CN105483060A - 一种高产瓜氨酸的谷氨酸棒杆菌及其在发酵生产瓜氨酸中的应用 - Google Patents
一种高产瓜氨酸的谷氨酸棒杆菌及其在发酵生产瓜氨酸中的应用 Download PDFInfo
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- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 title claims abstract description 37
- RHGKLRLOHDJJDR-UHFFFAOYSA-N Ndelta-carbamoyl-DL-ornithine Natural products OC(=O)C(N)CCCNC(N)=O RHGKLRLOHDJJDR-UHFFFAOYSA-N 0.000 title claims abstract description 37
- 229960002173 citrulline Drugs 0.000 title claims abstract description 37
- 235000013477 citrulline Nutrition 0.000 title claims abstract description 37
- 241000186226 Corynebacterium glutamicum Species 0.000 title claims abstract description 22
- 238000004519 manufacturing process Methods 0.000 title abstract description 7
- 244000005700 microbiome Species 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 15
- 238000010564 aerobic fermentation Methods 0.000 claims description 14
- 230000001580 bacterial effect Effects 0.000 claims description 10
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 9
- 240000008042 Zea mays Species 0.000 claims description 9
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 9
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 9
- 239000004202 carbamide Substances 0.000 claims description 9
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- 239000000843 powder Substances 0.000 claims description 9
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 7
- 229930003756 Vitamin B7 Natural products 0.000 claims description 7
- 235000011912 vitamin B7 Nutrition 0.000 claims description 7
- 239000011735 vitamin B7 Substances 0.000 claims description 7
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- 239000008103 glucose Substances 0.000 claims description 6
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 5
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- 229910052799 carbon Inorganic materials 0.000 claims description 5
- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 claims description 5
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- 239000001103 potassium chloride Substances 0.000 claims description 3
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- 150000003751 zinc Chemical class 0.000 claims description 3
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 2
- 235000013343 vitamin Nutrition 0.000 claims description 2
- 229930003231 vitamin Natural products 0.000 claims description 2
- 239000011782 vitamin Substances 0.000 claims description 2
- 229940088594 vitamin Drugs 0.000 claims description 2
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 13
- 241000894006 Bacteria Species 0.000 abstract description 7
- 239000006227 byproduct Substances 0.000 abstract description 7
- 238000000855 fermentation Methods 0.000 abstract description 7
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- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
