CN1084217A - 生物素的生产方法 - Google Patents
生物素的生产方法 Download PDFInfo
- Publication number
- CN1084217A CN1084217A CN93116830A CN93116830A CN1084217A CN 1084217 A CN1084217 A CN 1084217A CN 93116830 A CN93116830 A CN 93116830A CN 93116830 A CN93116830 A CN 93116830A CN 1084217 A CN1084217 A CN 1084217A
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- Prior art keywords
- vitamin
- sphinx
- microorganism
- substratum
- culture
- Prior art date
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- ZKQFHRVKCYFVCN-UHFFFAOYSA-N ethoxyethane;hexane Chemical compound CCOCC.CCCCCC ZKQFHRVKCYFVCN-UHFFFAOYSA-N 0.000 description 1
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- 230000002349 favourable effect Effects 0.000 description 1
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- 235000010355 mannitol Nutrition 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
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- 230000004899 motility Effects 0.000 description 1
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- 229910052757 nitrogen Inorganic materials 0.000 description 1
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- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
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- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
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- 239000001632 sodium acetate Substances 0.000 description 1
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- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
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- 229940001516 sodium nitrate Drugs 0.000 description 1
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- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
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- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
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Abstract
本发明公开了一种生产生物素的方法,其中在培
养基中制备具有产生生物素能力的斯芬克斯小杆菌
属微生物的培养物,并收集培养基中产生和积累的生
物素。本发明还公开了具有产生生物素能力的斯芬
克斯小杆菌属的微生物,其可用于本发明的这种生产
方法中。
Description
本发明涉及生物素的生产方法以及该方法中所使用的微生物。
生物素是一种维生素,是人类、动物、蔬菜和某些种类的微生物所需要的。作为使用微生物生产生物素的方法,存在着使用微生物的多种已知方法,例如使用链霉菌属或小单孢菌属(JP-B 41-21756)、掷孢酵母菌属(JP-B 42-3074)、芽孢杆菌属(JP-A.56-160998)、埃希氏杆菌属(JP-A 61-149091)、沙雷氏菌属(JP-A 2-27980)和短杆菌属(JP-A 3-240489)的方法。
不过,由于这些微生物产生生物素的产量低,这些常规方法并不总是令人满意的。因此,需要开发一种使用高效率微生物生产生物素的方法,以适应于工业规模的生物素生产。
在这些情形之下,本发明的发明人在自然界和保存在有利于微生物贮藏的典型培养物保藏所中广泛寻找了具有增加的生物素生产能力的微生物。结果,他们发现斯芬克斯小杆菌属的微生物可以在培养基中积累大量的生物素,,从而完成了本发明。
这样,本发明提供了一种生物素的生产方法,其特征在于:在适当的培养基中培养一种能产生生物素的斯芬克斯小杆菌属的微生物和收集培养基中积累的生物素。本发明还提供了该方法中所使用的多种微生物。
本发明方法中所使用的微生物是斯芬克斯小杆菌属的微生物,该属具有产生生物素的能力,其例子有S.adhaeshiva、S.parapaucimobilis和S.paucimonilis。在这些微生物中,斯芬克斯小杆菌SC-42405是优选的。通常,斯芬克斯小杆菌属的微生物具有以下特点:
1.它们是革兰氏阴性杆菌;
2.它们在触酶试验中呈阳性;
3.主要形成黄色菌落;
4.它们含有作为磷脂的斯芬克斯脂类;
5.类异戊二烯醌类型是Q10;
6.它们含有作为细菌脂肪酸的2-羟基酸类;
7.其DNA中G+C的摩尔百分比为60~70不等。
斯芬克斯小杆菌SC-42405是一种由发明人从日本大分县的土壤中分离的细菌中分离出来的微生物,发现它能够在培养基中积累大量的生物素。这种微生物已按照布达佩斯条约贮存在工业科学及技术机构中的发酵研究所(更名为“国家生物科学和人类技术研究所”)中,保藏日为1992年9月3日,登记号码为FERM BP-3995。
斯芬克斯小杆菌SC-42405的细菌学特征如下:
(a)形态学
1.细胞形状与大小
形状:杆状
大小:0.4-0.8μm×1.5~3.0μm
2.多形性:无
3.游动性:活泼、极性及围毛性鞭毛
4.孢子形成:无
5.革兰氏染色:阴性
6.抗酸性:无
(b)生长性质
1.营养琼脂平板培养:圆,凸,黄色
2.营养琼脂斜面培养:平滑表面,略带光泽,黄色
3.营养肉汤液体培养:混浊生长
4.营养肉汤明胶穿刺培养:不液化
5.石蕊牛乳:无变化
(c)生理学特性
1.硝酸盐还原作用(营养肉汤硝酸盐培养基):-
2.反硝化作用:-
3.MR试验:-
4.VP试验:-
5.吲哚形成:-
6.硫化氢形成:+
7.淀粉水解作用:-
8.柠檬酸利用
科泽氏培养基:+
克里斯坦森培养基:+
9.无机氮源利用
铵盐:+
硝酸盐:+
10.色素形成
细胞色素:+(黄色)
水溶性色素(金氏A型培养基):-
水溶性色素(金氏B型培养基):-
11.脲酶:±
12.氧化酶:+
13.触酶:+
14.生长条件
PH值:5.1~7.9
温度:10~33℃
15.对氧气的反应:略偏重需氧性
16.氧化-发酵试验:发酵(弱)
17.来自糖的酸及气体形成
酸 气体
(1)L-阿拉伯糖: + -
(2)D-木糖: + -
(3)D-葡萄糖: ± -
(4)D-甘露糖: ± -
(5)D-果糖: + -
(6)D-半乳糖: + -
(7)麦芽糖: ± -
(8)蔗糖: ± -
(9)乳糖: ± -
(10)海藻糖: - -
(11)D-山梨糖醇: - -
(12)D-甘露糖醇: - -
(13)肌醇: - -
(14)甘油: - -
(15)淀粉: ± -
(d)其它特性:
1.七叶苷水解作用:+
2.精氨酸水解作用:-
3.赖氨酸脱羧作用:-
4,鸟氨酸脱羧作用:-
5.DNA酶:-
6.吐温80水解作用:-
7.0/129敏感性:-
8.固氮酶:-
9.聚-β-羟基丁酸盐的积累:+
10.DNA中G+C的摩尔百分比:64.8
11.类异戊二烯醌的分子类型:辅酶Q10
12.斯芬克斯磷酯:+
