CN116334052B - 一种巴曲酶的发酵培养基及发酵方法 - Google Patents
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Abstract
本发明公开了一种重组巴曲酶的酵母发酵方法及使用的培养基,包括优化后基础发酵培养基,优化后甘油补料培养基以及优化后诱导培养基,发酵步骤主要包括一级种子培养、二级种子培养以及发酵培养,在发酵培养阶段,通过在培养基中补加多种营养成分,如甘油、casamino acids以及PTM1无机盐等,并调节合适的发酵培养基的pH值,通过甲醇诱导培养,极大的提高了发酵的重组蛋白的活性。本发明通过casamino acids的加入对发酵产物中蛋白酶的抑制作用,山梨醇与甲醇作为共用碳源提高目的蛋白表达的活性,经过后期的纯制备较高的巴曲酶重组蛋白,其活性基本能够达到与市售的天然巴曲酶类似的活性。
Description
技术领域
本发明涉及生物工程领域,具体涉及一种巴曲酶的发酵培养基及发酵方法。
背景技术
早在1963年Van Klobusitzky等就从巴西矛头蛇的毒液中纯化出一种酶性止血剂Batroxobin,在国内又称“巴曲酶”。它是一种单链糖蛋白,总氨基酸数为231个,分子量39~43kDa。巴曲酶属于丝氨酸蛋白酶类分子,是一种单链糖蛋白,它能专一作用于纤维蛋白原分子Aα链N末端的Arg16-Gly17肽键,释放血纤维蛋白肽A,生成不稳定的可溶性纤维蛋白Ⅰ单体,进而交联聚合成纤维蛋白Ⅰ多聚体,促进出血部位血小板聚集产生止血效应,并激活血管内皮释放组织型纤维蛋白溶酶原激活物(t-PA),发挥抗血栓的作用。
随着血栓栓塞性疾病患者的增加,抗血栓药物的研制已成为热点之一。人们一方面采用分子生物学技术对现有药物进行分子结构改造,以期获得更为优良的溶栓药物。另一方面,积极的寻找一些安全性好、疗效好、副作用小的天然来源的溶栓药物。据报道,巴曲酶在临床上已被广泛应用于不稳定性心绞痛、高黏血症等多种疾病的治疗和闭塞性心脑血管血栓性疾病的预防,疗效确切,不良反应低微。
因为大量生产从蛇中纯化的巴曲酶是有限的,产生重组蛋白质的方法已由众多研究者进行了深入研究。
P.pastoris表达系统兼具原核和真核体系遗传背景清楚、操控简便和正确产生糖基化的特点,外源基因可以稳定整合到酵母基因组,高效表达目的蛋白,外源蛋白可以进行糖基化等蛋白翻译后修饰;胞外高分泌目的蛋白也有利于产物保持生物活性和后期纯化,避免了原核表达破碎细胞壁等烦琐步骤,提高了产量。韩国的You Weon-Kyoo于2004年报道了巴曲酶在毕氏酵母中进行表达,表达量达到3.431NIH/mL,从每升发酵液中可纯化得到7mg。
由于巴曲酶自身酶结构的特性,在采用基因工程发酵生产的过程中,巴曲酶的活性有下降趋势,主要原因是由于酵母宿主系统中蛋白酶可能对发酵液中累积的巴曲酶产生降解作用,从而影响巴曲酶的产量以及酶活性。重组巴曲酶的基因工程表达通常使用毕赤酵母系统,但从已有的文献来看,表达量都不高。
因此,现有技术中存在发酵制备获得的巴曲酶的活性偏低,以及酶降解的不足。
发明内容
本发明的目的在于针对现有技术中纯化的巴曲酶产量的偏低,酶活性不高的问题,提供一种巴曲酶的发酵培养基及发酵方法。
