Isolation Of Cellulase-Producing Strains And Catalytic Efficiency Improvement Of Endo-β-1, 4-Glucanase From Bacillus Subtilis

In the past 50 years much effort had gone into the studies of cellulases as a potential means to obtain sustainable biobased products to replace depleting fossil fuels from an abundant, renewable energy resource, plant biomass. However, the high cost of cellulases production seemed to be a very important and difficult challenge in the cellulose bioconversion process. The way to increase enzyme volumetric productivity was to isolate hyperproducers and to improve the necessary characteristics of cellulases.In this research, from more than 450 marine microorganisms and terrestrial soil samples, 46 and 99 isolates were found positive for cellulase production, respectively.Among these, cellulase-producing strains were mainly identified as Bacillus,Halomonasand Pseudomonas. Eight endo-β-1, 4-glucanase (EG) genes were cloned from Bacillus strains and showed different homology by DNA or protein phylogenetic tree analysis. Furthermore, the colony which harbored Bacillus subtilis BME-15 EG gene (cel5A) showed the highest halo-forming activity on CMC plates and was chose to be studied.Using directed evolution techniques of error-prone PCR and DNA shuffling, several Cel5A variants with improved catalytic activity had been screened from the mutant library, which contained 71,000 colonies. Compared with the wild-type enzyme, the variants (M44-11, S75 and S78) showed 2.03 to 2.68 folds increased activities toward sodium carboxymethyl cellulose (CMC), while the M44-11 also exhibited a wider pH tolerance and higher thermostability. Structural models of M44-11, S75, S78 and WT proteins revealed that most of the substitutions were not located in the strictly conserved regions, except the mutation V255A of S75, which was closed to the nucleophile Glu257 in the catalytic center of the enzyme.In order to study the functions of substitutions in the mutants, site-directed mutagenesis of K120 and D272 were constructed by using cycled PCR. By analyzing the halo-forming activities of the mutants and parents, substitutions K120E and D272G were found to show slight increased activity compared with wild type enzyme. This phenomenon implied that the increased activity of S78 and M44-11 was not due to the contribution of the single substitution, but due to the synergistic effect of multi-site substitutions. And it also revealed that mutations outside of the catalytic center or the binding sites resulted in increased catalytic activity by making new hydrogen bonds and repositioning of catalytic residues in the active site. Moreover, saturation mutagenesis of W69 and A263-S264-G265 sites were also constructed to study the reaction between Cel5A and substrate. The result showed that substitutions of W69 slight decreased the halo-forming activity of the enzyme, while substitutions of A263-S264-G265 resulted in most activity losing and this revealed that A263-S264-G265 played some very important fuction in binding substrate.This study provided useful references for directed evolution of the enzymes belonged to glycoside hydrolase family 5 (GH5).