The recovered mycelia were washed with Tris buffer for three times and used for another bioconversion cycle. Molecular docking The deacylase homology protein structure model was built based on the crystal structure (PDB ID 5C9I; 39.1% identity) using Discovery Studio 2016, and the model with the lowest DOPE score (-79981) was selected for docking. 24 Substrates polymyxin B1 for deacylase were modeled in Chem3D. Substrate docking was performed using Autodock4. The substrate was docked into the binding pocket using flexible docking. AutoDock tools were used for enzyme and substrate preparations. To encompass the entire substrate-binding pocket, the docking box was set to 60 × 60 × 60 grid points with a grid spacing of 0.375 Å. The box center was set as X = 19.336, Y = -0.931, and Z = 52.155. Results And Discussion Over-expression of deacylase gene in Streptomyces hosts Enzymatic modification is more efficient than chemical modification, but deacylase was rate-limiting in the antibiotics production process, as the bioconversion efficiency of deacylases in the original strain is low. Streptomyces strains are suitable heterologous hosts for producing enzymes, particularly GC-rich deacylases, for industrial applications. 25,26 To investigate the effect of deacylase gene, pDS701 plasmid consist of deacylase gene was introduced into six Streptomyces species ( S. lividans TK24, S. coelicolor , S. albus , S. avermitilis K139, S. ambofaciens 2283 and S. griseus ), yielding recombinant strains SLI701, SCO701, SAL701, SAV701, SAM701 and SGR701. 13 There were no apparent phenotypic differences between Streptomyces and its recombinant strains. Genotypes of six recombinant strains were verified by polymerase chain reaction (PCR). The 3.3-Kb DNA fragments were amplified. Sequencing and alignment confirmed that all six segments had 100% identity with the deacylase gene of recombinant plasmid pDS701 (Fig. 2). As negative controls, PCR products could not be amplified from S. albus host and wild-type A. utahensis . Deacylation of different recombinant Streptomyces strains Six recombinant Streptomyces strains and A. utahensis NRRL 12052 were inoculated, the mycelia were collected to determine the bioconversion efficiency. The reaction mixture containing 2 g/L polymyxin B1 in 0.1 M Tris buffer pH8.0, was incubated at 30℃, 5 h and then analysed by high-performance liquid chromatography (HPLC). All seven samples showed the new peak, which almost having the same retention time with the standard cyclic heptapeptide moiety of polymyxin B1 (4.56 min, Fig. 3b), and a peak of remaining polymyxin B1 substrate (31.10 min, Fig. 3a). The overlapped peak also obtained by injecting the mixture of SAL701 sample and standard cyclic heptapeptide polymyxin B1. So we preliminary verified that all six recombinant species and wild-type strain could deacylate polymyxin B1, with different activity dependent on the host strains. As shown in Table 1, five of the six recombinant strains showed similar bioconversion efficiencies, and although SAV701 showed a lower conversion efficiency (Fig. 3g), it was still higher than that of the wild-type strain (34.0% compared with 23.3%). SAL701 (Fig. 3f) showed the highest efficiency with the highest percentage of bioconversion rate (65.2%). No polymyxin B1 or its cyclic heptapeptide moiety was detected in the whole-cells of all seven species examined. Therefore, the S. albus were considered as the preferred host for enzymatic deacylation. SAL701 strain has many advantages such as easy-cultivating, rapid-growing and high bioconversion efficiency, so it could be applied to bioconversion of polymyxin B1 in a short time. Table 1 Bioconversion rate by wild-type and different recombinant Streptomyces strains Strains Description Bioconversion rate (%) A. utahensis NRRL 12052 wild-type 23.3 ± 3.2 SLI701 pDS701 integrated into S. lividans TK24 58.1 ± 2.1 SCO701 pDS701 integrated into S. coelicolor 60.7 ± 0.4 SAL701 pDS701 integrated into S. albus 65.2 ± 4.5 SAV701 pDS701 integrated into S. avermitilis K139 34.0 ± 3.9 SAM701 pDS701 integrated into S. ambofaciens 2283 59.8 ± 0.9 SGR701 pDS701 integrated into S. griseus 52.3 ± 1.0 S. albus Streptomyces without plasmid as negative control 0 Qualitative analysis of polymyxin B1 and its cyclic heptapeptide Polymyxin B1 and its cyclic heptapeptide moiety were extracted from the reaction mixture, and their identities were confirmed. The structure of the polymyxin B1 cyclic heptapeptide moiety was verified by using electrospray ionization-mass spectrometry (ESI-MS). The parent ion was observed at m/z 784.4441 [M+Na] + (calculated 784.4440), and its mass spectrum was consistent with the molecular formula C 35 H 59 N 11 O 8 (Fig. 4a). Polymyxin B1 showed parent ions at m/z 602.3822 [M+2H] 2+ (calculated 602.3822) and m/z 1203.7566 [M+H] + (calculated 1203.7572), and its mass spectrum was consistent with the molecular formula C 56 H 98 N 16 O 13 (Fig. 4b). 7 Impure preparations, polymyxin B containing polymyxin B1, were also successfully deacylated but at a slower rate. Effect of reaction buffer on bioconversion of polymyxin B1 by SAL701 The buffer of reaction mixture strongly affects the bioconversion rate. Hydrolysis of polymyxin B1 catalyzed by SAL701 was evaluated in different 0.1 M buffer pH range of 3.0-10.0 at 30 °C for 5 h. As shown in Table 2, the strain displayed the highest activity at pH 8.0 in Tris buffer (65.2%), which was selected as the standard buffer for deacylation. The same pH value (pH 7.5 or 8.0) in three different buffers were compared, and found that Tris buffer (60.8% and 65.2%) was more suitable for reaction than phosphate buffer (60.5% and 62.9%) and disodium hydrogen phosphate-citrate buffer (53.2% and 52.3%). Table 2 Effect of reaction buffer pH Bioconversion rate (%) disodium hydrogen phosphate-citrate buffer phosphate buffer Tris buffer 3.0 21.2 ± 1.2 3.5 19.3 ± 3.4 - - 4.0 23.6 ± 0.9 - - 4.5 25.9 ± 4.6 - - 5.0 26.9 ± 3.0 5.5 33.3 ± 2.7 31.9 ± 2.2 - 6.0 42.8 ± 3.5 41.3 ± 4.0 - 6.5 41.4 ± 2.2 50.9 ± 3.1 - 7.0 42.9 ± 4.1 55.6 ± 3.7 - 7.5 53.2 ± 0.8 60.5 ± 0.8 60.8 ± 3.0 8.0 52.3 ± 1.2 62.9 ± 0.9 65.2 ± 4.0 8.5 64.7 ± 1.2 9.0 - - 61.5 ± 1.9 9.5 - - 62.2 ± 2.1 10.0 60.3 ± 2.0 Also, the activities were increased when the ionic strength of buffer was increased (0.05-0.3 M phosphate buffer and Tris buffer). The bioconversion efficiency increased from 56.3% to 70.1% in Tris buffer and from 52.8% to 61.2% in phosphate buffer (Table 3). Table 3 Effect of ionic and ionic strength Buffer Ionic Bioconversion rate (%) 0.05 M 0.1 M 0.2 M 0.3 M phosphate buffer - 52.8 ± 1.7 55.6 ± 3.9 59.0 ± 2.2 61.2 ± 1.6 Tris buffer - 56.3 ± 4.2 65.2 ± 1.1 70.1 ± 2.9 69.2 ± 1.0 Tris buffer K + - - 73.9 ± 3.0 - Tris buffer Na + - - 72.1 ± 1.5 - Tris buffer Mg 2+ - - 74.7 ± 2.1 - Tris buffer Cu 2+ - - 69.2 ± 2.0 - Tris buffer Fe 2 + - - 72.7 ± 1.3 - Deacylase activity did not require metal ions as previously studied. But the inclusion of an ion (0.2 M K + , Na + , Mg 2+ or Fe 2+ ), enhanced the deacylase activity (73.9%, 72.1%, 74.7% and 72.7% respectively, compared to 70.1% for enzyme alone, Table 3). Addition of Cu 2+ did not increase the activity. Therefore, 0.2 M Tris buffer pH 8.0 with 0.2 M Mg 2+ was selected as the standard condition for the deacylation reaction. Effect of reaction temperature and time on bioconversion of polymyxin B1 by SAL701 The enzymatic activity varies over a temperature range of 25-70°C. Maximum deacylase activity was achieved at 40°C under the optimized reaction buffer (0.2 M Tris buffer, pH 8.0 with 0.2 M Mg 2+ ), the conversion rate of polymyxin B1 was 76.9% (Fig. 5). The ability of SAL701 to deacylate polymyxin B1 was examined during 96 h. The time course was determined under the optimized buffer conditions. As shown in Fig. 6, the deacylated product occurred at the earliest time tested (5 h), and the bioconversion efficiency was 74.8%, and then gradually increased to 77.7% at 96 h. Effect of substrate concentration on bioconversion of polymyxin B1 by SAL701 The optimum concentration of polymyxin B1 in the bioconversion mixture was confirmed using SAL701, SCO701, SAM701 recombinant strains and wild-type A. utahensis NRRL 12052, the relatively highly active recombinant Streptomyces strains. The optimal substrate concentration of SAL701 was increased from 2.0 to 6.0 g/L, under conditions in which the bioconversion efficiency was more than 60%, and SCO701, SAM701 were increased from 2.0 to 4.0 g/L (Table 4). Table 4 Effect of substrate concentration polymyxin B1 concentration (g/L) Bioconversion rate (%) SAL701 SCO701 SAM701 A. utahensis NRRL 12052 1.0 78.4 ± 3.0 74.8 ± 2.5 75.1 ± 2.1 39.4 ± 2.2 2.0 73.9 ± 1.7 72.4 ± 1.7 72.3 ± 2.8 31.8 ± 1.8 3.0 70.2 ± 1.3 69.4 ± 1.5 68.9 ± 0.6 26.4 ± 3.0 4.0 70.7 ± 1.3 66.4 ± 2.5 64.3 ± 2.8 22.5 ± 0.3 6.0 61.2 ± 0.8 58.7 ± 3.1 56.1 ± 0.5 21.0 ± 0.7 8.0 43.7 ± 0.3 43.6 ± 3.2 50.4 ± 1.5 23.0 ± 1.7 10.0 47.3 ± 1.3 36.8 ± 2.7 41.4 ± 1.7 19.5 ± 1.2 12.0 33.9 ± 1.6 37.9 ± 2.8 30.1 ± 3.9 21.2 ± 3.9 T hermostability of SAL701 The thermostability of the mycelia of recombinant S. albus was evaluatedat different temperatures under the optimized conditions above (Fig. 7). The enzyme showed no decrease inactivity after storage at 4°C and 30°C for at least 48 h. However, the mycelia also maintained 90% of its activity following storage at 50°C for at least 12 h. Recycling of mycelia of SAL701 The mycelia was used to bioconvert polymyxin B1, the number of times required for the mycelia to be reused was investigated. The mycelia of SAL701 could be recycled at least three times without loss of catalytic activity on the basis of maintaining the bioconversion efficiency at 70.2% under the optimized conditions above (Table 5). Table 5 Number of times of reutilizing the mycelia Number of times Bioconversion rate (%) 1 72.4 ± 0.2 2 73.5 ± 3.1 3 70.2 ± 3.0 4 50.3 ± 3.7 5 30.9 ± 1.8 6 23.4 ± 1.6 7 6.3 ± 1.2 8 6.9 ± 2.4 Substrate spectrum of SAL701 The aforementioned results established the characteristic of SAL701 and necessitated further research about their substrate spectrum. The chemical structure of β-lactam antibiotics also consists a β-lactam ring and a side chain. Different β-lactam substrates were used to determine the bioconversion rate. But no enzymatic deacylation was observed for the β-lactam compounds penicillin G and cephalosporin C. Hydrolysis of specific amide bonds of SAL701 Enzyme-catalyzed reactions are specific. Amidohydrolase from E. coli N.C.I.B. 8743 is stereospecific for the deacylation of α-amino acids, acylated L-α-amino acids but not D-α-amino acids are hydrolyzed, whereas DL-amino acids are only 50% hydrolyzed. 