本发明公开了用于重组巴曲酶发酵的培养基,包括:优化基础发酵培养基、优化甘油补料培养基以及优化甲醇诱导培养基,所述优化基础发酵培养基组分:K2SO4:0.91%;MgSO4:0.36%;CaSO4.2H2O:0.059%;85%H3PO4:2.5%(V/V);KOH:0.206%;甘油:4%;泡敌:0.05%-0.1%;PTM1:0.4%(V/V),casamino acids:0.5%;所述优化甘油补料培养基组分:50%甘油+12mL/L PTM1+0.5%casamino acids;所述优化甲醇诱导培养基组分:(100%甲醇+12mL/L PTM1)95%+4.5%山梨醇+0.5%casamino acids。
本发明公开了一种巴曲酶的发酵方法,包括如下步骤:
1)一级种子培养,取保存菌株接种至一级种子培养基中,所述一级种子培养基配方为:酵母粉,1.0%;聚蛋白胨2.0%;葡萄糖,2.0%,采用纯化水配制后灭菌备用;
2)二级种子培养,在无菌的条件下,一级种子液接入含有二级培养基的种子罐中,接种比例为5%-11%,所述二级种子培养基为配方为:酵母粉,1.0%;聚蛋白胨2.0%;葡萄糖,2.0%,采用纯化水配制后灭菌备用;
3)发酵培养,待二级种子液OD600在5-7之间,在无菌的条件下加入优化基础发酵培养基(4ml/L)至发酵罐中,在无菌的条件下将二级种子液移入发酵罐中,培养4小时后,向发酵罐中优化甘油补料培养基,并调节pH值,甲醇诱导;
4)收集上清,纯化获得重组巴曲酶。
优选的,所述优化后基础发酵培养基组分:K2SO4:0.91%;MgSO4:0.36%;CaSO4 .2H2O:0.059%;85%H3PO4:2.5%(V/V);KOH:0.206%;甘油:4%;泡敌:0.05%-0.1%;PTM1:0.4%(V/V),casamino acids:0.5%。
优选的,所述优化甘油补料培养基组分:50%甘油+12mL/L PTM1+0.5%casaminoacids。
优选的,甲醇诱导阶段采用优化甲醇诱导培养基进行诱导,所述优化甲醇诱导培养基组分:(100%甲醇+12mL/L PTM1)95%+4.5%山梨醇+0.5%casamino acids。
优选的,调节的pH范围为5.0-7.0。
优选的,所述PTM1的配方为:0.6%CuSO4·5H2O,0.008%NaI,0.3%MnSO4·H2O,0.02%Na2MoO4·2H2O,0.02%H3BO3,0.05%CoCl2·6H2O,6.5%FeSO4·7H2O,2%ZnCl2,0.02%Biotin,0.0005%(V/V)H2SO4。
优选的,所述优化基础发酵培养基、优化甘油补料培养基以及优化甲醇诱导培养基分别单独配制
本发明公开了一种重组巴曲酶的酵母发酵方法及使用的培养基,包括优化后基础发酵培养基,优化后甘油补料培养基以及优化后诱导培养基,发酵步骤主要包括一级种子培养、二级种子培养以及发酵培养,在发酵培养阶段,通过在培养基中补加多种营养成分,如甘油、casamino acids以及PTM1无机盐等,并调节合适的发酵培养基的pH值,通过甲醇诱导培养,极大的提高了发酵的重组蛋白的活性。本发明通过casamino acids的加入对发酵产物中蛋白酶的抑制作用,山梨醇与甲醇作为共用碳源提高目的蛋白表达的活性,经过后期的纯化制备较高活性的巴曲酶重组蛋白,其活性基本能够达到与市售的天然巴曲酶类似的活性,值的推广应用。
附图说明
图1.PCR对转化的酵母菌落的筛选鉴定图。
图2.甲醇诱导不同阶段的重组巴曲酶的活性测定。
图3.优化基础培养基组分对发酵蛋白活性的影响。
图4.优化甲醇诱导培养基对发酵蛋白活性的影响。
图5.添加甘油补料培养基对发酵蛋白活性的影响。
图6.优化甘油补料培养基对发酵蛋白活性的影响。
图7.发酵培养基不同pH值对于发酵重组巴曲酶的活性测定。
