CN114703160A - 一种发酵生产聚磷酸盐激酶1突变体的方法 - Google Patents
一种发酵生产聚磷酸盐激酶1突变体的方法 Download PDFInfo
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- CN114703160A CN114703160A CN202210321774.9A CN202210321774A CN114703160A CN 114703160 A CN114703160 A CN 114703160A CN 202210321774 A CN202210321774 A CN 202210321774A CN 114703160 A CN114703160 A CN 114703160A
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- polyphosphate kinase
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Abstract
本发明公开了一种发酵生产聚磷酸盐激酶1突变体的方法,包括以下步骤:(1)将聚磷酸盐激酶1突变体生产菌的种子液接种至发酵培养基中进行发酵培养,发酵培养至发酵液稀释100倍后的OD600值为0.40‑0.60时,降温至21‑23℃,向体系中加入IPTG进行诱导培养,诱导培养20‑24h;培养过程监测体系的残糖含量,当体系的残糖含量≤1.0g/L时开始添加补料,通过流加补料使体系中的残糖浓度保持在0.5‑1.0g/L;(2)将诱导培养后的培养物进行破菌处理,分离收集上清,即生产得到含有聚磷酸盐激酶1突变体的培养液。本发明通过对聚磷酸盐激酶1进行突变处理,提高了聚磷酸盐激酶1的酶活;并构建了聚磷酸盐激酶1突变体的发酵生产体系,实现了聚磷酸盐激酶1突变体的工业化生产。
Description
技术领域
本发明涉及生物工程技术领域,具体涉及一种发酵生产聚磷酸盐激酶1突变体的方法。
背景技术
多聚磷酸盐(polyphosphate,poly P)作为一种广泛存在的线性多聚体,由几个至数百个磷酸盐残基通过与ATP磷酸酐键相同的的高能磷酸键相互聚合形成。近年来研究发现,poly P不仅是生物体中不可或缺的生物能和磷酸基团储存库、负电荷密度高的生物大分子,还在微生物中参与调节基因表达、调控细菌离子通道和DNA摄取、代谢等,并与哺乳动物中瞬时电位的调节、线粒体代谢、细胞钙化等生理过程有着密切的关系。
聚磷酸盐激酶1(polyphosphate kinase 1,PPK1)是poly P合成代谢的关键酶。PPK1通过催化ATP末端的磷酸基团转移到磷酸盐上而形成多聚磷酸盐这一可逆反应的发生,从而将磷酸盐聚集形成多聚磷酸盐。此外,PPK1还是生产腺嘌呤核苷酸(AMP)、鸟嘌呤核苷酸(GMP)、胞嘧啶核苷酸(CMP)、尿嘧啶核苷酸(UMP)、胸腺嘧啶核苷酸(TMP)等产品都会用到的一种酶。因此,PPK1具有广阔的应用前景,市场需求较大。
但是,现有PPK1的酶活一般在650U/mg左右,仍有待进一步的提高;并且PPK1对pH控制较为严格,在生产5'-单磷酸核苷酸(特别是腺苷单磷酸)时,PPK1和腺苷激酶的最适pH不同但需要混合投料,这进一步影响了PPK1在实际应用中酶活的发挥。此外,目前还很少见有实现工业化发酵生产PPK1的报道。
发明内容
针对上述现有技术,本发明的目的是提供一种发酵生产聚磷酸盐激酶1突变体的方法。本发明通过对聚磷酸盐激酶1进行突变处理得到聚磷酸盐激酶1突变体,提高了聚磷酸盐激酶1的酶活;并构建了聚磷酸盐激酶1突变体的发酵生产体系,实现了聚磷酸盐激酶1突变体的工业化生产。
为实现上述目的,本发明采用如下技术方案:
一种发酵生产聚磷酸盐激酶1突变体的方法,包括以下步骤:
(1)将聚磷酸盐激酶1突变体生产菌的种子液接种至发酵培养基中进行发酵培养,发酵培养的温度为32-34℃,pH为6.8-7.2,溶氧(DO)为20-40%;发酵培养至发酵液稀释100倍后的OD600值为0.40-0.60时,降温至21-23℃,向体系中加入IPTG进行诱导培养,诱导培养20-24h;
培养过程监测体系的残糖含量,当体系的残糖含量≤1.0g/L时开始添加补料,通过流加补料使体系中的残糖浓度保持在0.5-1.0g/L;
(2)将诱导培养后的培养物进行破菌处理,分离收集上清,即生产得到含有聚磷酸盐激酶1突变体的培养液。
优选的,步骤(1)中,所述聚磷酸盐激酶1突变体生产菌由如下方法构建而成:
将pQE-60质粒用BspEⅠ和AflⅢ双酶切,再连接上序列如SEQ ID NO.5所示的片段,构建得到质粒pQE-N,所述质粒pQE-N的核苷酸序列如SEQ ID NO.6所示;用AccⅢ和SphⅠ对质粒pQE-N双酶切,再将编码聚磷酸盐激酶1突变体的基因连接到酶切后的质粒上,获得重组表达载体pQE-ppk1,所述重组表达载体pQE-ppk1的核苷酸序列如SEQ ID NO.7所示;
将获得的重组表达载体pQE-ppk1导入到大肠杆菌中,构建得到聚磷酸盐激酶1突变体生产菌。
优选的,步骤(1)中,聚磷酸盐激酶1突变体生产菌的种子液的接入量为发酵培养基重量的4-8%。
优选的,步骤(1)中,所述发酵培养基的组成为:蛋白胨2.5g/L、脱脂豆粉20g/L、葡萄糖5g/L、甜菜糖蜜5g/L,酵母膏8g/L、氯化钠3g/L、硫酸铵2.5g/L、三水磷酸氢二钾4g/L、柠檬酸铁铵0.3g/L、柠檬酸2.1g/L、七水硫酸镁0.5g/L、氨苄青霉素100ppm。
优选的,步骤(1)中,加入IPTG,使IPTG在体系中的终浓度为0.2mmol/L。
优选的,步骤(1)中,所述补料的组成为:葡萄糖200g/L、甜菜糖蜜200g/L、酵母膏80g/L、脱脂豆粉60g/L。
优选的,步骤(2)中,采用均质机进行破菌处理,均质处理的条件为:均质压力12,000PSI,均质流量200-300L/Hr。
优选的,步骤(2)中,所述聚磷酸盐激酶1突变体的氨基酸序列如SEQ ID NO.3所示。
本发明的有益效果:
(1)本发明首先对大肠杆菌聚磷酸盐激酶1进行了突变改造处理,将野生型聚磷酸盐激酶1第246位的L突变为P,第247位的M突变为V,获得了聚磷酸盐激酶1突变体。本发明的聚磷酸盐激酶1突变体具有如下突出的优势:
①本发明的聚磷酸盐激酶1突变体的酶活性达800-850U/mg,与野生型聚磷酸盐激酶1相比,酶活提高了23%-33%。
②本发明的聚磷酸盐激酶1突变体对pH的适应范围广,能够在pH5.0-pH9.3之间均能保持较高的酶活性,更适合工业化生产。