27-29 SAL701 catalyzes hydrolysis of amide bond 1 (Fig. 1), which is the closest amide bond to the cyclic heptapeptide of polymyxin B1, rather than at other amide bonds, suggesting its high specificity for the catalytic site. The specificity of deacylase could avoid the existence of impurities, reduce the difficulty in the final purification process, and improve the economic benefits of the process. An enzyme’s specificity depends on its structure. As a representative functional deacylase, the sequence similarity to many other deacylases. Although a few deacylase structures have been reported, that of a functional deacylase has not been determined. Homology protein structure models for deacylase was constructed to identify the structural basis for the programming of this distinct specificity (Fig. 8) based on the known crystal structure of the MacQ (PDB ID 5C9I; resolution: 1.80 Å, and identity of 39.1% with deacylase). MacQ derived from Acidovorax sp. strain MR-S7 are comprised of 806 amino acid residues, and exhibits acylase activity against β-lactam antibiotics and N-acylhomoserine lactones (AHLs). Structural comparison with MacQ revealed that deacylase has a similar structure and active site organization, indicating that the key enzyme-co-product interactions among MacQ are well-conserved, deacylase and similar enzymes such as the catalytic site specificity. Ser/Thr/Cys is the necessary residue of catalysis, and initiates amide bond cleavage of various substrate compounds as a nucleophile. Similar to Ser1β, which act as a catalytic residue in MacQ, the docking results indicated that Thr190 in deacylase from A.utahensis NRRL 12052, which located near amide bond 1, can facilitate hydrolysis with high regioselectivity. 24,30,31 The relatively close distance of the hydroxyl group to the amide carbon atom of the peptide bond, suggests an important role for the residues in catalysis, as revealed by substrate docking. Conclusion We previously reported the hydrolytic activity for the side chains of echinocandin B and daptomycin by the recombinant Streptomy ces, this drove us to explore different antibiotics of great significance. 13,21 In this study, we efficiently bioconversion polymyxin B1 to its cyclic heptapeptide, a starting material synthesizing antibiotics, which has not been reported before, and deacylase may be useful in the antibiotic industry for catalyzing the hydrolysis of polymyxin B1. We also tried to hydrolyze other important antibiotics with a similar structure, to expand the application of recombinant strain in the future. Furthermore, we have determined the optimal reaction conditions for this process, 40 °C in 0.2 M Tris buffer (pH 8.0) with 0.2 M Mg 2+ . Also the mycelia of SAL701 was high thermostability and reusability. Additionally, catalytic site specificity of the deacylase was described and predicted by homology modeling and docking, which showed Thr190 facilitates hydrolysis with high regioselectivity. All above results show that recombinant Streptomyces strain SAL701 is effective in catalysing polymyxin B1 to its cyclic heptapeptide moiety. The recombinant strain may be used for the industrial production of polymyxin antibiotics. Declarations Funding This research was financially supported by the Natural Science Foundation of Shanghai (20ZR1424600), the National Natural Science Foundation of China (81773616), the Shanghai Excellent Technology Leader Program (17XD1423200), and Nature Science Foundation of Jiangsu Higher Education Institutions of China (20KJB180002). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions Xiaojing Wang, Kai Wu and Hanzhi Zhang performed the experiments and data analyses, and wrote the manuscript. 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Appl Environ Microb 79(4):1126–1133. https://doi.org/10.1128/AEM.02792-12 Debono M, Abbott BJ, Turner JR, Howard LC, Gordee RS, Hunt AS, Barnhart M, Molloy RM, Willard KE, Fukuda D, Butler TF, Zeckner DJ (1988) Synthesis and evaluation of LY121019, a member of a series of semisynthetic analogues of the antifungal lipopeptide echinocandin B. Ann N Y Acad Sci 544:152–167. https://doi.org/10.1111/j.1749-6632.1988.tb40398.x Gandolfi R, Marinelli F, Ragg E, Romano D, Molinari F (2012) Chemoenzymatic deacylation of ramoplanin. Bioorg Med Chem Lett 22(16):5283–5287. https://doi.org/10.1016/j.bmcl.2012.06.046 Cudic P, Behenna DC, Kranz JK, Kruger RG, Wand AJ, Veklich YI, McCafferty DG (2002) Functional analysis of the lipoglycodepsipeptide antibiotic ramoplanin. Chem Biol 9(8):897–906. https://doi.org/10.1016/S1074-5521(02)00191-6 McCafferty DG, Cudic P, Frankel BA, Barkallah S, Kruger RG, Li W (2002) Chemistry and biology of the ramoplanin family of peptide antibiotics. Biopolymers 66:261–284. https://doi.org/10.1002/bip.10296 Di PS, Gandolfi R, Jovetic S, Marinelli F, Romano D, Molinari F (2007) A new bacterial mannosidase for the selective modification of ramoplanin and its derivatives. Enzyme Microb Technol 41(6–7):806–811. https://doi.org/10.1016/j.enzmictec.2007.07.013 Ciabatti R, Maffioli SI, Panzone G, Canavesi A, Michelucci E, Tiseni PS, Marzorati E, Checchia A, Giannnone M, Jabes D (2007) Synthesis and preliminary biological characterization of new semisynthetic derivatives of ramoplanin. J Med Chem 50(13):3077–3085. https://doi.org/10.1021/jm070042z Petraitiene R, Petraitis V, Groll AH, Candelario M, Sein T, Bell A, Lyman CA, McMillian CL, Bacher J, Walsh TJ (1999) Antifungal activity of LY303366, a novel echinocandin B, in experimental disseminated candidiasis in rabbits. Antimicrob Agents Chemother 43:2148–2155. https://doi.org/10.1128/AAC.43.9.2148 Wang XJ, Liu J, Zhang HZ, Wu K, Yang ZJ, Ning RN, Huang JH, Shao L (2021) Efficient bioconversion of daptomycin to its nucleus by heterologous expression of deacylase genes in Streptomyces . J Chem Technol Biotechnol 96(11):3066–3073. https://doi.org/10.1002/jctb.6858 Luzhetskii AN, Ostash BE, Fedorenko VA (2001) Intergeneric conjugation Escherichia coli-Streptomyces globisporus 1912 using integrative plasmid pSET152 and its derivatives. Russ J Genet 37:1123–1129. https://doi.org/10.1023/a:1012344319564 Tabakov V, Voeikova TA, Tokmakova IL, Bolotin AP, Vavilova E, Lomovskaia D (1994) Intergeneric Escherichia coli - Streptomyces conjugation as a means for the transfer of conjugative plasmids into producers of antibiotics chlortetracycline and bialaphos. Genetika 30:57–61. https://doi.org/10.1111/1468-5965.00207 Yasutake Y, Kusada H, Ebuchi T, Hanada S, Kamagata Y, Tamura T, Kimura N (2017) Bifunctional quorum-quenching and antibiotic-acylase MacQ forms a 170-kDa capsule-shaped molecule containing spacer polypeptides. Sci rep 7(1):1–11. https://doi.org/10.1038/s41598-017-09399-4 Ueda S, Shibata T, Ito K, Oohata N, Yamashita M, Hino M, Yamada M, Isogai Y, Hashimoto S (2011) Cloning and expression of the FR901379 acylase gene from Streptomyces sp. no. 6907. J Antibiot (Tokyo) 64:169–175. https://doi.org/10.1038/ja.2010.151 Junji I, Hideo T, Haruo IM (1993) Efficient production of aculeacin a acylase in recombinant Streptomyces strains. Appl Microbiol Biotechnol 39:532–536. https://doi.org/10.1007/BF00205046 Parmar VS, Prasad AK, Singh PK (1992) Lipase-catalysed transesterifications using 2, 2, 2-trifluoroethyl butyrate: effect of temperature on rate of reaction and enantioselectivity. Tetrahedron Asymmetry 3(11):1395–1398. https://doi.org/10.1016/0957-4166(92)80015-O M Cole () (1969) Deacylation of acylamino compounds other than penicillins by the cell-bound penicillin acylase of Escherichia coli . Biochem J 115(4):741–745. https://doi.org/ 10.1042/bj1150741 Liu X, Yang M, Liu Y, Ge F, Zhao J (2020) Structural and Functional Insights into a Lysine Deacylase in the Cyanobacterium Synechococcus sp. PCC 7002 Plant Physiol 184(2):762–776. https://doi.org/10.1104/pp.20.00583 Seemüller E, Lupas A, Stock D, Löwe J, Huber R, Baumeister W (1995) Proteasome from Thermoplasma acidophilum : a threonine protease. Science 268(5210):579–582. https://doi.org/10.1126/science.7725107 Duggleby HJ, Tolley SP, Hill CP, Dodson EJ, Dodson G, Moody PC (1995) Penicillin acylase has a single-amino-acid catalytic centre. Nature 373(6511):264–268. https://doi.org/10.1038/373264a0 Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 01 Jun, 2022 Reviewers invited by journal 01 Jun, 2022 Editor assigned by journal 16 May, 2022 First submitted to journal 14 May, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1655594","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":110437958,"identity":"4ac45aca-4434-48c5-b40f-af1f7b5e708a","order_by":0,"name":"Xiaojing Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYHCCxAeJf/7JsbE3HyBaS7LBx4YDxnw8xxKI1sImObPhQOI8iRwF4tQb3Eh4IM274056G0MOA8OPim1EaDlzIMGY98yz3DaGswcYe87cJqzF7HhDQjIPG3NuG2NfAjNjGzFaDjMkHAZqSWdj5jEgUsvxhsTGmW2HE9jYiNVif+ZAMsOHM2mGbTxsCQeJ8ovkjJz0HwkVNvLy8x8ffPCjgggtDAw8CXDmAWLUAwE7sQpHwSgYBaNgxAIA0KxA6nteU98AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-9636-6511","institution":"Shanghai University of Medicine and Health Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xiaojing","middleName":"","lastName":"Wang","suffix":""},{"id":110437959,"identity":"c73b80f5-ad5c-4468-b5aa-d639936aca94","order_by":1,"name":"Kai Wu","email":"","orcid":"","institution":"Shanghai University of Medicine and Health Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Wu","suffix":""},{"id":110437960,"identity":"0fd1de82-d335-4e8b-beca-7da4b231c709","order_by":2,"name":"Hanzhi Zhang","email":"","orcid":"","institution":"Abiochem biotechnology Co., Ltd","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hanzhi","middleName":"","lastName":"Zhang","suffix":""},{"id":110437961,"identity":"30b47c53-0259-4c18-8715-93f3a677656c","order_by":3,"name":"Jing Liu","email":"","orcid":"","institution":"Shanghai University of Medicine and Health Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Liu","suffix":""},{"id":110437962,"identity":"bf2e5e73-ffff-4269-9037-1b0b4e5ba5ff","order_by":4,"name":"Zhijun Yang","email":"","orcid":"","institution":"Shanghai University of Medicine and Health Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhijun","middleName":"","lastName":"Yang","suffix":""},{"id":110437963,"identity":"82a08944-5e3c-4741-8278-924607e91778","order_by":5,"name":"Jing Bai","email":"","orcid":"","institution":"Suzhou University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Bai","suffix":""},{"id":110437964,"identity":"fb0fc97b-0abf-489a-88c9-7f18b7ba630a","order_by":6,"name":"Hao Liu","email":"","orcid":"","institution":"Shanghai institute for food and drug control","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Liu","suffix":""},{"id":110437965,"identity":"d583b28b-be9a-4233-ac6f-02e96f317226","order_by":7,"name":"Lei Shao","email":"","orcid":"https://orcid.org/0000-0003-4130-2817","institution":"Shanghai University of Medicine and Health Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Shao","suffix":""}],"badges":[],"createdAt":"2022-05-14 08:34:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1655594/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1655594/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":22242749,"identity":"2ce4d529-e221-41ed-9d65-939b2531fb45","added_by":"auto","created_at":"2022-06-03 22:20:10","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":28868,"visible":true,"origin":"","legend":"\u003cp\u003eBioconversion of polymyxin B1 to its cyclic heptapeptide by deacylase.\u003c/p\u003e","description":"","filename":"Fig.1B.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1655594/v1/72d15178a1bfb75edd9a9cc1.jpg"},{"id":22242756,"identity":"2f7dacba-a47e-4fec-aefa-99672b58c879","added_by":"auto","created_at":"2022-06-03 22:20:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":49286,"visible":true,"origin":"","legend":"\u003cp\u003eGenotype\u0026nbsp;identification\u0026nbsp;of six \u003cem\u003eStreptomyces\u003c/em\u003e recombinant strains by PCR reaction. Lane 1-6, 3.3 kb products for SLI701, SCO701, SAL701, SAV701, SAM701 and SGR701. Marker: 100, 250, 500, 750, 1000, 1500, 2000, 3000, 5000 and 10000 bp.\u003c/p\u003e","description":"","filename":"Fig.2C.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1655594/v1/869492fda1302a99863692be.jpg"},{"id":22242753,"identity":"6a52991d-188f-4fb0-aeff-6a083010e16d","added_by":"auto","created_at":"2022-06-03 22:20:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":205560,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC profiles of deacylation reaction mixtures by \u003cem\u003eA. utahensis\u003c/em\u003e NRRL 12052 or different recombinants. Standard polymyxin B1 and its cyclic heptapeptide were shown as control. (A) Standard polymyxin B1; (B) standard polymyxin B1 cyclic heptapeptide; (C) \u003cem\u003eA. utahensis\u003c/em\u003e NRRL 12052; (D-I) SLI701, SCO701, SAL701, SAV701, SAM701 and SGR701.\u003c/p\u003e","description":"","filename":"Fig.3C.png","url":"https://assets-eu.researchsquare.com/files/rs-1655594/v1/c5ec2d039bbb845747e5f10e.png"},{"id":22242752,"identity":"1978bc91-f566-4cf6-a90c-f3f6ff8bc2a7","added_by":"auto","created_at":"2022-06-03 22:20:12","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":61578,"visible":true,"origin":"","legend":"\u003cp\u003eMS data of polymyxin B1 (4a) and its cyclic heptapeptide (4b)\u003c/p\u003e","description":"","filename":"Fig4B.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1655594/v1/f5557fdd627160d29066676c.jpg"},{"id":22242755,"identity":"d666e811-c9af-4cf7-9a15-2c0c88505848","added_by":"auto","created_at":"2022-06-03 22:20:14","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":21678,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of temperature on bioconversion of polymyxin B1 by SAL701\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1655594/v1/7bfc1f9f553a35dfde3ef815.jpg"},{"id":22242754,"identity":"c2d9c042-8e52-41ab-8bbf-76ab994c9ce6","added_by":"auto","created_at":"2022-06-03 22:20:13","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":20489,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of reaction time on bioconversion of polymyxin B1 by SAL701\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1655594/v1/ad7e93c795ad7d1d4fbad4ea.jpg"},{"id":22242751,"identity":"ed702f59-540e-42f2-abf7-ea4d5bcf61ec","added_by":"auto","created_at":"2022-06-03 22:20:11","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":25639,"visible":true,"origin":"","legend":"\u003cp\u003eThermostability of SAL701 at 4 (●), 30 (◆) and 50℃ (▲)\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1655594/v1/f51fa8c24ec9d0569e70f3c8.jpg"},{"id":22242750,"identity":"2ab5677c-23ce-4c6d-91a7-71d6fae9bc81","added_by":"auto","created_at":"2022-06-03 22:20:11","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":155641,"visible":true,"origin":"","legend":"\u003cp\u003eSubstrate docking based on structure model of deacylase from\u003cem\u003e A.utahensis\u003c/em\u003e NRRL 12052. The substrate is colored in yellow and potential catalytic residue Thr190 is colored in purple.\u003c/p\u003e","description":"","filename":"Fig.8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1655594/v1/16ed1978a83b0e67b4e90a6e.jpg"},{"id":22242787,"identity":"7f97a64d-832c-412a-b453-4337766cc1a0","added_by":"auto","created_at":"2022-06-03 22:20:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":667877,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1655594/v1/23f11a0f-2ed7-4ae9-945b-360447712f58.pdf"}],"financialInterests":"","formattedTitle":"Efficient side-chain deacylation of polymyxin B1 in recombinant Streptomyces strains","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePolymyxins are antibacterial polypeptides first isolated from cultures of \u003cem\u003eBacillus polymyxa\u003c/em\u003e strains, and polymyxin B1 is one of the main components, which is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e The core scaffold contains a cyclic heptapeptide linked to the side chain of a linear tripeptide with an N-terminal fatty acyl.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e They have been reused as \u0026ldquo;last resort\u0026rdquo; therapy option for multidrug-resistant Gram-negative bacteria.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e The situation is worsen by the emergence of polymyxin-resistant infections. The development of novel polymyxin derivatives increase its activity and reduce toxicity.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e The antibacterial activity of polymyxin B1 is mainly affected by changing the amino acid of the side chain.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Numerous novel compounds have been semi-synthesized by deacylating and replacing their side chains with a variety of different chains.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Cui replaced the amino acid l-Dab in position 3 of polymyxin B1 with d-Ser to synthesize polymyxin S2, which showed enhanced activity against \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e and \u003cem\u003eKlebsiella pneumonia\u003c/em\u003e, and reduced toxicity.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Therefore, polymyxin B1 analogues with increased antibacterial activity can generate by direct modifications of the cyclic peptide.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEnzyme modification is an efficient strategy for deacylation. Deacylase from \u003cem\u003eActinoplanes utahensis\u003c/em\u003e hydrolyzes the amide bonds of cyclic lipopeptides, which has broad substrate specificity for both the acyl side chain and cyclic peptide analogs of echinocandins, ramoplanin, daptomycin and other important antibiotics.