WO2017031838A1 - 一种利用重组枯草芽孢杆菌全细胞转化生产l-鸟氨酸的方法 - Google Patents

一种利用重组枯草芽孢杆菌全细胞转化生产l-鸟氨酸的方法 Download PDF

Info

Publication number
WO2017031838A1
WO2017031838A1 PCT/CN2015/094424 CN2015094424W WO2017031838A1 WO 2017031838 A1 WO2017031838 A1 WO 2017031838A1 CN 2015094424 W CN2015094424 W CN 2015094424W WO 2017031838 A1 WO2017031838 A1 WO 2017031838A1
Authority
WO
WIPO (PCT)
Prior art keywords
ornithine
arginine
bacillus subtilis
conversion
recombinant
Prior art date
Application number
PCT/CN2015/094424
Other languages
English (en)
French (fr)
Inventor
饶志明
王梅洲
徐美娟
张显
杨套伟
Original Assignee
江南大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江南大学 filed Critical 江南大学
Publication of WO2017031838A1 publication Critical patent/WO2017031838A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/10—Citrulline; Arginine; Ornithine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus

Definitions

  • the invention relates to a method for producing L-ornithine by whole cell transformation of recombinant Bacillus subtilis, belonging to the fields of bioengineering and biotechnology.
  • L-ornithine is an important non-protein amino acid and an important precursor for the synthesis of arginine and proline.
  • L-ornithine is an important intermediate metabolite of urea cycle in human body, so it has an important guarantee for the detoxification function of the liver.
  • L-ornithine stimulates the secretion of growth hormone in the body, promotes protein synthesis and catabolism of sugars and lipids.
  • the compound amino acid prepared by the combination of L-ornithine and other amino acids has a good function of protecting the liver and protecting the liver and stimulating the vitality of the diseased liver.
  • the combination of L-ornithine and phenylacetic acid can effectively treat hepatic encephalopathy.
  • L-ornithine can stably reduce blood ammonia in patients by promoting the synthesis and excretion of glutamine. concentration.
  • Ornithine ⁇ -ketoglutarate is a good clinical nutrient to promote recovery of surgical trauma patients, improve chronic malnutrition, and improve immune function.
  • the combination of ornithine and malate and citrate can improve the taste of foods and beverages and reduce bitterness.
  • L-ornithine is marketed as a dietary drug.
  • Ornithine is a basic amino acid which is easily soluble in water and ethanol and slightly soluble in organic solvents such as ether. Ornithine accepts two molecules of NH 4 + and one molecule of CO 2 to form a molecule of L-arginine. L-arginine can also be hydrolyzed to ornithine and urea by the action of arginase.
  • L-ornithine includes chemical synthesis, microbial fermentation, and L-arginine hydrolysis.
  • the production of L-ornithine by microbial fermentation mainly obtains L-ornithine high-yield strains by mutagenesis or genetic engineering, and synthesizes L-ornithine with relatively cheap glucose or even starch as a starting material.
  • Japan started earlier, and the study of ornithine production began in the 1950s and 1960s, mainly based on mutagenesis screening.
  • Kinoshita et al. first reported the production of ornithine by fermentation using a mutant strain of Corynebacterium glutamicum. Since then, Okumurahe Shibuya et al.
  • L-ornithine by L-arginine hydrolysis includes alkaline hydrolysis and enzymatic hydrolysis, especially enzymatic hydrolysis, which has received increasing attention due to its high reaction efficiency and high product specificity.
  • some people in China have begun to try to produce L-ornithine by enzymatic conversion with arginine as a substrate.
  • Xu Wei Beijing University of Chemical Technology, obtained a strain of Bacillus thuringiensis with high arginase activity, and used this cell as a biocatalyst to produce L-ornithine with L-arginine as a substrate, and finally obtained 43.57 g / L L-ornithine.
  • the invention firstly provides a recombinant bacterium producing arginase, Bacillus subtilis argI, using pMA5 as an expression vector and Bacillus subtilis 168 as a host, and constructing a genetic engineering strain B.subtilis168/pMA5 -argI.
