Vibrio natriegens几丁质酶基因的异源表达及酶学性质研究

Heterologous Expression and Catalytic Properties of Chitinase Gene from Vibrio Natriegens

几丁质是自然界中除纤维素外第二多的多糖聚合物,在几丁质酶的作用下生成的几丁寡糖具有较好的生物活性及应用价值。通过分子生物学手段获得高活性重组几丁质酶,有利于实现虾壳等几丁质资源的高值化利用。对来源于Vibrio natriegens中的几丁质酶基因进行克隆及诱导表达,获得重组几丁质酶VnChit4,然后通过亲和层析对该酶进行纯化,并测定其酶学性质及酶解产物。结果表明,VnChit4纯酶液的酶活力为2.66 U/mL,最适温度和最适pH值分别为50℃、pH7.0。在低于40℃、pH6~9范围内保持较好的稳定性;Ca^(2+)、Fe^(2+)、Co^(2+)对重组酶VnChit4的活力有促进作用,Mn^(2+)、Ni^(+)、Cu^(2+)和EDTA对该酶有抑制作用。以胶体几丁质为底物时,VnChit4的米氏常数K_(m)值为2.82 mg/mL,最大反应速率V_(max)为6.56 g/min·L。该酶对几丁质、虾壳粉、几丁四糖、几丁五糖和几丁六糖也表现出一定的水解作用,酶解产物主要为几丁二糖,是一种内切几丁质酶。该研究构建的重组几丁质酶表现出较好的酶活性,对其酶学性质的研究,有利于该几丁质酶的开发及虾壳几丁质的高值化利用。

As the second most abundant polysaccharides following cellulose,chitin can be hydrolyzed by chitinase to become chitin oligosaccharide,which shows better biological activity and application values than chitin.The recombinant chitinase with high activity would promote the high-value application of chitin from shrimp shells.In our work,chitinase gene from Vibrio natriegens was cloned,induced,and further expressed in Escherichia coli.The recombinant chitinase VnChit4 was purified by affinity chromatography.Then,the enzymatic properties and products were analyzed.The activity of VnChit4 was 2.66 U/mL.The optimum enzymatic temperature was 50℃,and pH 7.0.It kept more stable in the range of stored temperature below 40℃and pH from 6 to 9.Ca^(2+),Fe^(2+),Co^(2+) had stimulation on the activity of VnChit4,while Mn^(2+),Ni^(+),Cu^(2+)and EDTA had inhibition.When the substrate was colloidal chitin,the kinetic parameters K_(m),V_(max) were 2.82 mg/mL and 6.56 g/min·L,respectively.VnChit4 could also hydrolyze chitin,shrimp shells,chitintetraose,chitinpentose and chitinhexaose,and most of the enzymatic product was chitinbiose,which indicated that it was an endochitinase.VnChit4 showed good catalytic activity,and the enzymatic properties analysis is helpful for the chitinase application and high-value use of chitin from shrimp shells.