\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e The enzyme hydrolyze the acyl moieties of antibiotic echinocandins, and forms a cyclic hexapeptide moiety and long-chain fatty acid.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e The resulting cyclic hexapeptide can be further reacylated to produce a series of analogs useful as therapeutic antibiotics in clinical practice, such as anidulafungin.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Ramoplanin is a lipoglycodepsipeptide with antibacterial activity against major Gram-positive bacteria.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Numerous novel ramoplanin derivatives have been semi-synthesized by deacylating and replacing their side chains with different kinds of carboxylic acids.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Among them, the 2-methylphenylacetic acyl group, displays increased tolerability and high activity, indicating its potential for overcoming the limitations of ramoplanin \u003cem\u003ein vivo\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Deacylation is the key step in generating the aforementioned antibiotics, and deacylase may be useful in the antibiotic industry for catalyzing the hydrolysis of not only echinocandins and ramoplanin, but also teicoplanin, daptomycin and its derivatives, as well as other related antibiotics with corresponding acyl side chains.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn this study, we attempted to expand the substrate spectrum of recombinant \u003cem\u003eStreptomyces\u003c/em\u003e with deacylase gene, and we found that the enzyme could hydrolyze polymyxin B1. In addition, we tried to predict the specificity of the catalytic site through structure analysis, and payed attention to its potential biotechnological application.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eStrains and \u003c/strong\u003e\u003cstrong\u003eculture medium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. lividans\u003c/em\u003e TK24, \u003cem\u003eS. coelicolor\u003c/em\u003e, \u003cem\u003eS. albus\u003c/em\u003e, \u003cem\u003eS. avermitilis \u003c/em\u003eK139, \u003cem\u003eS. ambofaciens \u003c/em\u003e2283, \u003cem\u003eS. griseus\u003c/em\u003e, and\u003cem\u003e A. utahensis\u003c/em\u003e NRRL 12052 were stored in our laboratory. \u003cem\u003eEscherichia coli\u003c/em\u003e ET12567/PUZ8002, a donor strain for conjugation between \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eStreptomyc\u003c/em\u003e\u003cem\u003ees\u003c/em\u003e, was purchased from Huayueyang Biological Technology Co., Ltd. (Beijing, China). Plasmids pDS701, pSET152 containing a deacylase gene under the control of PermE, were constructed in our laboratory as described earlier.\u003csup\u003e21 \u003c/sup\u003eAll chemicals, biochemicals, restriction enzymes, media and molecular biological reagents were of analytical grade and obtained from standard commercial sources. \u003c/p\u003e\n\u003cp\u003eGauserime synthetic agar plates (2% soluble starch, 0.05% NaCl, 0.05%K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u0026middot;3H\u003csub\u003e2\u003c/sub\u003eO,0.1% KNO\u003csub\u003e3\u003c/sub\u003e, 0.05% MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 0.001% FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, and 1.5% agar powder [pH 7.4]) were used to culture strains for sporulation. Seed medium (2.5% sucrose, 2.0% oatmeal, 0.25% yeast powder, 0.1% K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 0.05% KCl, 0.05%MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, and 0.0002% FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO) and fermentation medium (2% sucrose, 1% peanut meal, 0.1% KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, and 0.025% MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO) were used to ferment \u003cem\u003eA. utahensis. \u003c/em\u003eAnd seed medium (1.0% glucose, 0.5% yeast powder, and 1% peptone) and fermentation medium (2.5% glucose, 1% beanflour, 0.3% NaCl, and 0.3% CaCO\u003csub\u003e3\u003c/sub\u003e) were used to ferment\u003cem\u003e Streptomyces.\u003c/em\u003e\u003csup\u003e21\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHeterologous over-expression of deacylase gene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasmid pDS701 was introduced into different \u003cem\u003eStreptomyces\u003c/em\u003e by intergeneric conjugation from E. coli ET12567 according to standard procedures.\u003csup\u003e22\u003c/sup\u003e Recombinant strains were cultured in medium supplemented with 50 \u0026mu;g/mL apramycin, and further confirmed using PCR amplification with universal primers M13F-47 and M13R-48. Wild-type and recombinant cultures were grown on Gauserime synthetic agar plates at 30\u0026deg;C for sporulation. An agar piece\u003csup\u003e \u003c/sup\u003ewas inoculated into 50 mL seed medium\u003cem\u003e \u003c/em\u003efor 30 h at 30\u0026deg;C on a shaker at 220 rpm, the resulting mycelial suspension was plused (2%) to fresh fermentation medium and further incubated for 48 h. \u003cem\u003eS. albus\u003c/em\u003e without the plasmid was fermented as a control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of bioconversion efficiency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe used the whole-cell reaction system for bioconversion, because it is convenience, high efficiency, reusability and safety. The wet mycelia were sedimented by centrifugation and washed twice with 0.1 M Tris buffer (pH 8.0). The 10 g cell pellet (20% m/v) was resuspended in a 50 mL Tris buffer. The enzymatic reaction was initiated by adding polymyxin B1 (2 g/L) and allowed to continue for 5 h at 30\u0026deg;C with shaking at 50 rpm. The reaction mixture was immersed in an ice bath and stopped by adding an equivalent volume of methanol. The mixtures were centrifuged, and the supernatants were filtered through a 0.22 \u0026mu;m pore diameter membrane.\u003c/p\u003e\n\u003cp\u003eAn analytical HPLC system was used to measure the cyclic heptapeptide polymyxin B1 released during the hydrolysis of polymyxin B1. Chromatographic separation was carried out on a Waters 2695 HPLC System (Milford, MA, USA) consisting of analytical C18 column (250 \u0026times; 4.6 mm; 5 \u0026mu;m; Agilent Technologies, Santa Clara, CA, USA) with a mixture of sodium sulfate solution (30 mM, pH was adjusted to 2.3 with phosphoric acid) and acetonitrile in a ratio of 77.5:22.5 V/V as the mobile phase.\u003csup\u003e7\u003c/sup\u003e HPLC was performed at a flow rate of 0.8 mL/min for 35 min with UV detection at 215 nm. The compounds were identified using ESI-MS analysis performed on an Agilent HPLC 1260 coupled with a 6550 quadrupole time-of-flight massspectrometry system. The method was linear at concentrations of 0.5-1500 \u0026mu;M with a correlation coefficient of 0.999. A standard curve was drawn using different amounts of cyclic heptapeptide polymyxin B1 and peak areas. The molar bioconversion rates were compared according to the following formula: bioconversion rate = ([product of daptomycin cyclic heptapeptide polymyxin B1]/substrate addition) \u0026times; 100%.\u003csup\u003e23\u003c/sup\u003e All enzyme measurements were performed in triplicate, and the maximum error was less than 5%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOptimization of bioconversion conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEnzymatic activity was determined using the standard assay conditions for each case but using different buffers. The effect of pH was examined at different pH values ranging from 3.0 to 8.0 in disodium hydrogen phosphate-citrate buffer, from pH 5.5 to 8.0 in phosphate buffer, and from pH 7.5 to 10.0 in Tris buffer at 0.1 M constant ionic strength and 30\u0026deg;C by 2 g/L polymyxin B1. The effect of metal ions was evaluated by adding 0.2 M KCl, NaCl, MgCl\u003csub\u003e2\u003c/sub\u003e, CuSO\u003csub\u003e4\u003c/sub\u003e or FeSO\u003csub\u003e4 \u003c/sub\u003eto the Tris buffer. The effect of the ion concentration in the phosphate buffer and Tris buffer was also tested from 0.05 to 0.3 M.\u003c/p\u003e\n\u003cp\u003eThe effect of temperature of SAL701 was measured at temperatures ranging from 25\u0026deg;C to 70\u0026deg;C in 0.1 M Tris buffer pH 8.0 by 2 g/L polymyxin B1. The reaction time was prolonged to 96 h to determine the biotransformation efficiency. \u003c/p\u003e\n\u003cp\u003eThe optimum concentration of substrate was determined by adding different amounts of polymyxin B1 (1-12 g/L) under the optimized conditions in a total reaction volume of 200 mL using wild-type \u003cem\u003eA. utahensis\u003c/em\u003e NRRL 12052, recombinant strains SAL701, SCO701 and SAM701.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003cstrong\u003ehermostability\u003c/strong\u003e\u003cstrong\u003e and recycling of SAL701\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mycelia of \u003cem\u003eStreptomyces\u003c/em\u003e strains carrying the deacylase gene were incubated at 4, 30 and 50\u0026deg;C under the optimized conditions. At different time points during incubation, mycelia samples were withdrawn and assayed to determine their deacylase activity using polymyxin B1 as a substrate. \u003c/p\u003e\n\u003cp\u003eThe mycelia were added to a 50 mL reaction buffer containing 2 g/L polymyxin B1 under the optimized conditions. After the enzymatic reaction for 5 h, the solution was centrifuged at 3000 \u0026times;\u003cem\u003eg\u003c/em\u003e for 30 min, and the supernatant was measured using HPLC as described above. The recovered mycelia were washed with Tris buffer for three times and used for another bioconversion cycle.