CN102586296A - 一种密码子优化的β-葡萄糖苷酶Mbg1基因及其表达 - Google Patents
一种密码子优化的β-葡萄糖苷酶Mbg1基因及其表达 Download PDFInfo
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Abstract
本发明公开了一种密码子优化的β-葡萄糖苷酶Mbg1基因及其表达,所述β-葡萄糖苷酶Mbg1基因的核苷酸序列如SEQ ID No 1,其编码的蛋白质序列如SEQ ID No 2。本发明采用连续延伸PCR方法合成所述β-葡萄糖苷酶Mbg1基因,通过将所述基因构建到大肠杆菌表达载体并转化入大肠杆菌EG50中表达出高比活的β-葡萄糖苷酶,其比活达到232umg-1min-1以上,可用于β-葡萄糖苷酶的工业生产中。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种密码子优化的β-葡萄糖苷酶Mbg1基因及其表达。
背景技术
β-葡萄糖苷酶(β-glucosidase,EC3.2.1.21),又称β-D-葡萄糖苷葡萄糖水解酶,是纤维素酶的一个关键组分,它能够催化水解芳基或烃基与糖基原子团之间的糖苷键,同时释放出β-D-葡萄糖和相应的配基。1837年,Liebig和Wohler首次在苦杏仁汁中发现了β-葡萄糖苷酶。后来的研究发现,β-葡萄糖苷酶广泛存在于植物、动物和微生物中,它参与生物体的糖代谢和胞内信号传导等,对维持生物体正常生理功能起着重要作用。β-葡萄糖苷酶在食品、饲料、医疗、生物能源等领域具有重要的应用价值。在食品工业中,β-葡萄糖苷酶能将水果、蔬菜、茶叶中的风味前体物质转化为具有浓郁天然风味的香气物质,用于食品增香;β-葡萄糖苷酶可以帮助多酚葡萄糖昔转化为游离多酚,以提高多酚的生物活性或者帮助分离分析多酚;水解含有氰醇与葡萄糖形成的糖苷,消除不稳定氰醇的毒性等。在饲料工业中,β-葡萄糖苷酶可以用于对饲料中纤维素的降解,促进猪、鸡等非反刍动物影响的吸收;在生物能源领域,β-葡萄糖苷酶是纤维素转化生成乙醇的关键酶之一,通过水解纤维素分解过程中的纤维二糖,可以有效地减少纤维二糖纤维素酶的抑制作用。此外,β-葡萄糖苷酶在合成低聚龙胆糖等寡糖和相关的糖复合物中有广泛的应用前景。
相对于真表达系统而言,大肠杆菌作为一种非常成熟的原核表达系统有诸多优点:1)对大肠杆菌的遗传背景和生理特性研究已相当彻底,已有很多不同抗药性、不同营养依赖型和不同校正突变型的菌种供选择应用,可以根据不同的载体而选择不同的菌种;2)尽管大肠杆菌细胞的空间小,但繁殖能力强,在营养条件充足时,20~30min即可繁殖一代,而且大规模发酵成本低,具有巨大的生产潜力;3)表达水平一般较真核系统高,某些外源基因在大肠杆菌中的表达量可达总蛋白的5%~30%,且下游工艺简单、易于控制。因此,大肠杆菌是生产β-葡萄糖苷酶较为理想的表达系统。
发明内容
本发明所要解决的技术问题在于提供一种密码子优化的β-葡萄糖苷酶Mbg1基因及其表达,所述β-葡萄糖苷酶Mbg1基因通过连续延伸PCR方法合成,并将本发明合成的β-葡萄糖苷酶Mbg1基因转化入大肠杆菌EG50中表达出高比活的β-葡萄糖苷酶。
为达到上述目的,本发明通过以下技术方案来实现:
所述β-葡萄糖苷酶Mbg1基因的核苷酸序列SEQ ID NO 1所示。
所述β-葡萄糖苷酶Mbg1基因的核苷酸序列编码的蛋白质序列如SEQ ID NO 2所示。
本发明按照偏爱密码子对野生型的Mbg1基因进行改造,改造后的基因序列与野生型序列(GenBank Accession No.CP000749)同源性为80%(参见图1),其编码的蛋白质共447个氨基酸。
本发明的经密码子优化的β-葡萄糖苷酶Mbg1基因合成采用连续延伸PCR方法。两侧引物分别加入BamHI和SacI酶切位点,使用的引物如下:
P1:GGA,TCC,ATG,TCT,ATC,ACT,CTT,CCA,TCT,CAC,TCC,AAG,ATG,CTG,ACC,TCC,GAC,TTC,ATC,TTT
P2:TGG,TAG,CAC,CTT,CAA,TCT,GGA,AAG,AGG,CAG,TAG,CAA,CAC,CAA,AGA,TGA,AGT,CGG,AGG,TCA
P3:CCA,GAT,TGA,AGG,TGC,TAC,CAC,TGC,TGA,CAA,CAG,ACT,CCC,ATC,CAT,CTG,GGA,TAC,CTT,CTG
