维氏气单胞菌几丁质酶ChiC的性质和与几丁质结合蛋白协同作用的研究

Studies on Character of a Chitinase ChiC from Aeromonas veronii B565 and its Synergism with Chitin Binding Protein

为了探索维氏气单胞菌B565低酶活几丁质酶ChiC的性质及突破其低酶活的应用瓶颈,构建了重组ChiC毕赤酵母工程菌GS115/PIC9-ChiC,进行甲醇诱导表达纯化,研究其基本酶学性质。结果表明,ChiC在毕赤酵母中分泌表达,甲醇诱导48 h后,达到摇瓶水平最大表达量232.6 mg/L。ChiC最适反应pH为8.0,温度为40℃。最适反应条件时比活力为7.6 U/mg。ChiC具有较宽的pH及温度适应性,在pH 3.0-10.0,40℃或者0-40℃,pH 7.0,均可以保持80%以上最适酶活力。同时几丁质结合蛋白CBP21于大肠杆菌中实现胞内可溶表达,其表现出对虾壳几丁质和胶体几丁质的具有不同的结合能力。但CBP21促进ChiC降解胶体几丁质的能力（提高约9倍）明显高于其促进ChiC降解虾壳几丁质的能力（提高约2倍）。上述结果为该几丁质酶基因的深入研究奠定了理论基础,为突破该低酶活几丁质酶的应用瓶颈提供了一种可能的解决方案。

To investigate the characteristics of low activity ChiC from A. veronii B565 and break the bottleneck of its application,this experiment established a recombined ChiC pichia engineering bacteria GS115 /PIC9-ChiC,carried out methanol induction expression and purification,and studied its basic enzymology properties. The result indicated that ChiC was expressed in P. pastoris with a yield of 232. 6 mg /L after 48 h methanol induction. The purified ChiC showed optimal specific activity of 7. 6 U /mg at pH 8. 0 and 40℃. ChiC had broad adaptability for pH and temperature. It retained over 80% optimal activity at pH 3. 0 - 10. 0 at 40℃ or the temperature 0 - 40℃ at pH 7. 0.The chitin binding protein CBP21 from Serratia marcescens GIM1. 217 was expressed in E. Coli. The affinity of CBP21 for shrimp shell chitin and colloidal chitin were different,the chitinase activity of ChiC was increased significantly from 2（ colloidal chitin as substrate） to 9（ shrimp shell chitin as substrate） fold,when acted synergistically with CBP21. This study laid a theoretical foundation for further studying chitinase gene,and provided a possible solution for breaking the bottleneck of this low enzyme activity of chitinase application.