CN115125235B - 一株高产溶葡萄球菌酶的大肠杆菌突变体 - Google Patents
一株高产溶葡萄球菌酶的大肠杆菌突变体 Download PDFInfo
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Abstract
本发明提供了一种大肠埃希氏菌(Escherichia coil)BL21/(DE3)/pOpt‑lys 4‑36,保藏日期为2022年5月23号,保藏机构为中国普通微生物菌种保藏管理中心,地址为北京市朝阳区北辰西路1号院3号,保藏编号CGMCC No.24960,本发明提供了大肠杆菌突变株,所产溶葡萄球菌酶为胞外分泌表达,且突变株的胞外溶葡萄球菌酶活性较原始菌株提高了3.1倍。
Description
技术领域
本发明属于微生物产酶技术领域,具体涉及一种高产溶葡萄球菌酶的大肠杆菌突变体。
背景技术
溶葡萄球菌酶(lysostaphin,Lys)可以专一性地作用于金黄色葡萄球菌细胞壁中的Gly五肽交联桥,从而裂解其细胞壁,对金黄色葡萄球菌具有很好的溶菌杀菌效果。尽管溶葡萄球菌酶在预防和治疗葡萄球菌感染方面有非常大的应用潜力,但由于其生产成本较高,目前仍没有被广泛的应用。
常压室温等离子体(Atmospheric Room Temperature Plasma,ARTP)诱变系统能够在大气压下产生温度在25-40℃之间的、具有高活性粒子(包括处于激发态的氦原子、氧原子、氮原子、 OH自由基等)浓度的等离子体射流,来改变目标生物细胞壁和质膜的结构和通透性,进而引起DNA损伤,快速有效地突变细菌、放线菌、微藻、真菌、酵母等微生物。ARTP因其具有温度低,活性粒子浓度高且种类多样、对环境无污染和危害、操作简易、运行成本低廉等优点,已经成为发酵工业获得高产突变菌株的高效方法。
发明内容
针对现有技术的不足,本发明提供了一株高产溶葡萄球菌酶的大肠杆菌突变体。
本发明结合ARTP诱变,对申请号为202210279685.2,专利申请名称为一种利用大肠杆菌产溶葡萄球菌酶的方法,该专利文献中记载的大肠杆菌工程菌 BL21(DE3)/pET22b(+)-Opt-Lys进行诱变获得了一株胞外溶葡萄球菌酶产量高的突变菌株。
本发明也提供了一种对诱变菌株进行高通量的筛选的方法。
本发明的技术方案如下:
一种大肠埃希氏菌(Escherichia coil)BL21/(DE3)/pOpt-lys 4-36,保藏日期为2022年5 月23号,保藏机构中国普通微生物菌种保藏管理中心,地址北京市朝阳区北辰西路1号院3 号,保藏编号CGMCC No.24960。
上述大肠埃希氏菌(Escherichia coil)BL21/(DE3)/pOpt-lys 4-36的培养方法,包括如下步骤:
将大肠埃希氏菌(Escherichia coil)BL21/(DE3)/pOpt-lys 4-36接种于含有氨苄青霉素的 LB培养基中,35-37℃,18-200rpm培养获得大肠埃希氏菌(Escherichia coil)BL21/(DE3)/pOpt -lys 4-36。
根据本发明优选的,LB培养基的成分组成为:蛋白胨10g/L、酵母粉5g/L、氯化钠10g/L,余量水,pH值6.7。
根据本发明优选的,氨苄青霉素的终浓度为100μg/mL。
上述大肠埃希氏菌(Escherichia coil)BL21/(DE3)/pOpt-lys 4-36生产溶葡萄球菌酶的方法,包括如下步骤:
①将大肠埃希氏菌(Escherichia coil)BL21/(DE3)/pOpt-lys 4-36培养于含有氨苄青霉素的LB培养基,35-37℃摇床180-200rpm培养11-13h获得种子液。