③本发明的聚磷酸盐激酶1突变体的亲水性增加,酶的溶解度上升,更有利于酶的工业化应用。
(2)本发明还对聚磷酸盐激酶1突变体的发酵培养基、发酵培养条件以及诱导培养条件进行了优化。其中,本发明的发酵培养基选择糖蜜作为碳源之一,具有以下好处:
①含有高浓度的腐熟型有机质、腐殖酸、生化类黄腐酸、氨基酸、高吸收率的NPK;
②含有在发酵过程中产生的UGF;
③降低粉尘及颗粒料的粉化率,减少了损失;
④促进菌体生长,改善部分生产性能;
⑤降低成本,糖蜜比葡萄糖便宜一半左右。
加入少量柠檬酸能减少菌种延滞期时间;加入脱脂豆粉、酵母膏都可降低成本。
综上,本发明通过生产菌的构建和发酵工艺的优化,实现了聚磷酸盐激酶1突变体的规模化、工业化生产,并显著提高了聚磷酸盐激酶1的酶活。
附图说明
图1:通过分子动力学模拟软件分析野生型聚磷酸盐激酶1的亲水性。
图2:通过分子动力学模拟软件分析聚磷酸盐激酶1突变体的亲水性。
图3:质粒pQE-N的结构示意图。
图4:重组表达载体pQE-ppk1的结构示意图。
图5:琼脂糖凝胶电泳验证图;泳道M:Marker;泳道1:pQE-N;泳道2:pQE-ppk1双酶切。
图6:诱导表达后蛋白的SDS-PAGE检测结果;图中,M:Marker;泳道1:未添加诱导剂IPTG;泳道2-7:分别为诱导表达6h、8h、10h、12h、14h、16h的检测结果。
图7:纯化蛋白的western blot检测结果。
图8:聚磷酸盐激酶1突变体与野生型聚磷酸盐激酶1的酶活与pH之间的关系;图中,1为聚磷酸盐激酶1突变体,2为野生型聚磷酸盐激酶1。
具体实施方式
应该指出,以下详细说明都是例示性的,旨在对本申请提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
为了使得本领域技术人员能够更加清楚地了解本申请的技术方案,以下将结合具体的实施例详细说明本申请的技术方案。
本发明实施例和对比例中所用的试验材料均为本领域常规的试验材料,如无特殊说明,均可通过商业渠道购买得到。其中:
本实施例和对比例中所使用的大肠杆菌为,菌株名称为E.coli K-12(ATCC25404)。
一级种子培养基:胰蛋白胨10g/L,酵母提取物5g/L,氯化钠10g/L,氨苄青霉素100ppm。
二级种子培养基:胰蛋白胨10g/L,酵母提取物5g/L,氯化钠10g/L,氨苄青霉素100ppm。
发酵培养基:蛋白胨2.5g/L、脱脂豆粉20g/L、葡萄糖5g/L、甜菜糖蜜5g/L,酵母膏8g/L、氯化钠3g/L、硫酸铵2.5g/L、三水磷酸氢二钾4g/L、柠檬酸铁铵0.3g/L、柠檬酸2.1g/L、七水硫酸镁0.5g/L、氨苄青霉素100ppm。
补料:葡萄糖200g/L、甜菜糖蜜200g/L、酵母膏80g/L、脱脂豆粉60g/L。
上述培养基中所使用的原料均为市售产品,其中所使用的“脱脂豆粉”为低温脱脂的黄豆粉,购自山东万德福生物科技有限公司。
实施例1:聚磷酸盐激酶1突变处理
发明人从现有数据库中获得野生型聚磷酸盐激酶1氨基酸序列如SEQ ID NO.1所示;编码基因的核苷酸序列如SEQ ID NO.2所示。对其进行拓扑并分析,构建盒子,在charmm力场下进行能量最小化,然后进行NVT平衡和NPT平衡,对成品进行1ns的MD模拟,最终选择将野生型聚磷酸盐激酶1第246位的L突变为P,第247位的M突变为V;突变后的聚磷酸盐激酶1突变体的氨基酸序列如SEQ ID NO.3所示。
通过分子动力学模拟软件分析突变前后聚磷酸盐激酶1的亲水性,结果分别如图1和图2所示。结果表明:突变后的聚磷酸盐激酶1突变体的亲水性提高。
实施例2:聚磷酸盐激酶1突变体生产菌的构建
(1)对pQE-60质粒用BspEⅠ和AflⅢ双酶切,再连接上序列如下的大小为124bp的片段
TCCGGACTCGAGAAATCATAAAAAATTTATTTGCTTTGTGAGCGGATAACAATTATAATAGATTCAATTGTGAGCGGATAACAATTTCACACAGAATTCATTAAAGAGGAGAAATTAAGCATGC;(SEQ ID NO.5)
构建得到质粒pQE-N(结构示意图如图3所示),其核苷酸序列如SEQ ID NO.6所示。
(2)用AccⅢ和SphⅠ对质粒pQE-N双酶切,把优化后的编码聚磷酸盐激酶1突变体的ppk1基因(核苷酸序列如SEQ ID NO.4所示)连接到酶切后的质粒上,获得重组表达载体pQE-ppk1(结构示意图如图4所示),其核苷酸序列如SEQ ID NO.7所示。
将构建的重组表达载体pQE-ppk1用AccⅢ和SphⅠ两个酶进行酶切验证,结果如图5所示。结果表明:ppk1基因(SEQ ID NO.4所示)已成功整合到质粒pQE-N上。
(3)将构建的重组表达载体pQE-ppk1导入到到大肠杆菌中,获得转化子。将转化子在含有100μg/ml氨苄青霉素(Amp)的LB平板上涂布,挑出能够长的单菌落,将其作为阳性转化子。
将阳性转化子接种至含有100μg/ml氨苄青霉素的LB液体培养基中,33℃培养至OD600=0.6,缓慢降温至22℃,加入IPTG(使IPTG的终浓度为0.2mmol/L),诱导培养16h。诱导培养结束后,超声破菌,离心,分离上清液,进行SDS-PAGE验证,其结果如图6所示。在80.3KDa处有表达条带,与外源插入的目的基因ppk1所表达蛋白的理论计算得到的分子量一致。
采用His-镍亲和层析柱对目的蛋白进行分离纯化,得到的纯化蛋白。对纯化蛋白进行western blot检测,结果如图7所示。
由图7可以看出,采用本发明的聚磷酸盐激酶1突变体生产菌可以成功表达聚磷酸盐激酶1突变体。
由此证明:本实施例已成功构建得到聚磷酸盐激酶1突变体生产菌。
实施例3:聚磷酸盐激酶1突变体的发酵生产
(1)菌种活化:
将实施例2构建的聚磷酸盐激酶1突变体生产菌划线接种到含有100μg/ml氨苄青霉素的LB平板中,33℃、培养12h。
(2)一级种培养:
从平板划取1接种环菌体接到一级种子培养基中,33℃、pH7.0,培养16h。
(3)二级种培养:
以1%(体积分数)接种量,将一级种子液接种于二级种子培养基中,33℃、溶氧(DO)30-40%,培养至OD600nm值0.3(稀释100倍)。
(4)发酵培养:
以4%接种量(即二级种子液的接入量为发酵培养基重量的4%),将聚磷酸盐激酶1突变体生产菌的二级种子液接种至发酵罐(10t发酵罐)中进行发酵培养,发酵培养的温度为33℃,pH为6.8-7.2,溶氧(DO)为20-40%;发酵培养至发酵液稀释100倍后的OD600值为0.5时,降温至22℃,向体系中加入IPTG至终浓度为0.2mmol/L进行诱导培养,诱导培养22h;
