γ-聚谷氨酸的产生菌分离鉴定及其生物合成条件的研究
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摘要
本研究旨在从多种样品中分离筛选出γ-聚谷氨酸(γ-PGA)高产菌种,并通过个体、菌落的形态及生理生化等特征,对目标菌株进行鉴定,命名为枯草芽孢杆菌纳豆亚种(Bacillus subtilis natto)QY-09。
本研究对发酵培养基和发酵工艺条件进行探讨,即对碳源、氮源、谷氨酸钠、氯化钠、无机离子的浓度以及初始pH、温度、种龄、接种量、溶氧量进行了研究,建立了发酵过程曲线,探讨菌体生长与γ-PGA合成的关系。结果表明,发酵培养基和发酵工艺条件对γ-PGA的产率和生产成本有较大影响。高产菌株QY-09为谷氨酸依赖型菌株,培养基适宜配方为葡萄糖50g/L,大豆蛋白胨40g/L,谷氨酸钠40g/L,氯化钠30g/L,MgSO_4 0.5g/L、CaCl_2 0.3g/L、K_2HPO_4 3.0g/L、KH_2PO_4 4.5g/L及适量生物素。适宜的培养条件为:初始pH7.0,发酵温度37℃,种龄15h,接种量3%(V/V),装液量40mL/250mL,摇床培养56h,其γ-PGA产量可达32.7 g/L。发酵过程曲线表明γ-PGA的合成与菌体生长并非完全同步。
本研究对纯化后产品(纯度为96.10%)的理化性质、结构特征、分子量测定、含量测定方法进行了探讨和分析。结果表明γ-PGA易溶于水,不溶于乙醇、丙酮等有机溶剂,由单一的谷氨酸聚合而成。建立紫外分光光度法测定γ-PGA含量的方法,线性方程为A=0.493 c+0.0108,相关系数r为0.9981,平均回收率为99.5%,相对标准偏差为1.0%。凝胶渗透色谱、SDS-聚丙烯酰胺凝胶电泳对γ-PGA分子量研究表明,不同发酵时间的发酵产物分子量不同,发酵56h时γ-PGA相对分子质量在409～473 kDa之间,且56 h后,其相对分子质量范围分布逐步变宽。
The purpose of this study was to isolate and,select high-yieldingγ-polyglutamic acid bacteria from multiple samples.The target strain could be identified and nominated as Bacillus subtilis natto QY-09 through individual and colony morphology,physiological and biochemical characteristics.
This study explored the culture medium and fermentation conditions. That was the concentration of carbon source,nitrogen source,sodium glutamate,NaCl,inorganic ions as well as the initial pH,temperature, species age,inoculum size,dissolved oxygen.We had established the curve of the fermentation process to study the relationship of bacteria growth and synthesis ofγ-PGA.The results showed that the culture medium and fermentation conditions had a greater impact on theγ-PGA yield and production costs.The strain,Bacillus subtilis natto QY-09,was a glutamate-dependent one,whose suitable medium was glucose 50g/L,soy peptone 40g/L,sodium glutamate 40g/L,NaC1 30g/L,MgSO_4 0.5g/L,CaCl_2 0.3g/L,K_2HPO_4 3.0g/L,KH_2PO_4 4.5g/L and suitable biotin.Fitting culture conditions were as follows:initial pH7.0,temperature 37℃,seed age 15h, inoculation 3%(V/V),the volume of liquid 40mL/250mL for 56h.The yield ofγ-PGA was up to 32.7g/L.We could see that the fermentation process ofγ-PGA synthesis and cell growth was not completely synchronized from the curve.
The physical and chemical properties,structural characteristics and molecular weight of the purified products(96.10%purity) were discussed. The results showed it was soluble in water,insoluble in ethanol,acetone and other organic solvents.It was composed of glutamic acid without other amino acids or proteins.We established UV spectrophotometry to determinate theγ-PGA content.Its linear equation was A= 0.493 c + 0.0108, correlation coefficient r 0.9981,the average recovery 99.5%,and relative standard deviation 1.0%.The study of molecular weight ofγ-PGA through GPC and SDS-PAGE had shown that the products had different molecular weight in different fermentation time.The molecular weight ofγ-PGA was between 409 and 473 kDa at 56h.Furthermore its molecular weight distribution became gradually wide since 56h.
