Heterologous Expression And Application Of Endo-β-1,4-glucanase Rfom Thermobifida Fusca

Endo-β-1,4-glucanase （EC3.2.1.4）, one major type of cellulase, cleaves the cellulose atrandom β-1,4linkages and releases cello-oligosaccharides. It has been found to catalyzechitosan hydrolysis effectively to produce chitosan oligosaccharides.In the present study, Thermobifida fusca endoglucanase Cel5A was cloned andexpressed in Escherichia coli BL21（DE3）. Moreover, it was used to depolymerize chitosanfor the production of COS. The main results were listed as follows:（1） T. fusca genome was used as template for amplifying endo-β-1,4-glucanases genecel5A by the polymerase chain reaction. After PCR, a1293-bp fragment was obtained. Therecombinant plasmids, pET-24a（+）/pelB-cel5A, were constructed and transformed into E.coli BL21（DE3）. The engineered cells were grown in TB medium. The carboxymethylcellulase （CMCase） activity in shake flasks reached46.8IU/mL.（2） With the aim to achieve high production of T. fusca Cel5A, the optimization ofculture medium was investigated. The optimized medium was listed as follows:16g/Lglycerol,45g/L yeast extract,4mmol/L Mg²⁺,30mmol/L PO43-. Under this condition, theenzyme activity increased from46.8IU/mL to72.6IU/mL. In addition, the yield ofrecombinant Cel5A by engineered E. coli was further explored in3L fermentor utilizingfed-batch strategy, and the highest enzyme activity reached656.6IU/mL, which representedthe highest yield of T. fusca Cel5A reported so far.（3） The recombinant Cel5A was purified and characterized in detail. The optimumtemperature of recombinant enzyme was80°C. The activity of recombinant Cel5A wasretained more than90%over the range of pH5.0-10.0with maximal activity at pH5.5.Using carboxymethyl cellulose as the substrate, the Km and Vmax values were5.1mg/mLand48.7IU/mg, respectively. The enzyme showed super stability in surfactants and thepotential mechanism of this stability was investigated by analysis of the electrostaticpotential of the surface of the enzyme.（4）. Cel5A produced by engineered E. coli BL21（DE3） was used to depolymerizechitosan. The optimum conditions for enzymatic hydrolysis were investigated. On theselected optimum conditions, chitosan was hydrolyzed for1h, and COS yield was60.3%and the products were mainly COS with degree of polymerization （DP） in the3⁸range.