培养过程监测体系的残糖含量,当体系的残糖含量≤1.0g/L时开始添加补料,通过流加补料使体系中的残糖浓度保持在0.5-1.0g/L。
(5)诱导培养结束后进行放罐,将放罐后的发酵液采用均质机进行破菌处理,均质处理的条件为:均质压力12,000PSI,均质流量300L/Hr;均质处理后离心,分离上清,即生产得到含有聚磷酸盐激酶1突变体的培养液。
上述发酵生产过程中,溶氧(DO)采用溶氧电极测定,溶氧以溶氧电极在空气中的溶氧水平设定为100%,以饱和亚硫酸钠溶液中的溶氧为0。OD600和pH值采用取样测定。
对比例1:
将SEQ ID NO.2所示的ppk1基因通过常规基因工程的手段整合到质粒pQE-N中,构建得到重组表达载体;然后将构建的重组表达载体导入到大肠杆菌中,获得转化子,筛选阳性转化子,并进行验证,构建得到生产菌A。
利用构建的生产菌A,按实施例3的发酵生产条件进行培养,为节省成本,将发酵罐的体积调整成10L,发酵罐中发酵培养基的添加量按比例相应的进行调整,生产得到培养液A。
对比例2:
将优化后的编码聚磷酸盐激酶1突变体的ppk1基因(核苷酸序列如SEQ ID NO.4所示)通过常规基因工程的手段整合到质粒pQE-60中,构建得到重组表达载体;然后将构建的重组表达载体导入到大肠杆菌中,获得转化子,按实施例3的方法筛选阳性转化子,并进行验证,构建得到生产菌B。
利用构建的生产菌B,按实施例3的发酵生产条件进行培养,为节省成本,将发酵罐的体积调整成10L,发酵罐中发酵培养基的添加量按比例相应的进行调整,生产得到培养液B。
对比例3:
将实施例3中的发酵培养基的组成调整为:
蛋白胨12g/L、葡萄糖10g/L、酵母膏8g/L、氯化钠3g/L、硫酸铵2.5g/L、三水磷酸氢二钾4g/L、柠檬酸铁铵0.3g/L、七水硫酸镁0.5g/L、氨苄青霉素100ppm。
将补料的组成调整为:葡萄糖40g/L、蛋白胨30g/L、酵母膏100g/L。
将发酵罐的体积调整成10L,发酵罐中发酵培养基的添加量按比例相应的进行调整。
培养过程中监测体系的葡萄糖含量。
其与条件同实施例3,生产得到培养液C。
试验例1:
对实施例3的到培养液,以及对比例1-对比例3得到的培养液A-培养液C中的蛋白进行纯化。计算目的蛋白的表达量并对目的蛋白的酶活进行检测。其中:
目的蛋白的纯化方法如下:
加样至预先用结合缓冲液(20mmol/LNa2HPO4,500mmol/L NaCl,20mmol/L咪唑,8mol/L尿素,pH7.4)平衡的His-镍亲和层析柱。用洗涤缓冲液A(20mmol/L Na2HPO4,500mmol/L NaCl,20mmol/L咪唑,8mol/L尿素,pH7.4)和洗涤缓冲液B(20mmol/L Na2HPO4,500mmol/L NaCl,30mmol/L咪唑,8mol/L尿素,pH7.4)洗脱杂蛋白,再用洗脱缓冲液(20mmol/L Na2HPO4,500mmol/LNaCl,500mmol/L咪唑,8mol/L尿素,pH7.4)洗脱目的蛋白,收集洗脱液,得到纯化蛋白。
目的蛋白的酶活检测方法如下:
参考“多聚磷酸盐激酶的原核表达、纯化及酶活性测定”(中国生物制品学杂志,2020年5月第33卷第5期)的方法对不同生产菌在相同条件下培养后得到的上清液中的聚磷酸盐激酶1的酶活进行检测,具体检测方法如下:
根据DAPI染色法,在波长为415nm处激发,550nm处检测DAPI-polyP复合物的荧光,测定PPK催化生成的polyP含量,从而计算出PPK酶活性浓度。
酶催化作用反应体系:50mmol/LHepes-KOH(pH 7.2),40mmol/L硫酸铵,4mmol/LMgCl,22mmol/L磷酸肌酸,20μg/mL肌酸激酶,10μgPPK。37℃孵育15或30min加入40mmol/LEDTA终止反应,使用10umol/L DAPI与纯化蛋白酶按1:1混合,于415nm处激发550nm处测定每个反应重复3次。以不同反应条件得到的最大值为蛋白酶活性。配制1000、500、250、125、62.5、0ng/mL系列浓度的polyP标准品采用DAPI染色法测定以poly P标准品浓度为横坐标以荧光强度为纵坐标建立poly P标准曲线,根据poly P标准曲线计算polyP的浓度取均值并按下式计算酶活性。PPK酶活性(U/mg)定义为在测定条件下每分钟每毫克酶催化1pmol磷酸盐掺入poly P中的量。
酶活性(U/mg)=poly P(pmol/L)*V/(m*T)
式中V为反应总体积(L),m为待测酶的质量(mg),T为反应时间(min)。
结果见表1。
表1:
组别 | 聚磷酸盐激酶1的表达量 | 聚磷酸盐激酶1的酶活(U/mg) |
实施例3 | 32g/L | 800-850 |
对比例1 | 30g/L | 630-645 |
对比例2 | 15g/L | 800-850 |
对比例3 | 13g/L | 800-850 |
试验例2:
将试验例1中得到的纯化蛋白(即聚磷酸盐激酶1突变体和野生型聚磷酸盐激酶1)在不同pH条件下测试其酶活,酶活测定方法参考试验例1。
结果如图8所示,结果表明:与野生型聚磷酸盐激酶1相比,本发明的聚磷酸盐激酶1突变体对pH的适应范围广,能够在pH5.0-pH9.3之间均能保持较高的酶活性,更适合工业化生产。
以上所述仅为本申请的优选实施例而已,并不用于限制本申请,对于本领域的技术人员来说,本申请可以有各种更改和变化。凡在本申请的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本申请的保护范围之内。
SEQUENCE LISTING
<110> 新泰市佳禾生物科技有限公司
<120> 一种发酵生产聚磷酸盐激酶1突变体的方法
<130> 2022
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Val Glu Val Leu Arg Glu Lys Leu Thr Ile Ser Arg Tyr Asp Ser Ile
275 280 285
Val Pro Gly Gly Arg Tyr His Asn Phe Lys Asp Phe Ile Asn Phe Pro
290 295 300
Asn Val Gly Lys Ala Asn Leu Val Asn Lys Pro Leu Pro Arg Leu Arg
305 310 315 320
