Mangrovibacterium sp. SH-52耐热内切型海藻酸裂解酶基因的克隆及酶学鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2020-0375

摘要/Abstract

摘要：

研究来自厌氧海藻酸分解菌的海藻酸裂解酶,表征其酶学特征,旨为丰富海藻酸裂解酶工业应用提供理论依据。从一株厌氧菌Mangrovibacterium sp. SH-52中克隆一个海藻酸裂解酶基因SHA-I,分析基因序列,并在大肠杆菌中进行异源表达,纯化后对其酶学性质进行分析。SHA-I由362个氨基酸构成,分子量大约为41 kD,与Lewinella cohaerens中的海藻酸裂解酶同源性最高,为80%,属于多聚糖裂解酶类（Polysaccharide lyase,PL）7家族。SHA-I最适pH为7.0,最适温度是50℃,在60℃时的活性超过最高活性的50%,在80℃时约为最高活性的40%,表明SHA-I是一种耐热性海藻酸裂解酶。SHA-I对polyG（聚甘露糖醛酸）有底物特异性,其降解海藻酸主要产生二糖和三糖,是一种内切型海藻酸裂解酶。

关键词: 厌氧海藻酸分解菌, Mangrovibacterium sp. SH-52, 异源表达, 内切型海藻酸裂解酶, 耐热性

Abstract:

The objective of this work is to investigate the alginate lyases isolated from anaerobic alginolytic bacteria,to characterize the novel alginate lyase,thus to provide a theoretical basis for the enriching industrial application of alginate lyase. A gene SHA-I encoding an alginate lyase was cloned from an anaerobic bacterium Mangrovibacterium sp. SH-52,sequenced and expressed in Escherichia coli. Its expressed enzyme was purified and the enzymatic characteristics were analyzed. The alginate lyase SHA-I was composed of 362 amino acids with molecular weight of approximately 41 kD. SHA-I belonged to polysaccharide lyase（PL）family 7 and showed 83% amino acid identity with alginate lyase from Lewinella agarilytica. The optimal pH for SHA-I activity was 7.0. SHA-I demonstrated the highest activity at 50℃and remained approximate 60% of the highest activity at 60℃,and 40% at 80℃,indicating that SHA-I was a thermo-tolerant alginate lyase. SHA-I presented substrate specificity to polyG（polymannuronic acid）. It degraded polyG blocks to alginate oligosaccharides as the main products.

Key words: anaerobic alginolytic bacteria, Mangrovibacterium sp. SH-52, heterologous expression, endotype alginate lyase, thermo-tolerant

李卫娜, 申冬玲, 张煜星, 刘学通, 伊日布斯. Mangrovibacterium sp. SH-52耐热内切型海藻酸裂解酶基因的克隆及酶学鉴定[J]. 生物技术通报, 2020, 36(12): 82-90.

LI Wei-na, SHEN Dong-ling, ZHANG Yu-xing, LIU Xue-tong, IRBIS Chagan. Cloning and Enzymatic Identification of Thermo-tolerant and Endotype Alginate Lyase Gene from Mangrovibacterium sp. SH-52[J]. Biotechnology Bulletin, 2020, 36(12): 82-90.

图/表 7

图1 菌株SH-52的16S rRNA序列系统进化树

图2 SHA-I与PL7家族氨基酸序列的比对黑色箭头代表PL7家族海藻酸裂解酶高度保守区SA3,SA4和SA5[30];红框代表 PL7家族海藻酸裂解酶保守催化残基[13,30-32];黑色环代表 PL7 表面活性裂缝残基[32];蓝色箭头代表Sphingomonas sp. A1- II’活性裂缝的两个盖环（L1和L2）[30,31]. SHA-I来自 Mangrovibacterium sp. SH-52;alginate lyase来自Zobellia galactanivorans（GenBank accession number,WP_013995454）;alginate lyase来自Lewinella cohaerens（GenBank accession number,WP_020539194.1）;alginate lyase来自Cellulophaga baltica（GenBank accession number,WP_029448962）;alginate lyase来自Flavobacteriales bacterium ALC-1（GenBank accession number,WP_008 271146）;alginate lyase来自Zobellia uliginosa（GenBank accession number,WP_03 8232734）;alginate lyase来自Flavobacterium sp. S20（GenBank accession number,AEB69783.1）;A1- II’来自 Sphingomonas sp. A1（GenBank accession number,BAD16656）

图3 SDS-PAGE检测重组酶SHA-I的纯化 M：蛋白marker;1：纯化前的细菌上清蛋白;2：纯化后的流穿液;3：纯化后的洗涤液;4：用还原性谷胱甘肽洗脱后的洗脱液

表1 重组海藻酸裂解酶SHA-I的纯化

Step	Total protein/（mg·L^-1）	Totalactivity/U	Specific activity/（U·mg^-1）	Yield/%	Purification（fold）
Cell extract	162.0	421.2	2.6	100.0	1
GST Sefinose Resin BSP091	3.2	41.6	13.0	9.8	5

图4 pH和温度对SHA-I酶活性和稳定性的影响 A：SHA-I最适pH;B：SHA-I的pH稳定性;C：SHA-I最适温度;D：SHA-I热稳定性

表2 金属离子对重组酶SHA-I活性的影响

Reagent	Concentration/（mmol·L^-1）	Relative activity/%
None	0	100
NaCl	1	101
	50	156
	100	158
KCl	1	115
MgCl₂	1	112
NiCl₂	1	7
ZnCl₂	1	114
CoCl₂	1	92
HgCl₂	1	8.5
CaCl₂	1	104
MnCl₂	1	85.1
EDTA	1	33.5

图5 SHA-I底物特异性和降解产物的TLC分析 M：Maker; 1：SHA-1降解产物

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