图8.纯化的巴曲酶蛋白的SDS电泳检测图。
具体实施方式
以下结合附图对本发明的具体实施方式进行详细说明。应当理解的是,此处所描述的具体实施方式仅用于说明和解释本发明,并不用于限制本发明。
实施例1含有巴曲酶基因的酵母表达载体的构建及转化
1)巴曲酶编码基因的克隆
采用GeneBank中公开的巴曲酶(J02684.1)的核酸序列,首先人工合成出该基因序列。根据该基因的信息以及基因中的酶切位点信息等,设计扩增巴曲酶编码基因的引物序列,并在引物两端添加XhoI以及SacII的酶切位点序列。设计的扩增引物序列如下:
正义引物:5’-aactcgaggtcattgga ggtgatgaat-3’;
反义引物:5’-aaccgcggcgggcaagt cgcagttt-3’。
采用上述扩增引物以合成的巴曲酶基因J02684.1进行PCR扩增。
PCR扩增反应条件如下:
95℃3min;95℃40s,53℃30s,72℃1min,35个循环;72℃15min。
获得的扩增产物4℃保存备用。
2)酵母表达载体的构建及转化
将步骤1)中的PCR扩增产物纯化回收后采用XhoI以及SacII进行双酶切,同时对酵母表达载体pPICZαA也采用XhoI以及SacII进行双酶切。分别对酶切的产物进行回收后,再用T4连接酶进行连接后,将连接产物转化大肠杆菌BL21。鉴定重组菌,提取重组质粒,经双酶切及PCR扩增目的基因筛选阳性克隆,重组质粒命名为pPICZαA-Batr。
提取pPICZαA-Batr质粒,用Sac I线性化后,采用电转化法转化毕赤酵母X-33所有转化子涂布在YPD+Zeocin(300μg/ml)培养基上30℃培养,挑选菌落进行菌落PCR鉴定,采用上述扩增引物进行PCR鉴定。其中,PCR对酵母菌落的鉴定图参见图1。对于鉴定的阳性菌落克隆进行保存备用。
实施例2工程酵母菌X-33/pPICZαA-Batr的发酵表达
1)阳性菌落酶表达量的初步筛选
挑取阳性克隆,首先接种于甘油复合培养基(BMGY)中振荡培养过夜,离心去除培养液,沉淀菌体重悬于BMMY中并稀释至OD600为1进行诱导表达,每约12小时取样并补加甲醇至终浓度为1%,72h后离心收集上清。每次取样经离心保留上清并初步测定凝血酶活性,测定结果参见表1。初步筛选获得凝血酶活性较高的菌落克隆以用于发酵罐发酵。其中,凝血酶活性测定的方法如下:
取人-枸橼酸凝血质控血浆0.2ml,加入96孔板中,置于37℃保温3min,加入37℃预热的样品溶液0.2ml,立即振荡混匀计时,于40s开始检查血浆凝固情况,记录初凝时间,同时测定3管,误差应<20s。若初凝时间<40s,则是当稀释公式样品溶液若干倍,记录(60±20)s内凝固的供试样品溶液浓度。在上述条件下,能使0.2ml人-枸橼酸抗凝血浆在60s凝固的酶量,定义为一个酶活单位。
表1:不同阳性克隆工程酵母菌X-33/pPICZαA-Batr诱导表达36小时的初步凝血酶活性测定
菌落编号 | 1 | 2 | 3 | 4 | 5 | 6 |
凝固时间(S) | 262 | 231 | 247 | 289 | 255 | 269 |
从测定结果来看,菌落2中表达的巴曲酶的活性最高,促凝固的时间最短,在后期的发酵操作中采用2号菌落来进行。
实施例3酵母菌X-33/pPICZαA-Batr-2的小规模发酵条件优化
1)小规模初级发酵的建立
①一级种子培养:取工作种子菌株1-2支,无菌条件下打开,接种于一级种子培养基,接种比例为0.06%-0.2%,30±1℃,220rpm±10rpm,当种子液的OD600为2-5时结束培养,培养时间为30±8h。