His Ile Trp Phe Asp Lys Ala Gln Phe Arg Asn Gly Phe Asp Ala Ile
325 330 335
Arg Glu Arg Asp Val Leu Leu Tyr Tyr Pro Tyr His Thr Phe Glu His
340 345 350
Val Leu Glu Leu Leu Arg Gln Ala Ser Phe Asp Pro Ser Val Leu Ala
355 360 365
Ile Lys Ile Asn Ile Tyr Arg Val Ala Lys Asp Ser Arg Ile Ile Asp
370 375 380
Ser Met Ile His Ala Ala His Asn Gly Lys Lys Val Thr Val Val Val
385 390 395 400
Glu Leu Gln Ala Arg Phe Asp Glu Glu Ala Asn Ile His Trp Ala Lys
405 410 415
Arg Leu Thr Glu Ala Gly Val His Val Ile Phe Ser Ala Pro Gly Leu
420 425 430
Lys Ile His Ala Lys Leu Phe Leu Ile Ser Arg Lys Glu Asn Gly Glu
435 440 445
Val Val Arg Tyr Ala His Ile Gly Thr Gly Asn Phe Asn Glu Lys Thr
450 455 460
Ala Arg Leu Tyr Thr Asp Tyr Ser Leu Leu Thr Ala Asp Ala Arg Ile
465 470 475 480
Thr Asn Glu Val Arg Arg Val Phe Asn Phe Ile Glu Asn Pro Tyr Arg
485 490 495
Pro Val Thr Phe Asp Tyr Leu Met Val Ser Pro Gln Asn Ser Arg Arg
500 505 510
Leu Leu Tyr Glu Met Val Asp Arg Glu Ile Ala Asn Ala Gln Gln Gly
515 520 525
Leu Pro Ser Gly Ile Thr Leu Lys Leu Asn Asn Leu Val Asp Lys Gly
530 535 540
Leu Val Asp Arg Leu Tyr Ala Ala Ser Ser Ser Gly Val Pro Val Asn
545 550 555 560
Leu Leu Val Arg Gly Met Cys Ser Leu Ile Pro Asn Leu Glu Gly Ile
565 570 575
Ser Asp Asn Ile Arg Ala Ile Ser Ile Val Asp Arg Tyr Leu Glu His
580 585 590
Asp Arg Val Tyr Ile Phe Glu Asn Gly Gly Asp Lys Lys Val Tyr Leu
595 600 605
Ser Ser Ala Asp Trp Met Thr Arg Asn Ile Asp Tyr Arg Ile Glu Val
610 615 620
Ala Thr Pro Leu Leu Asp Pro Arg Leu Lys Gln Arg Val Leu Asp Ile
625 630 635 640
Ile Asp Ile Leu Phe Ser Asp Thr Val Lys Ala Arg Tyr Ile Asp Lys
645 650 655
Glu Leu Ser Asn Arg Tyr Val Pro Arg Gly Asn Arg Arg Lys Val Arg
660 665 670
Ala Gln Leu Ala Ile Tyr Asp Tyr Ile Lys Ser Leu Glu Gln Pro Glu
675 680 685
<210> 4
<211> 2074
<212> DNA
<213> 聚磷酸盐激酶1突变体
<400> 4
ccggaatggg tcaggaaaaa ctgtacatcg aaaaagaact gtcttggctg tctttcaacg 60
aacgtgttct gcaggaagct gctgacaaat ctaacccgct gatcgaacgt atgcgtttcc 120
tgggtatcta ctctaacaac ctggacgaat tctacaaagt tcgtttcgct gaactgaaac 180
gtcgtatcat catctctgaa gaacagggtt ctaactctca ctctcgtcac ctgctgggta 240
aaatccagtc tcgtgttctg aaagctgacc aggaattcga cggtctgtac aacgaactgc 300
tgctggaaat ggctcgtaac cagatcttcc tgatcaacga acgtcagctg tctgttaacc 360
agcagaactg gctgcgtcac tacttcaaac agtacctgcg tcagcacatc accccgatcc 420
tgatcaaccc ggacaccgac ctggttcagt tcctgaaaga cgactacacc tacctggctg 480
ttgaaatcat ccgtggtgac accatccgtt acgctctgct ggaaatcccg tctgacaaag 540
ttccgcgttt cgttaacctg ccgccggaag ctccgcgtcg tcgtaaaccg atgatcctgc 600
tggacaacat cctgcgttac tgcctggacg acatcttcaa aggtttcttc gactacgacg 660
ctctgaacgc ttactctatg aaaatgaccc gtgacgctga atacgacctg gttcacgaaa 720
tggaagcttc tccggttgaa ccggtttctt cttctctgaa acagcgtctg accgctgaac 780
cggttcgttt cgtttaccag cgtgacatgc cgaacgctct ggttgaagtt ctgcgtgaaa 840