P4:CTC,ACC,ATT,GTC,CAT,ACC,TTT,GAC,CTT,GCC,TGG,AGT,AGC,ACA,GAA,GGT,ATC,CCA,GAT,GGA
P5:AAG,GTA,TGG,ACA,ATG,GTG,AGA,TTG,CTT,GTG,ACC,ACT,ATC,ATC,TCT,GGG,AAC,AAG,ACA,TTC
P6:GAG,AGT,CTG,TAG,GCA,TCA,ACA,CCA,AGG,TCT,TTG,ATC,AGC,TGA,ATG,TCT,TGT,TCC,CAG,AGA
P7:GTT,GAT,GCC,TAC,AGA,CTC,TCC,ATT,GCT,TGG,CCA,AGA,GTC,ATG,GAC,AAG,AAA,GGT,GAA,GCT
P8:TCT,TGA,GCA,GGT,TTC,TGT,AGA,AGT,CCA,GAC,CAG,CTT,GGT,TAG,CTT,CAC,CTT,TCT,TGT,CCA
P9:CTA,CAG,AAA,CCT,GCT,CAA,GAA,GCT,CAA,GGC,TGA,AGG,TCT,CAC,TGT,CTT,TGC,TAC,TCT,CTA
P10:ACC,ACC,TTT,GTC,TTC,CAG,GTG,CTG,TGG,CAG,ATC,CCA,ATG,GTA,GAG,AGT,AGC,AAA,GAC,AGT
P11:ACC,TGG,AAG,ACA,AAG,GTG,GTT,GGC,TCA,ACA,GAG,AGA,CTG,CCT,ACC,AGT,TCA,AGA,ACT,ATG
P12:GAG,TCA,ACC,CAC,TCA,GCA,AGC,TCT,TTA,GTG,ACC,AGG,TCA,GCA,TAG,TTC,TTG,AAC,TGG,TAG
P13:CTT,GCT,GAG,TGG,GTT,GAC,TCT,TGG,GCT,ACC,TTC,AAC,GAA,CCA,TTC,TGT,GCT,GCC,ATC,CTT
P14:GTT,TGG,ACA,AAC,CAG,GAG,CAT,GGA,TAC,CCA,GCT,CAT,AGC,CAA,GGA,TGG,CAG,CAC,AGA,ATG
P15:TGC,TCC,TGG,TTT,GTC,CAA,ACC,AGC,CTT,TGG,TAG,ACA,AGC,TGC,TCA,CCA,CAT,CTT,GCT,CGC
P16:TGG,AGC,GTT,CTT,TCT,GAT,GAC,TGG,AAG,AGC,CAA,GCC,ATG,AGC,GAG,CAA,GAT,GTG,GTG,AGC
P17:TCA,TCA,GAA,AGA,ACG,CTC,CAA,AGT,CTC,AAG,TTG,GTA,TCG,TTC,TCA,ACA,TGA,ACA,GAT,CCT
P18:AAG,CAA,GCA,AAC,TGG,TCT,TCG,GTC,TTC,TCA,GAG,GCA,GCA,TAG,GAT,CTG,TTC,ATG,TTG,AGA
P19:GAA,GAC,CAG,TTT,GCT,TGC,TTG,ATG,AGA,GAG,ACT,CTC,GAC,AAC,CAG,TTC,TTC,ATC,GAA,CCA
P20:CAA,CAG,TCT,TCA,GCA,ACT,GTG,GGT,ACT,GAC,CCT,TCA,TCA,GTG,GTT,CGA,TGA,AGA,ACT,GGT
P21:ACA,GTT,GCT,GAA,GAC,TGT,TGC,TCC,ACA,GTA,CTT,GCC,AAC,CAT,CCT,TCC,AGG,TGA,CAT,GGA
P22:GAA,GTT,CAT,GCC,AAG,GAA,GTC,GAT,TGG,TTG,AGA,GAT,GAT,GTC,CAT,GTC,ACC,TGG,AAG,GAT
P23:ACT,TCC,TTG,GCA,TGA,ACT,TCT,ACA,CTT,GCA,ATC,ACA,ATG,CCT,ACG,ATG,CTG,ATG,ACA,TGT
P24:TCA,GTG,TAC,TCA,ACA,GTC,TGA,GAG,TTC,TGG,ACG,TTC,TTG,AAC,ATG,TCA,TCA,GCA,TCG,TAG
P25:CAG,ACT,GTT,GAG,TAC,ACT,GAC,ATC,GGT,TGG,GAG,ATT,GCT,CCA,CAT,GCC,TTC,ACT,GAA,CTG
P26:AGA,TTG,GTG,GAA,GAG,AGT,ACT,GCT,TGT,GGA,GGT,TGA,CCA,ACA,GTT,CAG,TGA,AGG,CAT,GTG