13.全部细菌脂肪酸的组成:C16∶0(13.1%),C16∶1(11.2%),C17∶1(1.1%),C18∶1(51.8%),2-OH-14∶0(14.1%),2-OH-16∶0(4.5%)
(d)项,10~13中所述的特性可由常规的化学分类程序加以确定,例如在“放线菌鉴定实验”中所描述的,该书由日本放线菌研究学会编辑,1985年由日本放线菌学会秘书处出版。这种程序的典型实例如下所述。
1.DNA的G+C的摩尔百分比
培养(100ml营养肉汤培养基在坂口瓶中在30℃下培养36小时)
/……通过离心法来收集细菌细胞。
通过离心法用盐水-乙二胺四乙酸(0.15M NaCl,0.1M乙二胺四乙酸,pH值8.0)冲洗两次。
/……在18ml的盐水-乙二胺四乙酸中悬浮细菌细胞。
细菌细胞的溶解〔溶菌酶处理(0.5mg/ml,37℃,处理1小时),随后加入十二烷基硫酸钠(达到最终浓度为12%),然后在60℃下加热15分钟〕。
/……混合物变成透明的,并因DNA增加了粘度。
/……冰冷却。
加入EtOH达到最终浓度为67%。
/……弃去上清液。
溶解于20ml的SSC(0.15M NaCl,0.015M柠檬酸钠,pH值7.0)中。
/
在SCC(SSC饱和液)中加入等量苯酚,并震荡塞封的瓶子。
/……离心(分离成三层,即水层、天然蛋白质层和有机溶剂层)。
用吸管吸出水层。
/……加入SSC,再次震荡、离心分离并除去水层。
/……冰冷却。
/……在4℃下以12,000转/分钟的速度离心10分钟,将上清液倾倒在大烧杯中以保持1cm的厚度。
DNA绕数
/……将95%的冷EtOH以两倍体积加入水中,用玻璃棒搅拌烧杯中的内容物。当浓度达到约30%EtOH时,DNA开始在玻璃棒上旋绕。使旋绕在玻璃棒上的DNA在含80%EtOH的试管中静置片刻(除去苯酚),随后用95%的EtOH进行同样处理。在空气中蒸发EtOH并将DNA加入0.1X SSC中。在冰箱中放置过夜后,得到基本均匀的溶液。
/……适度离心以去除混浊。
RNA酶处理(加入RNA酶A*至50μg/ml,在37℃下处理1小时)。
/…*在80℃下处理1-2mg/ml SCC10分钟(DNA酶失活)。
/……加入1/10量的10X SSC并冷却。
当加入EtOH时DNA在玻璃棒上旋绕(如果是纯DNA,它变成透明的)。
/……将DNA加入0.1X SSC中并在冰箱中静置过夜(如果第二天仍然混浊,则重复离心、RNA酶处理和旋绕)。
/……使A260达到10~20。
将100μl纯DNA/0.1X SSC溶液加入微离心管中。
/……在100℃下保持5分钟,随后进行冰冷却。
/←100μl 0.1mg/ml核酸酶P1(Yamasa Shoyu有限公司),40mM NaAc和2mMZnSO4(pH5.3)。
50℃下反应1小时。
1←100μl碱性磷酸酶(BAP,Takara Shuzo有限公司)
/(在0.1M Tris-HCl中2.4U/ml,pH8.1)
在37℃下反应1小时。
/
HPLC分析
柱:Gμ C18+μBondasphere C18(3.9mmφ×150mm),
流动相:0.2M NH4H2PO4/MeOH(100∶8),
流速:0.9ml/分,
检测:紫外260nm,
样品注入:5μL(4-7μL)
2.类异戊二烯醌的分子类型
100mg冻干的细菌细胞(在营养肉汤中制成液体培养物)
/……在搅拌条件下用20mL的CHCl3-MeOH(2∶1)萃取过液。
硅胶薄层色谱分析
/……用苯展开色谱并确定哪些是泛醌或甲基萘醌类(当用苯展开色谱时,如Rf=0.3,是泛醌,如Rf=0.7,是甲基萘醌类)。
刮下硅胶薄层色谱中的产物并纯化
/用苯来展开泛醌,而用正己烷-苯-CHCl3(5∶2∶1)来展开甲基萘醌类。
用丙酮从硅胶上洗脱去除
/……配制100μl丙酮溶液。
反相HPLC分析
柱:YMC-Pack AM-303(4.6mmφ×250mm),
洗脱:2-PrOH-MeOH(25∶75),
流速:1.0mL/分,
检测:270nm。
3.斯芬克斯磷脂的存在
0.5g湿细菌细胞
/……用5mL CHCl3-MeOH(2∶1)萃取磷脂。
将CHCl3层分离成两部分并加入88μl的6N KOH。
/……40℃下水解1小时。
/……用乙酸中和。
/……干燥。
薄层色谱分析(色谱展开之后,显色是由钼试剂引起的)
4.全部细菌脂肪酸的组成
(1)全部细菌脂肪酸的分析程序
25mg冻干的细菌细胞(于30℃下,在营养肉汤中制成液体培养物)
/
/←2mL 5%的无水HCl-MeOH(Wako Pure Chemical Industries,Ltd.)