②将种子液以体积分数2-3%的比例转接至含有氨苄青霉素的LB培养基中,培养到 OD600至0.6-0.7。
③添加异丙基-β-D-硫代半乳糖苷(IPTG),终浓度为0.45-0.55mM,并在180-200rpm、 35-37℃下诱导培养35-37h,获得溶葡萄球菌酶发酵液。
根据本发明优选的,步骤①、步骤②中氨苄青霉素的终浓度为100μg/mL。
一种筛选胞外分泌溶葡萄球菌酶菌株的方法,包括如下步骤:
Ⅰ将待筛选菌株点接于下层添加异丙基-β-D-硫代半乳糖苷(IPTG)、氨苄青霉素抗性的LB平板,35-37℃静置培养11h以上,制得含筛选菌株的平板;
Ⅱ将金黄色葡萄球菌过夜培养,用磷酸盐缓冲液稀释至OD600为1.0的菌悬液,将菌悬液与LB固体培养基以体积比1:(95-100)的比例混合,作为含指示菌的上层培养基,将含有指示菌的上层培养基倒入步骤I制备的含筛选菌株的平板上,制备上层平板,在35-37℃下孵育22-24h;
Ⅲ通过步骤Ⅱ平板上产生的抑菌圈筛选胞外分泌溶葡萄球菌酶菌株。
根据本发明优选的,步骤Ⅰ中氨苄青霉素的终浓度为100μg/mL。
根据本发明优选的,步骤Ⅰ中异丙基-β-D-硫代半乳糖苷(IPTG)的终浓度为0.45-0.55 mM。
根据本发明优选的,步骤Ⅰ中培养12-24h。
根据本发明优选的,步骤Ⅱ中磷酸盐缓冲液为100mM,pH7.0。
有益效果
1、本发明提供了一株产溶葡萄球菌酶的大肠杆菌突变株,所产溶葡萄球菌酶为胞外分泌表达,且突变株的胞外酶活性较原始菌株提高了3.1倍。
2、本发明提供了一株高产溶葡萄球菌酶菌株的筛选方法,利用双层平板法上抑菌圈的大小来判断菌株的溶葡萄球菌酶活性,简单易行,且可以进行高通量的筛选操作。
附图说明
图1为溶葡萄球菌酶高产突变株的高通量筛选方法示意图。
图2为不同菌株在筛选平板上产抑菌圈的图片;
图中:mutant 4为突变菌株BL21(DE3)/pET22b(+)-Opt-Lys 4-36。
图3为高产溶葡萄球菌酶突变株的SDS-PAGE分析图;
图中:1为未诱变菌株BL21(DE3)/pET22b(+)-Opt-Lys,2为突变菌株 BL21(DE3)/pET22b(+)-Opt-Lys 4-36。
具体实施方式
下面通过实施例并结合附图对本发明作进一步说明,但本发明内容的保护范围不限于此。
实施例中未详加说明的均按本领域现有技术。
大肠杆菌BL21(DE3)/pET22b(+)-Opt-Lys 4-36即为大肠埃希氏菌(Escherichiacoil) BL21/(DE3)/pOpt-lys 4-36。
实施例1
ARTP诱变仪突变大肠杆菌BL21(DE3)/pET22b(+)-Opt-Lys
采用ARTP-ⅡS诱变育种仪(四川四清源生物科技有限公司,无锡,中国)。主要参数包括:电源P(P=100W)、气体流量G(G=10.0slm)、等离子体温度T(T=20.0℃)。
将20μL大肠杆菌BL21(DE3)/pET22b(+)-Opt-Lys菌悬液(106-108cells/mL)均匀地铺在已灭菌的金属载玻片上并分别处理0、15、30、45、60、75、90、105和120s,每次处理后,将载玻片转移到含有1mL生理盐水的无菌EP管中洗脱。在获得理想的致死率处理时间条件下,将洗脱液涂布至含100μg/mL氨苄青霉素(Amp)的LB平板,在37℃静置培养12h。挑取菌落用于ARTP突变体的初步筛选。结果显示处理时间为90s时能够获得90%的致死率,因此处理时间选定为90s。
实施例2
突变体高通量筛选方法的建立
利用溶葡萄球菌酶的胞外分泌特性,本发明建立一种简单的突变体筛选方法,来获得溶葡萄球菌酶高产菌株。此方法的示意图如图1所示。