P27:GTA,CTC,TCT,TCC,ACC,AAT,CTA,CAT,CAC,TGA,GAA,TGG,TGC,TGC,TTG,CGC,TGA,TCA,GAT,CAT
P28:CAG,GTA,TCT,GAC,TCT,CTG,TTC,GTC,GTT,GAT,CTC,ACC,ATC,GAT,GAT,CTG,ATC,AGC,GCA,AGC
P29:AAC,AGA,GAG,TCA,GAT,ACC,TGG,ATG,GTC,ACA,TCA,ACG,CTG,TCA,ATC,AGG,CTA,TTG,AGT,CTG
P30:TCC,ATC,AGA,GAC,CAA,GCG,AAG,TAA,CCT,CTG,ATG,TCA,ACA,CCA,GAC,TCA,ATA,GCC,TGA,TTG
P31:TTC,GCT,TGG,TCT,CTG,ATG,GAC,AAC,TTC,GAA,TGG,GCT,GAA,GGT,TAC,TCC,AAG,AGA,TTC,GGT
P32:TGA,TGG,TCC,TCT,CTT,GAG,TCT,GAT,AGT,CAA,CGT,AGG,TGA,GAC,CGA,ATC,TCT,TGG,AGT,AAC
P33:GAC,TCA,AGA,GAG,GAC,CAT,CAA,GAG,AAG,TGG,TCA,TGC,TTA,CCA,GAC,TCT,TCT,CTC,TTC,CAG
P34:GAGCTCTTA,ATG,GTG,ATG,GTG,ATG,ATG,ACC,CTT,TCT,GGA,AGA,GAG,AAG,AGT,CTG
回收PCR扩增产物,使其与pMD18-T载体相连(Takara),4℃连接过夜,高效转化DH5α感受态中,获得阳性克隆。抽取该阳性克隆质粒,经BamHI和SacI双酶切后,通过T4DNA连接酶与大肠杆菌表达载体pYPG251连接,酶切鉴定和序列测定获得了含有目的基因的重组质粒pYMbg1。
包括所述β-葡萄糖苷酶Mbg1基因的pYMbg1载体,用于大肠杆菌表达中。
利用电击法将上述pYMbg1质粒转入大肠杆菌EG50中,在含有X-Gal的2YT板上有蓝色菌落出现,表明β-葡萄糖苷酶Mbg1基因有降解X-Gal的活性(参见图4)。
以邻硝基苯-β-D-吡喃葡萄糖苷(ONPG)为底物,Mbg1表达产物的比活可达到232umg-1min-1以上。
本发明的有益效果:
本发明按大肠杆菌偏爱密码子优化合成的β-葡萄糖苷酶Mbg1基因与野生型Mbg1基因的同源性高达80%,并且包含本发明所述β-葡萄糖苷酶Mbg1基因的pYMbg1载体可在大肠杆菌中表达出高比活的β-葡萄糖苷酶,且Mbg1表达产物的比活可达到232u mg-1min-1以上,在食品、医药、生物能源等领域有广泛的应用潜力。
附图说明
图1为本发明采用连续延伸PCR方法合成密码子优化的β-葡萄糖苷酶Mbg1基因的步骤。
图2为本发明密码子优化的β-葡萄糖苷酶Mbg1基因和野生型β-葡萄糖苷酶Mbg1基因序列比对图,其中,上行为野生型β-葡萄糖苷酶Mbg1基因;下行为本发明密码子优化的β-葡萄糖苷酶Mbg1基因,两者同源性=80%。
图3为本发明的用于大肠杆菌表达的pYMbg1载体。
图4为本发明密码子优化的β-葡萄糖苷酶Mbg1基因在大肠杆菌中表达的功能性验证。
具体实施方式
以下结合具体实施例进一步详细描述本发明的技术方案。
本发明实施中未注明的实验方法,如连接、转化、相关培养基的配制等参照《分子克隆实验指南》第三版(黄培堂等译,中国,科学出版社,2002)中的方法进行。未注明的化学药品为分析纯级,购自上海国药集团有限公司。
相关的DNA回收试剂盒、载体、连接酶、核酸内切酶均购自Takara公司,DH5α感受态、大肠杆菌EG50由上海市农业科学院生物技术研究所植物基因工程研究室保存。
实施例1密码子优化β-葡萄糖苷酶基因的合成
本发明根据来源于海洋单胞菌(Marinomonas MWYL1)的Mbg1序列(GenBank AccessionNo.CP000749),采取连续延伸PCR方法合成密码子优化的β-葡萄糖苷酶Mbg1基因。由上海生物工程有限公司合成34条引物,用于β-葡萄糖苷酶Mbg1基因的合成。合成方法如图1所示,合成使用的Taq DNA聚合酶为KOD FX taq酶(Toyobo公司日本),在50μL反应体系中进行。50μL反应体系为:ddH2O 11μL;2×PCR缓冲液25μL;2mM dNTPs 10μL;KOD FX1μL;两侧引物各100ng,内侧引物各10ng,补足无菌水至50μL。扩增条件为:94℃预热10min;94℃,30s,54℃,30s,72℃,40s,30个循环;最后72℃延伸10min。第一步,分别以P1、P12,P13、P24,P25、P34为两侧引物合成片段1、片段2、片段3;第二步,各取1μL片段1、片段2、片段3为模板,用引物P1、P34扩增得到目的基因Mbg1。