/……在沸水中保持3小时。
放置使样品冷却
/←1mL水
/←3mL正己烷
萃取脂肪酸甲基酯(震荡10分钟)
/……用移液管移出正己烷层
/……加入3mL水并震荡(酸去除)
/……去除正乙烷层并用无水Na2SO4脱水。
/……干燥。
溶解于200μL CH3CN中。
/
气相色谱分析
柱:10%DEGS铬目砂W80/100(AW-DMCS),3mmφ×5.2m,
载体:40mL/分N2,
注入温度:220℃,
柱温:180℃,
检测:FID。
(2)鉴定不饱和脂肪酸甲酯的程序
全部细菌脂肪酸甲酯样品20μl
/……干燥。
溶解于200μl乙醚中。
1←在乙醚中加入5μl 2%的Br2。
/……封盖并在室温下静置30分钟。
通入氮气而蒸发溶剂和残留的Br2。
/……溶解于60μl的CH3CN中。
气相色谱分析〔同(1)项中所述的方法〕
(3)鉴定羟基脂肪酸甲酯的程序
全部细菌脂肪酸甲酯样品20μl
/……应用于硅胶薄层色谱。
色谱展开(正己烷-乙醚,85∶15)
/……喷雾0.02%(W/V)的2′,7′-二氯荧光素-乙醇溶液。
刮下显色的斑点。
/如Rf=0.2,斑点是3-羟基脂肪酸甲酯。
/如Rf=0.3,斑点是2-羟基脂肪酸甲酯。
/如Rf=0.8,斑点是非极性脂肪酸甲酯。
用乙醚萃取,随后干燥并溶于20μl的CH3CN中。
/
气相色谱〔同(1)项所述方法〕
(4)鉴定环化脂肪酸甲酯的程序
全部细菌脂肪酸甲酯样品20μl
/……干燥。
100℃下在2mL 5%的无水HCl-MeOH中加热1小时。
/
如(1)项所述方法萃取脂肪酸甲酯并溶于20μl CH3CN中。
/
气相色谱分析〔同(1)项中所述方法〕
将上述特点与下列资料中公开的数据进行比较:“Bergey's Manual of Systematic Bacteriology”(1984),Yabuuchi et al.,Microbiol.Immunol.,34,99-119(1990),Validation list No.34,Int.J.Syst,Bacteriol.,40,320-321(1990),本发明的发明人所发现的细菌菌株被鉴定为斯芬克斯小杆菌属的微生物并定名为斯芬克斯小杆菌SC-42405。应该注意是,尚不知道任何具有产生生物素能力的斯芬克斯小杆菌属的微生物。在这方面,斯芬克斯小杆菌SC-42405被认为是一种新菌株。此外,这个菌株的任何变种(即斯芬克斯小杆菌SC-42405的任何突变体)、任何细胞融合菌株或任何重组菌株也可用于本发明的方法。
本发明方法中所用的微生物培养物可以在多种类型的培养基中制备,这些培养基含有碳源和氮源、有机和/或无机盐等等,所有这些都广泛应用于制备普通细菌培养物中。
碳源的例子为葡萄糖、麦芽糖、甘油、淀粉、糊精、蔗糖、动植物油和糖蜜。
氮源的例子为天然有机氮源,例如肉汤、胨、酵母膏、麦芽汁、大豆粉、玉米浆、棉籽粉、干酵母和酪蛋白氨基酸;其它有机或无机氮源例如氨、氯化铵、硝酸钠、硝酸铵、硫酸铵、乙酸铵和尿素。天然有机氮源也可以用作碳源。
有机和无机盐的例子为氯化物、硫酸盐、乙酸盐、碳酸盐和磷酸盐,如钾、钠、镁、铁、锰、钴和锌等元素的上述盐。具体的例子是氯化钾、氯化钠、硫酸镁、硫酸亚铁、硫酸锰、氯化钴、硫酸锌、硫酸铜、乙酸钠、碳酸钙、碳酸钠、磷酸一氢钾、磷酸二氢钾、磷酸一氢钠和磷酸二氢钠。
本发明方法中所使用的微生物具有在多种培养基中产生生物素的优良能力,这对于工业规模的生产是很有利的。为了增强本发明方法中所使用的微生物产生生物素的能力,可以在培养基中加入氨基酸(如丙氨酸)或脂肪酸(如庚二酸)。例如,这些化合物的用量可以是每1000mL培养基中约1mg到约5mg。
