CN111117897B - 一种高产D-泛酸内酯水解酶的串珠镰刀菌JHpharm 2-1及其发酵方法 - Google Patents
一种高产D-泛酸内酯水解酶的串珠镰刀菌JHpharm 2-1及其发酵方法 Download PDFInfo
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Abstract
本发明公开了一种高产D‑泛酸内酯水解酶的串珠镰刀菌JHpharm 2‑1,其保藏号为CGMCC No.19023；已于2019年11月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心。本发明的高产D‑泛酸内酯水解酶的串珠镰刀菌JHpharm 2‑1发酵48h时生物量45g/L，酶活性150IU/g，通过固定化试剂将酶进行固定化后，套用10次，酶活性只降低了5％。不仅酶活比其他菌种高出50％以上，而且可以重复套用多次。
Description
技术领域
本发明涉及一种高产D-泛酸内酯水解酶的串珠镰刀菌JHpharm 2-1及其发酵方法。
背景技术
DL-泛酸内酯是外消旋化合物，通过D-泛酸内酯水解酶可以将 DL-泛酸内酯拆分为D-泛酸内酯，用于合成D-泛酸钙。D-泛酸钙是泛酸的右旋体钙盐。D-泛酸又称遍多酸、维生素B5，是辅酶A的组成部分，参与糖、脂肪、蛋白质代谢。辅酶A是泛酸与3-磷酸腺苷、焦磷酸和β-巯基乙胺结合的复合分子。因此，泛酸的作用即是辅酶A 的生理功能：参与体内脂肪酸降解、脂肪酸合成、柠檬酸循环、胆碱乙酰化(一种神经冲动传导物质)、抗体的合成等代谢，泛酸的存在有利于各种营养成分的吸收和利用。
DL-泛酸内酯是通过手性拆分试剂如D-樟脑磺酸、麻黄素、奎宁等与DL-泛酸内酯碱解物形成复盐，利用复盐在溶剂中溶解度不用将 D-体和L-体的复盐分离，然后通过化学处理，得到D-泛酸内酯。但是由于手性拆分试剂价格昂贵，而且化学拆分过程繁琐，不适合工业化生产。目前报道较多的是微生物酶法拆分，通过酶拆分DL-泛酸内酯得到D-泛酸内酯。该方法具有成本低、反应条件温和、对环境友好等优点。目前通过水解酶来进行拆分的工艺相对成熟，但现有的发酵法所产生的酶量及酶活不高、酶使用后难以过滤、重复使用次数低等缺点使得生产成本降不下来。
发明内容
本发明要解决的技术问题是克服现有技术中DL-泛酸内酯酶活不高，重复使用次数低的缺陷，提供一种高产D-泛酸内酯水解酶的串珠镰刀菌JHpharm 2-1及其发酵方法。
为了解决上述技术问题，本发明提供了如下的技术方案：
菌株来源：分别采于山东临沂地区、山西吕梁地区、湖北黄石地区的棉花或者玉米中分离纯化后得到的三株串珠镰刀菌。
将这三种菌株进行基础培养基培养，28℃培养三天后测定生物量和D-泛酸内酯水解酶的酶活性，得到一株活力最为旺盛的菌株。将该菌株的发酵液分散均匀后，按照10倍稀释法稀释至细胞数107/ml, 取菌液0.1ml，均匀的涂布在无菌的空平板中，风干后进行N+离子束注入，N+离子束注入剂量为(90、135、180、225、270)*2.6*1013N+/cm2,N+离子束注入能量为15KeV，辐射结束后用1ml的无菌水洗涤细胞，按 10倍稀释法稀释后涂入培养基，28℃培养一天，挑取菌丝，摇瓶培养三天后，检测生物量和酶活性，筛选出生长最快且酶活性最高的菌株。并命名为JHpharm 2-1。该菌株建议的分类命名：串珠镰刀菌 Fusariummoniliforme。
该菌株已于2019年11月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心。其保藏号为CGMCC No.19023，保藏地址为北京市朝阳区北辰西路1号院3号。
该菌株采用好氧培养方式，用于培养该菌株的碳源可以是葡萄糖、甘油、蔗糖、乳糖、可溶性淀粉、玉米浆等，用于该菌株的氮源可以是酵母膏、蛋白胨、豆饼粉等。该菌株生长的最佳温度范围是 26～30℃，pH范围是5～9，最佳pH范围是7.0～8.0。该菌株平板培养形态：薄膜状、无色、菌丝致密、纤细，丛卷毛状至毛毡状，白葡萄酒色至毛毡色。由于小型分生孢子的形成，经常呈粉状外观。
该串珠镰刀菌JHpharm 2-1的发酵方法，包括如下步骤：
S1、种子培养：从活化的平板上接一环菌到液体种子培养基中， 28℃培养24h，种子培养基组成：碳源：15～30g/L、氮源：15～45g/L、其余为水，pH：7～8；
S2、接种后，在26～30℃下好氧发酵24～60h，装液量为100ml 发酵液/500ml三角瓶，转速为150rpm；
发酵48h产量达到最高。生物量45g/L，酶活性150IU/g。通过固定化试剂将酶进行固定化后，套用10次，酶活性只降低了5％。
其中，酶活的定义为：在一定条件下，每分钟水解1umol D-泛酸内酯或D-泛解酸的酶量定义为1个酶活力单位(IU)
方法：取5ml发酵液，加入5ml 1mol/L泛酸内酯，再加40ml Tris-HCl缓冲液，28℃,200rpm摇床中摇1h，过滤去除菌丝体，高效液相色谱测定泛酸内酯消耗量并计算每克干细胞的酶活单位 (IU/g)
其中，S1中碳源选自葡萄糖、甘油、蔗糖、乳糖、可溶性淀粉、玉米浆等中的至少一种，所述的氮源选自酵母膏、蛋白胨、豆饼粉等中的至少一种。