  • the arginase gene is derived from Bacillus cereus and the sequence is set forth in SEQ ID NO.
  • the invention also provides a method for producing L-ornithine by using the recombinant Bacillus subtilis B. subtilis 168/pMA5-argI whole cell to transform L-arginine, and constructing a transformation by using whole cells of the recombinant bacteria as a biocatalyst.
  • Method for producing L-ornithine conversion system the conversion system uses 0.25-0.3M carbonate buffer with pH 9.0, containing 0-0.5 mM Mn 2+ , 100-200 g/L L-spermine
  • the acid has a conversion temperature of 35-40 ° C, and the substrate L-arginine is added in time to maintain the substrate concentration between 120-200 g/L.
  • the total cell dosage is 3-5 g/L in the transformation system.
  • the conversion system uses a 0.25 M carbonate buffer having a pH of 9.0, containing 0.5 mM of Mn 2+ , 200 g/L of L-arginine, and a transformation temperature of 40 ° C. And add the substrate L-arginine in time to maintain the substrate concentration between 120-200g / L.
  • the arginase gene argI derived from Bacillus cereus was cloned and expressed in B. subtilis 168, and the arginase activity was increased by 26.7 times compared with the original strain.
  • the present invention directly expresses arginase directly under the control of the Hpa II promoter by the shuttle vector pMA5, and does not require induction by an expensive inducer such as IPTG.
  • the present invention uses recombinant Bacillus subtilis B. subtilis 168/pMA5-argI whole cell to transform L-arginine to produce L-ornithine, and obtains 148.7 g/L of L-ornithine in 4 hours.
  • the molar conversion of the amino acid reached 100%; by adding the substrate L-arginine, the yield of L-ornithine was as high as 378.9 g/L within 12 h, and the substrate molar conversion reached 99.9%.
  • Enzyme activity assay method 0.2 M substrate L-arginine (pH 9.0, 0.2 M carbonate buffer) was prepared, 0.9 ml of the substrate solution was taken, 0.1 ml of the enzyme solution was added, and the reaction was carried out at 40 ° C for 10 min.
  • the enzyme reaction solution was diluted to a corresponding multiple, and 1 ml of the diluted reaction solution was taken, and the content of L-ornithine in the reaction solution was measured by Chinard colorimetric method.
  • the enzyme activity defines the amount of enzyme required to catalyze the conversion of 1 umol L-arginine to L-ornithine in 1 min.
  • PCR primers P1 and P2 of the arginase gene were designed based on the argI gene sequence in the Bacillus cereus genome-wide nucleic acid sequence in NCBI.
  • the total DNA of Bacillus cereus was used as a template, and the primers provided above were used for PCR amplification.
  • the amplification conditions were: 94 °C pre-denaturation, 5 min, one cycle; 94 °C denaturation, 1 min, 56 °C annealing, 1 min, 72 ° C extension, 45 s , 35 cycles; end extension at 72 ° C for 10 min.
  • PCR amplification system template 1 ⁇ L, upstream and downstream primers 0.4 ⁇ L, dNTP Mix 4 ⁇ L, 10 ⁇ Ex Taq Buffer 5 ⁇ L, sterilized double distilled water 37 ⁇ L, Ex Taq DNA polymerase 1 ⁇ L.
  • the PCR product was purified and recovered by a gel recovery kit, and the concentration of the recovered product was examined by electrophoresis.
  • the recovered product was stored in a 1.5 mL centrifuge tube and stored in a -20 ° C refrigerator for later use.
  • the recovered product was ligated to pMD18-T Vector, the ligation product was transformed into E.coil JM109, and the transformed product was coated with ampicillin-containing LB plate, cultured at 37 ° C overnight, and the colony was picked up to 10 mL of liquid LB.
  • the plasmid was extracted after overnight incubation at 37 °C, and named as pMD18-T-argI.
  • the glycerol was added to a final concentration of 15% to 20% (w/v), and stored at -70 °C in the refrigerator. .
