CN102212588B - 酵母展示脂肪酶催化合成淀粉磷酸单酯的方法 - Google Patents
酵母展示脂肪酶催化合成淀粉磷酸单酯的方法 Download PDFInfo
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Abstract
本发明公开了一种酵母展示脂肪酶催化合成淀粉磷酸单酯的方法,包括:将谷物淀粉溶于水,吸涨后加入NaH2PO4·2H2O、Na2HPO4·12H2O和酵母展示脂肪酶,在55~65℃下搅拌反应1~1.5小时,分离、纯化制得淀粉磷酸单酯;本发明通过将脂肪酶展现在细胞外,利用该生物催化剂催化合成淀粉醋酸酯,不仅提高了转化效率,而且缩短了反应时间,降低了生产成本。
Description
技术领域
本发明涉及生物工程技术领域,尤其涉及一种酵母表面脂肪酶催化合成淀粉磷酸单酯的方法。
背景技术
淀粉磷酸单酯是由原淀粉与磷酸盐发生酯化而生成的一种淀粉衍生物,属阴离子变性淀粉,与原淀粉相比,淀粉磷酸单酯具有分散液透明、粘度高、抗老化、稳定性好的特性和良好的保水性能,在低温长期储存或重复冷冻、融化时,也无水分析出。不同取代度的淀粉磷酸单酯已广泛应用于食品、医药、化工和其它工业。特别是在食品工艺中,作为一种食品添加剂,中低取代度的淀粉磷酸单酯广泛应用于冷冻食品的加工等方面。
相较于化学法合成淀粉磷酸单酯,生物酶法可以在较短时间内获得高取代度的反应产物。就相同反应体系而言,生物酶法相比化学法显著提升反应效率和产率,均一度和稳定度也有显著提高。同时,采用生物酶法避免了化学法反应条件苛刻(强酸、碱催化、高温高压)、无特异性、产物复杂、副产物多、分离难、安全性差等弊端。脂肪酶是目前酯化合成中使用最广泛的酶,但是生产成本高、固定化过程繁杂费时大大局限了其商业化应用。
发明内容
本发明提供了一种酵母展示脂肪酶催化合成淀粉醋酸酯的方法,该方法可提高粉磷酸单酯合成的酯化反应效率和产率,降低成本。
一种酵母展示脂肪酶催化合成淀粉磷酸单酯的方法,包括:
将谷物淀粉溶于水,吸涨后加入NaH2PO4·2H2O、Na2HPO4·12H2O和酵母展示胞脂肪酶,在55~65℃下搅拌反应1~1.5小时,分离、纯化制得淀粉磷酸单酯;
以重量份计,反应各原料配比可以如下:水100份、谷物淀粉35~40份、醋酸酐1~2份、酵母全细胞脂肪酶生物催化剂1~2份,
优选的,所述的分离、纯化方法为:将反应液pH值调节至5~6进行沉淀,沉淀经洗涤,抽滤,干燥制得淀粉醋酸酯。
优选的,所述的搅拌速率为200~300转/分钟。
所述的酵母展示脂肪酶通过如下方法制备:
将经线性化处理的重组质粒转入毕赤酵母(Pichia pastoris)GS115,将所得转化子接种于BMMY培养基中,诱导培养72~144小时后离心收集菌体,菌体经冲洗、生物印迹和冷冻干燥制得酵母展示脂肪酶;
所述的重组质粒由原始载体pPIC9K和依次插入原始载体pPIC9K中MFα1信号肽下游的脂肪酶基因和毕赤酵母GS115的细胞壁α凝集素基因组成。
所述的脂肪酶基因可选用Genbank号为AF229435的序列,毕赤酵母(Pichia pastoris)GS115为商业化产品,可从Invitrogen公司购得。它的细胞壁α凝集素基因序列的Genbank号为M28164。
载体pPIC9K为商业化产品(如Invitrogen公司),它存在MFα1信号肽序列(Genbank号:M17301),同时该载体内信号肽序列上游存在AOX1启动子(Genbank号:Z46233)。重组质粒线性化处理的目的是为了与胞内基因组发生同源重组,提高表达稳定性。
优选的,所述生物印迹所用配体为油酸,它可以诱导酶结构改变,提高转化率。
本发明通过将脂肪酶基因和细胞壁α凝集素基因导入毕赤酵母细胞GS115,毕赤酵母细胞诱导培养后,脂肪酶表达并分泌到胞外,同时利用细胞壁α凝集素将该脂肪酶固定在细胞表面。利用该酵母展现脂肪酶对酯化反应进行催化,不仅提高了操作稳定性、耐热性以及重复性,反应时间也大大缩短,从原来的15小时下降到2~3小时,另外该酵母表面展现脂肪酶生物催化剂生产成本低,催化合成转化效率高。