本研究对发酵培养基和发酵工艺条件进行探讨,即对碳源、氮源、谷氨酸钠、氯化钠、无机离子的浓度以及初始pH、温度、种龄、接种量、溶氧量进行了研究,建立了发酵过程曲线,探讨菌体生长与γ-PGA合成的关系。结果表明,发酵培养基和发酵工艺条件对γ-PGA的产率和生产成本有较大影响。高产菌株QY-09为谷氨酸依赖型菌株,培养基适宜配方为葡萄糖50g/L,大豆蛋白胨40g/L,谷氨酸钠40g/L,氯化钠30g/L,MgSO_4 0.5g/L、CaCl_2 0.3g/L、K_2HPO_4 3.0g/L、KH_2PO_4 4.5g/L及适量生物素。适宜的培养条件为:初始pH7.0,发酵温度37℃,种龄15h,接种量3%(V/V),装液量40mL/250mL,摇床培养56h,其γ-PGA产量可达32.7 g/L。发酵过程曲线表明γ-PGA的合成与菌体生长并非完全同步。
本研究对纯化后产品(纯度为96.10%)的理化性质、结构特征、分子量测定、含量测定方法进行了探讨和分析。结果表明γ-PGA易溶于水,不溶于乙醇、丙酮等有机溶剂,由单一的谷氨酸聚合而成。建立紫外分光光度法测定γ-PGA含量的方法,线性方程为A=0.493 c+0.0108,相关系数r为0.9981,平均回收率为99.5%,相对标准偏差为1.0%。凝胶渗透色谱、SDS-聚丙烯酰胺凝胶电泳对γ-PGA分子量研究表明,不同发酵时间的发酵产物分子量不同,发酵56h时γ-PGA相对分子质量在409～473 kDa之间,且56 h后,其相对分子质量范围分布逐步变宽。
The purpose of this study was to isolate and,select high-yieldingγ-polyglutamic acid bacteria from multiple samples.The target strain could be identified and nominated as Bacillus subtilis natto QY-09 through individual and colony morphology,physiological and biochemical characteristics.
This study explored the culture medium and fermentation conditions. That was the concentration of carbon source,nitrogen source,sodium glutamate,NaCl,inorganic ions as well as the initial pH,temperature, species age,inoculum size,dissolved oxygen.We had established the curve of the fermentation process to study the relationship of bacteria growth and synthesis ofγ-PGA.The results showed that the culture medium and fermentation conditions had a greater impact on theγ-PGA yield and production costs.The strain,Bacillus subtilis natto QY-09,was a glutamate-dependent one,whose suitable medium was glucose 50g/L,soy peptone 40g/L,sodium glutamate 40g/L,NaC1 30g/L,MgSO_4 0.5g/L,CaCl_2 0.3g/L,K_2HPO_4 3.0g/L,KH_2PO_4 4.5g/L and suitable biotin.Fitting culture conditions were as follows:initial pH7.0,temperature 37℃,seed age 15h, inoculation 3%(V/V),the volume of liquid 40mL/250mL for 56h.The yield ofγ-PGA was up to 32.7g/L.We could see that the fermentation process ofγ-PGA synthesis and cell growth was not completely synchronized from the curve.
The physical and chemical properties,structural characteristics and molecular weight of the purified products(96.10%purity) were discussed. The results showed it was soluble in water,insoluble in ethanol,acetone and other organic solvents.It was composed of glutamic acid without other amino acids or proteins.We established UV spectrophotometry to determinate theγ-PGA content.Its linear equation was A= 0.493 c + 0.0108, correlation coefficient r 0.9981,the average recovery 99.5%,and relative standard deviation 1.0%.The study of molecular weight ofγ-PGA through GPC and SDS-PAGE had shown that the products had different molecular weight in different fermentation time.The molecular weight ofγ-PGA was between 409 and 473 kDa at 56h.Furthermore its molecular weight distribution became gradually wide since 56h.
引文
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