  • the plasmids pMD18-T-argI and pMA5 stored in E.coli JM109 were extracted and double-digested with BamH I and Nde I, respectively, and recovered by gel recovery kit, and ligated. 7 ⁇ L, 1 ⁇ L of pMA5 digested product, 1 ⁇ L of T4 DNA ligase buffer, 1 ⁇ L of T4 DNA ligase, and ligated overnight at 16 °C.
  • the ligated recombinant plasmid pMA5-argI was transformed into competent E. coil JM109, and positive colonies were picked with ampicillin LB plates. After 37 °C shaker overnight culture, the plasmid was extracted and named as pMA5-argI. After correct digestion, the glycerol was added to a final concentration of 15% to 20% (w/v), and stored in a -70 °C refrigerator for later use.
  • B. subtilis 168 was inoculated into a 5 mL LB liquid medium test tube, and cultured overnight at 37 ° C on a shaker. 100 ⁇ L of the overnight culture solution was taken, inoculated into 5 mL SPI Medium, and shaken at 37 ° C for 5 h. OD 600 , when the culture was grown to the end of the logarithm, 200 ⁇ L was quickly inoculated into 2 mL of SPII Medium, and cultured at 37 ° C for 100 h/min shaker for 1.5 h.
  • the recombinant strain B. subtilis 168/pMA5-argI constructed in Example 4 and the starting strain B. subtilis 168 were inoculated separately into 10 mL of LB medium containing kanamycin, cultured overnight at 37 ° C with shaking, and inoculated with 1% the next day. The amount was transferred to 50 mL of LB medium, and cultured at 37 ° C for 12 h. The fermentation broth was centrifuged at 4 ° C, 10000 r / min for 10 min, and the collected cells were washed 3 times with pH 7.0 Tris-HCl buffer, and finally with 5 mL of pH 7. 0 Tris-HCl buffer to suspend cells. The crude enzyme solution was prepared by ultrasonication treatment.
  • a 0.2 M substrate L-arginine (pH 9.0, 0.2 M carbonate buffer) was prepared, 0.9 ml of the substrate solution was taken, 0.1 ml of the enzyme solution was added, and the reaction was carried out at 40 ° C for 10 min.
  • the enzyme reaction solution was diluted to a corresponding multiple, and 1 ml of the diluted reaction solution was taken, and the content of L-ornithine in the reaction solution was measured by Chinard colorimetric method.
  • the enzyme activity defines the amount of enzyme required to catalyze the conversion of 1 umol L-arginine to L-ornithine in 1 min.
  • Recombinant B. subtilis168/pMA5-argI was inoculated in 50ml LB medium for 12h (OD 600 ⁇ 3.5), and the recombinant cells were obtained by centrifugation. 0.2g of recombinant cells were resuspended in 50ml buffer to 100g/L. L-arginine is a substrate and is converted to produce L-ornithine.
  • Buffer The effects of phosphate buffer, carbonate buffer, borax buffer, Tris-HCl buffer, citrate buffer and disodium hydrogen phosphate-citrate buffer on the conversion rate were determined. Optimal buffer concentration. The results are shown in Table 1.
  • the carbonate buffer has a strong pH buffering ability under alkaline conditions, and is converted to L-ornithine by using a carbonate buffer as compared with other buffers. The conversion rate is the fastest.
  • Mn 2+ 0.1-5 mM Mn 2+ was added to the conversion solution, and the influence on the conversion rate was examined. The results showed that the concentration of Mn 2+ in the conversion system was 0-0.5 mM, and the conversion rate was accompanied by Mn. The increase of 2+ concentration increases, and the increase of Mn 2+ concentration does not promote the increase of conversion rate. On the contrary, Mn 2+ produces Mn(OH) 2 under alkaline conditions (pH 9.0). Conducive to the progress of the conversion.
