Box-Behnken响应面法优化司替霉素B产生菌发酵条件

Optimization of fermentation medium for steffimycin B production by Box-Behnken response surface methodology

摘要:

【背景】粗糙链霉菌(Streptomyces scabrisporus) HBERC-53204是本中心自主分离的一株链霉菌，经鉴定，其产生一种活性化合物司替霉素B (steffimycin B，SMB)，对多种动植物重要病原菌具有良好生物活性。【目的】提高SMB发酵水平，拓宽放线菌活性天然产物在农牧业领域的研究及应用。【方法】以本实验室筛选出的一株产SMB的粗糙链霉菌HBERC-53204为研究对象，运用单因素试验筛选培养基的主效碳源、氮源、无机盐及各营养成分最适浓度，并基于单因素试验结果，通过Plackett-Burman (PB)试验设计筛选出显著影响因素，再结合最陡爬坡试验、Box-Behnken (BB)响应面法拟合显著因子与产量的非线性方程求解，进一步优化菌株产SMB的最佳发酵培养基配方。【结果】优化后最佳培养基配方为：葡萄糖36.22 g/L，蛋白胨8.00 g/L，酵母粉8.51 g/L，酸水解酪蛋白1.50 g/L，MgSO₄ 0.68 g/L，KNO₃ 1.00 g/L。经摇瓶验证，优化后SMB效价达到477.26 mg/L，比初始配方产量提高1 773.08%。在20 L发酵罐培养120 h，目标化合物产量达到214.48 mg/L。【结论】通过对菌株HBERC-53204发酵培养基优化，显著提高了SMB产量，并在20 L发酵罐中得到验证，在此基础上获得了克级纯品。

Abstract:

[Background] Streptomyces scabrisporus HBERC-53204 is an actinomycete strain isolated from a soil sample collected in Hunan province.It produces an active compound steffimycin B (SMB) with strong biological activity against a variety of animal and plant pathogens.[Objective] To improve the productivity of the strain for SMB and broaden the research and application of SMB in agriculture and animal husbandry.[Methods] We determined the optimum concentrations of main components including carbon-,nitrogen-containing nutrients and inorganic salts in the fermentation medium to increase the production of SMB by S. scabrisporus HBERC-53204 through single factor experiments.On the basis of the results of single factor experiments,the factors significantly affecting the production of SMB were screened out through Plackett-Burman (PB) design.On the basis of the steepest ascent design and Box-Behnken (BB) design,the medium for the production of SMB was optimized by response surface methodology through fitting the nonlinear equation of significant factors with the yield of SMB.[Results] The composition of the medium for the production of SMB by HBERC-53204 was optimized as follows:glucose 36.22 g/L,peptone 8.00 g/L,yeast powder 8.51 g/L,acid hydrolyzed casein 1.50 g/L,MgSO₄ 0.68 g/L,and KNO₃ 1.00 g/L.The yield of SMB produced in the optimized medium reached 477.26 mg/L in shake flask fermentation,which was 1 773.08% higher than that of the original medium.At the time point of 120 h of fermentation in a 20 L fermenter,the yield of SMB reached a maximum of 214.48 mg/L.[Conclusion] After optimization of the fermentation medium of strain HBERC-53204,the yield of SMB was significantly increased,which was scaled up in a 20 L fermenter.Based on this study,grams of purified SMB compound were obtained.