表2:一级种子配方(固体W/V,配制体积2L,用纯化水补齐)
物料 | 酵母粉 | 聚蛋白胨 | 葡萄糖 |
配比 | 1.0% | 2.0% | 2.0% |
②二级种子培养
二级种子接种:在无菌的条件下,一级种子液接入含有二级培养基的种子罐中,接种比例为5%~11%。
控制种子罐温度为30±1℃,转速≥100rpm(初始转速100rpm),罐压<0.08MPa,通过控制转速、通气量(初始通气量20L/min)保证此阶段溶氧>20%,培养4小时后,每间隔2小时取样一次,镜检,测OD600,并记录各参数。当种子液的OD600为5-7时结束培养,培养时间为10±4h。
表3二级种子配方(固体W/V,配制体积2L,用纯化水补齐)
成分 | 聚蛋白胨 | 酵母粉 | 葡萄糖 |
含量 | 2.0% | 1.0% | 2.0% |
③发酵培养
待二级种子液OD600在5~7之间,在无菌的条件下加入原始基础发酵培养基(4ml/L)至发酵罐中,在无菌的条件下将二级种子液移入50L发酵罐中。控制温度在30±1℃、pH5.0±0.2(氨水)、罐压<0.08MPa,通过控制转速(初始转速100rpm)、通气量(初始通气量100L/min)、通氧量保证此阶段溶氧>20%。当DO迅速上升,此阶段结束,此阶段菌体湿重为90-150g/L,所需时间约为16-24h。所述原始基础发酵培养基组分:K2SO4:0.91%;MgSO4:0.36%;CaSO4·2H2O:0.059%;85%H3PO4:2.5%(V/V);KOH:0.206%;甘油:4%;泡敌:0.05%-0.1%;PTM1:0.4%(V/V)。
甲醇诱导阶段:控制温度在20-30℃、pH6.0±0.2(氨水)、罐压<0.08MPa,通过控制甲醇补加速度、转速、通气量、通氧量保证此阶段溶氧>20%。初始加入甲醇量不超过初始发酵体积的0.5%,待溶氧快速上升至峰值时(通常在1min之内),开始匀速流加甲醇,并逐步提高流速,甲醇最高流速不得高于12mL/h.L(每升发酵液每小时补料12mL)发酵液体积。如果DO不能保持在20%以上,应停止流加,等待DO达到峰值后再继续流加甲醇溶液。此阶段菌体湿重为350-600g/L,甲醇诱导时间不得低于60h。所述原始甲醇诱导培养基组分:100%甲醇+12mL/L PTM1。
在甲醇诱导后的不同阶段取诱导培养基上清按照实施例2中的方法检测表达的巴曲霉的凝血酶活性。发酵结果参见图2。从图2的测定结果可以看出,在小规模发酵中,甲醇诱导时间在60小时左右,培养基中的重组巴曲霉的活性达到最高。在60小时之后,巴曲霉的活性趋于稳定,但略有下降,推测可能是由于培养基中的杂蛋白对于重组酶的降解作用加强。
2)优化基础发酵培养基组分进行发酵培养
对于上述“③发酵培养”中的原始基础发酵培养基组分进行优化,即在二级种子液转入发酵罐加入优化后基础发酵培养基(4ml/L),其他发酵条件同上述1)。所述优化后基础发酵培养基组分:K2SO4:0.91%;MgSO4:0.36%;CaSO4 .2H2O:0.059%;85%H3PO4:2.5%(V/V);KOH:0.206%;甘油:4%;泡敌:0.05%-0.1%;PTM1:0.4%(V/V),casamino acids:0.5%。
在甲醇诱导后的不同阶段取诱导培养基上清按照实施例2中的方法检测表达的巴曲霉的凝血酶活性。发酵结果参见图3。从图3的测定结果可以看出,通过对基础发酵培养基组分的优化,在甲醇诱导的各个阶段,培养基中的重组巴曲霉的活性均有一定的提高。
