CN107099487B - 一种高分泌纳豆激酶的枯草芽孢杆菌及其应用 - Google Patents
一种高分泌纳豆激酶的枯草芽孢杆菌及其应用 Download PDFInfo
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Abstract
本发明公开了一种高分泌纳豆激酶枯草芽孢杆菌及其应用,所述菌株的分类命名为枯草芽孢杆菌(Bacillus subtilis)gs‑11061,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,其保藏编号是CGMCC No.13932。本发明还公开了利用上述高产纳豆激酶的枯草芽孢杆菌生产纳豆激酶的方法。本发明方法与其他发酵方法相比,在发酵方面,该菌可以在高温条件下发酵,同时明显地缩短发酵周期;在培养基成分方面,利用木糖母液粗原料降低成本,能耗低,污染小;与原始菌相比,纳豆激酶的分泌量明显提高,且发酵产物易处理,副产物少。
Description
技术领域
本发明属于生物发酵技术领域,具体涉及一种高分泌纳豆激酶的枯草芽孢杆菌。
背景技术
纳豆芽孢杆菌(Bacillus natto)是从日本传统食品中分离出来的菌种,其原始菌株与枯草芽孢杆菌相同,是枯草芽孢杆菌的一个亚种。纳豆芽孢杆菌不仅具有分解蛋白质、碳水化合物、脂肪等大分子物质的性能,使发酵产品中富含氨基酸、有机酸、寡聚糖等多种易被人体吸收的成分,而且在发酵过程中在发现了一些生理活性物质而使纳豆具有多种保健功能,如抗肿瘤、降血压、抗菌等作用,还可预防骨质疏松、提高蛋白质的消化率、抗氧化等。最主要的是,纳豆菌能产生具有溶栓活性的纳豆激酶,而且在酸性胃环境中能保持高度的稳定性。因此,对于纳豆芽孢杆菌的研究市场前景仍非常广阔。
纳豆激酶具有很强的溶解血栓功能,与目前临床所用的尿激酶、链激酶等溶栓药物相比,具有安全性好,易被人体吸收,作用直接迅速,药效持续时间长,可由纳豆枯草芽抱杆菌直接发酵生产因而造价低廉等优点。因此,纳豆激酶是一种很有潜力的新型溶栓药物。纳豆激酶的发酵工艺一般分为两种,分别为固态发酵和液态发酵。相比较于固态发酵易染菌、难散热、回收率低、气味难以接受、很难满足严格药品特别是生物制剂的生产要求等问题。液体发酵具有成本低廉、纯度高、环境污染小等优点,因此,本实验室仍采用传统的液体发酵的方式,但在发酵过程中,优化培养基及培养条件,极大地节约了成本,并且提高了纳豆激酶的生产效率和酶活。目前用于产纳豆激酶的菌株有保加利亚乳杆菌(Lactobacillus bulgaricus)、枯草芽孢杆菌(Bacillus subtilis)、链霉菌(Streptomyces)、乳酸菌(lactic acid bacteria)、大肠杆菌(Escherichia coli)、巴斯德毕赤酵母(Pichia pastoris)等。在液体发酵产纳豆激酶中,其副产物中含有部分的嘌呤的产生。目前,专利201110099838.7《一种产纳豆激酶枯草芽孢杆菌及该菌种发酵生产纳豆激酶的方法》中记载,获得的纳豆激酶酶活最高达到5670FU/g。通常情况下,纳豆激酶液态发酵的时间为72 h左右。
发明内容
本发明的目的在于提供一种高分泌纳豆激酶枯草芽孢杆菌及其应用。该枯草芽孢杆菌的纳豆激酶活性高,且可有效利用木糖母液等物质发酵生产纳豆激酶,降低生产成本且安全环保。
为实现上述目的,本发明采用的技术方案如下:
一种高分泌纳豆激酶枯草芽孢杆菌,所述菌株的分类命名为枯草芽孢杆菌(Bacillus subtilis) gs-11061,已于2017年3月27日保藏于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,中国科学院微生物研究所,保藏号为CGMCCNO.13932。
