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

doi:10.13523/j.cb.20190511

中国生物工程杂志

2019, Vol. 39

Issue (5): 96-104 DOI: 10.13523/j.cb.20190511

研究报告

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 ^*

杜立^1,^2,³,宿玲恰^1,^2,³,吴敬^1,^2,^3,^**(

)

1 江南大学食品科学与技术国家重点实验室无锡 214122
2 江南大学生物工程学院工业生物技术教育部重点实验室无锡 214122
3 江南大学教育部食品安全国际合作联合实验室无锡 214122

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

Li DU^1,^2,³,Ling-qia SU^1,^2,³,Jing WU^1,^2,^3,^**(

)

1 State Key Laboratory of Food Science and Technology, Jiangnan University,Wuxi 214122,China
2 School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, Wuxi 214122, China
3 International Joint Laboratory on Food Safety,Jiangnan University,Wuxi 214122,China

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摘要：

将B. circulans 251 β-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 251 β-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pH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%。

关键词： β-CGTase; 易错PCR; 分子改造; 亲和性; 海藻糖转化

Abstract:

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 251 β-CGTase mutants with improved affinity for maltose as a disproportionation acceptor were screened thorugh error-prone PCR and high-throughput screening. Mutant M234I with higher affinity for maltose was selected with a low concentration of 4,6-ethylidene-p-nitrophenyl-indole-D-maltoheptaose (EPS) chromogenic method. The wild-type β-CGTase and the mutant enzyme M234I were purified, and the enzymatic properties were characterized. The specific activity of the mutant M234I was 345.25U/mg (disproportionation activity), while that of the wild type was 357.63U/mg. The maltose K_m vaule of the mutant M234I was 0.258 2mmol/L, which was only 54.4% of the wild type (0.474 9mmol/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 M234I 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.

Key words: β-CGTase Error-prone PCR Molecular modification Affinity Trehalose conversion

收稿日期: 2018-10-08 出版日期: 2019-06-04

ZTFLH:

Q819

基金资助: * 国家自然科学基金资助项目(31771916);国家自然科学基金资助项目(31425020)

通讯作者: 吴敬 E-mail: jingwu@jiangnan.edu.cn

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引用本文:

杜立,宿玲恰,吴敬. 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 ^*[J]. 中国生物工程杂志, 2019, 39(5): 96-104.

Li DU,Ling-qia SU,Jing WU. Enhancing Maltose Affinity of Bacillus circulans 251 β-CGTase and its Application in Trehalose Preparation. China Biotechnology, 2019, 39(5): 96-104.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190511 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I5/96

表1 突变体测序结果

图1 野生型和突变体酶M234I的SDS-PAGE分析

表2 野生型和突变体M234I β-CGTase的纯化结果

表3 野生型和突变体M234I β-CGTase的酶学动力学参数

图2 野生型和突变体M234I β-CGTase的最适温度

图3 野生型和突变体M234I β-CGTase的温度稳定性

图4 野生型和突变体M234I β-CGTase的最适pH

图5 野生型和突变体M234I β-CGTase的pH稳定性

图6 不同加酶量下的海藻糖转化率

表4 糖化前的反应体系中的葡萄糖、麦芽糖和海藻糖含量

图7 野生型与突变体M234I β-CGTase 动力学轨迹的平均结构,绿色表示野生型的loop区域和残基,蓝色表示突变体M234I的loop区域和残基

图8 野生型(a)与突变体M234I(b) β-CGTase 平均结构的受体底物对接模型

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