使用的引物如下:
P1:GGA,TCC,ATG,TCT,ATC,ACT,CTT,CCA,TCT,CAC,TCC,AAG,ATG,CTG,ACC,TCC,GAC,TTC,ATC,TTT
P2:TGG,TAG,CAC,CTT,CAA,TCT,GGA,AAG,AGG,CAG,TAG,CAA,CAC,CAA,AGA,TGA,AGT,CGG,AGG,TCA
P3:CCA,GAT,TGA,AGG,TGC,TAC,CAC,TGC,TGA,CAA,CAG,ACT,CCC,ATC,CAT,CTG,GGA,TAC,CTT,CTG
P4:CTC,ACC,ATT,GTC,CAT,ACC,TTT,GAC,CTT,GCC,TGG,AGT,AGC,ACA,GAA,GGT,ATC,CCA,GAT,GGA
P5:AAG,GTA,TGG,ACA,ATG,GTG,AGA,TTG,CTT,GTG,ACC,ACT,ATC,ATC,TCT,GGG,AAC,AAG,ACA,TTC
P6:GAG,AGT,CTG,TAG,GCA,TCA,ACA,CCA,AGG,TCT,TTG,ATC,AGC,TGA,ATG,TCT,TGT,TCC,CAG,AGA
P7:GTT,GAT,GCC,TAC,AGA,CTC,TCC,ATT,GCT,TGG,CCA,AGA,GTC,ATG,GAC,AAG,AAA,GGT,GAA,GCT
P8:TCT,TGA,GCA,GGT,TTC,TGT,AGA,AGT,CCA,GAC,CAG,CTT,GGT,TAG,CTT,CAC,CTT,TCT,TGT,CCA
P9:CTA,CAG,AAA,CCT,GCT,CAA,GAA,GCT,CAA,GGC,TGA,AGG,TCT,CAC,TGT,CTT,TGC,TAC,TCT,CTA
P10:ACC,ACC,TTT,GTC,TTC,CAG,GTG,CTG,TGG,CAG,ATC,CCA,ATG,GTA,GAG,AGT,AGC,AAA,GAC,AGT
P11:ACC,TGG,AAG,ACA,AAG,GTG,GTT,GGC,TCA,ACA,GAG,AGA,CTG,CCT,ACC,AGT,TCA,AGA,ACT,ATG
P12:GAG,TCA,ACC,CAC,TCA,GCA,AGC,TCT,TTA,GTG,ACC,AGG,TCA,GCA,TAG,TTC,TTG,AAC,TGG,TAG
P13:CTT,GCT,GAG,TGG,GTT,GAC,TCT,TGG,GCT,ACC,TTC,AAC,GAA,CCA,TTC,TGT,GCT,GCC,ATC,CTT
P14:GTT,TGG,ACA,AAC,CAG,GAG,CAT,GGA,TAC,CCA,GCT,CAT,AGC,CAA,GGA,TGG,CAG,CAC,AGA,ATG
P15:TGC,TCC,TGG,TTT,GTC,CAA,ACC,AGC,CTT,TGG,TAG,ACA,AGC,TGC,TCA,CCA,CAT,CTT,GCT,CGC
P16:TGG,AGC,GTT,CTT,TCT,GAT,GAC,TGG,AAG,AGC,CAA,GCC,ATG,AGC,GAG,CAA,GAT,GTG,GTG,AGC
P17:TCA,TCA,GAA,AGA,ACG,CTC,CAA,AGT,CTC,AAG,TTG,GTA,TCG,TTC,TCA,ACA,TGA,ACA,GAT,CCT
P18:AAG,CAA,GCA,AAC,TGG,TCT,TCG,GTC,TTC,TCA,GAG,GCA,GCA,TAG,GAT,CTG,TTC,ATG,TTG,AGA
P19:GAA,GAC,CAG,TTT,GCT,TGC,TTG,ATG,AGA,GAG,ACT,CTC,GAC,AAC,CAG,TTC,TTC,ATC,GAA,CCA
P20:CAA,CAG,TCT,TCA,GCA,ACT,GTG,GGT,ACT,GAC,CCT,TCA,TCA,GTG,GTT,CGA,TGA,AGA,ACT,GGT