所述菌株是通过如下方法筛选得到的:从市面上买来的纳豆产品中分离得到五株枯草芽孢杆菌,利用液态LB培养基进行基础培养,35℃培养1天之后,同时测定生物量和纳豆激酶的分泌量,得到一株活力最为旺盛的菌株,命名为best-1,作为优势菌株。
将best-1接于液态LB种子培养基中培养,将培养好的枯草芽孢杆菌的菌液按10倍稀释法稀释至细胞数为109/mL,取菌液0.1 mL 均匀涂于无菌的空平板中,待风干后进行N+离子束注入,N+离子束注入剂量为(95、140、185、230、275)×2.6×1013N+/cm2,N+离子束注入能量为15keV。辐照结束后用1 mL 无菌水洗涤细胞,按10倍稀释法稀释后涂入平板培养基,37 ℃倒置培养1 d,待挑取单菌落,摇瓶检测,筛选出菌落生长速度快且纳豆激酶分泌率最高的菌株,命名为枯草芽孢杆菌(Bacillus subtilis) gs-11061。
所述枯草芽孢杆菌(Bacillus subtilis) gs-11061的培养条件为:
菌种采用好氧培养的方式,用于培养该菌株的碳源可以是葡萄糖,蔗糖、果糖、乳糖、可溶性淀粉、木糖母液等材料;用于培养该菌株的氮源可以是酵母膏、牛肉膏、胰蛋白胨、大豆蛋白胨、全豆粉、豆渣等材料。该菌株生长的最佳温度范围为25-58℃,pH范围5-9,最佳pH范围为6-8。在菌株培养过程中还可添加磷酸氢二钾、磷酸二氢钾、硫酸镁、磷酸镁、硫酸锰、氯化亚铁等无机盐类。
所述枯草芽孢杆菌(Bacillus subtilis) gs-11061的生理形态特征为:
枯草芽胞杆菌,在LB平板上生长旺盛,明显观察到单菌落着色均匀,且芽孢呈现椭圆形或柱状,孢囊膨大,有鞭毛,颜色呈乳白色。孢子耐热性强。菌落表面粗糙不透明,污白色或微黄色,在液体培养基中生长时,常形成皱醭。
所述的枯草芽孢杆菌(Bacillus subtilis) gs-11061在液体发酵生产纳豆激酶中的应用。包括如下步骤:
1)种子培养:从活化的平板上取一环菌接入液体种子培养基中,25-58℃培养11h;
2)取步骤1)的种子液接入液体培养基中进行发酵培养,接种量为2.5%,培养温度为25℃~58℃,液体发酵。
步骤1)中所述液体种子培养基包括如下质量百分比的组分:碳源10~50g/L,氮源15~35g/L,无机盐0~1g/L,其余为水,pH5.5~8.0。其中所述碳源选自葡萄糖、蔗糖、果糖、乳糖、可溶性淀粉、木糖母液等中的至少一种;所述氮源选自牛肉膏、蛋白胨、酵母膏、全豆粉、豆渣、胰蛋白胨等中的至少一种,所述无机盐选自磷酸氢二钾、磷酸二氢钾、硫酸镁、磷酸镁、硫酸锰、氯化亚铁中的至少一种。
最优选的生产方式为:将菌株活化后,在37℃下好氧发酵24-60h,装液量为80ml发酵液/500ml三角瓶,转速为180rpm。
其中,所述的好氧发酵培养基为:木糖母液15g/L,蛋白胨 20g/L,K2HPO4•3H2O1.2g/L,KH2PO4 1.6g/L,MgSO4•7H2O 0.45g/L,Cacl2 0.21g/L,MnSO40.001g/L,FeCl20.002g/L,其余为水,pH=7。
有益效果:本发明与现有技术相比具有如下优势:
1.与其他菌种相比,本发明的纳豆芽孢杆菌具有很高的代谢活力,在高温条件下产酶高,发酵周期明显的缩短,从原来的发酵时长96小时缩短了36小时,发酵生产纳豆激酶活性高。
2.与传统的液体发酵法相比,该菌株发酵可利用木糖母液,从成本上来说,得到了很大幅度的降低,而且可以减少废料的排放,在节约成本的同时,降低环境污染。
具体实施方式
通过下述实施例,可以更好地理解本发明。然后,本领域的技术人员容易理解,实施例所描述的具体的物料比,工艺条件及其结果仅用于说明本发明,而不应当也不会限制权利要求书说所详细描述的本发明。
实施例1 枯草芽孢杆菌 (Bacillus subtilis) gs-11061的诱变筛选
