Cloning and Enzymatic Identification of Thermo-tolerant and Endotype Alginate Lyase Gene from Mangrovibacterium sp. SH-52

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

Abstract

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

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.

Figures/Tables 7

References 38

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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

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

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

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