干酪乳杆菌(Lactobacillus casei)HDS-01甘露聚糖酶基因异源表达及功能验证

杨若兮, 张鑫, 赵丹

中国农学通报. 2023, 39(32): 15-21

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中国农学通报 ›› 2023, Vol. 39 ›› Issue (32) : 15-21. DOI: 10.11924/j.issn.1000-6850.casb2023-0276
生物科学

干酪乳杆菌(Lactobacillus casei)HDS-01甘露聚糖酶基因异源表达及功能验证

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Mannanase Gene in Lactobacillus casei HDS-01: Heterologous Expression and Functional Verification

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摘要

为实现甘露聚糖酶基因异源表达并验证功能,本研究以产甘露聚糖酶的副干酪乳杆菌(Lactobacillus casei) HDS-01为供试菌株,将基因组序列中功能预测为甘露糖苷酶的基因M1在大肠杆菌中表达,分别以pET-28a和Escherichia coli BL21(DE3)为载体和宿主菌构建M1基因工程菌株,纯化重组蛋白并验证甘露聚糖降解功能。结果表明:重组蛋白M1在表达过程中以包涵体形式存在,经Ni柱亲和层析纯化后,SDS-PAGE显示蛋白为98 kDa,复性后酶活和蛋白浓度分别为19.24±0.55 U/mL和0.51±0.01 mg/mL。高效液相色谱(High pressure liquid chromatography, HPLC)检测重组蛋白M1降解魔芋粉的产物,反应前3 h,甘露三糖或四糖浓度显著高,此后,甘露糖和甘露二糖浓度逐渐升高。本研究为产甘露聚糖酶乳酸菌直接用于食品级领域奠定了基础,也为乳酸菌甘露聚糖酶蛋白的定向改造提供了依据。

Abstract

In order to realize heterologous expression of mannanase and verify its functions, the mannanase-producing Lactobacillus casei HDS-01 was used as the test strain in this study. The M1 predicted to be mannosidase was expressed in Escherichia coli. M1 gene engineering strains were constructed using pET-28a as the vector and E. coli BL21 (DE3) as the host. The recombinant protein was purified and the degradation function of mannan was verified. The results indicated that the recombinant protein M1 existed as inclusion body during expression. After purification by Ni affinity chromatography, the size of the protein was 98 kDa. After renaturation, mannanase activity and protein concentration were 19.24±0.55 U/mL and 0.51±0.01 mg/mL, respectively. High pressure liquid chromatography (HPLC) was used to detect the degradation product of Konjac powder. The concentration of mannotriose or tetrasaccharide was significantly higher in the first 3 hours, and mannose and mannobiose gradually increased as the reaction progressing. This study laid the foundation for the direct application of mannanase-producing lactic acid bacteria in food-grade field and provided the basis for the selective modification of mannanase in lactic acid bacteria.

关键词

β-甘露聚糖酶 / 干酪乳杆菌 / 异源表达 / 重组蛋白 / 功能验证

Key words

β-1,4-mannanase / Lactobacillus casei / heterologous expression / recombinant protein / functional verification

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杨若兮 , 张鑫 , 赵丹. 干酪乳杆菌(Lactobacillus casei)HDS-01甘露聚糖酶基因异源表达及功能验证. 中国农学通报. 2023, 39(32): 15-21 https://doi.org/10.11924/j.issn.1000-6850.casb2023-0276
YANG Ruoxi , ZHANG Xin , ZHAO Dan. Mannanase Gene in Lactobacillus casei HDS-01: Heterologous Expression and Functional Verification. Chinese Agricultural Science Bulletin. 2023, 39(32): 15-21 https://doi.org/10.11924/j.issn.1000-6850.casb2023-0276

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基金

国家自然科学基金“干酪乳杆菌-魔芋葡甘聚糖合生元协同效应及其对肠道菌群的调节研究”(32072189)
国家自然科学基金“乙酸溢流与群体感应的协同效应对副干酪乳杆菌HD1.7种群稳定性影响机制的探究”(32071519)
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