Molecular Modification And Fermentation Optimization Of Catalase From Bacillus Subtilis

Catalase (EC1.11.1.6) is a kind of terminal oxidase widely distributed in almost allaerobes. It could catalyze H2O2into H2O and O2to protect cells from over oxidation anddamage. Catalase is a key enzyme in bio-defense system.Catalase plays an important role in industry, especially in textile industry. To meet theneed of industrial production and application, genetic engineering was used to mutate keyamino acids of catalase from Bacillus subtilis WSHDZ-01. Mutated genes were expressed inBacillus subtilis. The variant with best properties was selected for further research onfermentation optimization. The main research results are as follows:(1) Site-directed mutagenesis on114K of catalase from B. subtilis WSHDZ-01wasperformed using pSTOP1622-katA as template, after which mutated genes were expressed inB. subtilis WB600. It was found that strains600Y and600V exhibited higher enzyme activity,which was2.3and2.5-fold of the wild strain, respectively. Enzymatic characteristics werestudied after purification of variants. As a result, K114Y exhibited highest t1/2value at60oC,indicating best thermostability. K114Y also showed highest catalytic efficiency, which was5.3-fold of the wild-type.(2) To further strengthen the thermostability of catalase and study the influence ofsite-directed mutagenesis on enzymatic properties, site-directed mutations on193G and123Dwere performed based on previous mutated plasmids KY. After mutagenesis, mutated geneswere expressed in B. subtilis WB600. Strains G600F, G600C, D600Y and D600F showedhigher catalase activity compared with600Y. Enzymatic characteristics of variants werestudied after purification. G193F showed higher thermostability whose t1/2value increased by23%compared with K114Y and D123Y showed highest catalytic efficiency which was1.4-fold of K114Y.(3) Catalase variants with higher thermostatbility and catalytic efficiency were obtainedafter mutations. To realize industrial production, fermentation of WB600WT and600Y on3Lfermentor were performed. As a result, the enzyme activity on the fermentor wasapproximately same with that of shaking flasks, indicating no obvious increase in larger-scaleproduction. Consequently, corresponding mutated plasmids were transformed to B. subtilisWSHDZ-01for production in3L fermentor. The enzyme activity of KY01, KY/GF01andKY/DY01was25423U·mL-1,27895U·mL-1and30487U·mL-1, which was1.9,1.8and1.5-fold of that of shaking flask, respectively. Taking thermostability and activity intoconsideration, KY/GF01was selected for fermentation optimization on the level of shakingflask. The optimized conditions were determined as follows, glucose25g·L-1, NaNO37.5g·L-1, xylose concentration0.1%. The fermentation on3L fermentor was performed withoptimized conditions. The enzyme activity could reach33898U·mL-1, which was1.2-fold ofthat before optimization.