目前利用糖苷磷酸化酶制备α-1,3-葡聚糖的前景广阔。该研究考察了一种类芽孢杆菌来源的α-1,3-葡聚糖磷酸化酶制备α-1,3-葡聚糖的能力并对其进行发酵优化。类芽孢杆菌(Paenibacillus sp.)HMSSN-139来源的α-1,3-葡聚糖磷酸化酶编码基因被克隆后转化至大肠杆菌(Escherichia coli BL21)和枯草芽孢杆菌(Bacillus subtilis)中进行重组表达,利用重组酶与麦芽糖磷酸化酶偶联催化制备α-1,3-葡聚糖。结果显示,以200 g/L麦芽糖为底物,在pH 7.0、37 ℃、磷酸盐终浓度20 mmol/L及加酶量均为0.2 mg/mL条件下,可生成聚合度为3~6的α-1,3-葡聚糖。反应达到312 h时,α-1,3-葡聚糖含量最高可达62.90%。此外优化了其启动子和发酵培养基碳氮源。将初始启动子PamyE替换为PamyQ`,酶活力可提高至2.35 U/mL,为初始重组菌酶活力的1.50倍;优化后的最优培养基的氮源为10 g/L大豆蛋白胨,碳源为10 g/L甘油,在此培养基中重组菌酶活力提高至5.41 U/mL,达到优化前的2.30倍。该研究获得了一种新型的α-1,3-葡聚糖磷酸化酶,并对其进行了发酵优化,为工业生产高聚合度的α-1,3-葡聚糖提供了新的参考。
Currently, the use of glycoside phosphorylases for the preparation of α-1,3-glucan is promising.In this study, the application ability to prepare α-1,3-glucan and the fermentation optimization of a novel α-1,3-glucan phosphorylase was investigated.The coding gene of α-1,3-glucan phosphorylase from Paenibacillus sp.HMSSN-139 was cloned and recombinantly expressed in Escherichia coli BL21 and Bacillus subtilis, and the recombinant enzyme was used to catalyze the preparation of α-1,3-glucan by coupling with maltose phosphorylase.When this reaction was carried out with 200 g/L maltose as substrate, α-1,3-glucan with a degree of polymerization of 3-6 was produced under the conditions of pH 7.0, 37 ℃, 20 mmol/L phosphate and enzyme addition both at 0.2 mg/mL.The content of α-1,3-glucan was up to 62.90% after the reaction for 312 h.In addition, this study optimized the promoter and fermentation medium carbon and nitrogen sources.The enzyme activity increased to 2.35 U/mL, which was 1.50 times of the initial enzyme activity, by replacing the initial promoter PamyE with PamyQ`.The optimal nitrogen and carbon source of the fermentation medium were 10 g/L soy peptone and 10 g/L glycerol, respectively, and the enzyme activity in this condition was increased to 5.41 U/mL, which was 2.30 times of that before optimization.In this study, a novel α-1,3-glucan phosphorylase was obtained and its fermentation was optimized, which provides a new reference for the industrial production of α-1,3-glucan with a high polymerization degree.
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