一种筛选胞外分泌溶葡萄球菌酶的菌株的方法,包括如下步骤:
(1)BL21(DE3)/pET22b(+)-Opt-Lys菌株经过实施例1中ARTP诱变后,获得的单菌落使用无菌牙签点种于下层添加IPTG终浓度为0.5mM、Amp终浓度为100μg/mL抗性的LB 平板,37℃静置培养12h,制得含筛选菌株的平板。
(2)将金黄色葡萄球菌过夜培养,用磷酸盐缓冲液(100mM,pH 7.0)稀释至OD600为1.0,该菌悬液与LB固体培养基以体积比1:100的比例混合,作为含指示菌的上层培养基。将含有指示菌的上层培养基倒入步骤(1)制备的含筛选菌株的平板上,制备上层平板,在37℃下孵育24h。
(3)通过步骤(2)平板上产生的抑菌圈大小进行随机诱变菌株胞外酶活的比较,筛选出高活性的突变株。结果如图2所示,mutant 4为突变菌株BL21(DE3)/pET22b(+)-Opt-Lys 4-36,大肠杆菌重组菌株BL21(DE3)/pET22b(+)-Lys胞外不产透明圈,重组菌株(即突变的原始菌株)BL21(DE3)/pET22b(+)-Opt-Lys可以产透明圈,而ARTP诱变后获得的突变株,其抑菌圈呈现不同水平的变化。
实施例3
与实施例2的不同之处在于,步骤(1)中点接突变菌种BL21(DE3)/pET22b(+)-Opt-Lys 4-36 后直接,培养24h后,将步骤(2)中含有指示菌的上层培养基倒入步骤(1)制备的含筛选菌株的平板上,制备上层平板,在37℃下孵育24h。结果显示,菌株所产透明圈大小和培养 12h的无明显区别。
对比例1
与实施例2的不同之处在于,步骤(1)中点接原始菌株BL21(DE3)/pET22b(+)-Opt-Lys、突变菌种BL21(DE3)/pET22b(+)-Opt-Lys 4-36后,不进行12h的培养,将步骤(2)中含有指示菌的上层培养基倒入步骤(1)制备的含筛选菌株的平板上,制备上层平板,在37℃下孵育24h。结果显示,菌株不能显现抑菌圈。
对比例2
与实施例2的不同之处在于,步骤(1)中点接原始菌株BL21(DE3)/pET22b(+)-Opt-Lys、突变菌种BL21(DE3)/pET22b(+)-Opt-Lys 4-36后,进行8h的培养,将步骤(2)中含有指示菌的上层培养基倒入步骤(1)制备的含筛选菌株的平板上,制备上层平板,在37℃下孵育24h。结果显示,菌株产抑菌圈较小,原始菌株BL21(DE3)/pET22b(+)-Opt-Lys与突变菌种BL21(DE3)/pET22b(+)-Opt-Lys 4-36根据透明圈无法区分。
实施例4
高产溶葡萄球菌酶的大肠杆菌突变体的特性
将所筛选的高产菌株大肠杆菌BL21(DE3)/pET22b(+)-Opt-Lys 4-36和未诱变的大肠杆菌 BL21(DE3)/pET22b(+)-Opt-Lys菌株,分别以体积比2%转接至含有100μg/mLAmp的LB液体培养基中,培养到OD600nm至0.6后,添加异丙基-β-D-硫代半乳糖苷(IPTG)终浓度为0.5mM,并在37℃下诱导36h。取诱导后的菌液在12000rpm离心5min后取上清,经过0.22μm滤膜过滤,制得胞外蛋白样品。进行12%(v/v)的SDS-PAGE分析比较,并进行酶活的测定。
结果如图3所示,BL21(DE3)/pET22b(+)-Opt-Lys 4-36胞外蛋白条带宽度明显大于未诱变菌株BL21(DE3)/pET22b(+)-Opt-Lys。此外,酶活测定结果显示未诱变菌株 BL21(DE3)/pET22b(+)-Opt-Lys胞外溶葡萄球菌酶的活性为11.07U/mL,而突变菌株BL21(DE3)/pET22b(+)-Opt-Lys 4-36胞外溶葡萄球菌酶的活性为34.32U/mL,相比提高了3.1 倍。
