Two heterologous expression systems using thioredoxin (trxA) as a gene fusion part in Escherichia coli were developed to produce recombinant pediocin PA-1.Pediocin PA-1 structural gene pedA was isolated from Pediococc...Two heterologous expression systems using thioredoxin (trxA) as a gene fusion part in Escherichia coli were developed to produce recombinant pediocin PA-1.Pediocin PA-1 structural gene pedA was isolated from Pediococcus acidilactici PA003 by the method of polymerase chain reaction (PCR),then cloned into vector pET32a(+),and expressed as thioredoxin-PedA fusion protein in the host strain E.coli BL21 (DE3).The fusion protein was in the form of inclusion body and was refolded before purification by nickel-iminodiacetic acid (Ni-IDA) agarose resin column.Biological activity of recombinant pediocin PA-1 was analyzed after cleavage of the fusion protein by enterokinase.Agar diffusion test revealed that 512-arbitrary unit (AU) recombinant pediocin PA-1 was obtained from 1 ml culture medium of E.coli (pPA003PED1) using Listeria monocytogenes as the indicator strain.Thioredoxin-PedA fusion gene was further cloned into pET20b(+).Thioredoxin-PedA fusion protein was detected in both the periplasmic and cytoplasmic spaces.The recombinant pediocin PA-1 from the soluble fraction attained 384 AU from 1 ml culture medium of E.coli (pPA003PED2).Therefore,biologically active pediocin PA-1 could be obtained by these two hybrid gene expression methods.展开更多
【目的】鉴定一株来源于泡菜能够产片球菌素的乳酸菌L131-1,并优化其产片球菌素的发酵条件。【方法】采用形态学观察、16S r DNA序列比对及生理生化实验鉴定;通过碳源、氮源、温度、初始p H、发酵时间的单因素优化实验以及响应面法获得...【目的】鉴定一株来源于泡菜能够产片球菌素的乳酸菌L131-1,并优化其产片球菌素的发酵条件。【方法】采用形态学观察、16S r DNA序列比对及生理生化实验鉴定;通过碳源、氮源、温度、初始p H、发酵时间的单因素优化实验以及响应面法获得最佳发酵条件。【结果】菌株L131-1被鉴定为乳酸片球菌(Pediococcus acidilactici),其最优发酵产酶条件为:蛋白胨16.59 g/L、酵母膏1.13g/L、牛肉膏16.37g/L、葡萄糖20g/L、无水乙酸钠2.6g/L、吐温801ml/L、七水硫酸镁0.58g/L、一水硫酸锰0.19g/L、三水磷酸氢二钾2.6g/L。培养基初始p H 6.0,37℃培养18h。在此条件下,片球菌素最大活性可达到820AU/m L,比优化前提高了近8倍。【结论】实验结果为片球菌素产生菌的筛选鉴定提供了经验,为片球菌素的应用提供了基础。展开更多
基金Project supported by the National Natural Science Foundation of China (No.30571378)the Tianjin Natural Science Foundation (No.08JCZDJC22500),China
文摘Two heterologous expression systems using thioredoxin (trxA) as a gene fusion part in Escherichia coli were developed to produce recombinant pediocin PA-1.Pediocin PA-1 structural gene pedA was isolated from Pediococcus acidilactici PA003 by the method of polymerase chain reaction (PCR),then cloned into vector pET32a(+),and expressed as thioredoxin-PedA fusion protein in the host strain E.coli BL21 (DE3).The fusion protein was in the form of inclusion body and was refolded before purification by nickel-iminodiacetic acid (Ni-IDA) agarose resin column.Biological activity of recombinant pediocin PA-1 was analyzed after cleavage of the fusion protein by enterokinase.Agar diffusion test revealed that 512-arbitrary unit (AU) recombinant pediocin PA-1 was obtained from 1 ml culture medium of E.coli (pPA003PED1) using Listeria monocytogenes as the indicator strain.Thioredoxin-PedA fusion gene was further cloned into pET20b(+).Thioredoxin-PedA fusion protein was detected in both the periplasmic and cytoplasmic spaces.The recombinant pediocin PA-1 from the soluble fraction attained 384 AU from 1 ml culture medium of E.coli (pPA003PED2).Therefore,biologically active pediocin PA-1 could be obtained by these two hybrid gene expression methods.
文摘【目的】鉴定一株来源于泡菜能够产片球菌素的乳酸菌L131-1,并优化其产片球菌素的发酵条件。【方法】采用形态学观察、16S r DNA序列比对及生理生化实验鉴定;通过碳源、氮源、温度、初始p H、发酵时间的单因素优化实验以及响应面法获得最佳发酵条件。【结果】菌株L131-1被鉴定为乳酸片球菌(Pediococcus acidilactici),其最优发酵产酶条件为:蛋白胨16.59 g/L、酵母膏1.13g/L、牛肉膏16.37g/L、葡萄糖20g/L、无水乙酸钠2.6g/L、吐温801ml/L、七水硫酸镁0.58g/L、一水硫酸锰0.19g/L、三水磷酸氢二钾2.6g/L。培养基初始p H 6.0,37℃培养18h。在此条件下,片球菌素最大活性可达到820AU/m L,比优化前提高了近8倍。【结论】实验结果为片球菌素产生菌的筛选鉴定提供了经验,为片球菌素的应用提供了基础。
