Study On Heterologous Expression And Substrate Specificity Of Glycosyl Hydrolases From Environmental Genomics

Lignocellulose, as the most abundant biomass in the world, exhibits competitive potential to produce commercial bioethanol which has dramatically relieved the pressure brought by the lack of fossil fuel. Enzymic hydrolysis efficiency is considered to be a key point in bioprogresses of lignocellulosic ethanol production. Vital is construction of efficient glycosyl hydrolase systems toward specific elements in lignocellulose.Contrary to traditional methods basing on microbial cultivation, the foundation of environmental genomics strategy is that all genetic material extracted from enviornmal samples is constructed directly into genomic libraries, from which enzymes expressed by uncultivated microorganisms can be screened. Environmental genomics has significantly widened hydrolase resources and accelerated enzymatic discovery. However, none environmental genomic enzyme has been reported for commercial application yet. The reason maybe lies in two aspects:one is the great challenge existing in heterologous protein expression; the other one is short of systematic researches on enzymic functional characters, specially including substrate specificity which is critical to recognize hydrolytic efficiency. Thus, the present study investigated codon usage bias between Umcel5e2 and E.coli Rossetta which perhaps had impeded heterologous expression of Umcel5e2 and factors influencing catalytic substrate specificity of Umcel5c2.In the previous study, five glycosyl hydrolases had been screened from environmental genomic libraries. The present research tried to express these enzymes in E. coli. Only two of them got expressed-umcel5c2 and umcel5e1. From the other three, umcel5e2 was selected randomly for codon usage analysis and a codon optimized gene umcel5e2op was synthetized. But expression of umcel5e2op failed. It demonstrated the fact that codon usage differences between a heterologous protein and its host did not play a crucial role in expression obstruction in some cases.Umce15c2 got purified. Its catalytic characters were tested and results indicated that among substrates such as carboxymethylcellulose (CMC), lichenan and so forth, Umcel5c2 presents the highest catalytic activity towards xyloglucan. It is also a Michaelis enzyme. Combined with homologous modeling and sequence alignment, causes of substrate specificity discrepancy existing between Umcel5c2 and its best template PpXG5 from P. pabuli were analyzed. To examine the hypothesis ratiocinated according to the analysis, three residue deletion mutants were constructed:G78-E85 (the loop G78-E85 of wild type Umce15c2 deleted), T170-A179 (the loop T170-A179 deleted) and E229-F234 (the loop E229-F234 deleted). Meanwhile, a mutant which electron prodonor E218 was mutated to Q218 was generated, too. The inactive mutant was utilized as a blank control. Comparison enzyme activities and kinetic parameters between wild type and mutants declared that all of these three loops participated in substrate specificity determination, though their impacts were different: without loop T170-A179, enzyme almost looses its activity; not only in the process of xyloglucan hydrolysis, but also loop E229-F234 was very effective in that of CMC hydrolysis. It inferred the mechanism that these two loops participated hydrolytic process directly or influenced the breakage of glucosidic hond by changing conformation of catalytic center. The loopG78-E85 increased hydrolytic ability of Umce15c2 when substrate was xyloglucan rather than CMC.