黄杆菌褐藻胶裂解酶的高效表达及其在褐藻寡糖制备中的应用

doi:10.7506/spkx1002-6630-20191213-149

食品科学 ›› 2021, Vol. 42 ›› Issue (4): 145-152.doi: 10.7506/spkx1002-6630-20191213-149

黄杆菌褐藻胶裂解酶的高效表达及其在褐藻寡糖制备中的应用

江君，刘军，杨绍青，马帅，闫巧娟，江正强

（1.中国农业大学食品科学与营养工程学院，中国轻工业食品生物工程重点实验室，北京 100083；2.中国农业大学工学院，北京 100083）

出版日期:2021-02-25 发布日期:2021-02-25
基金资助:
“十三五”国家重点研发计划重点专项（2017YFD0400204）

High Level Expression of Alginate Lyase from Flavobacterium sp. and Its Application in Preparation of Alginate Oligosaccharides

JIANG Jun, LIU Jun, YANG Shaoqing, MA Shuai, YAN Qiaojuan, JIANG Zhengqiang

(1. Key Laboratory of Food Bioengineering (China National Light Industry), College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China; 2. College of Engineering, China Agricultural University, Beijing 100083, China)

Online:2021-02-25 Published:2021-02-25

摘要/Abstract

摘要： 研究黄杆菌褐藻胶裂解酶基因（rFlAlyA）在枯草芽孢杆菌中的高效表达，高密度发酵48 h最高酶活力为2 550 U/mL，蛋白质量浓度达4.5 mg/mL。经(NH4)2SO4沉淀和SP强阳离子交换柱纯化后得到电泳级纯酶，分子质量约为30 kDa。褐藻胶裂解酶rFlAlyA最适pH值为7.5，在pH 4.0～11.0范围内可保持80%以上酶活力；最适温度为50 ℃，在50 ℃及以下可保持80%以上酶活力。底物特异性分析表明，rFlAlyA特异性降解聚甘露糖醛酸片段，最短链底物为甘露糖醛酸三糖。rFlAlyA在最适条件下酶解褐藻酸钠4 h后还原糖质量浓度达33 mg/mL，底物转化率为70.1%，电喷雾电离-质谱分析酶解产物的聚合度（degree of polymerization，DP）为1～8，离子色谱分析显示DP=3时相对含量最高，为34.0%。结果表明，经枯草芽孢杆菌高效表达的rFlAlyA可用于制备褐藻寡糖。

关键词: 褐藻胶裂解酶；黄杆菌；枯草芽孢杆菌；高密度发酵；褐藻寡糖

Abstract: High-efficiency expression of the alginate lyase gene (rFlAlyA) from Flavobacterium sp. in Bacillus subtilis was studied. After high cell-density fermentation for 48 h, the enzymatic activity reached the highest level of 2 550 U/mL with a protein content of 4.5 mg/mL. The rFlAlyA, with a molecular mass of about 30 kDa, was purified to electrophoretic homogeneity after fractionation with (NH4)2SO4 and ion exchange chromatography on SP strong cation exchange column. Maximal enzymatic activity was observed at pH 7.5 and 50 ℃. The enzyme showed good stability at pH 4.0?11.0 and was thermostable up to 50 ℃, retaining over 80% of its activity. The substrate specificity indicated that rFlAlyA was a polymannuronate lyase with trisaccharide being the shortest-chain substrate. For alginate oligosaccharides production, sodium alginate was reacted with rFlAlyA under the optimal conditions for 4 h, giving a final concentration of reducing sugar of 33 mg/mL in the reaction mixture and a conversion rate was 70.1%. Electrospray ionization tandem mass spectrometry showed that the degree of polymerization (DP) of the hydrolysates was 1 to 8 and trisaccharide was the most abundant, which accounted for 34.0% of the total components when the DP was 3. The findings of this study indicated that the recombinant alginate lyase could be applied for the production of alginate oligosaccharides.

Key words: alginate lyase; Flavobacterium sp.; Bacillus subtilis; high cell-density fermentation; alginate oligosaccharides

中图分类号:

Q814

江君，刘军，杨绍青，马帅，闫巧娟，江正强. 黄杆菌褐藻胶裂解酶的高效表达及其在褐藻寡糖制备中的应用[J]. 食品科学, 2021, 42(4): 145-152.

JIANG Jun, LIU Jun, YANG Shaoqing, MA Shuai, YAN Qiaojuan, JIANG Zhengqiang. High Level Expression of Alginate Lyase from Flavobacterium sp. and Its Application in Preparation of Alginate Oligosaccharides[J]. FOOD SCIENCE, 2021, 42(4): 145-152.

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黄杆菌褐藻胶裂解酶的高效表达及其在褐藻寡糖制备中的应用

High Level Expression of Alginate Lyase from Flavobacterium sp. and Its Application in Preparation of Alginate Oligosaccharides

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