P21:ACA,GTT,GCT,GAA,GAC,TGT,TGC,TCC,ACA,GTA,CTT,GCC,AAC,CAT,CCT,TCC,AGG,TGA,CAT,GGA
P22:GAA,GTT,CAT,GCC,AAG,GAA,GTC,GAT,TGG,TTG,AGA,GAT,GAT,GTC,CAT,GTC,ACC,TGG,AAG,GAT
P23:ACT,TCC,TTG,GCA,TGA,ACT,TCT,ACA,CTT,GCA,ATC,ACA,ATG,CCT,ACG,ATG,CTG,ATG,ACA,TGT
P24:TCA,GTG,TAC,TCA,ACA,GTC,TGA,GAG,TTC,TGG,ACG,TTC,TTG,AAC,ATG,TCA,TCA,GCA,TCG,TAG
P25:CAG,ACT,GTT,GAG,TAC,ACT,GAC,ATC,GGT,TGG,GAG,ATT,GCT,CCA,CAT,GCC,TTC,ACT,GAA,CTG
P26:AGA,TTG,GTG,GAA,GAG,AGT,ACT,GCT,TGT,GGA,GGT,TGA,CCA,ACA,GTT,CAG,TGA,AGG,CAT,GTG
P27:GTA,CTC,TCT,TCC,ACC,AAT,CTA,CAT,CAC,TGA,GAA,TGG,TGC,TGC,TTG,CGC,TGA,TCA,GAT,CAT
P28:CAG,GTA,TCT,GAC,TCT,CTG,TTC,GTC,GTT,GAT,CTC,ACC,ATC,GAT,GAT,CTG,ATC,AGC,GCA,AGC
P29:AAC,AGA,GAG,TCA,GAT,ACC,TGG,ATG,GTC,ACA,TCA,ACG,CTG,TCT,ATC,AGG,CTA,TTG,AGT,CTG
P30:TCC,ATC,AGA,GAC,CAA,GCG,AAG,TAA,CCT,CTG,ATG,TCA,ACA,CCA,GAC,TCA,ATA,GCC,TGA,TTG
P31:TTC,GCT,TGG,TCT,CTG,ATG,GAC,AAC,TTC,GAA,TGG,GCT,GAA,GGT,TAC,TCC,AAG,AGA,TTC,GGT
P32:TGA,TGG,TCC,TCT,CTT,GAG,TCT,GAT,AGT,CAA,CGT,AGG,TGA,GAC,CGA,ATC,TCT,TGG,AGT,AAC
P33:GAC,TCA,AGA,GAG,GAC,CAT,CAA,GAG,AAG,TGG,TCA,TGC,TTA,CCA,GAC,TCT,TCT,CTC,TTC,CAG
P34:GAGCTCTTA,ATG,GTG,ATG,GTG,ATG,ATG,ACC,CTT,TCT,GGA,AGA,GAG,AAG,AGT,CTG
按密码子优化合成的β-葡萄糖苷酶Mbg1基因开放阅读框为1344bp,不包含酶切位点。PCR反应得到1344bp的片段即为本发明的β-葡萄糖苷酶Mbg1基因,其核苷酸序列如SEQ ID No 1所示,其编码的蛋白质序列如SEQ ID NO 2所示。通过DNAMAN软件进行同源性比对,结果发现本发明合成基因序列与原基因序列同源性为80%(参见图2),其编码的蛋白质共447个氨基酸。
实施例2Mbg1基因在大肠杆菌中的表达和纯化
2.1大肠杆菌表达载体的构建
1.5%琼脂糖胶回收实例1中的PCR扩增产物,取10μL回收产物与pMD18-T载体相连。4℃连接过夜,高效转化DH5α感受态中,获得阳性克隆。抽取阳性克隆质粒,经BamHI和SacI双酶切后,以正确的阅读框插入到大肠杆菌表达载体pYPG251中,测序验证,得到重组质粒pYMbg1(参见图3)。
2.2Mbg1基因的功能性表达
利用电击法将pYMbg1质粒转入大肠杆菌EG50中,涂布于含有Ap(氨苄青霉素,100μg/ml)和X-gal(5-溴-4-氯-3-吲哚基-B-D-半乳糖甘,100μg/ml)的2YT固体培养基中,37℃培养过夜至长出蓝色菌落(参见图4)。
挑取新鲜转化的含有重组质粒pYMbgl的单克隆,接入50ml含有Ap(100μg/ml)抗生素的LB液体培养基中,用250ml摇瓶,37℃震摇培养至OD600值达到0.6-1.0。将摇瓶置于冰上5分钟,5000rpm 4℃离心5分钟收集菌体。重悬细胞于5ml预冷的破碎缓冲液(50mM磷酸缓冲液,0.3M NaCl,0.5mg/ml溶菌酶,1mM PMSF,1mM MgCl2)中。把混合菌体在冰水中用超声破碎仪破碎,破碎好的菌液经12000rpm,4℃离心30分钟后,上清用0.45μm的滤膜过滤后,收集滤液。