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe deacylase homology protein structure model was built based on the crystal structure (PDB ID 5C9I; 39.1% identity) using Discovery Studio 2016, and the model with the lowest DOPE score (-79981) was selected for docking.\u003csup\u003e24\u003c/sup\u003e Substrates polymyxin B1 for deacylase were modeled in Chem3D. Substrate docking was performed using Autodock4. The substrate was docked into the binding pocket using flexible docking. AutoDock tools were used for enzyme and substrate preparations. To encompass the entire substrate-binding pocket, the docking box was set to 60 \u0026times; 60 \u0026times; 60 grid points with a grid spacing of 0.375 \u0026Aring;. The box center was set as X = 19.336, Y = -0.931, and Z = 52.155.\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003e\u003cstrong\u003eOver-expression of deacylase gene in \u003cem\u003eStreptomyces \u003c/em\u003ehosts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEnzymatic modification is more efficient than chemical modification, but deacylase was rate-limiting in the antibiotics production process, as the bioconversion efficiency of deacylases in the original strain is low. \u003cem\u003eStreptomyces\u003c/em\u003e strains are suitable heterologous hosts for producing enzymes, particularly GC-rich deacylases, for industrial applications.\u003csup\u003e25,26 \u003c/sup\u003eTo investigate the effect of deacylase gene, pDS701 plasmid consist of deacylase gene was introduced into six \u003cem\u003eStreptomyces\u003c/em\u003e species (\u003cem\u003eS. lividans\u003c/em\u003e TK24, \u003cem\u003eS. coelicolor\u003c/em\u003e, \u003cem\u003eS. albus\u003c/em\u003e, \u003cem\u003eS. avermitilis \u003c/em\u003eK139, \u003cem\u003eS. ambofaciens \u003c/em\u003e2283 and \u003cem\u003eS. griseus\u003c/em\u003e), yielding recombinant strains SLI701, SCO701, SAL701, SAV701, SAM701 and SGR701.\u003csup\u003e13\u003c/sup\u003e There were no apparent phenotypic differences between\u003cem\u003e \u003c/em\u003e\u003cem\u003eStreptomyces\u003c/em\u003e and its recombinant strains. Genotypes of six recombinant strains were verified by polymerase chain reaction (PCR). The 3.3-Kb DNA fragments were amplified. Sequencing and alignment confirmed that all six segments had 100% identity with the deacylase gene of recombinant plasmid pDS701 (Fig. 2). As negative controls, PCR products could not be amplified from \u003cem\u003eS. albus\u003c/em\u003e host and wild-type \u003cem\u003eA. utahensis\u003c/em\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeacylation of different recombinant \u003cem\u003eStreptomyces\u003c/em\u003e strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix recombinant \u003cem\u003eStreptomyces\u003c/em\u003e strains and \u003cem\u003eA. utahensis\u003c/em\u003e NRRL 12052 were inoculated, the mycelia were collected to determine the bioconversion efficiency. The reaction mixture containing 2 g/L polymyxin B1 in 0.1 M Tris buffer pH8.0, was incubated at 30℃, 5 h and then analysed by high-performance liquid chromatography (HPLC). All seven samples showed the new peak, which almost having the same retention time with the standard cyclic heptapeptide moiety of polymyxin B1 (4.56 min, Fig. 3b), and a peak of remaining polymyxin B1 substrate (31.10 min, Fig. 3a). The overlapped peak also obtained by injecting the mixture of SAL701 sample and standard cyclic heptapeptide polymyxin B1. So we preliminary verified that all six recombinant species and wild-type strain could deacylate polymyxin B1, with different activity dependent on the host strains. As shown in Table 1, five of the six recombinant strains showed similar bioconversion efficiencies, and although SAV701 showed a lower conversion efficiency (Fig. 3g), it was still higher than that of the wild-type strain (34.0% compared with 23.3%). SAL701 (Fig. 3f) showed the highest efficiency with the highest percentage of bioconversion rate (65.2%). No polymyxin B1 or its cyclic heptapeptide moiety was detected in the whole-cells of all seven\u003cem\u003e \u003c/em\u003especies examined. Therefore, the \u003cem\u003eS. albus\u003c/em\u003e were considered as the preferred host for enzymatic deacylation. SAL701 strain has many advantages such as easy-cultivating, rapid-growing and high bioconversion efficiency, so it could be applied to bioconversion of polymyxin B1 in a short time.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1 Bioconversion rate by wild-type and different recombinant \u003cem\u003eStreptomyces\u003c/em\u003e strains \u003c/strong\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"26.55367231638418%\"\u003e\n \u003cp\u003eStrains\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"49.90583804143126%\"\u003e\n \u003cp\u003eDescription\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.540489642184557%\"\u003e\n \u003cp\u003eBioconversion rate (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"26.55367231638418%\"\u003e\n \u003cp\u003e\u003cem\u003eA. utahensis NRRL \u003c/em\u003e12052\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"49.90583804143126%\"\u003e\n \u003cp\u003ewild-type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.540489642184557%\"\u003e\n \u003cp\u003e23.3 \u0026plusmn; 3.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"26.55367231638418%\"\u003e\n \u003cp\u003eSLI701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"49.90583804143126%\"\u003e\n \u003cp\u003epDS701 integrated into \u003cem\u003eS. lividans\u003c/em\u003eTK24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.540489642184557%\"\u003e\n \u003cp\u003e58.1 \u0026plusmn; 2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"26.55367231638418%\"\u003e\n \u003cp\u003eSCO701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"49.90583804143126%\"\u003e\n \u003cp\u003epDS701 integrated into \u003cem\u003eS. coelicolor \u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.540489642184557%\"\u003e\n \u003cp\u003e60.7 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"26.55367231638418%\"\u003e\n \u003cp\u003eSAL701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"49.90583804143126%\"\u003e\n \u003cp\u003epDS701 integrated into \u003cem\u003eS. albus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.540489642184557%\"\u003e\n \u003cp\u003e65.2 \u0026plusmn; 4.5\u003cem\u003e \u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"26.55367231638418%\"\u003e\n \u003cp\u003eSAV701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"49.90583804143126%\"\u003e\n \u003cp\u003epDS701 integrated into \u003cem\u003eS. avermitilis \u003c/em\u003eK139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.540489642184557%\"\u003e\n \u003cp\u003e34.0 \u0026plusmn; 3.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"26.55367231638418%\"\u003e\n \u003cp\u003eSAM701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"49.90583804143126%\"\u003e\n \u003cp\u003epDS701 integrated into \u003cem\u003eS. ambofaciens \u003c/em\u003e2283\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.540489642184557%\"\u003e\n \u003cp\u003e59.8 \u0026plusmn; 0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"26.55367231638418%\"\u003e\n \u003cp\u003eSGR701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"49.90583804143126%\"\u003e\n \u003cp\u003epDS701 integrated into \u003cem\u003eS. griseus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.540489642184557%\"\u003e\n \u003cp\u003e52.3 \u0026plusmn; 1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"26.55367231638418%\"\u003e\n \u003cp\u003e\u003cem\u003eS. albus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"49.90583804143126%\"\u003e\n \u003cp\u003e\u003cem\u003eStreptomyces\u003c/em\u003e without plasmid as negative control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.540489642184557%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eQualitative analysis of polymyxin B1 and its cyclic heptapeptide\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePolymyxin B1 and its cyclic heptapeptide moiety were extracted from the reaction mixture, and their identities were confirmed. The structure of the polymyxin B1 cyclic heptapeptide moiety was verified by using electrospray ionization-mass spectrometry (ESI-MS). The parent ion was observed at m/z 784.4441 [M+Na]\u003csup\u003e+\u003c/sup\u003e (calculated 784.4440), and its mass spectrum was consistent with the molecular formula C\u003csub\u003e35\u003c/sub\u003eH\u003csub\u003e59\u003c/sub\u003eN\u003csub\u003e11\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e (Fig. 4a). Polymyxin B1 showed parent ions at m/z 602.3822 [M+2H]\u003csup\u003e2+\u003c/sup\u003e (calculated 602.3822) and m/z 1203.7566 [M+H]\u003csup\u003e+\u003c/sup\u003e (calculated 1203.7572), and its mass spectrum was consistent with the molecular formula C\u003csub\u003e56\u003c/sub\u003eH\u003csub\u003e98\u003c/sub\u003eN\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e13\u003c/sub\u003e (Fig. 4b).