按照普通细菌所使用的常规程序制备本发明方法中所用的微生物培养物,可以是固体培养,也可以是液体培养,如使用试管、往复式摇动器或旋转式摇动器的震荡培养,和使用罐式发酵器或发酵罐的其它培养。
微生物的培养物通常是在需氧条件下培养。特别是,当使用发酵罐时,有必要引入无菌空气,通常是以每分钟约0.1至约2倍于培养物体积的速度通气。
培养温度可在一定范围内变化,其中所用微生物在培养物中是活的。例如,可以在约20℃~40℃,优选约25°~35℃,更好是28°~33℃下培养。培养基的pH值最好控制在约6~8之间。
培养时间依多种条件而不同,一般为约1-7天,优选为约3-4天。
培养完成后,可按照从天然来源中提取和纯化所需化合物的常规程序,利用生物素的性质从培养物中收集生物素。例如,利用离心等技术从培养物中除去细菌细胞,培养物中积累的生物素被吸附于活性炭或多孔非离子交换树脂上,随后,利用生物素与杂质间溶解度的不同,用离子交换树脂或经重结晶进行纯化。
本发明可通过下述实施例来进一步加以阐述,但这些实施例并不限制本发明的范围。
实施例1
通过震荡培养生产生物素
将一菌环斯芬克斯小杆菌SC-42405(FERM BP-3995)接种到一个大试管(22mmφ×220mm)中,试管中含有10mL预培养基(1%的甘油、2%的多胨、0.15%的K2HPO4、0.15%的MgSO4·7H2O,pH值7.2),在30℃下以200转/分的速度震荡培养3天。然后,将2mL这种预培养物接种到500mL的坂口烧瓶中,烧瓶中含50mL的培养基(2%的甘油、2%的酵母膏、0.5%的无维生素酪蛋白氨基酸、0.15%的K2HPO4、0.05%KCl、0.05%的MgSO4·7H2O、0.001%的FeSO4·7H2O、0.001%的MnSO4·4-6H2O,2μg/L的盐酸硫胺素,pH值为7.0),在30℃下以每分钟130转的速度震荡培养4天。培养后的细菌浓度(OD650)为22.0,此时培养基中所产生和积累的生物素浓度由使用植物乳杆菌IFO3070的定量微生物检测而确定(Izumi and Yamada,Methods of Vitaminological Experiments,Ⅱ,Water-Soluble Vitamine,pp.481-499,1985年由日本维生素学学会主编,东京Kagaku Dohjin),结果为3.3mg/L。
实施例2
通过需氧搅拌培养生产生物素
在一个大试管中预先培养斯芬克斯小杆菌SC-42405的培养物,方法同实施例1中所述。预培养物以2mL为单位逐份接种到3个500mL的坂口烧瓶中,每个烧瓶中含有100mL相同的预培养基,在30℃下以130转/分的速度震荡培养3天。然后,将300ml这种培养物接种到30L的发酵罐中,发酵罐中含有15L的培养基(2%的甘油、2%的酵母膏、0.5%的无维生素酪蛋白氨基酸、0.15%的K2HPO4、0.05%的KCl、0.05%的MgSO4·7H2O、0.001%的FeSO4·7H2O、0.001%的MnSO4·4-6H2O,20μg/L的盐酸硫胺素,pH值为7.0),在通气、搅拌及30℃下培养4天。通气速度为15L/分,搅拌速度为200~500转/分(将其控制到培养基中所溶解的氧的浓度为2ppm或更高一些)。通过加入消泡剂Einol(Biotto公司)达到自动消泡。培养完毕后细菌浓度(OD650)为38.0,此时培养基中所产生和积累的生物素浓度可由与实施例1中所述相同的定量微生物检测来确定,结果为2.3mg/L。
实施例3
产物的分离和鉴定
