A novel high performance metagenome-derived alkali-thermostable endo-β-1,4-glucanase for lignocellulosic biomass hydrolysis in the harsh conditions

Abstract Background: Lignocellulosic biomass, is a great resource for the production of bio-energy and bio-based material since it is largely abundant, inexpensive and renewable. The requirement of new energy sources has led to a wide search for novel effective enzymes to improve the exploitation of lignocellulose, among which the importance of thermostable and halotolerant cellulase enzymes with high pH performance is significant. Results: The primary aim of this study was to discover a novel alkali-thermostable endo-β-1,4-glucanase from the sheep rumen metagenome. Using a multi-step in-silico analysis, primary candidates with desired properties were found and subjected to cloning, expression, and purification followed by functional and structural characterization. The enzymes' kinetic parameters, including V max , Km, and specific activity, were calculated. The PersiCel4 demonstrated its optimum activity at pH 8.5 and a temperature of 85°C and was able to retain more than 70% of its activity after 150 hours of storage at 85°C. Furthermore, this enzyme was able to maintain its catalytic activity in the presence of different concentrations of NaCl, MgCl 2 , CaCl 2 , and MnCl 2 . Our results showed that treatment with MnCl 2 could enhance the enzyme’s activity by 89%. PersiCel4 was ultimately used for enzymatic hydrolysis of autoclave pretreated rice straw, the most abundant agricultural waste with rich cellulose content. In autoclave treated rice straw, enzymatic hydrolysis with the PersiCel4 increased the release of reducing sugar up to 260% after 72 hours in the harsh condition ( T= 85°C, pH = 8.5). Conclusion: Considering the urgent demand for stable cellulases that are operational on extreme temperature and pH conditions and due to several proposed distinctive characteristics of PersiCel4, it can be used in the harsh condition for bioconversion of lignocellulosic biomass.