一株产低温β-半乳糖苷酶微杆菌的筛选鉴定、产酶条件及其酶学特性

Isolation and identification of a Microbacterium sp. LW106 strain producing cold-active β-galactosidase, and study on its enzyme production conditions and enzymatic properties

摘要:

【背景】低温β-半乳糖苷酶能在低温下仍保持较高的乳糖水解活性，筛选酶学特性适合在牛乳体系中高效水解乳糖的β-半乳糖苷酶生产菌株，是低乳糖牛乳加工产业关注的焦点。【目的】对天山中国一号冰川沉积物中分离的一株产低温β-半乳糖苷酶菌株的产酶条件和酶学特性进行研究。【方法】结合X-gal平板法初筛和测定粗酶液酶活复筛，获得产低温β-半乳糖苷酶的菌株。通过形态学、生理生化试验及16S rRNA基因测序分析对筛选菌株进行鉴定，单因素摇瓶实验优化菌株的产酶条件，硫酸铵分级沉淀初步纯化β-半乳糖苷酶并对其酶学特性进行分析。【结果】通过形态学、生理生化特征和16S rRNA基因鉴定，确定菌株LW106为微杆菌属(Microbacterium)菌株；该菌株最适产酶温度为25 °C，最佳产酶碳源为可溶性淀粉，培养基初始pH为7.0，接种量为3%；对初步纯化的低温β-半乳糖苷酶酶学性质的研究表明，LW106所产β-半乳糖苷酶的最适pH为6.0，最适反应温度为35 °C，4 °C时酶活为最大酶活的78%，4 °C和pH 7.0时的稳定性最好，10 mmol/L的Na+对酶活性基本没有抑制作用，Ca2+对酶活性具有一定的激活作用。【结论】菌株LW106所产低温β-半乳糖苷酶的酶学特性表明该酶在乳品低温加工领域具有进一步研究和应用的价值。

Abstract:

[Background] Cold-active β-galactosidases are typically characterized by a high lactose hydrolysis activity at low temperature. Such a characteristic makes cold-active β-galactosidase highly attractive for application in low-lactose milk production industry, and how to mine novel cold-active β-galactosidase with enzymatic properties perfectly suitable for lactose hydrolysis in milk solution condition becomes a research focus. [Objective] This study is aiming to screen novel strains producing cold-active β-galactosidase from glacier sediments at China No.1 glacier in Tianshan Mountains and to evaluate if the enzymatic properties of the cold-active β-galactosidase is suitable for further application in low-lactose milk production. [Methods] Primary screening using X-gal culture plate, and secondary screening by assaying the relative β-galactosidase activity at low temperature were carried out. The target strain selected was identified by physiological, biochemical and 16S rRNA gene sequencing. The growth condition and enzyme production condition of the isolate were optimized by single-factor experiment. Enzymatic properties of the partially purified β-galactosidase produced by the isolate were also investigated. [Results] LW106, a strain with high β-galactosidase activity, was identified as Microbacterium sp. LW106. The optimal temperature for LW106 producing β-galactosidase is 25 °C. Soluble starch was the optimal carbon source for LW106 to produce β-galactosidase, the optimum medium initial pH was 7.0, and the optimal inoculum size was 3%. Preliminary study on its enzymatic properties showed that the optimal pH of the β-galactosidase of strain LW106 was 6.0, and the optimal reaction temperature was 35 °C with a relative activity of 78% at 4 °C. The β-galactosidase produced by LW106 showed the best stability at 4 °C and pH 7.0. Metal ion Na+ at a level of 10 mmol/L did not inhibit the activity of β-galactosidase of LW106, while 10 mmol/L of Ca2+ activated the β-galactosidase activity. [Conclusion] The enzymatic properties of β-galactosidase produced by Microbacterium sp. LW106 indicated it may be suitable for lactose hydrolysis at low temperature, showing good application potential in low-lactose milk production industry.