基因選殖與表現耐熱飼料添加酵素α-amylase及β-glucanase

Abstract

In feed industry, amylases and glucanases are in common use in animal dietary as additives to improve the utilization of nutrients. Because there will be a short time and high temperature step in the feed processing, feed additive enzymes must be thermostable and maintain high activity in the gut of pigs. If so, it will get more utilization, commercialization and competitiveness. In this study, several genes encoded hydrolytic enzymes from thermophilic microorganism were cloned and expressed. We hope to obtain hydrolytic enzymes which have better thermostability. The thermophilic microorganisms, Bacillus licheniformis ATCC 14580, Bacillus stearothermophilus ATCC 29609, Geobacillus thermoleovorans ATCC 43509, Clostridium thermocellum ATCC 27405 and Thermotoga neapolitana ATCC 49049, who obtained from American Type Culture Collection (ATCC) and Bioresource Collection and Research Center (BCRC). These genes were amplified by polymerase chain reaction (PCR) using thermophilic microorganisms genomic DNA as the templates and subcloned into the expression vectors (pET, pBBad system). The recombinant vectors were transformed into Escherichia coli strains. The amyL gene from B. licheniformis was expressed in BL21 (DE3), and the most of the recombinant proteins were insoluble form (inclusion bodies). The inclusion body of recombinant amyL proteins were dissolved in 6 mol/L urea and purified according to Ni-NTA purification system. There is no enzymatic activity detected in purified recombinant amyL, and it indicates that recombinant protein does not return to its correct conformation in the renaturation procedure. However, the recombinant aggregates displayed α-amylase activity. Therefore, the phenomenon of aggregation probably occurred by specific interaction of mature recombinant protein molecules rather than by the misfolded intermediates. Furthermore, we adopt a more moderate way by glycerol extraction and inclusion body dissolution ratio is about 5%. After evaluation of a variety of purification, we found they were not conformed the economic efficiency. Therefore, we use the crude extract of recombinant protein directly for enzyme biochemical characterization and stability analysis. The thermal stability of the amyL, amyS, and amy recombinant enzymes were accorded with the industrial requirements; these recombinant enzymes retained the 70%, 90% and 70% activity at 80℃ for 5 minutes, respectively. The thermostability of amyS is preferred at 90℃ for 5 minutes retained 90% of the activity. The β-glucanase thermostability is lower to reach the requirement for feed additives. We expected to change the enzymes characteristics and improve the utilization of the industry by genetic engineering. The recombinant β-glucanase eg1314 (B. subtilis) has the best hydrolytic activity at 37℃, but its thermostability is poor; The recombinant β-glucanase licMB (C. thermocellum) has better thermostability, but lower hydrolytic activity. A fusion gene eg-licMB, encoded the β-glucanase eg1314 and licMB, was constructed via end-to-end fusion and expressed in E. coli. Fusion protein biochemical characteristics are more accorded with the applicable material than the original enzymes, but did not fully accord with high thermostability and high activity at 37℃. In the future, these genes being cloned in this study would subject to site-directed mutagenesis and Error-prone PCR to improve the enzyme hydrolysis activity and thermostability.