3)优化甲醇诱导培养基组分进行发酵培养
按照上述2)的发酵诱导方法,所不同的是,在甲醇诱导阶段,使用优化后甲醇诱导培养基组分:(100%甲醇+12mL/L PTM1)95%+4.5%山梨醇+0.5%casamino acids进行诱导。
在甲醇诱导后的不同阶段取诱导培养基上清按照实施例2中的方法检测表达的巴曲霉的凝血酶活性。发酵结果参见图4。从图4的测定结果可以看出,通过对甲醇诱导培养基组分的优化,在诱导的各个阶段,培养基中的重组巴曲霉的活性均有一定的提高。
4)增加甘油补料步骤对于发酵的蛋白的活性的影响
菌体发酵的起始阶段对于培养基中的碳源、氮源以及无机盐等的需求巨大,因此,在发酵初期进行适当的补料,增加培养基中的营养成分,以提高重组蛋白的表达量。
按照上述3)的发酵诱导方法,所不同的是,在菌体发酵的起始阶段,控制温度在30±1℃、pH5.0±0.2、罐压<0.08MPa,培养4小时左右。每升发酵培养基加不同体积的(5mL、10mL、15mL、20mL、30mL)的原始甘油补料培养基,所述原始甘油补料培养基组分:50%甘油+12mL/L PTM1。
在甲醇诱导后的60小时左右取诱导培养基上清按照实施例2中的方法检测表达的巴曲霉的凝血酶活性。发酵结果参见图5。从图5的测定结果可以看出,通过补料甘油的步骤,对于甲醇诱导后的发酵蛋白的活性均有一定程度的提高。其中所述原始甘油补料培养基甘油从5mL增加到10mL体积的量,对于巴曲酶的活性提高明显,在此基础上,虽然进一步增加甘油的量,但是活性未明显提升。
5)优化甘油补料培养基对于发酵的蛋白的活性的影响
按照上述4)的发酵诱导方法,所不同的是,对添加的甘油补料培养基进行优化,且在诱导的不同阶段取上清。
在菌体发酵的起始阶段,控制温度在30±1℃、pH5.0±0.2、罐压<0.08MPa,培养4小时左右。每升发酵培养基加10mL的优化甘油补料培养基,所述优化甘油补料培养基组分:50%甘油+12mL/L PTM1+0.5%casamino acids
在诱导后的不同阶段取诱导培养基上清按照实施例2中的方法检测表达的巴曲霉的凝血酶活性。发酵结果参见图6。从图6的测定结果可以看出,通过优化甘油补料培养基的步骤,对于甲醇诱导后的发酵蛋白的活性均有一定程度的提高。
6)发酵培养基不同pH值条件下的诱导
按照上述5)的发酵诱导方法,所不同的是,调整发酵培养基的起始pH值。分别调整发酵培养基的pH值为5.0、5.5、6.0、6.5、7.0,甲醇诱导约60小时后检测培养基中重组巴曲霉的活性。
在甲醇诱导后的不同阶段取诱导培养基上清按照实施例2中的方法检测表达的巴曲霉的凝血酶活性。发酵结果参见图7。从图7的测定结果可以看出,通过调整发酵培养基的pH值,对于甲醇诱导后的发酵蛋白的活性均有影响,其中在pH值为6.5时,发酵的重组蛋白表现出较高的活性,较低的pH值反而对蛋白的表达活性具有抑制作用。
实施例4酵母菌X-33/pPICZαA-Batr-2的发酵纯化
收集实施例3中按照条件6)的发酵(pH值选择6.5),收集培养基上清按照如下方法进行纯化。
1)取巴曲酶酵母发酵液上清,调节pH值和电导率;2)过阳离子树脂进行层析;洗脱操作为先用低盐缓冲液将杂蛋白去除,使用缓冲液pH调整为8.0的PB,将色素洗脱去除,最后采用缓冲液pH为9.0的Tris-HCl洗脱。3)过阴离子树脂层析;采用洗脱缓冲液为50mMTris-HCl+0.5M NaCl,pH9.0。4)单向切向流过滤浓缩;5)将浓缩的蛋白液进行凝胶过滤层析,采用洗脱缓冲液为20mM PB+0.15M NaCl,pH6.0。6)洗脱收集目的蛋白峰,获得纯化的巴曲酶。经纯化获得的重组巴曲酶进行SDS电泳检测纯度,检测结果参见图8。