从市面上买来的纳豆产品中分离得到五株枯草芽孢杆菌,利用液态LB培养基进行基础培养,37℃培养1天之后,同时测定生物量和纳豆激酶的分泌量,得到一株活力最为旺盛的菌株,命名为best-1,作为优势菌株。
将best-1接于液态LB种子培养基中培养。将培养好的枯草芽孢杆菌的菌液按10倍稀释法稀释至细胞数为109/mL,取菌液0.1 mL 均匀涂于无菌的空平板中,待风干后进行N+离子束注入,N+离子束注入剂量为(95、140、185、230、275)×2.6×1013N+/cm2,N+离子束注入能量为15keV。辐照结束后用1 mL 无菌水洗涤细胞,按10倍稀释法稀释后涂入平板培养基,37 ℃倒置培养1 d,待挑取单菌落,摇瓶检测,筛选出菌落生长速度快且纳豆激酶分泌率最高的菌株,命名为枯草芽孢杆菌(Bacillus subtilis) gs-11061。
实施例2 枯草芽孢杆菌(Bacillus subtilis) gs-11061的培养及生理学特征
菌种采用好氧培养的方式。用于培养该菌株的碳源可以是葡萄糖,蔗糖、果糖、乳糖、可溶性淀粉等材料;用于培养该菌株的氮源可以是酵母膏、牛肉膏、胰蛋白胨、大豆蛋白胨、全豆粉、豆渣等材料。该菌株生长的最佳温度范围为25-50℃,pH范围5-9,最佳pH范围为6-8。在菌株培养过程中还可添加磷酸氢二钾,磷酸二氢钾,硫酸镁,磷酸镁等无机盐类。
所述枯草芽孢杆菌(Bacillus subtilis) gs-11061的生理形态特征为:
枯草芽胞杆菌,在LB平板上生长旺盛,明显观察到单菌落着色均匀,且芽孢呈现椭圆形或柱状,孢囊膨大,有鞭毛,颜色呈乳白色。孢子耐热性强。菌落表面粗糙不透明,污白色或微黄色,在液体培养基中生长时,常形成皱醭。
实施例3 枯草芽孢杆菌(Bacillus subtilis) gs-11061发酵产纳豆激酶
平板培养基:蛋白胨 15 g/L,酵母膏 7.5g/L,NacL15g/L,琼脂 20 g/L,pH 自然。
好氧发酵培养基:木糖母液15g/L,蛋白胨 20g/L,K2HPO4•3H2O 1.2g/L,KH2PO41.6g/L,MgSO4•7H2O 0.45g/L,Cacl2 0.21g/L,MnSO40.001g/L,FeCl20.002g/L,其余为水,初始pH 7.4。500mL三角瓶装液量80mL,121℃灭菌20分钟。木糖母液购买自巨鹿县威科食品厂。
分别将初始菌株及CGMCC No .13932菌首先于35℃平板活化菌种,24h后接两环生长良好的菌体入80mL发酵培养基,35℃,180rpm培养11h,然后按照2.5%( v/v )接种量转接入装液量为80ml发酵液/500ml三角瓶,35℃培养,转速为180rpm。发酵60 h后CGMCCNo.13932的发酵液产纳豆激酶为11710.5 Fu/g较初始菌株相比,纳豆激酶产量提高了4.61倍。
实施例4 枯草芽孢杆菌(Bacillus subtilis) gs-11061发酵产纳豆激酶
好氧发酵培养基:木糖母液15g/L,K2HPO4•3H2O 1.2g/L,蛋白胨 20g/L,KH2PO41.6g/L,MgSO4•7H2O 0.45g/L,Cacl2 0.21g/L,MnSO4 0.001g/L,FeCl20.002g/L,其余为水,pH=7.4。7 L发酵罐装液量3 L,121℃灭菌20分钟。
分别将初始菌株CGMCC No .13932菌首先于35℃平板活化菌种,24h后接两环生长良好的菌体入80mL发酵培养基,35℃,180rpm培养11 h,然后按照8%( v/v )接种量转接入3L发酵培养基,通空气1.1v/vm,转速300rpm,35℃培养。发酵24h后CGMCC No .13932的发酵产酶为14644.5 Fu/g。较初始菌株相比,纳豆激酶的产酶量提高了 5.78倍。