相关培养基的配制:
2YT固体培养基:胰化蛋白胨8g;酵母提取物5g;NaCl 2.5g;琼脂粉12g,用蒸馏水定容至1L。
LB液体培养基:胰化蛋白胨5g;酵母提取物10g;NaCl 10g,用蒸馏水定容至1L。
2.3表达产物的纯化
表达产物的纯化采用Sigma公司的Ni2+-NTA亲和层吸柱,具体纯化步骤为:
1)用6ml缓冲液A平衡亲和柱;
2)取实施例2.2所得的滤液进行上样;
3)用10ml缓冲液B洗去层析柱中未结合蛋白;
4)每次用1ml缓冲液C洗脱,收集含有目的蛋白的洗脱液。
其中,缓冲液A:50mM磷酸缓冲液,500mM NaCl,10mM咪唑,1%甘油,1%吐温,1%triton,1%β-巯基乙醇,pH值8.0。
缓冲液B:50mM磷酸缓冲液,500mM NaCl,50mM咪唑,1%甘油,1%吐温,1%triton,1%β-巯基乙醇,pH值8.0。
缓冲液C:50mM磷酸缓冲液,500mM NaCl,250mM咪唑,1%甘油,1%吐温,1%triton,1%β-巯基乙醇,pH值8.0。
实施例3Mbg1基因在大肠杆菌表达产物的酶活测定
3.1邻硝基苯酚标准曲线的绘制
准确吸取0.5mM邻硝基苯酚溶液20μL、30μL、40μL、50μL、60μL、70μL,用Zm缓冲液补足到200μL,分别加入60μL 1M Na2CO3终止显色,反应完成后测定420nm的光吸收值。以邻硝基苯酚浓度为Y轴,光吸收值为X轴,绘制标准曲线。
3.2Mbg1β-葡萄糖苷酶酶活力测定
酶活测定采用分光光度法,具体实施方法为:取实施例2纯化所得的浓度为0.01μg/μL的酶液20μL,20mM邻硝基苯-β-D-吡喃葡萄糖苷(ONPG)20μL,Zm缓冲液(pH 7.0)160μL,充分混匀后置37℃水浴保温10min,然后加入60μL 1M Na2CO3终止显色,使用infiniteM200多功能酶标仪(TECAN)测定420nm处比色测定光吸收值的变化。根据邻硝基苯酚标准曲线计算β-葡萄糖苷酶的活力。其中,酶活力单位(U)的定义:在该反应条件下,以每分钟产生1μmol邻硝基苯酚所需的酶量定义为1个单位。
经过测定,Mbg1表达产物在以20mM邻硝基苯-β-D-吡喃葡萄糖苷(ONPG)为底物时,比活达到232u mg-1min-1。
相关试剂的配制:
Zm缓冲液(50mL):0.80g Na2HPO4·7H2O(0.06M);0.28g NaH2PO4·H2O(0.04M);0.5mL1M KCl(0.01M);0.05mL 1M MgSO4(0.001M);0.135mL β-巯基乙醇(BME)(0.05M);溶解到40mL蒸馏水中,调节pH到7.0,最后定容到50mL,4℃保存。
最后应当说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围,其均应涵盖在本发明的权利要求范围中。
序列表
Claims (6)
1.一种密码子优化的β-葡萄糖苷酶Mbg1基因,其特征在于,其核苷酸序列如SEQ IDNo.1所示。
2.根据权利要求1所述的β-葡萄糖苷酶Mbg1基因,其特征在于,其编码的蛋白质序列如SEQ ID No 2所示。
3.根据权利要求1或2所述的β-葡萄糖苷酶Mbg1基因,其特征在于,所述基因采用连续延伸PCR方法合成。
4.根据权利要求3所述的β-葡萄糖苷酶Mbg1基因,其特征在于,所述连续延伸PCR方法中所用的引物具体如下:
P1:GGA,TCC,ATG,TCT,ATC,ACT,CTT,CCA,TCT,CAC,TCC,AAG,ATG,CTG,ACC,TCC,GAC,TTC,ATC,TTT
P2:TGG,TAG,CAC,CTT,CAA,TCT,GGA,AAG,AGG,CAG,TAG,CAA,CAC,CAA,AGA,TGA,AGT,CGG,AGG,TCA
P3:CCA,GAT,TGA,AGG,TGC,TAC,CAC,TGC,TGA,CAA,CAG,ACT,CCC,ATC,CAT,CTG,GGA,TAC,CTT,CTG
P4:CTC,ACC,ATT,GTC,CAT,ACC,TTT,GAC,CTT,GCC,TGG,AGT,AGC,ACA,GAA,GGT,ATC,CCA,GAT,GGA
P5:AAG,GTA,TGG,ACA,ATG,GTG,AGA,TTG,CTT,GTG,ACC,ACT,ATC,ATC,TCT,GGG,AAC,AAG,ACA,TTC