\u003csup\u003e7 \u003c/sup\u003eImpure preparations, polymyxin B containing polymyxin B1, were also successfully deacylated but at a slower rate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of \u003c/strong\u003e\u003cstrong\u003ereaction\u003c/strong\u003e\u003cstrong\u003e buffer on bioconversion of polymyxin B1 by SAL701\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe buffer of reaction mixture strongly affects the bioconversion rate. Hydrolysis of polymyxin B1 catalyzed by SAL701 was evaluated in different 0.1 M buffer pH range of 3.0-10.0 at 30 \u0026deg;C for 5 h. As shown in Table 2, the strain displayed the highest activity at pH 8.0 in Tris buffer (65.2%), which was selected as the standard buffer for deacylation. The same pH value (pH 7.5 or 8.0) in three different buffers were compared, and found that Tris buffer (60.8% and 65.2%) was more suitable for reaction than phosphate buffer (60.5% and 62.9%) and disodium hydrogen phosphate-citrate buffer (53.2% and 52.3%). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2 Effect of reaction buffer\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" width=\"83.87096774193549%\"\u003e\n \u003cp\u003eBioconversion rate (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"45.51282051282051%\"\u003e\n \u003cp\u003edisodium hydrogen phosphate-citrate buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003ephosphate buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.153846153846153%\"\u003e\n \u003cp\u003eTris buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.17204301075269%\"\u003e\n \u003cp\u003e21.2 \u0026plusmn; 1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.956989247311828%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.741935483870968%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.17204301075269%\"\u003e\n \u003cp\u003e19.3 \u0026plusmn; 3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.956989247311828%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.741935483870968%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.17204301075269%\"\u003e\n \u003cp\u003e23.6 \u0026plusmn; 0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.956989247311828%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.741935483870968%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.17204301075269%\"\u003e\n \u003cp\u003e25.9 \u0026plusmn; 4.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.956989247311828%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.741935483870968%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.17204301075269%\"\u003e\n \u003cp\u003e26.9 \u0026plusmn; 3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.956989247311828%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.741935483870968%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.17204301075269%\"\u003e\n \u003cp\u003e33.3 \u0026plusmn; 2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.956989247311828%\"\u003e\n \u003cp\u003e31.9 \u0026plusmn; 2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.741935483870968%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.17204301075269%\"\u003e\n \u003cp\u003e42.8 \u0026plusmn; 3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.956989247311828%\"\u003e\n \u003cp\u003e41.3 \u0026plusmn; 4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.741935483870968%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e6.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.17204301075269%\"\u003e\n \u003cp\u003e41.4 \u0026plusmn; 2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.956989247311828%\"\u003e\n \u003cp\u003e50.9 \u0026plusmn; 3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.741935483870968%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e7.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.17204301075269%\"\u003e\n \u003cp\u003e42.9 \u0026plusmn; 4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.956989247311828%\"\u003e\n \u003cp\u003e55.6 \u0026plusmn; 3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.741935483870968%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.17204301075269%\"\u003e\n \u003cp\u003e53.2 \u0026plusmn; 0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.956989247311828%\"\u003e\n \u003cp\u003e60.5 \u0026plusmn; 0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.741935483870968%\"\u003e\n \u003cp\u003e60.8 \u0026plusmn; 3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.17204301075269%\"\u003e\n \u003cp\u003e52.3 \u0026plusmn; 1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.956989247311828%\"\u003e\n \u003cp\u003e62.9 \u0026plusmn; 0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.741935483870968%\"\u003e\n \u003cp\u003e65.2 \u0026plusmn; 4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e8.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.17204301075269%\"\u003e\n \u003cp\u003e\u003cem\u003e \u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.956989247311828%\"\u003e\n \u003cp\u003e\u003cem\u003e \u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.741935483870968%\"\u003e\n \u003cp\u003e64.7 \u0026plusmn; 1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e9.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.17204301075269%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.956989247311828%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.741935483870968%\"\u003e\n \u003cp\u003e61.5 \u0026plusmn; 1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e9.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.17204301075269%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.956989247311828%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.741935483870968%\"\u003e\n \u003cp\u003e62.2 \u0026plusmn; 2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e10.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.17204301075269%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.956989247311828%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.741935483870968%\"\u003e\n \u003cp\u003e60.3 \u0026plusmn; 2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eAlso, the activities were increased when the ionic strength of buffer was increased (0.05-0.3 M phosphate buffer and Tris buffer). The bioconversion efficiency increased from 56.3% to 70.1% in Tris buffer and from 52.8% to 61.2% in phosphate buffer (Table 3). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3 Effect of ionic and ionic strength \u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"23.68972746331237%\"\u003e\n \u003cp\u003eBuffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"12.368972746331236%\"\u003e\n \u003cp\u003eIonic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" width=\"63.941299790356396%\"\u003e\n \u003cp\u003eBioconversion rate (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0.05 M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0.1 M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0.2 M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0.3 M\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"23.73949579831933%\"\u003e\n \u003cp\u003ephosphate buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.394957983193278%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e52.8 \u0026plusmn; 1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e55.6 \u0026plusmn; 3.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e59.0 \u0026plusmn; 2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e61.2 \u0026plusmn; 1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"23.73949579831933%\"\u003e\n \u003cp\u003eTris buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.394957983193278%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e56.3 \u0026plusmn; 4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e65.2 \u0026plusmn; 1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e70.1 \u0026plusmn; 2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e69.2 \u0026plusmn; 1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"23.73949579831933%\"\u003e\n \u003cp\u003eTris buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.394957983193278%\"\u003e\n \u003cp\u003eK\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e73.9 \u0026plusmn; 3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"23.73949579831933%\"\u003e\n \u003cp\u003eTris buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.394957983193278%\"\u003e\n \u003cp\u003eNa\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e72.1 \u0026plusmn; 1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"23.73949579831933%\"\u003e\n \u003cp\u003eTris buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.394957983193278%\"\u003e\n \u003cp\u003eMg\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e74.7 \u0026plusmn; 2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"23.73949579831933%\"\u003e\n \u003cp\u003eTris buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.394957983193278%\"\u003e\n \u003cp\u003eCu\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e69.2 \u0026plusmn; 2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"23.73949579831933%\"\u003e\n \u003cp\u003eTris buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.394957983193278%\"\u003e\n \u003cp\u003eFe\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e72.7 \u0026plusmn; 1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.966386554621849%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eDeacylase activity did not require metal ions as previously studied. But the inclusion of an ion (0.2 M K\u003csup\u003e+\u003c/sup\u003e, Na\u003csup\u003e+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e or Fe\u003csup\u003e2+\u003c/sup\u003e), enhanced the deacylase activity (73.9%, 72.1%, 74.7% and 72.7% respectively, compared to 70.1% for enzyme alone, Table 3). Addition of Cu\u003csup\u003e2+ \u003c/sup\u003edid not increase the activity. Therefore, 0.2 M Tris buffer pH 8.0 with 0.2 M Mg\u003csup\u003e2+ \u003c/sup\u003ewas selected as the standard condition for the deacylation reaction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of \u003c/strong\u003e\u003cstrong\u003ereaction\u003c/strong\u003e\u003cstrong\u003e temperature and time on bioconversion of polymyxin B1 by SAL701\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe enzymatic activity varies over a temperature range of 25-70\u0026deg;C. Maximum deacylase activity was achieved at 40\u0026deg;C under the optimized reaction buffer (0.2 M Tris buffer, pH 8.0 with 0.2 M Mg\u003csup\u003e2+\u003c/sup\u003e), the conversion rate of polymyxin B1 was 76.9% (Fig. 5).