实施例5纯化的重组巴曲酶与市售的天然巴曲酶的酶活力的测定
取市售巴曲酶,溶于缓冲液(20mM PB+0.15M NaCl,pH6.0),并常规方法测定蛋白浓度与实施例4中方法制备的纯化重组巴曲酶一致。
酶活力的测定方法参考实施例2中的方法:凝血酶活性测定的方法如下:
取人-枸橼酸凝血质控血浆0.2ml,加入96孔板中,置于37℃保温3min,加入37℃预热的样品溶液0.2ml,立即振荡混匀计时,于40s开始检查血浆凝固情况,记录初凝时间,同时测定3管,误差应<20s。若初凝时间<40s,则是当稀释公式样品溶液若干倍,记录(60±20)s内凝固的供试样品溶液浓度。在上述条件下,能使0.2ml人-枸橼酸抗凝血浆在60s凝固的酶量,定义为一个酶活单位。测定结果参见表4。
表4:重组巴曲酶的酶活力测定
酶类型 | 重组巴曲酶 | 市售巴曲酶 |
凝血时间(S) | 183 | 174 |
本发明通过上述实施例来说明本发明的工艺方法,但本发明并不局限于上述工艺步骤,即不意味着本发明必须依赖上述工艺步骤才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明所选用原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
Claims (2)
1.一种巴曲酶的发酵方法,其特征在于,包括如下步骤:
1)一级种子培养,取保存菌株接种至一级种子培养基中,所述一级种子培养基配方为:酵母粉,1.0%;聚蛋白胨2.0%;葡萄糖,2.0%,采用纯化水配制后灭菌备用;
2)二级种子培养,在无菌的条件下,一级种子液接入含有二级培养基的种子罐中,接种比例为5%-11%,所述二级种子培养基为配方为:酵母粉,1.0%;聚蛋白胨2.0%;葡萄糖,2.0%,采用纯化水配制后灭菌备用;
3)发酵培养,待二级种子液OD600在5-7之间,在无菌的条件下加入优化基础发酵培养基4ml/L至发酵罐中,在无菌的条件下将二级种子液移入发酵罐中,培养4小时后,向发酵罐中优化甘油补料培养基,并调节pH值为6.5,甲醇诱导;所述甘油补料培养基的浓度为10ml/L;
4)收集上清,纯化获得重组巴曲酶;
所述优化基础发酵培养基组分:K2SO4:0.91%;MgSO4:0.36%;CaSO4 .2H2O:0.059%;
85%H3PO4:2.5%V/V;KOH:0.206%;甘油:4%;泡敌:0.05%-0.1%;PTM1:0.4%V/V,casamino acids:0.5%;所述优化甘油补料培养基组分:50%甘油+12mL/L PTM1+0.5%casamino acids;甲醇诱导阶段采用优化甲醇诱导培养基进行诱导,所述优化甲醇诱导培养基组分:(100%甲醇+12mL/L PTM1)95%+4.5%山梨醇+0.5%casamino acids。
2.根据权利要求1所述的发酵方法,其特征在于,所述PTM1的配方为:0.6%CuSO4·5H2O,0.008%NaI,0.3%MnSO4·H2O,0.02%Na2MoO4·2H2O,0.02%H3BO3,0.05%CoCl2·6H2O,6.5%FeSO4·7H2O,2%ZnCl2,0.02%Biotin,0.0005%V/V H2SO4。
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