酶活测定方法:采用分光光度法测定溶葡萄球菌酶的溶菌活性。将金黄色葡萄球菌在LB 中培养过夜,收集菌体后使用PBS洗涤1次。然后在PBS中重悬细胞,稀释OD600至1.0。向其中加入50μL的胞外发酵液,37℃水浴10min,测定金黄色葡萄球菌菌悬液浊度。溶葡萄球菌酶酶活定义为:pH7.5条件下在37℃水浴10min,金黄色葡萄球菌菌悬液浊度降低0.1所需的量为1U。
进一步对突变菌株的遗传稳定性进行了测定,将菌株BL21(DE3)/pET22b(+) -Opt-Lys 4-36连续传代8次,每代菌株以下诱导操作,转接至含有100μg/mL Amp的LB液体培养基中,培养到OD600nm至0.6后,添加异丙基-β-D-硫代半乳糖苷(IPTG)终浓度为0.5mM,并在37℃下诱导36h。取胞外发酵液进行溶葡萄球菌酶的活性测定,结果如表1所示,连续传代8次,突变菌种的产酶活性仍然稳定。
表1突变株不同传代次数产酶特性
代数 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
酶活(U/mL) | 34.32 | 35.55 | 35.29 | 34.83 | 34.17 | 33.60 | 34.25 | 33.81 |
本发明提供了一株产溶葡萄球菌酶的大肠杆菌BL21(DE3)/pET22b(+)-Opt-Lys4-36,所产溶葡萄球菌酶为胞外分泌表达,且大肠杆菌BL21(DE3)/pET22b(+)-Opt-Lys 4-36的胞外溶葡萄球菌酶的活性较原始菌株提高了3.1倍,大肠杆菌BL21(DE3)/pET22b(+)-Opt-Lys 4-36胞外溶葡萄球菌酶的活性为34.32U/mL。本发明还提供了一株高产溶葡萄球菌酶菌株的筛选方法,利用双层平板法上抑菌圈的大小来判断菌株的溶葡萄球菌酶活性,简单易行,且可以进行高通量的筛选操作。
本发明提供的大肠杆菌BL21(DE3)/pET22b(+)-Opt-Lys 4-36,也称为大肠埃希氏菌 (Escherichia coil)BL21/(DE3)/pOpt-lys 4-36,保藏日期为2022年5月23号,保藏机构为中国普通微生物菌种保藏管理中心,地址为北京市朝阳区北辰西路1号院3号,保藏编号CGMCC No.24960。
Claims (3)
1. 一种大肠埃希氏菌(Escherichia coil)BL21/(DE3)/pOpt-lys 4-36,保藏日期为2022.5.23,保藏机构为中国普通微生物菌种保藏管理中心,地址为北京市朝阳区北辰西路1号院3号,保藏编号CGMCC No.24960。
2. 权利要求1所述大肠埃希氏菌(Escherichia coil)BL21/(DE3)/pOpt-lys 4-36生产溶葡萄球菌酶的方法,其特征在于,包括如下步骤:
① 将大肠埃希氏菌(Escherichia coil)BL21/(DE3)/pOpt-lys 4-36培养于含有氨苄青霉素的LB培养基,35-37℃摇床180-200rpm培养11-13 h获得种子液;
② 将种子液以体积分数2 -3%的比例转接至含有氨苄青霉素的LB培养基中,培养到OD600至0.6-0.7;
③ 添加异丙基-β-D-硫代半乳糖苷,终浓度为0.45-0.55 mM,并在180-200rpm、35-37℃下诱导培养35-37 h,获得溶葡萄球菌酶发酵液。
3.如权利要求2所述方法,其特征在于,步骤①、步骤②中氨苄青霉素的终浓度为100μg/mL。
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