| Nowadays, with problem of population explosion, energy exhaustion and environmental degradation, searching for renewable resources has become an urgent issue around the world. Natural cellulose is considered to be the hope to solve problems of food, energy and environment. At present, cellulose microbial degradation has earned much more attention. However, cellulase which have been reported have common weakness of low activity and few species. Therefore, searching and developing novel cellulase with features of high activity, broad substrate and adaptability to extreme environment has been the most important issue. In the cellulase system, P-glucosidase which is used for hydrolysis cellobiose and short chain cellulose oligosaccharides, is the key enzyme in the complete degradation of cellulose. Therefore, development of novel and efficient P-glucosidase has great significance for the whole industry.There is a wide range of microorganism in nature, but only 1-10%of them have been known. At the present, research of microorganisms has entered in the molecular and genetic period, and a series approaches have been established, such as metagenomics. Metagenomics is a research method for microbial diversity, population structure, evolutionary relationship, functional activity and relation with the environment.This research is based on a large number of cellulose-degrading microorganisms in black liquor and the significant value ofβ-glucosidase. We constructed the library of genomic DNA isolated directly from black liquor, and got a novel 6-phospho-p-glucosidase gene. Then the putative 6-phospho-β-glucosidase was overexpressed and purified for characterization.The main contents of this research are as follows:1. Construction and screening of the metagenomic library from black liquorIn this study, to construct library, metagenomic DNA was directly isolated from black liquor. Transformation of Escherichia coli DH5b resulted in the metagenomic library with a average insert size of 6.0-8.0 kb by using the pUC118 vector. The library was screened forβ-glucosidase activity, and one clone showed high 6-phospho-β-glucosidase activity. The 6-phospho-β-glucosidase gene pbg125-217 has a length of 1428 bp, it encodes 6-phospho-β-glucosidase Pbg125-217, which contains 476 amino acids. The enzyme is a hydrophilic protein, its molecular weight (MW) and isoelectric point (pI) is 54798.73 Da and 5.14. Currently, there are few studies about 6-phospho-β-glucosidase, and amino acid sequence of Pbg125-217 has low homology with those reported 6-phospho-β-glucosidase. Therefore, the research of Pbg125-217 has theoretical value.2. Cloning and expression of 6-phospho-β-glucosidase gene pbgl25-217In this study,6-phospho-β-glucosidase gene pbg125-217 was cloned and inserted into pETDuet-1 vertor. Then transformation of Escherichia coli Rosetta resulted in the recombinant plasmid pbg125-217-pETDuet. Ultimately, heterologous 6-phospho-β-glucosidase Pbg125-217 was expressed successfully.3. Purification and characterization of 6-phospho-p-glucosidase Pbg125-217In this study, Pbg125-217 was purified by affinity chromatography and anion exchange chromatography. Optimal conditions for the enzyme activity of Pbg125-217 were 37℃and pH 7, and stable conditions for the enzyme activity of Pbgl25-217 were<20℃and pH 5-9. Ca2+, Mg2+, Mn2+stabilized the activity of Pbg125-217, and Ni2+, Fe2+, Zn2+, Cu2+, Fe3+inhibited the activity of Pbgl25-217. The Km and Vmax value of Pbg125-217 were 4.80 mM and 5187.04 U·mg-1.4. Site-directed mutagenesis of 6-phospho-β-glucosidase Pbgl25-217In this study, base changes effected by site-directed mutagenesis were confirmed by sequence analysis. There are ten mutant forms of Pbgl25-217, S427A, S427C contained measurable activity of 6-phospho-β-glucosidase; W420F, S427D, S427E, Y437F contained partial activity; and E179A, E372A, Y311F, K435L contained no activity. |
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