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- 230000001954 sterilising effect Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 244000270200 Citrullus vulgaris Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 240000007049 Juglans regia Species 0.000 description 2
- 235000009496 Juglans regia Nutrition 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
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- 206010039073 rheumatoid arthritis Diseases 0.000 description 2
- 235000020234 walnut Nutrition 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 description 1
- 235000012840 Citrullus vulgaris Nutrition 0.000 description 1
- 241000424760 Corynebacterium crenatum Species 0.000 description 1
- 241000194032 Enterococcus faecalis Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 208000010513 Stupor Diseases 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
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- 238000003745 diagnosis Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-M dihydrogenphosphate Chemical compound OP(O)([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-M 0.000 description 1
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/15—Corynebacterium
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Abstract
本发明公开了一种高产瓜氨酸的谷氨酸棒杆菌及其在发酵生产瓜氨酸中的应用,所述菌株的分类命名为谷氨酸棒杆菌(<i>Corynebacterium</i><i>?glutamicum</i>)CIT002<i></i>,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.11567。本发明还公开了利用上述高产瓜氨酸的谷氨酸棒杆菌生产瓜氨酸的方法。本发明方法与化学法相比,能耗低,污染小,使用非化石资源作为原料,成本低廉,过程易操控,产量高。本发明菌株与原始菌相比,发酵产物单一,副产物少,产量高。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种高产瓜氨酸的谷氨酸棒杆菌及其在发酵生产瓜氨酸中的应用。
背景技术
瓜氨酸(Citrulline)是一种α-氨基酸,因1914年右贺太郎等第一次从西瓜中获取而得名为“瓜氨酸”。后来游离态的瓜氨酸在野生西瓜叶片、核桃仁、核桃幼苗等葫芦科植物的种子中被发现。瓜氨酸在生物体代谢中起到重要作用,主要参与人体尿素循环,是尿素形成的重要中间产物,对于维持人体血氨平衡具有重要的作用。此外瓜氨酸被广泛应用于健康和营养方面,如参与重要的信使分子NO的生成,肝脏昏迷的治疗,类风湿关节炎(rheumatoidarthritis,RA)的诊断,作为异体排斥效应的指示剂和天然高效的抗氧化剂等。
瓜氨酸的制备方法主要包括化学合成法、酶催化法及微生物发酵法。相比化学法难以控制、产率不高、纯度低;酶法催化效率不高且杂酶易对反应产生干扰等问题,微生物发酵法合成瓜氨酸具有成本低廉、产品纯度高、环境污染小等特点,因此生物法制备瓜氨酸成为今年来的研究热点。目前生物法合成瓜氨酸的主要菌种包括:Corynebacteriumhydrocarboclastus、Enterococcusfaecalis、Escherichiacoli、Corynebacteriumcrenatum等。其中谷氨酸棒杆菌Corynebacteriumglutamicum因其生长迅速、培养条件简单、耐受性强、发酵条件成熟,成为最具潜力的生产瓜氨酸的工业菌种。但是野生谷氨酸棒杆菌在发酵生产瓜氨酸的过程中,总是伴随着副产物L-赖氨酸和L-谷氨酸,影响了发酵产物的质量,增加了后续分离的负荷。
发明内容
本发明所要解决的技术问题是提供一株高产瓜氨酸的谷氨酸棒杆菌及其在发酵生产瓜氨酸中的应用。
为解决上述技术问题,本发明采用的技术方案如下:
发明人通过低能离子束注入技术,对谷氨酸棒杆菌(Corynebacteriumglutamicum)ATCC13032进行诱变,经三轮筛选得到一株谷氨酸棒杆菌,其好氧下可高产瓜氨酸,同时不产生副产物L-赖氨酸和L-谷氨酸。该菌株,其分类命名为谷氨酸棒杆菌(Corynebacteriumglutamicum)CIT002,目前该菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC),保藏单位地址: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,邮政编码100101,登记入册的编号是CGMCCNo.11567,保藏日期是2015年11月3日。以此菌作为生产菌株。
CGMCCNo.11567菌株具有下述性质:
1、菌落形态学特征:
在营养琼脂培养基上,30℃划线培养呈中度生长,菌苔线状,24h为白色,48h后稍呈淡黄色,随培养时间的延长颜色稍增深。表面湿润、光滑,有光泽,无黏性。
2、生理与生化特性:
a.培养温度:28~39℃,最适温度为30℃;
b.在pH5.0~8.5范围内生长;
c.色素产生情况:不产色素;
3、营养特征:
谷氨酸棒杆菌可在合成培养基上生长,培养基成分明确且价格低廉。该培养基碳源一般为葡萄糖或玉米糖化液,氮源可选用玉米浆干粉、牛肉膏、蛋白胨、酵母膏、尿素以及(NH4)2SO4等,培养基中还包括钾盐、钠盐、钙盐、镁盐、锌盐、铁盐、锰盐、磷酸盐、磷酸二氢盐和盐酸盐等常用的无机盐类,根据不同的氮源而定,以硫酸铵或尿素为唯一氮源时需全添加,在以玉米浆干粉、牛肉膏、蛋白胨、酵母膏时可以少量或不加入无机盐。
