一株产褐藻胶裂解酶的需钠弧菌筛选及酶解产物分析

Screening of a Vibrio natriegens SK42.001 strain for producing alginate lyase and enzymatically degraded product analyses

[背景]褐藻胶裂解酶种类丰富、降解机制多样,是高效环保降解褐藻胶、制备褐藻寡糖的工具酶,成为褐藻植物高值化开发利用的研究热点。[目的]从海泥中筛选获得褐藻胶裂解酶高效产酶菌株,确定菌株发酵产酶最优条件,鉴定和分析酶降解产物,进而解析该酶的降解特性。[方法]以褐藻胶为唯一碳源,从海带养殖场附近海泥中筛选菌株,通过形态学观察、生理生化实验和16S rRNA基因序列比对进行菌种鉴定。优化发酵培养基并利用筛选菌株进行发酵产酶。利用红外和阴离子交换色谱分析不同程度酶解产物以确定该酶的降解偏好,利用薄层色谱和液相色谱-质谱鉴定酶降解的终产物。[结果]筛选鉴定得到一株产褐藻胶裂解酶的需钠弧菌(Vibrio natriegens)SK42.001,最优产酶培养基为海藻酸钠8.0 g/L、(NH_(4))2SO_(4)8.0 g/L、NaCl 30.0 g/L、MgSO_(4)·7H_(2)O 5.0 mmol/L和K_(2)HPO_(4)·2H_(2)O 10.0 mmol/L,发酵酶活为(5.20±0.14)U/mL。菌株所产酶偏好于降解褐藻胶中的古罗糖醛酸片段,并且终产物聚合度较为单一,主要为褐藻三糖。[结论]筛选到褐藻胶裂解酶高效产酶菌株Vibrio natriegens SK42.001,该菌株所产酶具有古罗糖醛酸降解偏好性且可特异性生产褐藻三糖,具有进一步开发用于大规模制备褐藻胶裂解酶或褐藻寡糖生物制品的潜力。

[Background]Alginate lyase has various types and degradation mechanisms,and has become a tool for the efficient and environmentally friendly production of alginate oligosaccharides,as well as a research hotspot for high-value development and utilization of algae.[Objective]This study aimed to isolate alginate lyase-producing strains,determine the optimal fermentation conditions for enzyme production,and analyze degradation products to reveal the degradation characteristics of the enzyme.[Methods]The strain was screened from the sea mud near kelp farms using alginate as the sole carbon source,and was identified according to morphological observation,physiological and biochemical tests,and 16 S rRNA sequence alignment.The fermentation medium was optimized and used for enzyme production.Products at different degradation degrees were analyzed by IR and anion exchange chromatography to explore the degradation preference of the enzyme,and the final products were characterized by TLC and LC-MS.[Results]An alginate lyase-producing Vibrio natriegens SK42.001 was screened and identified.The optimized fermentation medium contained 8.0 g/L alginate,8.0 g/L(NH_(4))_(2)SO_(4),30.0 g/L NaCl,5.0 mmol/L MgSO_(4)·7 H_(2)O and 10.0 mmol/L K_(2)HPO_(4)·2 H_(2)O,and the fermentation activity was(5.20±0.14)U/mL.The alginate lyase produced by strain SK42.001 had a preference to degrade guluronic acid blocks,and the final products present a relatively simple degree of polymerization,which were predominantly trisaccharides.[Conclusion]A V.natriegens SK42.001 with high yield of alginate lyase was screened.The enzyme produced by the strain has a preference to degrade guluronic acid blocks and specifically produces trisaccharides,which has the potential to be further developed for large-scale production of alginate lyase and alginate oligosaccharide biological products.