S2中，好氧发酵的培养基为：葡萄糖40/L、蛋白胨20g/L、酵母粉10g/L、豆饼粉10g/L，其余为水，pH＝7。
本发明所达到的有益效果是：本发明的高产D-泛酸内酯水解酶的串珠镰刀菌JHpharm 2-1发酵48h时生物量45g/L，酶活性150IU/g，通过固定化试剂将酶进行固定化后，套用10次，酶活性只降低了5％。不仅酶活比其他菌种高出50％以上，而且可以重复套用多次。
具体实施方式
以下对本发明的优选实施例进行说明，应当理解，此处所描述的优选实施例仅用于说明和解释本发明，并不用于限定本发明。
实施例
实施例1：串珠镰刀菌(Fusarium moniliforme Sheld JHpharm 2-1)的诱变筛选
将分别采于山东临沂地区、山西吕梁地区、湖北黄石地区的棉花或者玉米中分离纯化后得到的三株串珠镰刀菌，利用PDA培养基， 28℃培养三天后测定生物量和D-泛酸内酯水解酶的酶活性，得到一株活力最为旺盛的菌株，作为优势菌株。
将该优势菌株的发酵液分散均匀后，按照10倍稀释法稀释至细胞数107/ml,取菌液0.1ml，均匀的涂布在无菌的空平板中，风干后进行N+离子束注入，N+离子束注入剂量为(90、135、180、225、270) *2.6*1013N+/cm2,N+离子束注入能量为15KeV，辐射结束后用1ml的无菌水洗涤细胞，按10倍稀释法稀释后涂入培养基，28℃培养一天，挑取菌丝，摇瓶培养三天后，检测生物量和酶活性，筛选出生长最快且酶活性最高的菌株。并命名为Fusariummoniliforme Sheld JHpharm 2-1。
实施例2：所述Fusarium moniliforme Sheld JHpharm 2-1的生理形态特征为：
该菌株平板培养形态：薄膜状、无色、菌丝致密、纤细，丛卷毛状至毛毡状，白葡萄酒色至毛毡色。由于小型分生孢子的形成，经常呈粉状外观，显微镜下可见管状菌丝。
实施例3：Fusarium moniliforme Sheld JHpharm 2-1发酵生产 D-泛酸内酯水解酶
种子培养：从活化的平板上接一环菌到液体种子培养基中，28℃培养24h。
种子培养基组成：葡萄糖30g/L、蛋白胨20g/L、酵母粉10g/L、酵母膏5g/L、豆饼粉5g/L，其余为水，pH：7～8。培养24h后取5ml 接入到发酵液中，在26～30℃下好氧发酵60h，装液量为100ml发酵液/500ml三角瓶，转速为150rpm。
好氧发酵的培养基为：葡萄糖40/L、蛋白胨20g/L、酵母粉10g/L、豆饼粉10g/L，其余为水，pH＝7。发酵结束，检测生物量45g/L，酶活性150IU/g。通过固定化试剂将酶进行固定化后，套用10次，酶活性只降低了5％。
固定化方法：水中加入50％戊二醛，再加入粗酶，降温至4℃，搅拌均匀后静置，静置完成后过滤，得固定化后酶。
D-泛酸内酯水解酶的测定方法
定义：在一定条件下，每分钟水解1umol D-泛酸内酯或D-泛解酸的酶量定义为1个酶活力单位(IU)
方法：取5ml发酵液，加入5ml 1mol/L泛酸内酯，再加40ml Tris-HCl缓冲液，30℃,200rpm摇床中摇1h，过滤去除菌丝体，高效液相色谱测定泛酸内酯消耗量并计算每克干细胞的酶活单位 (IU/g)。
最后应说明的是：以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (6)
1.一种高产D-泛酸内酯水解酶的串珠镰刀菌(Fusarium moniliforme)JHpharm 2-1,其保藏号为CGMCC No.19023；已于2019年11月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心。
2.如权利要求1所述的串珠镰刀菌JHpharm 2-1，其特征在于，串珠镰刀菌JHpharm 2-1平板培养时呈薄膜状、无色、菌丝致密、纤细，丛卷毛状至毛毡状，白葡萄酒色至毛毡色。
3.权利要求1所述的串珠镰刀菌JHpharm 2-1的发酵方法，其特征在于，培养温度为26～30℃，pH 5～9。
4.权利要求1所述的串珠镰刀菌JHpharm 2-1的发酵方法，其特征在于，包括如下步骤：
S1、种子培养：从活化的平板上接一环菌到液体种子培养基中，28℃培养24h，种子培养基组成：碳源：15～30g/L、氮源：15～45g/L、其余为水，pH：7～8；
S2、接种后，在26～30℃下好氧发酵24～60h，装液量为100ml发酵液/500ml三角瓶，转速为150rpm。
5.如权利要求4所述的串珠镰刀菌JHpharm 2-1的发酵方法，其特征在于，S1中碳源选自葡萄糖、甘油、蔗糖、乳糖、可溶性淀粉和玉米浆中的至少一种，所述的氮源选自酵母膏、蛋白胨和豆饼粉中的至少一种。
6.如权利要求4所述的串珠镰刀菌JHpharm 2-1的发酵方法，其特征在于，S2中，好氧发酵的培养基为：葡萄糖40g/L、蛋白胨20g/L、酵母粉10g/L、豆饼粉10g/L，其余为水，pH＝7。
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