P6:GAG,AGT,CTG,TAG,GCA,TCA,ACA,CCA,AGG,TCT,TTG,ATC,AGC,TGA,ATG,TCT,TGT,TCC,CAG,AGA
P7:GTT,GAT,GCC,TAC,AGA,CTC,TCC,ATT,GCT,TGG,CCA,AGA,GTC,ATG,GAC,AAG,AAA,GGT,GAA,GCT
P8:TCT,TGA,GCA,GGT,TTC,TGT,AGA,AGT,CCA,GAC,CAG,CTT,GGT,TAG,CTT,CAC,CTT,TCT,TGT,CCA
P9:CTA,CAG,AAA,CCT,GCT,CAA,GAA,GCT,CAA,GGC,TGA,AGG,TCT,CAC,TGT,CTT,TGC,TAC,TCT,CTA
P10:ACC,ACC,TTT,GTC,TTC,CAG,GTG,CTG,TGG,CAG,ATC,CCA,ATG,GTA,GAG,AGT,AGC,AAA,GAC,AGT
P11:ACC,TGG,AAG,ACA,AAG,GTG,GTT,GGC,TCA,ACA,GAG,AGA,CTG,CCT,ACC,AGT,TCA,AGA,ACT,ATG
P12:GAG,TCA,ACC,CAC,TCA,GCA,AGC,TCT,TTA,GTG,ACC,AGG,TCA,GCA,TAG,TTC,TTG,AAC,TGG,TAG
P13:CTT,GCT,GAG,TGG,GTT,GAC,TCT,TGG,GCT,ACC,TTC,AAC,GAA,CCA,TTC,TGT,GCT,GCC,ATC,CTT
P14:GTT,TGG,ACA,AAC,CAG,GAG,CAT,GGA,TAC,CCA,GCT,CAT,AGC,CAA,GGA,TGG,CAG,CAC,AGA,ATG
P15:TGC,TCC,TGG,TTT,GTC,CAA,ACC,AGC,CTT,TGG,TAG,ACA,AGC,TGC,TCA,CCA,CAT,CTT,GCT,CGC
P16:TGG,AGC,GTT,CTT,TCT,GAT,GAC,TGG,AAG,AGC,CAA,GCC,ATG,AGC,GAG,CAA,GAT,GTG,GTG,AGC
P17:TCA,TCA,GAA,AGA,ACG,CTC,CAA,AGT,CTC,AAG,TTG,GTA,TCG,TTC,TCA,ACA,TGA,ACA,GAT,CCT
P18:AAG,CAA,GCA,AAC,TGG,TCT,TCG,GTC,TTC,TCA,GAG,GCA,GCA,TAG,GAT,CTG,TTC,ATG,TTG,AGA
P19:GAA,GAC,CAG,TTT,GCT,TGC,TTG,ATG,AGA,GAG,ACT,CTC,GAC,AAC,CAG,TTC,TTC,ATC,GAA,CCA
P20:CAA,CAG,TCT,TCA,GCA,ACT,GTG,GGT,ACT,GAC,CCT,TCA,TCA,GTG,GTT,CGA,TGA,AGA,ACT,GGT
P21:ACA,GTT,GCT,GAA,GAC,TGT,TGC,TCC,ACA,GTA,CTT,GCC,AAC,CAT,CCT,TCC,AGG,TGA,CAT,GGA
P22:GAA,GTT,CAT,GCC,AAG,GAA,GTC,GAT,TGG,TTG,AGA,GAT,GAT,GTC,CAT,GTC,ACC,TGG,AAG,GAT
P23:ACT,TCC,TTG,GCA,TGA,ACT,TCT,ACA,CTT,GCA,ATC,ACA,ATG,CCT,ACG,ATG,CTG,ATG,ACA,TGT
P24:TCA,GTG,TAC,TCA,ACA,GTC,TGA,GAG,TTC,TGG,ACG,TTC,TTG,AAC,ATG,TCA,TCA,GCA,TCG,TAG
P25:CAG,ACT,GTT,GAG,TAC,ACT,GAC,ATC,GGT,TGG,GAG,ATT,GCT,CCA,CAT,GCC,TTC,ACT,GAA,CTG
P26:AGA,TTG,GTG,GAA,GAG,AGT,ACT,GCT,TGT,GGA,GGT,TGA,CCA,ACA,GTT,CAG,TGA,AGG,CAT,GTG
P27:GTA,CTC,TCT,TCC,ACC,AAT,CTA,CAT,CAC,TGA,GAA,TGG,TGC,TGC,TTG,CGC,TGA,TCA,GAT,CAT
P28:CAG,GTA,TCT,GAC,TCT,CTG,TTC,GTC,GTT,GAT,CTC,ACC,ATC,GAT,GAT,CTG,ATC,AGC,GCA,AGC