  • the recombinant B. subtilis 168/pMA5-argI was inoculated in 2L fermentation medium for 16h (OD 600 ⁇ 14.6-15.2), centrifuged to obtain cells, washed twice with pH 7.0 Tris-HCl, and resuspended in 2L substrate buffer. (0.25 M carbonate buffer, 200 g/L L-arginine, 0.5 mM Mn 2+ , pH 9.0), conversion was carried out at 40 °C. The substrate L-arginine was added during the conversion to maintain a substrate concentration of between 120 and 200 g/L.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种利用重组枯草芽孢杆菌全细胞转化生产L-鸟氨酸的方法，包括构建表达来源于Bacillus cereus的精氨酸酶的Bacillus subtilis 168工程菌，然后以此工程菌作为生物催化剂，以L-精氨酸作为底物，采用分批补加底物L-精氨酸的方法进行转化法生产L-鸟氨酸。

Description

[根据细则26改正29.12.2016]　一种利用重组枯草芽孢杆菌全细胞转化生产L-鸟氨酸的方法 技术领域
本发明涉及一种利用重组枯草芽孢杆菌全细胞转化生产L-鸟氨酸的方法，属于生物工程和生物技术领域。
背景技术
L-鸟氨酸是一种重要的非蛋白质氨基酸，是精氨酸和脯氨酸合成的重要前体物质。同时L-鸟氨酸是人体内尿素循环的重要中间代谢产物，因此其对肝脏的解毒功能具有重要的保障作用。L-鸟氨酸能够刺激体内生长激素的分泌，促进蛋白质的合成以及糖类和脂质的分解代谢作用。L-鸟氨酸和其他氨基酸一起制成的复方氨基酸有很好的保肝护肝以及激发病态下肝脏的活力的作用。联合使用L-鸟氨酸和苯乙酸能有效治疗肝性脑病，特别是对于鸟氨酸循环障碍的患者，L-鸟氨酸能通过促进谷氨酰胺的合成和排泄来稳定的降低患者血氨浓度。鸟氨酸α-酮戊二酸盐是很好的临床营养剂，促进外科创伤病人的恢复，改善慢性营养不良，改善免疫功能。同时鸟氨酸和苹果酸盐和柠檬酸盐合用能够改善食品和饮料的口味，减少苦味。在欧洲、美国以及日本，L-鸟氨酸都是作为膳食药物来销售的。
鸟氨酸是一种碱性氨基酸，易溶于水和乙醇，微溶于乙醚等有机溶剂。鸟氨酸接受二分子NH4 +和一分子CO2形成一分子L-精氨酸。L-精氨酸亦可以在精氨酸酶的作用下被水解成鸟氨酸和尿素。
工业上L-鸟氨酸的合成包括化学合成法、微生物发酵法以及L-精氨酸水解法。微生物发酵生产L-鸟氨酸主要是通过诱变或者基因工程的方法获得L-鸟氨酸高产菌株，以较为便宜的葡萄糖甚至是淀粉等为初始原料合成L-鸟氨酸。在这方面日本起步较早，上世纪五六十年代就开始鸟氨酸生产的研究，主要以诱变筛选为主。1957年kinoshita等首先报道了利用谷氨酸棒状杆菌突变株发酵生产鸟氨酸。此后，Okumurahe Shibuya等人在这方面也做了大量的工作，将具有精氨酸缺陷型及精氨酸氧肟酸盐抗性的谷氨酸棒杆菌，以及具有精氨酸和鸟氨酸以及霉酚酸抗性等标记的柠檬酸节杆菌突变株用于工业生产中。国内陈宁、刘树庆等人在1999年开始研究鸟氨酸的生产，并以谷氨酸棒杆菌为出发菌株，通过硫酸二乙酯和紫外线诱变定向选育出一种鸟氨酸生产菌，并通过发酵优化使得鸟氨酸产量达到9.85g/L。近几年，随着代谢工程技术的兴起，通过代谢工程改造的方法，鸟氨酸的发酵生产取得一定的进展，其中，中 山大学蒋玲艳等人以谷氨酸棒杆菌为出发菌株，通过表达异源的谷氨酸脱氢酶和甘油醛-3-磷酸脱氢酶，增加代谢流中NADPH的含量，使得L-鸟氨酸产量得到提高，最终到14.8g/L，而后蒋又通过代谢进化和代谢工程的方法，最后得到鸟氨酸的产量为24.1g/L。通过发酵法进行鸟氨酸生产可以利用较简单的碳源，如葡萄糖等，成本非常低廉，适合于工业的发展，但发酵周期一般都相对较长，而且发酵的产量较低对于后期分离需要较高的技术需求。