利用上述高产瓜氨酸的谷氨酸棒杆菌生产瓜氨酸的方法,将保藏号为CGMCCNo.11567的菌株活化后,在28~39℃下好氧发酵48~72h;
其中,好氧发酵培养基为完全培养基:碳源20~80g/L,氮源10~40g/L,无机盐0~3g/L,生长因子0~0.3mg/L,其余为水,pH5.0~7.5;
其中,所述的碳源为葡萄糖。
其中,所述的氮源为硫酸铵。
其中,所述的无机盐为钾盐、锰盐、铁盐、镁盐、锌盐、铜盐、钙盐、硫酸盐、磷酸盐或盐酸盐中的任意一种或几种的组合。
其中,所述的生长因子为生物素和维生素中的任意一种或两种的组合。
最优选的生产方式为:将保藏号为CGMCCNo.11567的菌株活化后,在30℃下好氧发酵48~72h,装液量为30ml发酵液/500ml三角瓶,转速为200rpm。
其中,所述的好氧发酵培养基为:葡萄糖40g/L,K2HPO4·3H2O1.5g/L,KH2PO40.5g/L,MgSO4·7H2O0.4g/L,FeSO4·7H2O20mg/L,MnSO4·H2O20mg/L,尿素5g/L,生物素100μg/L,维生素B1200μg/L,其余为水,pH7。
有益效果:本发明与现有技术相比,具有如下优势:
(1)与化学法及酶催化法相比,能耗低,污染小,使用非化石资源作为原料,无需使用贵重金属作为催化剂,成本低廉,过程易操控,产量高。
(2)与原始菌相比,发酵产物单一,副产物少,产量高。
附图说明
图1为本发明菌株CGMCCNo.11567好氧发酵终点的高效液相色谱氨基酸检测图谱。其中,标号1的峰为瓜氨酸的响应峰。
具体实施方式
根据下述实施例,可以更好地理解本发明。然而,本领域的技术人员容易理解,实施例所描述的仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1
平板培养基:蛋白胨10g/L,酵母膏5g/L,NaCl10g/L,琼脂20g/L,pH7.0。
好氧发酵培养基:葡萄糖40g/L,K2HPO4·3H2O1.5g/L,KH2PO40.5g/L,MgSO4·7H2O0.4g/L,FeSO4·7H2O20mg/L,尿素5g/L,生物素100μg/L,维生素B1200μg/L,初始pH7.0。500mL三角瓶装液量30mL,115℃灭菌10分钟。
分别将保藏号为ATCC13032及CGMCCNo.11567菌首先于30℃平板活化菌种,24h后接两环生长良好的菌体入30mL发酵培养基,30℃,200rpm培养12h,然后按照3%(v/v)接种量转接入装液量为30ml发酵液/500ml三角瓶,30℃培养,转速为200rpm。发酵48h后CGMCCNo.11567的发酵液中含有5.43g/L瓜氨酸。较ATCC13032相比,瓜氨酸产量提高了10.24倍,没有副产物L-赖氨酸和L-谷氨酸。
实施例2
平板培养基:同实施例1;
好氧发酵培养基:玉米糖化液500mL/L(玉米糖化液的含糖量约为100g/L),K2HPO4·3H2O1.5g/L,KH2PO40.5g/L,MgSO4·7H2O0.4g/L,FeSO4·7H2O20mg/L,尿素2.5g/L,生物素100μg/L,维生素B1200μg/L,初始pH7.0。5L发酵罐装液量4L,115℃灭菌10分钟。
分别将保藏号为ATCC13032及CGMCCNo.11567菌首先于30℃平板活化菌种,24h后接两环生长良好的菌体入30mL发酵培养基,30℃,200rpm培养12h,然后按照3%(v/v)接种量转接入4L发酵培养基,通空气1v/vm,转速800rpm,30℃培养。发酵60h后CGMCCNo.的发酵液中含有12.8g/L瓜氨酸。较ATCC13032相比,瓜氨酸产量提高了24.2倍,没有副产物L-赖氨酸和L-谷氨酸。
实施例3
平板培养基:同实施例1;
好氧发酵培养基:蔗糖40g/L,玉米浆干粉10g/L,初始pH7.0。5L发酵罐装液量4L,115℃灭菌10分钟。
分别将保藏号为ATCC13032及CGMCCNo.11567菌首先于30℃平板活化菌种,24h后接两环生长良好的菌体入30mL发酵培养基,30℃,200rpm培养12h,然后按照3%(v/v)接种量转接入4L发酵培养基,通空气1v/vm,转速800rpm,30℃培养。发酵60h后CGMCCNo.11567的发酵液中含有11.8g/L瓜氨酸。较ATCC13032相比,瓜氨酸产量提高了22.2倍,没有副产物L-赖氨酸和L-谷氨酸。
Claims (9)
1.一种高产瓜氨酸的谷氨酸棒杆菌,其分类命名为谷氨酸棒杆菌(Corynebacteriumglutamicum)CIT002,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CGMCCNo.11567。
2.权利要求1所述高产瓜氨酸的谷氨酸棒杆菌在发酵生产瓜氨酸中的应用。
3.根据权利要求2所述的应用,其特征在于,将保藏号为CGMCCNo.11567的菌株活化后,在28~39℃下好氧发酵48~72h;
其中,好氧发酵培养基为完全培养基:碳源20~80g/L,氮源10~40g/L,无机盐0~3g/L,生长因子0~0.3mg/L,其余为水,pH5.0~7.5。
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或者玉米糖化液。
5.根据权利要求3所述的应用,其特征在于,所述的氮源为玉米浆干粉、牛肉膏、蛋白胨、酵母膏、尿素或者(NH4)2SO4中的至少一种。
6.根据权利要求3所述的应用,其特征在于,所述的无机盐为钾盐、锰盐、铁盐、镁盐、锌盐、铜盐、钙盐、硫酸盐、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,所述的生长因子为生物素和维生素中的任意一种或两种的组合。
8.根据权利要求3所述的应用,其特征在于,将保藏号为CGMCCNo.11567的菌株活化后,在30℃下好氧发酵48~72h,转速为200rpm;
其中,所述的好氧发酵培养基为:葡萄糖40g/L,K2HPO4·3H2O1.5g/L,KH2PO40.5g/L,MgSO4·7H2O0.4g/L,FeSO4·7H2O20mg/L,MnSO4·H2O20mg/L,尿素5g/L,生物素100μg/L,维生素B1200μg/L,其余为水,pH7。
9.根据权利要求3所述的应用,其特征在于,将保藏号为CGMCCNo.11567的菌株活化后,在30℃下好氧发酵48~72h,转速为800rpm;
其中,所述的好氧发酵培养基为:玉米糖化液500mL/L,K2HPO4·3H2O1.5g/L,KH2PO40.5g/L,MgSO4·7H2O0.4g/L,FeSO4·7H2O20mg/L,尿素2.5g/L,生物素100μg/L,维生素B1200μg/L,初始pH7.0。
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