P29:AAC,AGA,GAG,TCA,GAT,ACC,TGG,ATG,GTC,ACA,TCA,ACG,CTG,TCA,ATC,AGG,CTA,TTG,AGT,CTG
P30:TCC,ATC,AGA,GAC,CAA,GCG,AAG,TAA,CCT,CTG,ATG,TCA,ACA,CCA,GAC,TCA,ATA,GCC,TGA,TTG
P31:TTC,GCT,TGG,TCT,CTG,ATG,GAC,AAC,TTC,GAA,TGG,GCT,GAA,GGT,TAC,TCC,AAG,AGA,TTC,GGT
P32:TGA,TGG,TCC,TCT,CTT,GAG,TCT,GAT,AGT,CAA,CGT,AGG,TGA,GAC,CGA,ATC,TCT,TGG,AGT,AAC
P33:GAC,TCA,AGA,GAG,GAC,CAT,CAA,GAG,AAG,TGG,TCA,TGC,TTA,CCA,GAC,TCT,TCT,CTC,TTC,CAG
P34:GAGCTCTTA,ATG,GTG,ATG,GTG,ATG,ATG,ACC,CTT,TCT,GGA,AGA,GAG,AAG,AGT,CTG。
5.包括权利要求1或2所述的β-葡萄糖苷酶Mbg1基因的pYMbg1载体。
6.权利要求5所述的pYMbg1载体在大肠杆菌表达中的应用。
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---|---|---|---|---|
CN108251403A (zh) * | 2016-12-29 | 2018-07-06 | 上海交通大学 | 一种新型鞘糖脂内切糖苷酶及其基因工程制备方法和应用 |
CN109234293A (zh) * | 2018-10-23 | 2019-01-18 | 怀化学院 | 一种编码β-葡萄糖苷酶基因及其表达载体和蛋白 |
CN114134094A (zh) * | 2021-12-07 | 2022-03-04 | 上海市农业科学院 | 一种从头合成核黄素的大肠杆菌工程菌的构建方法 |
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Non-Patent Citations (3)
Title |
---|
HO-WON CHANG ET AL.: "Marinomonas basaltis sp. nov., a marine bacterium isolated from black sand.", 《INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY》 * |
S. NAZ ET AL.: "Enhanced Production and Characterization of a b-Glucosidase from Bacillus halodurans Expressed in Escherichia coli.", 《BIOCHEMISTRY (MOSCOW)》 * |
任雪芸等: "南极海单胞菌BSw10005代谢活性物质及对植物病原菌抑制作用", 《植物病理学报》 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108251403A (zh) * | 2016-12-29 | 2018-07-06 | 上海交通大学 | 一种新型鞘糖脂内切糖苷酶及其基因工程制备方法和应用 |
CN108251403B (zh) * | 2016-12-29 | 2021-05-28 | 上海交通大学 | 一种新型鞘糖脂内切糖苷酶及其基因工程制备方法和应用 |
CN109234293A (zh) * | 2018-10-23 | 2019-01-18 | 怀化学院 | 一种编码β-葡萄糖苷酶基因及其表达载体和蛋白 |
CN109234293B (zh) * | 2018-10-23 | 2020-08-04 | 怀化学院 | 一种编码β-葡萄糖苷酶基因及其表达载体和蛋白 |
CN114134094A (zh) * | 2021-12-07 | 2022-03-04 | 上海市农业科学院 | 一种从头合成核黄素的大肠杆菌工程菌的构建方法 |
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