甲壳素酶Chisb的定向进化及生物转化合成几丁寡糖

Directed evolution of chitinase Chisb and biosynthesis of chitooligosaccharides

甲壳素酶具有广泛的工业应用前景,如可将虾壳、蟹壳和其他甲壳废物降解成以几丁寡糖为主的高附加值产品,但野生型甲壳素酶催化效率低,大大限制了几丁寡糖的生产。笔者在前期研究中表达了一个具有较高效催化效率的甲壳素酶Chisb,并对其酶学性质进行了初步研究。为进一步提高甲壳素酶Chisb的催化效率,以R13NprB-C-SP-H为亲本,采用易错PCR(Error-pronePCR)技术构建随机突变体文库,对甲壳素酶Chisb进行定向进化。经过96孔板初筛和摇瓶复筛,获得了两个催化效率进一步提高的突变体C43D和E336R。对突变体的酶学性质进行分析, C43D和E336R的最适催化温度为55℃, C43D的最适pH为5.0,E336R的最适pH为9.0;其催化效率相比对照分别提高了1.35倍和1.57倍;而E336R和C43D催化产几丁寡糖的含量分别为2.53 g/L和2.06 g/L,相比对照(0.89 g/L)分别提高了2.84倍和2.31倍;底物转化率分别为84.3%和68.7%,相比对照(29.7%)分别提高了54.6%和39%。研究表明,通过易错PCR引入随机突变的方法能够有效提高甲壳素酶Chisb的催化效率。上述研究获得的催化效率提高的正向突变体及其酶学性质分析对生物转化合成几丁寡糖具有重要研究意义和应用价值。

Chitinase has a wide industrial application prospect.For example,it can degrade shrimp shells,crab shells and other crustacean waste into high value-added chitooligosaccharides.However,the low catalytic efficiency of chitinase greatly limits the production of chitooligosaccharides.In previous study,the we expressed a chitinase Chisb with high catalytic efficiency and studied its enzymatic properties.In order to further improve the catalytic efficiency of Chisb,with R13 NprB-C-SP-H as the parent,here error-prone PCR was used to construct random mutant library to conduct directed evolution of chitinase Chisb.Two mutants C43D and E336R were obtained with 96-well plate primary screening and shaker-screening,and their enzymatic properties were also studied.The optimum temperature of C43D and E336R was 55°C,and the optimum pH of C43D was 5.0,while that of E336R was 9.0.The catalytic efficiency of C43D and E336R was 1.35 times and 1.57 times higher than that of control.The chitooligosaccharide concentration of E336R and C43D was 2.53 g/L and 2.06 g/L,improved by 2.84 times and 2.31 times compared with the control(0.89 g/L),respectively.In addition,the substrate conversion rate of mutants E336R and C43D was 84.3%and 68.7%,improved by 54.6%and 39%compared with the control(29.7%),respectively.In summary,the study indicates that random mutation introduced by error-prone PCR can effectively improve the catalytic efficiency of chitinase Chisb.The positive mutants with higher catalytic efficiency obtained in the above study and their enzymatic property analysis have important research significance and application value for the biosynthesis of chitooligosaccharides.