\u003c/p\u003e\n\u003cp\u003eThe ability of SAL701 to deacylate polymyxin B1 was examined during 96 h. The time course was determined under the optimized buffer conditions. As shown in Fig. 6, the deacylated product occurred at the earliest time tested (5 h), and the bioconversion efficiency was 74.8%, and then gradually increased to 77.7% at 96 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of substrate concentration on bioconversion of polymyxin B1 by SAL701\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe optimum concentration of polymyxin B1 in the bioconversion mixture was confirmed using SAL701, SCO701, SAM701 recombinant strains and wild-type \u003cem\u003eA. utahensis\u003c/em\u003e NRRL 12052, the relatively highly active recombinant \u003cem\u003eStreptomyces\u003c/em\u003e strains. The optimal substrate concentration of SAL701 was increased from 2.0 to 6.0 g/L, under conditions in which the bioconversion efficiency was more than 60%, and SCO701, SAM701 were increased from 2.0 to 4.0 g/L (Table 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4 Effect of substrate concentration\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"22.2007722007722%\"\u003e\n \u003cp\u003epolymyxin B1 concentration (g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" width=\"77.7992277992278%\"\u003e\n \u003cp\u003eBioconversion rate (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"20.843672456575682%\"\u003e\n \u003cp\u003eSAL701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.843672456575682%\"\u003e\n \u003cp\u003eSCO701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.843672456575682%\"\u003e\n \u003cp\u003eSAM701\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"37.468982630272954%\"\u003e\n \u003cp\u003e\u003cem\u003eA. utahensis\u003c/em\u003e NRRL 12052\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.2007722007722%\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e78.4 \u0026plusmn; 3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e74.8 \u0026plusmn; 2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e75.1 \u0026plusmn; 2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.15057915057915%\"\u003e\n \u003cp\u003e39.4 \u0026plusmn; 2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.2007722007722%\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e73.9 \u0026plusmn; 1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e72.4 \u0026plusmn; 1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e72.3 \u0026plusmn; 2.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.15057915057915%\"\u003e\n \u003cp\u003e31.8 \u0026plusmn; 1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.2007722007722%\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e70.2 \u0026plusmn; 1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e69.4 \u0026plusmn; 1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e68.9 \u0026plusmn; 0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.15057915057915%\"\u003e\n \u003cp\u003e26.4 \u0026plusmn; 3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.2007722007722%\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e70.7 \u0026plusmn; 1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e66.4 \u0026plusmn; 2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e64.3 \u0026plusmn; 2.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.15057915057915%\"\u003e\n \u003cp\u003e22.5 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.2007722007722%\"\u003e\n \u003cp\u003e6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e61.2 \u0026plusmn; 0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e58.7 \u0026plusmn; 3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e56.1 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.15057915057915%\"\u003e\n \u003cp\u003e21.0 \u0026plusmn; 0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.2007722007722%\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e43.7 \u0026plusmn; 0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e43.6 \u0026plusmn; 3.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e50.4 \u0026plusmn; 1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.15057915057915%\"\u003e\n \u003cp\u003e23.0 \u0026plusmn; 1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.2007722007722%\"\u003e\n \u003cp\u003e10.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e47.3 \u0026plusmn; 1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e36.8 \u0026plusmn; 2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e41.4 \u0026plusmn; 1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.15057915057915%\"\u003e\n \u003cp\u003e19.5 \u0026plusmn; 1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.2007722007722%\"\u003e\n \u003cp\u003e12.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e33.9 \u0026plusmn; 1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e37.9 \u0026plusmn; 2.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.216216216216218%\"\u003e\n \u003cp\u003e30.1 \u0026plusmn; 3.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.15057915057915%\"\u003e\n \u003cp\u003e21.2 \u0026plusmn; 3.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003cstrong\u003ehermostability\u003c/strong\u003e\u003cstrong\u003e of SAL701\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe thermostability of the mycelia of recombinant \u003cem\u003eS. albus\u003c/em\u003e was evaluatedat different temperatures under the optimized conditions above (Fig. 7). The enzyme showed no decrease inactivity after storage at 4\u0026deg;C and 30\u0026deg;C for at least 48 h. However, the mycelia also maintained 90% of its activity following storage at 50\u0026deg;C for at least 12 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRecycling of mycelia of SAL701\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mycelia was used to bioconvert polymyxin B1, the number of times required for the mycelia to be reused was investigated. The mycelia of SAL701 could be recycled at least three times without loss of catalytic activity on the basis of maintaining the bioconversion efficiency at 70.2% under the optimized conditions above (Table 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5 \u003c/strong\u003e\u003cstrong\u003eNumber of \u003c/strong\u003e\u003cstrong\u003etimes of reutilizing the mycelia\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"48.80952380952381%\"\u003e\n \u003cp\u003eNumber of times\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"51.19047619047619%\"\u003e\n \u003cp\u003eBioconversion rate (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"48.80952380952381%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"51.19047619047619%\"\u003e\n \u003cp\u003e72.4 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"48.80952380952381%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"51.19047619047619%\"\u003e\n \u003cp\u003e73.5 \u0026plusmn; 3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"48.80952380952381%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"51.19047619047619%\"\u003e\n \u003cp\u003e70.2 \u0026plusmn; 3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"48.80952380952381%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"51.19047619047619%\"\u003e\n \u003cp\u003e50.3 \u0026plusmn; 3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"48.80952380952381%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"51.19047619047619%\"\u003e\n \u003cp\u003e30.9 \u0026plusmn; 1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"48.80952380952381%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"51.19047619047619%\"\u003e\n \u003cp\u003e23.4 \u0026plusmn; 1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"48.80952380952381%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"51.19047619047619%\"\u003e\n \u003cp\u003e6.3 \u0026plusmn; 1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"48.80952380952381%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"51.19047619047619%\"\u003e\n \u003cp\u003e6.9 \u0026plusmn; 2.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eSubstrate spectrum of SAL701\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe aforementioned results established the characteristic of SAL701 and necessitated further research about their substrate spectrum. The chemical structure of \u0026beta;-lactam antibiotics also consists a \u0026beta;-lactam ring and a side chain. Different \u0026beta;-lactam substrates were used to determine the bioconversion rate. But no enzymatic deacylation was observed for the \u0026beta;-lactam compounds penicillin G and cephalosporin C. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHydrolysis of specific amide bonds of SAL701\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEnzyme-catalyzed reactions are specific. Amidohydrolase from \u003cem\u003eE. coli \u003c/em\u003eN.C.I.B. 8743 is stereospecific for the deacylation of \u0026alpha;-amino acids, acylated L-\u0026alpha;-amino acids but not D-\u0026alpha;-amino acids are hydrolyzed, whereas DL-amino acids are only 50% hydrolyzed.\u003csup\u003e27-29\u003c/sup\u003e SAL701 catalyzes hydrolysis of amide bond 1 (Fig. 1), which is the closest amide bond to the cyclic heptapeptide of polymyxin B1, rather than at other amide bonds, suggesting its high specificity for the catalytic site. The specificity of deacylase could avoid the existence of impurities, reduce the difficulty in the final purification process, and improve the economic benefits of the process.\u003c/p\u003e\n\u003cp\u003eAn enzyme\u0026rsquo;s specificity depends on its structure. As a representative functional deacylase, the sequence similarity to many other deacylases. Although a few deacylase structures have been reported, that of a functional deacylase has not been determined. Homology protein structure models for deacylase was constructed to identify the structural basis for the programming of this distinct specificity (Fig. 8) based on the known crystal structure of the MacQ (PDB ID 5C9I; resolution: 1.80 \u0026Aring;, and identity of 39.1% with deacylase). MacQ derived from \u003cem\u003eAcidovorax\u003c/em\u003e sp. strain MR-S7 are comprised of 806 amino acid residues, and exhibits acylase activity against \u0026beta;-lactam antibiotics and N-acylhomoserine lactones (AHLs). Structural comparison with MacQ revealed that deacylase has a similar structure and active site organization, indicating that the key enzyme-co-product interactions among MacQ are well-conserved, deacylase and similar enzymes such as the catalytic site specificity. Ser/Thr/Cys is the necessary residue of catalysis, and initiates amide bond cleavage of various substrate compounds as a nucleophile. Similar to Ser1\u0026beta;, which act as a catalytic residue in MacQ, the docking results indicated that Thr190 in deacylase from \u003cem\u003eA.utahensis \u003c/em\u003eNRRL 12052, which located near amide bond 1, can facilitate hydrolysis with high regioselectivity.\u003csup\u003e24,30,31\u003c/sup\u003e The relatively close distance of the hydroxyl group to the amide carbon atom of the peptide bond, suggests an important role for the residues in catalysis, as revealed by substrate docking.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe previously reported the hydrolytic activity for the side chains of echinocandin B and daptomycin by the recombinant \u003cem\u003eStreptomy\u003c/em\u003eces, this drove us to explore different antibiotics of great significance.\u003csup\u003e13,21 \u003c/sup\u003eIn this study, we efficiently bioconversion polymyxin B1 to its cyclic heptapeptide, a starting material synthesizing antibiotics, which has not been reported before, and deacylase may be useful in the antibiotic industry for catalyzing the hydrolysis of polymyxin B1. We also tried to hydrolyze other important antibiotics with a similar structure, to expand the application of recombinant strain in the future.\u003c/p\u003e\n\u003cp\u003eFurthermore, we have determined the optimal reaction conditions for this process, 40 \u0026deg;C in 0.2 M Tris buffer (pH 8.0) with 0.2 M Mg\u003csup\u003e2+\u003c/sup\u003e. Also the mycelia of SAL701 was high thermostability and reusability. Additionally, catalytic site specificity of the deacylase was described and predicted by homology modeling and docking, which showed Thr190 facilitates hydrolysis with high regioselectivity. All above results show that recombinant \u003cem\u003eStreptomyces\u003c/em\u003e strain SAL701 is effective in catalysing polymyxin B1 to its cyclic heptapeptide moiety. The recombinant strain may be used for the industrial production of polymyxin antibiotics.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThis research was financially supported by the Natural Science Foundation of Shanghai (20ZR1424600), the National Natural Science Foundation of China (81773616), the Shanghai Excellent Technology Leader Program (17XD1423200), and Nature Science Foundation of Jiangsu Higher Education Institutions of China (20KJB180002).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests \u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp;\u003c/strong\u003eXiaojing Wang, Kai Wu and Hanzhi Zhang performed the experiments and data analyses, and wrote the manuscript. Lei Shao and Hao Liu designed the study plan. Jing Liu, Zhijun Yang and Jing Bai revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBrown P, Dawson M (2017) Development of new polymyxin derivatives for multi-drug resistant Gram-negative infections. J Antibiot 70:386\u0026ndash;394. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ja.2016.146\u003c/span\u003e\u003cspan address=\"10.1038/ja.2016.146\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe J, Abdelraouf K, Ledesma KR, Chow DSL, Tam VH (2013) Pharmacokinetics and efficacy of liposomal polymyxin B in a murine pneumonia model. 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Nature 373(6511):264\u0026ndash;268. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/373264a0\u003c/span\u003e\u003cspan address=\"10.1038/373264a0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biotechnology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bile","sideBox":"Learn more about [Biotechnology Letters](https://www.springer.com/journal/10529)","snPcode":"10529","submissionUrl":"https://submission.nature.com/new-submission/10529/3","title":"Biotechnology Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Deacylase, polymyxin B1, Streptomyces host, bioconversion, antibiotic","lastPublishedDoi":"10.21203/rs.3.rs-1655594/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1655594/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjectives: \u003c/strong\u003ePolymyxins are antibacterial polypeptides used as “last resort”\u0026nbsp;therapy option for multidrug-resistant Gram-negative bacteria. The expansion of polymyxin-resistant infections has inspired development of novel polymyxin derivatives, and deacylation is one of the critical steps in generating those antibiotics. Deacylase from \u003cem\u003eActinoplanes utahensis\u003c/em\u003e hydrolyze the acyl moieties of echinocandins, and also efficiently deacylates daptomycin, ramoplanin and other important antibiotics. Here, deacylase was studied considering its potential usefulness in deacylating polymyxin B1.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eAll the six recombinant strains containing the deacylase gene catalysed hydrolysis of polymyxin B1, yielding cyclic heptapeptide. The efficiency of recombinant \u003cem\u003eS. albus\u003c/em\u003e (SAL701) was higher than others, whereby deacylation was the most efficient at 40\u0026nbsp;°C in 0.2 M Tris buffer (pH 8.0) with 0.2 M Mg\u003csup\u003e2+\u003c/sup\u003e. The optimal substrate concentration of SAL701 was increased from 2.0 to 6.0 g/L. SAL701 was highly thermostable, showing no loss of activity at 50°C for 12 h, and the mycelia could be recycled at least three times without loss of catalytic activity. SAL701 could not deacylate β-lactam substrate such as penicillin G and cephalosporin C. Deacylase catalyzes the amide bond 1 closest to the nucleus of polymyxin B1 rather than the other bond, suggesting that it has high catalytic site specificity. Homology modeling and the docking results implied Thr190 in deacylase can facilitate hydrolysis with high regioselectivity.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThese results show that SAL701 is effective in increasing cyclic heptapeptide moiety of polymyxin B1. These properties of the biocatalyst may enable its development in the industrial production of polymyxins antibiotics.\u003c/p\u003e","manuscriptTitle":"Efficient side-chain deacylation of polymyxin B1 in recombinant Streptomyces strains","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-06-03 22:20:05","doi":"10.21203/rs.3.rs-1655594/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2022-06-01T15:13:57+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-06-01T15:08:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-05-16T15:11:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biotechnology Letters","date":"2022-05-14T04:32:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biotechnology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bile","sideBox":"Learn more about [Biotechnology Letters](https://www.springer.com/journal/10529)","snPcode":"10529","submissionUrl":"https://submission.nature.com/new-submission/10529/3","title":"Biotechnology Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ac45f405-1454-47b9-b0ef-295ffe7cabdc","owner":[],"postedDate":"June 3rd, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-08-04T07:52:25+00:00","versionOfRecord":[],"versionCreatedAt":"2022-06-03 22:20:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1655594","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1655594","identity":"rs-1655594","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}