Isolation Of A Cellulase-producing Bacterium And Cloning Of Its β-glucosidase Gene

Cellulose is the most abundant biomass in the world.It is estimated that annually about 10¹¹tons of cellulose produced by plants'photosynthesis.However,due to its complicated and its insolubility structure,most of cellulose can't be used and is discarded by human.Researches on the powerful cellulases and the high efficient degradation of the cellulose have been slow,mainly due to its intricated structure.So, the screening and research of high efficient cellulolytic microorganisms is significant.In the biological degradation of cellulose,cellulases secreted by microorganism play an important role.Cellulose can be degraded to glucose through the synergistical hydrolysis of three classes of cellulase,including endo-β-1,4-glucanase, cellobiohydrolase andβ-glucosidase.The former two hydrolyze cellulose polymers into oligomers,mainly dimer cellobiose.β-glucosidase is a key component of the cellulase system.It further hydrolyzes cellobiose into glucose.In addition,β-glucosidase can regulate the velocity of the cellulose degradation.Therefore,high efficientβ-glucosidase have a profit of the high velocity of the cellulose degradation.This thesis was based on the significance of cellulase andβ-glucosidase,and screened and researched the strain which produced the cellulase to include the high efficientβ-glucosidase.In this paper,I report the study on anβ-glucosidase-producing bacterium, including its isolation,identification,and cloning of anβ-glucosidase gene.Partially characterization of theβ-glucosidase will also be reported.1.Isolation of a cellulases-producing bacteriumTo get a cellulase-high-yielding bacterium,a CMC_Naplate screening method was performed,and five cellulase-producing strains were obtained.Then,using the screeningβ-glucosidase plate method,one strain,termed as SWU-27,was selected for further study because of its high activity ofβ-glucosidase.The secretion of this strain exhibited activities of endoglucanase,cellubiohydrolase andβ-glucosidase,among which the highest activity was theβ-glucosidase.The activity of endoglucanase, cellubiohydrolase andβ-glucosidase is 10.663U/mL,15.910U/mL and 20.107 U/mL, respectively.The activity of theβ-glucosidase accounts for 43.07%of the total activities.2.Identification of the strain SWU-27According to the characteristics of morphology,cultivation,physiology and 16S rDNA sequence,we can identify the strain.SWU-27 was gram-negative rod bacteria, without spore,without flagellum and immobility.12 physiological and biochemical characteristics have been identified,and the identification results accord with those of the Klebsiella sp.strain.Bioinformatics analysis of 16S rDNA gene indicated that 99%identity was shared between SWU-27 strain and Klebsiella sp.strain zlmy.And the analysis on phylogenesis was performed,and the result showed that SWU-27 strain was high homology with many Klebsiella sp.strain.According to the identification results, the strain SWU-27 was identified as Klebsiella sp.And the strain SWU-27 was named Klebsiella sp.SWU-27.3.Cloning and analysis of theβ-glucosidase geneBy the shotgun cloning method and with Escherichia coli as the host cell,I isolated a novelβ-glucosidase gene,named as nglu02,from strain SWU-27.The open reading frame(ORF)of the nglu02.was 1,230 bp,encoding a peptide of 409 amino acid residues with a predicted isoelectric point value(pI)of 8.90.The hydrophobic cluster analysis and phylogenetic assay revealed that this gene belonged to cellulase family I and showed high homology to Ruminococcus flavefaciens celA.In the conservative and nonconservative fields of the polypeptide,activity residues Glu and Asp were also found.4.Partially study on the characters of theβ-glucosidaseTheβ-glucosidase was found in the culture supernatant of the same fermentation broth,and its properties were studied with the culture supernatant.The optimum pH of the enzyme activity was pH 10.0.When the pH was 11.0,about 80%enzyme activity remained.And the enzyme was stable form pH7.0 to pH11.0.The optimal temperature for the enzyme activity was 30℃,and the remain enzyme was 50%in the rang of 5℃-70℃.Different metal ions and surfactant show different effects on the enzyme activity. The enzyme was notably inhibited by 1 mM Ca²⁺,Mg²⁺and Zn²⁺,but was not so obviously effect by 1 mM K⁺,Na⁺,Cu²⁺,Fe³⁺,EDTA and Urea.Finally,theβ-glucosidase was stable in the high temperature and the alkaline environment,and endures some metal ions and surfactant,which means that this enzyme has great potential in the cellulase production,the textile biofinishing and detergent industry.