L-精氨酸水解法生产L-鸟氨酸包括，碱水解法和酶水解法，特别是酶水解，由于其反应效率高，产物转专一性高而受到越来越多的重视。近几年，国内有人开始尝试以精氨酸为底物进行酶转化法生产L-鸟氨酸。北京化工大学徐焘，通过筛选得到一株精氨酸酶活力较高的苏云金芽孢杆菌，以此细胞作为生物催化剂，以L-精氨酸为底物进行L-鸟氨酸生产，并最终得到43.57g/L L-鸟氨酸。而后张涛等人提取苏云金芽孢杆菌的精氨酸酶，以精氨酸为底物，精氨酸酶纯酶作催化剂，并最终得到72.1g/L的鸟氨酸。宋伟等人构建表达外源精氨酸酶的E.coli BL21，并以重组细胞为催化剂，L-精氨酸为底物，最终得到112.3g/L的L-鸟氨酸。然而，无论以产精氨酸酶的菌株作为催化剂，还是以纯化的精氨酸酶为催化剂转化L-精氨酸生产L-鸟氨酸，都还存在效率比较低，产量低的问题。特别是用纯化的精氨酸酶进行转化生产L-鸟氨酸，需要消耗较大的成本在精氨酸酶的分离纯化上，不利于工业化的应用。
发明内容
本发明首先提供了一株产精氨酸酶的重组枯草芽孢杆菌，是将精氨酸酶基因argI，以pMA5为表达载体，以Bacillus subtilis 168为宿主，构建了基因工程菌株B.subtilis168/pMA5-argI。
在本发明的一种实施方式中，所述精氨酸酶基因来源于Bacillus cereus，序列如SEQ ID NO.1所示。
本发明还提供了应用所述重组枯草芽孢杆菌B.subtilis 168/pMA5-argI全细胞转化L-精氨酸生产L-鸟氨酸的方法，是以重组菌全细胞作为生物催化剂，构建了转化法生产L-鸟氨酸的转化体系；所述转化体系采用pH为9.0的0.25-0.3M碳酸盐缓冲液，含有0-0.5mM的Mn2+、100-200g/L的L-精氨酸，转化温度为35-40℃，并适时补加底物L-精氨酸，使得底物浓度维持在120-200g/L之间。
在本发明的一种实施方式中，转化体系中，全细胞用量为3-5g/L。
在本发明的一种实施方式中，所述转化体系采用pH为9.0的0.25M碳酸盐缓冲液，含有0.5mM的Mn2+、200g/L的L-精氨酸，转化温度为40℃，并适时补加底物L-精氨酸，使 得底物浓度维持在120-200g/L之间。
本发明的优点和积极效果是：
(1)本发明首次在B.subtilis 168中克隆表达了来源于Bacillus cereus的精氨酸酶基因argI，相比出发菌株，精氨酸酶酶活提高了26.7倍。
(2)本发明通过穿梭载体pMA5，在Hpa II启动子控制下，精氨酸酶直接高效表达，不需要通过IPTG等价格昂贵的诱导剂下进行诱导表达。
(3)本发明应用重组枯草芽孢杆菌B.subtilis 168/pMA5-argI全细胞转化L-精氨酸生产L-鸟氨酸，在4h内获得了148.7g/L的L-鸟氨酸，精氨酸的摩尔转化率达到100％；通过补加底物L-精氨酸，在12h内L-鸟氨酸的产量高达378.9g/L，底物摩尔转化率达到99.9％。
具体实施方式
重组菌株酶活力测定
酶活测定方法：配制0.2M底物L-精氨酸(pH 9.0，0.2M碳酸盐缓冲液)，取0.9ml底物溶液，加入0.1ml酶液，40℃反应10min。将酶反应液稀释相应的倍数，取1ml稀释后的反应液，用Chinard比色法测定反应液中L-鸟氨酸的含量。酶活定义单位为1min催化1umol L-精氨酸转化成L-鸟氨酸所需的酶量。