对实施例2中得到的15升培养物进行连续离心,再加入HCl使pH值调到3后,使上清液通过装有500g活性炭(Wako Pure Chemical Industries,Ltd.)的柱。用15L水冲洗该柱,然后用5L 0.1N的氨水进行洗脱。浓缩洗脱物,然后使其通过装有100ml Dowex离子交换树脂1×8(The Dow Chemical Co.)的柱,接着用2L水冲洗并用2L 1N的乙酸洗脱。将洗脱物浓缩,使其通过装有100ml Dowex 50W-8(The Dow Chemical Co.)的柱,然后用2L水洗脱。将洗脱液浓缩,使其通过装有50g活性炭(Wako Pure Chemical Industries,Ltd.)的柱。用500mL水冲洗柱,再用500mL的0.1N氨水洗脱。使洗脱液蒸发至干,并将残留物溶解于5ml 0.1N的氢氧化钠水溶液中,然后通过使用装有50ml Sephadex G-10(Pharmacia AB)的柱凝胶过滤色谱进行纯化。合并含有洗脱的生物素的级分,通过加入0.05N的HCl将pH值调到3,然后使合并的级分在冷处放置过夜。收集沉淀的晶体,并在减压下进行干燥,得到13mg的生物素晶体。这样得到的生物素表现出的生理化学特性(如质谱、红外吸收光谱等)与生物素的标准制剂相同。
实施例4
多种微生物之间生物素生产的比较
将一菌环多种与斯芬克斯小杆菌属相似的微生物的每一种接种到一个大试管(22mmφ×22mm)中,试管中含有5ml预培养基(1%的甘油、2%的多胨、0.15%的K2HPO4、0.15%的MgSO4·7H2O,pH值为7.2),然后在30℃下以250转/分的速度震荡培养3天。培养完毕后,培养基中所产生和积累的生物素浓度可用与实施例1中所述相同的定量微生物检测来确定。结果如表1中所示。
表1
表1(续)
*公开于Agr.Biol.Chem.,29,10,889-894(1965)中的数据。
**公开于Agr.Biol.Chem.,45,9,1983-1989(1981)中的数据。
Claims (5)
1、一种生产生物素的方法,包括以下步骤:
(a)在培养基中制备具有产生生物素能力的斯芬克斯小杆菌属微生物的培养物;和
(b)收集在培养基中产生和积累的生物素。
2、根据权利要求1的方法,其中具有产生生物素能力的斯芬克斯小杆菌属微生物是斯芬克斯小杆菌SC-42405(FERM BP-3995)或其突变体。
3、具有产生生物素能力的斯芬克斯小杆菌属微生物在生产生物素中的应用。
4、根据权利要求3的用于生产生物素的微生物的应用,其中微生物是斯芬克斯小杆菌SC-42405(FERM BP-3995)或其突变体。
5、斯芬克斯小杆菌SC-42405(FERM BP-3995)或其突变体。
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JP3428078B2 (ja) | 2003-07-22 |
ES2126619T3 (es) | 1999-04-01 |
CN1050153C (zh) | 2000-03-08 |
TW255913B (zh) | 1995-09-01 |
US5432067A (en) | 1995-07-11 |
DK0589285T3 (da) | 1999-08-23 |
HUT67685A (en) | 1995-04-28 |
HU9302568D0 (en) | 1993-11-29 |
ATE174966T1 (de) | 1999-01-15 |
EP0589285A3 (en) | 1995-02-15 |
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