具体实施方式
实施例1制备酵母展示胞脂肪酶
通过人工合成的方法,合成米根霉(Rhizopus oryzae)的脂肪酶基因(Genbank号:AF229435)和毕赤酵母GS115的细胞壁α凝集素基因(Genbank号为M28164),同时在脂肪酶基因C端加上连接肽序列GSSGGSGGSGGSGGSGS(linker),连接后得到核苷酸序列pro-ROL-linker-α-agglutinin,同时在序列两端添加EcoR I和Not I酶切位点,其中pro-ROL为脂肪酶基因,α-agglutinin为细胞壁α凝集素基因。
以上述人工合成序列为模板,利用下述引物对,进行PCR扩增,
上游引物:5’-AAGGAAAAAAGAATTCGTTCCAGTTTCTGG-3’;
下游引物:5’-TTTTCCTTTTGCGGCCGCTAATGAAACG-3’
PCR反应体系为:模板DNA为1μl,高保真DNA聚合酶0.5μl,dNTP(50mM)0.4μl,上下游引物各0.5μl,10×PCR缓冲液5μl,加水至50μl。
PCR运行条件为:94℃3分钟,35个循环(94℃30秒,60℃1分钟,72℃30秒),72℃10分钟。
用EocR I和Not I同时酶切PCR产物和pPIC9K质粒,并在T4连接酶的作用下过夜连接形成pPIC9K-ROL质粒,通过电泳检验并回收质粒。为使目的基因与毕赤酵母GS115发生His 4单位点置换重组,用Sal I对pPIC9K-ROL质粒进行酶切线性化处理。将酶切线性化处理好的目的基因约15μl加入到事先准备好的毕赤酵母GS115感受态细胞中,转入电转杯中,冰浴15min,然后在1500V,400Ω,25uF条件下电击10ms,并加入约1ml预冷的山梨醇。将上述混匀的电转产物约400μl涂于MD平板上,筛选阳性转化子,然后将阳性转化子涂于不同浓度的G418平板上,根据阳性转化子对G418的抗性筛选出Mut表型的多拷贝阳性重组菌株。
将多拷贝阳性重组菌株接种在BMGY培养基中发酵培养30h,离心收集细胞;再将细胞置于含有0.5%(体积百分比)甲醇的BMMY培养基中诱导培养144h,离心收集细胞,用水冲洗后,接种至YGC培养基中30℃培养120h,然后3000g离心分钟收集菌体,蒸馏水洗涤后再用50mM pH7.0磷酸缓冲液洗涤,然后与2倍体积油酸混合,-80℃下预冻后再经德国Christ真空冷冻干燥机干燥24h,用己烷洗涤除去油酸,再进行再真空干燥去除己烷,即得到经生物印迹处理的酵母展示脂肪酶。
实施例2酵母展示脂肪酶催化合成淀粉磷酸单酯
实例1取350g谷物淀粉用水配制成35%淀粉乳(淀粉干基)、于65℃下吸水处理15min使淀粉乳吸水膨胀,冷却后加入1g上述制备的酵母展示脂肪酶,然后分批加入10g NaH2PO4·2H2O与Na2HPO4·12H2O(重量比3∶1)的混合物,置于85-1型磁力搅拌器中搅拌开始反应,转速为200转/分钟,反应温度保持在60℃,反应1h后停止搅拌,调节p H值为7.0,离心取沉淀,用水多次洗涤沉淀,喷雾干燥。
实例2取400g谷物淀粉用水配制成40%淀粉乳(淀粉干基)、于65℃下吸水处理15min使淀粉乳吸水膨胀,冷却后加入2g上述制备的酵母展示脂肪酶,然后分批加入15g NaH2PO4·2H2O与Na2HPO4·12H2O(重量比2∶1)的混合物,置于85-1型磁力搅拌器中搅拌开始反应,转速为250转/分钟,反应温度保持在62℃,反应1.5h后停止搅拌,调节pH值为7.0,离心取沉淀,用水多次洗涤沉淀,喷雾干燥。
实施例3采用传统化学法合成淀粉磷酸单酯
实例1取350g谷物淀粉用水配制成35%淀粉乳(淀粉干基)、于65℃下吸水处理15min使淀粉乳吸水膨胀,然后分批加入10g NaH2PO4·2H2O与Na2HPO4·12H2O(重量比3∶1)的混合物,置于85-1型磁力搅拌器中搅拌开始反应,转速为200转/分钟,反应温度保持在60℃,反应1h后停止搅拌,调节p H值为7.0,离心取沉淀,用水多次洗涤沉淀,喷雾干燥。
实施例4测定取代度和乙酰基含量
