南极交替单胞菌R11-5产卡拉胶酶的发酵条件优化

【背景】极地寒冷环境中发现了大量具有潜在应用前景的冷适应酶,同时也存在种类繁多的海藻多糖降解菌,因此极端环境微生物是筛选获得新颖、高效多糖降解酶的重要新源泉。由于筛选培养基通常并非野生菌发酵产酶的最优条件,为了使野生菌的产酶效率达到最高,需要对其培养条件进行优化,从而为其深入研究及开发利用提供依据。【目的】对一株产卡拉胶酶的南极菌株进行种属鉴定,并采用响应面法对该菌的发酵产酶条件进行优化。【方法】通过16SrRNA基因对产卡拉胶酶的南极菌株进行种属鉴定,采用响应面法优化南极菌株产酶发酵条件。【结果】该南极菌属于交替单胞菌属(Alteromonas),命名为交替单胞菌R11-5。发酵条件优化结果显示,7个环境因子影响交替单胞菌R11-5的产酶量。利用Design-Expert软件中的Plackett-Burman设计实验,筛选出影响交替单胞菌R11-5产酶量的4个主要因素分别为培养温度、牛肉膏浓度、卡拉胶浓度和Ca~(2+)浓度。通过Box-Behnken设计和响应面分析得到交替单胞菌R11-5最佳产酶发酵条件为:温度15.0°C,牛肉膏浓度11.0 g/L,卡拉胶浓度3.0 g/L,Ca~(2+)浓度5.0 mmol/L。优化后发酵上清液酶产量达到87.193 U/mL,与优化前相比提高了1.8倍。【结论】响应面法提高了南极交替单胞菌R11-5卡拉胶酶的产量,为其开发应用提供了科学依据。

Optimization of carrageenase fermentation conditions of Antarctic bacterium Alteromonas sp. R11-5

[Background] Cold-adapted enzymes from Antarctic and Arctic microorganisms offer several advantages over their mesophilic and thermophilic counterparts, and their properties make them valuable alternatives with industrial and biotechnological applications. Recent studies have shown that these cold environments represent an important reservoir of cold-adapted, complex polysaccharide-degrading marine bacteria that are a major source of novel polysaccharidases and diverse biological active compounds. In general, screening medium is not the best fermentation condition for producing enzyme bacteria, in order to improve enzyme production for research and application, the culture conditions of enzyme producing bacteria need to be optimized. [Objective] To identify a producing carrageenase Antarctic bacterium and optimize the fermentation conditions for carrageenase production. [Methods] The producing carrageenase Antarctic bacterium R11-5 was identified by 16S rRNA gene sequence and the fermentation conditions for carrageenase production of R11-5 was optimized using the method of response surface methodology. [Results] The 16S rRNA gene sequence analysis results showed that the Antarctic bacterium belonged to Alteromonas sp. and was named as Alteromonas sp. R11-5. In order to optimize the fermentation conditions of Alteromonas sp. R11-5, 7 single factors (inoculation amount, temperature, pH, carbon source, nitrogen source, carrageenan concentration and metal ions) were selected by single test. Four important factors (temperature, beef extract, carrageenan and calcium ion) influencing carrageenase production, which identified by initial experimental design of Plackett-Burman by Design-Expert software. Then Box-Behnken design and response surface analysis were adopted to further study the interaction among the variables and obtain optimal values that bring maximum carrageenase production. The optimal fermentation conditions were as follows: Temperature 15.0 °C, beef extract concentration 11.0 g/L, carrageenan concentration 3.0 g/L, Ca2+ concentration 5.0 mmol/L. The supernatant carrageenanse activity of optimized fermentation reached 87.193 U/mL, 1.8 times higher than that before optimization. [Conclusion] Carrageenanse production of Antarctic bacterium Alteromonas sp. R11-5 was improved after the fermentation conditions optimized using the method of response surface methodology. R11-5 was a promising candidate for industrial application.