提高源自Bacillus circulans 251的β-CGTase对麦芽糖亲和性及其在生产海藻糖中的应用

Enhancing Maltose Affinity of Bacillus circulans 251 β-CGTase and its Application in Trehalose Preparation

将B. circulans 251β-CGTase应用于海藻糖制备,海藻糖转化率从50. 4%提高至71. 9%。为进一步提高底物的转化率,运用易错PCR-高通量筛选技术筛选对以麦芽糖为歧化反应受体的亲和性提高的B. circulans 251β-CGTase突变体。利用低底物浓度的96孔板4,6-亚乙基-对硝基苯-α-D-麦芽七糖苷(EPS)显色法,最终筛选得到了一株对麦芽糖亲和性提高的突变体M234I。将野生型β-CGTase和突变体酶M234I进行蛋白质纯化,测定其酶学性质。结果表明,突变体的比活为345. 25U/mg,野生型则为357. 63U/mg;突变体M234I对麦芽糖的K_m为0. 258 2mmol/L,仅为野生型(0. 474 9mmol/L)的54. 4%,对麦芽糖的亲和性显著提高;突变体的最适温度、最适p H较野生型未发生较大变化。以麦芽糊精(DE值16)为底物,将突变体M234I用于多酶复配体系生产海藻糖,酶反应结果表明海藻糖的转化率最高达74. 9%,较野生型β-CGTase提高约3%。

B. circulans 251 β-CGTase was applied to trehalose preparation,and the trehalose yield was increased from 50. 4% to 71. 9%. In order to further improve the conversion rate of substrates,B. circulans 251β-CGTase mutants with improved affinity for maltose as a disproportionation acceptor were screened thorugh errorprone PCR and high-throughput screening. Mutant M234 I with higher affinity for maltose was selected with a low concentration of 4,6-ethylidene-p-nitrophenyl-indole-D-maltoheptaose( EPS) chromogenic method. The wildtype β-CGTase and the mutant enzyme M234 I were purified,and the enzymatic properties were characterized.The specific activity of the mutant M234 I was 345. 25 U/mg( disproportionation activity),while that of the wild type was 357. 63 U/mg. The maltose K_m vaule of the mutant M234 I was 0. 258 2 mmol/L,which was only 54. 4%of the wild type( 0. 474 9 mmol/L),which indicates that its affinity for maltose was significantly improved. The optimum temperature and the optimum pH of the mutant did not change much compared with those of the wild type. The mutant M234 I was used in the multi-enzyme complex system to produce trehalose with maltodextrin( DE 16) as substrate,the result showed that the trehalose yield was up to 74. 9%,which was 3% higher than that of the wild-type β-CGTase.