用水洗涤低取代度的淀粉磷酸单酯,除去游离磷(以无机盐存在)后用硫酸/硝酸混合酸处理,使结合磷游离出来,加入钼酸按和抗坏血酸,使之形成磷钼蓝,在波长为825nm上用分光光度计测定吸光度,通过标准曲线查出结合磷含量,即通过如下公式可计算出取代度(DS)。
磷含量wp=(m×100×100-6)/样品质量×100%
实际取代度:DSA=(5.32×wp)/(100-3.32wp)
[m-样品中磷的含量(ug);wp-淀粉磷酸酯中总的磷含量]
取代反应效率=DSA/DST×100%,
理论取代度:DST=(5.32×WP)/(100-3.32WP)
磷含量WP=(m×100×100-6)/样品质量×100%
[m-样品中磷的含量(ug);WP-所加入NaH2PO4·2H2O与Na2HPO4·12H2O中总的磷含量]
根据上述方法对制得的淀粉磷酸单酯样品进行检测,实施例2中,实例1产品的取代度和反应效率分别为0.0176和89.62%,实例2产品的取代度和反应效率分别为0.0153和87%。而实施例3利用传统化学方法合成淀粉醋酸酯的取代度和反应效率仅为0.0138和75%。
Claims (1)
1.一种酵母展示脂肪酶催化合成淀粉磷酸单酯的方法,包括:
人工合成米根霉(Rhizopus oryzae)的脂肪酶基因和毕赤酵母GS115的细胞壁α凝集素基因,接着在脂肪酶基因C端加上编码连接肽序列GSSGGSGGSGGSGGSGS的基因片段,连接得到核苷酸序列pro-ROL-linker-α-agglutinin,pro-ROL代表脂肪酶基因,Genbank号为:AF229435,α-agglutinin代表细胞壁α凝集素基因,Genbank号为:M28164,linker代表编码连接肽的基因片段;
以人工合成序列为模板,利用以下引物对,进行PCR扩增,使得在序列两端添加EcoRⅠ和NotⅠ酶切位点;
上游引物:5’-AAGGAAAAAAGAATTCGTTCCAGTTTCTGG-3’;
下游引物:5’-TTTTCCTTTTGCGGCCGCTAATGAAACG-3’;
PCR反应体系为:模板DNA为1μl,高保真DNA聚合酶0.5μl,50mM的dNTP0.4μl,上下游引物各0.5μl,10×PCR缓冲液5μl,加水至50μl;
PCR运行条件为:94℃3分钟;35个循环,每个循环为:94℃30秒,60℃1分钟,72℃30秒;72℃10分钟;
用EocR I和Not I同时酶切PCR产物和pPIC9K质粒,并在T4连接酶的作用下过夜连接形成pPIC9K-ROL质粒,通过电泳检验并回收质粒,用Sal I对pPIC9K-ROL质粒进行酶切线性化处理,将酶切线性化处理好的目的基因15μl加入到毕赤酵母GS115感受态细胞中,转入电转杯中,冰浴15min,然后在1500V,400Ω,25μF条件下电击10ms,并加入1mL预冷的山梨醇;
将混匀的电转产物400μl涂于MD平板上,筛选阳性转化子,然后将阳性转化子涂于不同浓度的G418平板上,根据阳性转化子对G418的抗性筛选出Mut表型的多拷贝阳性重组菌株;
将多拷贝阳性重组菌株接种在BMGY培养基中发酵培养30h,离心收集细胞;再将细胞置于含有体积百分比0.5%甲醇的BMMY培养基中诱导培养144h,离心收集细胞,用水冲洗后,接种至YGC培养基中30℃培养120h,然后3000g离心分钟收集菌体,蒸馏水洗涤后再用50mM pH7.0磷酸缓冲液洗涤,然后与2倍体积油酸混合,-80℃下预冻后再经德国Christ真空冷冻干燥机干燥24h,用己烷洗涤除去油酸,再进行再真空干燥去除己烷,即得到经生物印迹处理的酵母展示脂肪酶;
取350g谷物淀粉用水配制成35%淀粉乳,于65℃下吸水处理15min,冷却后加入1g上述制备的酵母展示脂肪酶,然后分批加入10g重量比为3:1的NaH2PO4·2H2O与Na2HPO4·12H2O的混合物,置于85-1型磁力搅拌器中搅拌开始反应,转速为200转/分钟,反应温度保持在60℃,反应1h后停止搅拌,调节pH值为7.0,离心取沉淀,用水洗涤沉淀,喷雾干燥。
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