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弧菌DS32褐藻胶裂解酶基因vralg1 的异源表达和酶学性质研究
李慧敏,邵宗泽,路瑶,杨江科,周梅先
0
(武汉轻工大学生命科学与技术学院,湖北 武汉 430023;自然资源部第三海洋研究所、自然资源部海洋生物遗传资源重点实验室,福建 厦门 361005)
摘要:
对从福建省东山湾沉积物样品中筛选到的菌株Vibrio sp. DS32的褐藻胶裂解酶基因vralg1进行克隆和异源表达,并对其酶学性质进行评估。以DS32基因组为模板,克隆褐藻胶裂解酶基因vralg1,构建了pETvralg1重组表达载体,并在大肠杆菌中实现了异源表达,对重组酶VRALG1的酶学性质、底物特异性和完全降解产物等进行了测定。结果表明:重组酶VRALG1最适温度为35 ℃,在5~50 ℃范围内相对酶活力达到80%以上,最适pH为6.5~7.5,在pH为6.0~9.0范围内保温1 h后相对酶活力在90%以上;重组酶VRALG1最大反应速率为5.919 mmol/(L·min),米氏常数为3.712 mmol/L,最适条件下比活力为5.874 U/mg;K+、Cs+、Na+、咪唑和乙醇对酶活性影响较小,5 mmol/L或50 mg/mL浓度下相对酶活力保持90%以上,EDTA对酶的抑制作用明显,1 mmol/L浓度下可使酶完全失活;重组酶VRALG1对海藻酸钠和聚古罗糖醛酸具有较高的降解活性,TLC分析显示产物主要为单糖、二糖和三糖混合物,结合底物特异性分析,推测重组酶VRALG1是具有明显聚古罗糖醛酸偏好性的内切型双功能褐藻胶裂解酶。本研究成功克隆了弧菌DS32中褐藻胶裂解酶基因并实现了其在大肠杆菌中的异源表达,所得重组酶VRALG1具有优良的海藻酸钠降解活性和明显的聚古罗糖醛酸偏好性,可以用于制备低聚合度的褐藻寡糖。
关键词:  海洋生物学  褐藻胶裂解酶  褐藻寡糖  克隆表达  酶学性质  聚古罗糖醛酸偏好性
DOI:10.3969/J.ISSN.2095-4972.20230303001
基金项目:国家自然科学基金(42030412);中国大洋协会项目(DY135-B2-01);自然资源部第三海洋研究所基本科研业务费(海三科2019021)
Heterologous expression and enzymatic properties of the alginate lyase gene vralg1 from Vibrio sp. DS32
LI Huimin,SHAO Zongze,LU Yao,YANG Jiangke,ZHOU Meixian
(College of Life Science and Technology, Wuhan Polytechnic University, Wuhan 430023, China;Key Laboratory of Marine Genetic Resources, Third Institute of Oceanography, MNR, Xiamen 361005, China)
Abstract:
Cloning, heterologous expression and enzymatic properties were studied using the alginate lyase gene vralg1 obtained from Vibrio sp. DS32 from the sediment samples of Dongshan Bay, Fujian Province. The alginate lyase gene vralg1was cloned from DS32 genome and the recombinant expression vector pETvralg1 was constructed and heterologously expressed in E.coli. The enzymatic properties, substrate specificity and complete degradation products of the recombinase were determined. The optimum temperature of the recombinant enzyme VRALG1 was 35 ℃ and the enzyme activity reached more than 80% in temperatures of 5~50 ℃. The optimum pH was 6.5~7.5 and the enzyme activity was above 90% after incubation at pH 6.0~9.0 for 1 h. The maximum reaction rate of the recombinant enzyme was 5.919 mmol/(L·min), the Michaelis constant was 3.712 mmol/L and the specific enzyme activity reached 5.874 U/mg under optimal conditions. K+, Cs+, Na+, imidazole and ethanol had little effect on the enzyme activity and the enzyme activity remained above 90% at 5 mmol/L(50 mg/mL) concentration. EDTA had obvious inhibitory effect on the enzyme, and the enzyme was completely inactivated at 1 mmol/L concentration. VRALG1 had high degradation activity for sodium alginate and PolyG. TLC analysis showed that it was mainly a mixture of monosaccharide, disaccharide and trisaccharide. Combined with substrate specificity analysis, it was speculated that the recombinant enzyme was an endo-type bifunctional alginate lyase with obvious polyguluronic acid preference. The alginate lyase gene was successfully cloned and heterologously expressed in E.coli.. The obtained recombinant enzyme VRALG1 had excellent sodium alginate degradation activity and obvious polyguluronic acid preference, which could be used to prepare alginate oligosaccharides with low degree of polymerization.
Key words:  marine biology  alginate lyase  alginate oligosaccharide  cloning and expression  enzymatic property  polygururonic acid preference

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