aactgaccat ctctcgttac gactctatcg ttccgggtgg tcgttaccac aacttcaaag 900
acttcatcaa cttcccgaac gttggtaaag ctaacctggt taacaaaccg ctgccgcgtc 960
tgcgtcacat ctggttcgac aaagctcagt tccgtaacgg tttcgacgct atccgtgaac 1020
gtgacgttct gctgtactac ccgtaccaca ccttcgaaca cgttctggaa ctgctgcgtc 1080
aggcttcttt cgacccgtct gttctggcta tcaaaatcaa catctaccgt gttgctaaag 1140
actctcgtat catcgactct atgatccacg ctgctcacaa cggtaaaaaa gttaccgttg 1200
ttgttgaact gcaggctcgt ttcgacgaag aagctaacat ccactgggct aaacgtctga 1260
ccgaagctgg tgttcacgtt atcttctctg ctccgggtct gaaaatccac gctaaactgt 1320
tcctgatctc tcgtaaagaa aacggtgaag ttgttcgtta cgctcacatc ggtaccggta 1380
acttcaacga aaaaaccgct cgtctgtaca ccgactactc tctgctgacc gctgacgctc 1440
gtatcaccaa cgaagttcgt cgtgttttca acttcatcga aaacccgtac cgtccggtta 1500
ccttcgacta cctgatggtt tctccgcaga actctcgtcg tctgctgtac gaaatggttg 1560
accgtgaaat cgctaacgct cagcagggtc tgccgtctgg tatcaccctg aaactgaaca 1620
acctggttga caaaggtctg gttgaccgtc tgtacgctgc ttcttcttct ggtgttccgg 1680
ttaacctgct ggttcgtggt atgtgctctc tgatcccgaa cctggaaggt atctctgaca 1740
acatccgtgc tatctctatc gttgaccgtt acctggaaca cgaccgtgtt tacatcttcg 1800
aaaacggtgg tgacaaaaaa gtttacctgt cttctgctga ctggatgacc cgtaacatcg 1860
actaccgtat cgaagttgct accccgctgc tggacccgcg tctgaaacag cgtgttctgg 1920
acatcatcga catcctgttc tctgacaccg ttaaagctcg ttacatcgac aaagaactgt 1980
ctaaccgtta cgttccgcgt ggtaaccgtc gtaaagttcg tgctcagctg gctatctacg 2040
actacatcaa atctctggaa cagccgtaag catg 2074
<210> 5
<211> 124
<212> DNA
<213> 人工序列
<400> 5
tccggactcg agaaatcata aaaaatttat ttgctttgtg agcggataac aattataata 60
gattcaattg tgagcggata acaatttcac acagaattca ttaaagagga gaaattaagc 120
atgc 124
<210> 6
<211> 2529
<212> DNA
<213> 人工序列
<400> 6
ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca 60
attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aaccatggga 120
ggatccagat cttaatagta attagctgag cttggactcc tgttgataga tccagtaatg 180
acctcagaac tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatt 240
ggtgagaatc caagctagct tggcgagatt ttcaggagct aaggaagcta aaatggagaa 300
aaaaatcact ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga 360
ggcatttcag tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc 420
ctttttaaag accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct 480
tgcccgcctg atgaatgctc atccggactc gagaaatcat aaaaaattta tttgctttgt 540
gagcggataa caattataat agattcaatt gtgagcggat aacaatttca cacagaattc 600
attaaagagg agaaattaag catgccggcc gtaatagtaa ttaacatgtg agcaaaaggc 660
cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 720
ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 780
ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 840
ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 900
agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 960
cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 1020
aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 1080
gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 1140
agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 1200
ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 1260
cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 1320
tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 1380
aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 1440
tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 1500
atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 1560
cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 1620
gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 1680
gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 1740
tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 1800
tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 1860
tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 1920
aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 1980
atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 2040
tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 2100
catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 2160
aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 2220
tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 2280
gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 2340
tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 2400
tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 2460
taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt 2520
cgtcttcac 2529
<210> 7
<211> 4481
<212> DNA
<213> 人工序列
<400> 7
ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca 60
attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aaccatggga 120
ggatccagat cttaatagta attagctgag cttggactcc tgttgataga tccagtaatg 180
acctcagaac tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatt 240
ggtgagaatc caagctagct tggcgagatt ttcaggagct aaggaagcta aaatggagaa 300
aaaaatcact ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga 360
ggcatttcag tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc 420
ctttttaaag accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct 480
tgcccgcctg atgaatgctc atccggaatg ggtcaggaaa aactgtacat cgaaaaagaa 540
ctgtcttggc tgtctttcaa cgaacgtgtt ctgcaggaag ctgctgacaa atctaacccg 600
ctgatcgaac gtatgcgttt cctgggtatc tactctaaca acctggacga attctacaaa 660
gttcgtttcg ctgaactgaa acgtcgtatc atcatctctg aagaacaggg ttctaactct 720
cactctcgtc acctgctggg taaaatccag tctcgtgttc tgaaagctga ccaggaattc 780
gacggtctgt acaacgaact gctgctggaa atggctcgta accagatctt cctgatcaac 840
gaacgtcagc tgtctgttaa ccagcagaac tggctgcgtc actacttcaa acagtacctg 900
cgtcagcaca tcaccccgat cctgatcaac ccggacaccg acctggttca gttcctgaaa 960
gacgactaca cctacctggc tgttgaaatc atccgtggtg acaccatccg ttacgctctg 1020
