利用琼胶酶生产琼胶寡糖具有良好的应用前景,然而目前产琼胶酶菌株的性状不稳定、酶活力低等因素限制了琼胶酶的工业化应用。从龙须菜表面筛选出1株具有高琼胶酶活力的菌株A8,利用VITEK 2 GN微生物鉴定系统和16S rRNA基因序列分析,分别进行生理生化和分子生物学鉴定,确定该菌为Vibrio fluvialis A8。通过单因素试验和正交试验确定该菌的最适产酶培养基为:在人工海水中添加2 g/L琼脂、3 g/L半乳糖、3 g/L 酵母浸粉、5 g/L NaCl;最适培养条件为:接种量2%、温度20 ℃、pH 7.0。优化后发酵液琼胶酶的活力为21.80 U/mL,是优化前的4.05倍。Pearson相关性分析表明,菌株生长对其产酶有正相关影响(P<0.01)。采用(NH4)2SO4分级沉淀和DEAE阴离子交换层析对琼胶酶进行分离纯化,琼胶酶的纯化倍数为3.26,比活力为141.52 U/mg,回收率为15.21%。SDS-PAGE凝胶电泳分析显示,琼胶酶的分子质量约为37 kDa。
Agar oligosaccharide produced by agarase has a good application prospect. However, the industrial application of agarase is limited by both unstable properties of the agarase-producing strains and low activity of enzyme. The strain A8 with high agarase activity was screened from the surface of Gracilaria lemaneiformis. The VITEK 2 GN microbial identification system and 16S rRNA gene sequence analysis were used for physiological, biochemical and molecular biological identification, and the strain was identified and renamed as Vibrio fluvialis A8. The optimum medium for enzyme production was determined as 2 g/L agar, 2 g/L galactose, 3 g/L yeast extract powder and 5 g/L NaCl in artificial seawater, and the optimum culture conditions were determined as temperature 20 ℃, inoculum size 2%, pH 7.0. The optimized enzyme activity in the fermentation medium was 21.80 U/mL, which was 4.05 times than that before optimization. Pearson correlation analysis showed that the strain growth had a positive correlation with its enzyme production (P<0.01). The agarase was isolated and purified bammonium sulfate fractionation and DEAE anion exchange chromatography. The purification ratio of agarase was 3.26, the specific activity 141.52 U/mg, and the recovery 15.21%. The SDS-PAGE gel electrophoresis analysis showed that the molecular weight of the agarase was about 37 kDa. These results provide technical support for further processing of agar.
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