实施例5 枯草芽孢杆菌(Bacillus subtilis) gs-11061高温条件下发酵产纳豆激酶
好氧发酵培养基:木糖母液15g/L,蛋白胨 20g/L,K2HPO4•3H2O 1.2g/L,KH2PO41.6g/L,MgSO4•7H2O 0.45g/L,Cacl2 0.21g/L,MnSO4 0.001g/L,FeCl20.002g/L其余为水,pH=7.4。7 L发酵罐装液量3 L,121℃灭菌20分钟。
分别将初始菌株CGMCC No .13932菌首先于58℃平板活化菌种,24h后接两环生长良好的菌体入80mL发酵培养基,58℃,180rpm培养11 h,然后按照8%( v/v )接种量转接入3L发酵培养基,通空气1.1v/vm,转速300rpm,58℃培养。发酵13 h时,补加无菌水500mL,发酵20 h结束后CGMCC No .13932的发酵产酶为10672.5 Fu/g 。较初始菌株相比,纳豆激酶的产酶量提高了4.21倍,发酵时长缩短了40小时。较传统液态发酵周期时长相比,发酵时长至少缩短了48小时。
纳豆激酶的检测方法:取0.7 mL 50 mmol·L-1Tris-HCl(pH 8.0)缓冲液与0.2 mL0.72%(w·v-1)的纤维蛋白原溶液混匀后,37℃放置5 min。向上述溶液中加入0.1 mL凝血酶溶液(20 U·mL-1)充分混匀,37℃放置10 min。再向上述反应体系中加入0.05 mL稀释的酶液充分混匀,于37℃水浴中保温反应,分别在反应开始后的20 min和40 min时,分别混匀10s。在准确计时60 min后加入1mL 0.2 mol·L-1的三氯乙酸终止反应,并在37℃水浴中再保温20 min。上述反应液于15000 r·min-1下离心10 min,测定离心上清液在275nm处的吸光值。一个酶活力单位(FU)定义为在37℃,pH 8.0的条件下,每分钟在275 nm处吸光值变化0.01所需的酶量。
比酶活定义:每mg蛋白所含的酶活力单位数,即比活力=活力FU·mg-1蛋白。
Claims (7)
1.一种高分泌纳豆激酶枯草芽孢杆菌,所述高分泌纳豆激酶枯草芽孢杆菌的分类命名为枯草芽孢杆菌(Bacillus subtilis) gs-11061,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,其保藏编号是CGMCC No.13932。
2.权利要求1所述高分泌纳豆激酶枯草芽孢杆菌在发酵生产纳豆激酶中的应用。
3.根据权利要求2所述的应用,其特征在于,将枯草芽孢杆菌gs-11061活化,在25~58℃下好氧发酵24~60h;
其中,好氧发酵培养基为完全培养基:碳源10~50g/L,氮源15~35g/L,无机盐0~10g/L,其余为水,pH5.5~8.0。
4.根据权利要求3所述的应用,其特征在于,所述的碳源为葡萄糖,蔗糖、果糖、乳糖、可溶性淀粉、木糖母液中的至少一种。
5.根据权利要求3所述的应用,其特征在于,所述的氮源为酵母膏、牛肉膏、胰蛋白胨、大豆蛋白胨、全豆粉、豆渣中的至少一种。
6.根据权利要求3所述的应用,其特征在于,所述的无机盐为镁盐、钾盐、钙盐、磷酸盐或盐酸盐中的任意一种或几种的组合。
7.根据权利要求3所述的应用,其特征在于,将枯草芽孢杆菌gs-11061活化后,在35℃下好氧发酵24 h,在发酵阶段通气比1.1v/vm、搅拌300rpm,
其中,所述的好氧发酵培养基为:木糖母液15g/L,蛋白胨 20g/L,K2HPO4•3H2O 1.2g/L,KH2PO41.6g/L,MgSO4•7H2O 0.45g/L,CaCl2 0.21g/L,其余为水,pH=7.4。
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