ctggaaatcc cgtctgacaa agttccgcgt ttcgttaacc tgccgccgga agctccgcgt 1080
cgtcgtaaac cgatgatcct gctggacaac atcctgcgtt actgcctgga cgacatcttc 1140
aaaggtttct tcgactacga cgctctgaac gcttactcta tgaaaatgac ccgtgacgct 1200
gaatacgacc tggttcacga aatggaagct tctccggttg aactgatgtc ttcttctctg 1260
aaacagcgtc tgaccgctga accggttcgt ttcgtttacc agcgtgacat gccgaacgct 1320
ctggttgaag ttctgcgtga aaaactgacc atctctcgtt acgactctat cgttccgggt 1380
ggtcgttacc acaacttcaa agacttcatc aacttcccga acgttggtaa agctaacctg 1440
gttaacaaac cgctgccgcg tctgcgtcac atctggttcg acaaagctca gttccgtaac 1500
ggtttcgacg ctatccgtga acgtgacgtt ctgctgtact acccgtacca caccttcgaa 1560
cacgttctgg aactgctgcg tcaggcttct ttcgacccgt ctgttctggc tatcaaaatc 1620
aacatctacc gtgttgctaa agactctcgt atcatcgact ctatgatcca cgctgctcac 1680
aacggtaaaa aagttaccgt tgttgttgaa ctgcaggctc gtttcgacga agaagctaac 1740
atccactggg ctaaacgtct gaccgaagct ggtgttcacg ttatcttctc tgctccgggt 1800
ctgaaaatcc acgctaaact gttcctgatc tctcgtaaag aaaacggtga agttgttcgt 1860
tacgctcaca tcggtaccgg taacttcaac gaaaaaaccg ctcgtctgta caccgactac 1920
tctctgctga ccgctgacgc tcgtatcacc aacgaagttc gtcgtgtttt caacttcatc 1980
gaaaacccgt accgtccggt taccttcgac tacctgatgg tttctccgca gaactctcgt 2040
cgtctgctgt acgaaatggt tgaccgtgaa atcgctaacg ctcagcaggg tctgccgtct 2100
ggtatcaccc tgaaactgaa caacctggtt gacaaaggtc tggttgaccg tctgtacgct 2160
gcttcttctt ctggtgttcc ggttaacctg ctggttcgtg gtatgtgctc tctgatcccg 2220
aacctggaag gtatctctga caacatccgt gctatctcta tcgttgaccg ttacctggaa 2280
cacgaccgtg tttacatctt cgaaaacggt ggtgacaaaa aagtttacct gtcttctgct 2340
gactggatga cccgtaacat cgactaccgt atcgaagttg ctaccccgct gctggacccg 2400
cgtctgaaac agcgtgttct ggacatcatc gacatcctgt tctctgacac cgttaaagct 2460
cgttacatcg acaaagaact gtctaaccgt tacgttccgc gtggtaaccg tcgtaaagtt 2520
cgtgctcagc tggctatcta cgactacatc aaatctctgg aacagccgta agcatgccgg 2580
ccgtaatagt aattaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 2640
ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 2700
gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 2760
gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 2820
ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 2880
tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 2940
gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 3000
tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 3060
tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 3120
tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 3180
ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 3240
ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 3300
gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 3360
aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 3420
aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 3480
cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 3540
ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 3600
cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 3660
ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 3720
ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 3780
ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 3840
gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 3900
ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 3960
ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 4020
gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 4080
ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 4140
cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 4200
ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 4260
aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 4320
gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 4380
gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa 4440
cctataaaaa taggcgtatc acgaggccct ttcgtcttca c 4481
Claims (7)
1.一种发酵生产聚磷酸盐激酶1突变体的方法,其特征在于,包括以下步骤:
(1)将聚磷酸盐激酶1突变体生产菌的种子液接种至发酵培养基中进行发酵培养,发酵培养的温度为32-34℃,pH为6.8-7.2,溶氧为20-40%;发酵培养至发酵液稀释100倍后的OD600值为0.40-0.60时,降温至21-23℃,向体系中加入IPTG进行诱导培养,诱导培养20-24h;
培养过程监测体系的残糖含量,当体系的残糖含量≤1.0g/L时开始添加补料,通过流加补料使体系中的残糖浓度保持在0.5-1.0g/L;
(2)将诱导培养后的培养物进行破菌处理,分离收集上清,即生产得到含有聚磷酸盐激酶1突变体的培养液。
2.根据权利要求1所述的方法,其特征在于,步骤(1)中,所述聚磷酸盐激酶1突变体生产菌由如下方法构建而成:
将pQE-60质粒用BspEⅠ和AflⅢ双酶切,再连接上序列如SEQ ID NO.5所示的片段,构建得到质粒pQE-N,所述质粒pQE-N的核苷酸序列如SEQ ID NO.6所示;用AccⅢ和SphⅠ对质粒pQE-N双酶切,再将SEQ ID NO.4所示的编码聚磷酸盐激酶1突变体的基因连接到酶切后的质粒上,获得重组表达载体pQE-ppk1,所述重组表达载体pQE-ppk1的核苷酸序列如SEQ IDNO.7所示;
将获得的重组表达载体pQE-ppk1导入到大肠杆菌中,构建得到聚磷酸盐激酶1突变体生产菌。
3.根据权利要求1所述的方法,其特征在于,步骤(1)中,聚磷酸盐激酶1突变体生产菌的种子液的接入量为发酵培养基重量的4-8%。
4.根据权利要求1所述的方法,其特征在于,步骤(1)中,所述发酵培养基的组成为:所述发酵培养基的组成为:蛋白胨2.5g/L、脱脂豆粉20g/L、葡萄糖5g/L、甜菜糖蜜5g/L,酵母膏8g/L、氯化钠3g/L、硫酸铵2.5g/L、三水磷酸氢二钾4g/L、柠檬酸铁铵0.3g/L、柠檬酸2.1g/L、七水硫酸镁0.5g/L、氨苄青霉素100ppm。
5.根据权利要求1所述的方法,其特征在于,步骤(1)中,加入IPTG,使IPTG在体系中的终浓度为0.2mmol/L。
6.根据权利要求1所述的方法,其特征在于,步骤(1)中,所述补料的组成为:葡萄糖200g/L、甜菜糖蜜200g/L、酵母膏80g/L、脱脂豆粉60g/L。
7.根据权利要求1-6任一项所述的方法,其特征在于,步骤(2)中,所述聚磷酸盐激酶1突变体的氨基酸序列如SEQ ID NO.3所示。
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CN108795958A (zh) * | 2018-07-11 | 2018-11-13 | 南京工业大学 | 一株表达多聚磷酸激酶的重组菌及其应用 |
CN109022328A (zh) * | 2018-09-05 | 2018-12-18 | 海南师范大学 | 一株聚磷菌及其多聚磷酸盐激酶基因在污水除磷中的应用 |
CN109609542A (zh) * | 2018-12-28 | 2019-04-12 | 博域环保技术研究院(南京)有限公司 | 多聚磷酸盐激酶基因ppk1在水稻中的基因工程应用 |
CN113265382A (zh) * | 2021-06-24 | 2021-08-17 | 洛阳华荣生物技术有限公司 | 多聚磷酸激酶突变体 |
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CN108795958A (zh) * | 2018-07-11 | 2018-11-13 | 南京工业大学 | 一株表达多聚磷酸激酶的重组菌及其应用 |
CN109022328A (zh) * | 2018-09-05 | 2018-12-18 | 海南师范大学 | 一株聚磷菌及其多聚磷酸盐激酶基因在污水除磷中的应用 |
CN109609542A (zh) * | 2018-12-28 | 2019-04-12 | 博域环保技术研究院(南京)有限公司 | 多聚磷酸盐激酶基因ppk1在水稻中的基因工程应用 |
CN113265382A (zh) * | 2021-06-24 | 2021-08-17 | 洛阳华荣生物技术有限公司 | 多聚磷酸激酶突变体 |
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