Chinard比色法：1ml标准液中依次加入1ml的冰醋酸，1ml混合酸(茚三酮溶液)，于沸水浴中反应1h，测定515nm下的吸光度值。
实施例1 精氨酸酶引物设计
根据NCBI中Bacillus cereus全基因组核酸序列中argI基因序列，设计精氨酸酶基因的PCR引物P1和P2。
P1：5’-ATCCATATGATGAAAAAAGAAATCTCAG-3’(Nde I)
P2：5’-ACCGGGATCCTTATTTTAGT TTTTCACCG-3’(BamH I)
实施例2 精氨酸酶基因的克隆
以Bacillus cereus总DNA为模板，利用上面提供的引物做PCR扩增，扩增条件为：94℃预变性，5min，一个循环；94℃变性，1min，56℃退火，1min，72℃延伸，45s，35个循环；72℃终延伸10min。PCR扩增体系：模板1μL，上下游引物各0.4μL，dNTP Mix 4μL，10×Ex Taq Buffer 5μL，灭菌的双蒸水37μL，Ex Taq DNA聚合酶1μL。采用凝胶回收试剂盒对PCR产物进行纯化和回收，电泳检验回收产物的浓度。回收产物存放在1.5mL的离心管中，-20℃冰箱保存备用。回收产物与pMD18-T Vector连接，连接产物转化E.coil JM109，转化产物涂布含氨苄青霉素的LB平板，经37℃培养过夜，挑取菌落到10mL液体LB培养 基中，37℃摇床过夜培养后提取质粒，命名为pMD18-T-argI，经酶切验证连接成功后，加入甘油至终浓度15％～20％(w/v)，-70℃冰箱保藏。
实施例3 重组质粒pMA5-argI的构建
提取保存于E.coli JM109中的质粒pMD18-T-argI和pMA5，并分别用BamH I和Nde I进行双酶切，利用凝胶回收试剂盒回收后进行连接，连接体系：目的基因酶切产物7μL，pMA5酶切产物1μL，T4 DNA连接酶buffer 1μL，T4 DNA连接酶1μL，16℃过夜连接。将连接好的重组质粒pMA5-argI转化到感受态E.coil JM109，用氨苄青霉素LB平板，挑取阳性菌落。37℃摇床过夜培养后提取质粒，命名为pMA5-argI，酶切验证正确后，加入甘油至终浓度15％～20％(w/v)，-70℃冰箱保藏备用。
实施例4 重组质粒pMA5-argI转化B.subtilis168
挑取B.subtilis 168接种于一支5mL LB液体培养基试管，37℃摇床培养过夜，取100μL过夜培养的菌液，接种至用5mL SPI Medium中，37℃摇床培养，5h后开始测OD600，当培养物生长到对数末期时，快速取200μL接种到2mL SPII Medium中，37℃100r/min摇床培养1.5h。加20μL 100×EGTA溶液，于37℃100r/min摇床培养10min，用1.5mL离心管分装成500μL每管，向管中加入适量的质粒pMA5-argI，轻轻混匀于37℃100r/min摇床培养30min。将离心管转移到250r/min摇床，37℃养1.5h，4000r/min离心收集菌体，弃部分上清液，留100μL重悬菌体，涂布于卡那霉素抗性平板，37℃过夜培养，挑取阳性菌落，提取质粒酶切验证，得到重组菌B.subtilis 168/pMA5-argI。
实施例5 重组菌B.subtilis168/pMA5-argI精氨酸酶活力测定
将实施例4构建的重组菌B.subtilis168/pMA5-argI，与出发菌株B.subtilis168分别接种于10mL含卡那霉素的LB培养基中，37℃振荡培养过夜，次日按1％的接种量转接于50mL LB培养基中，37℃培养12h，取发酵液于4℃，10000r/min离心10min，收集的细胞用pH7.0的Tris-HCl缓冲液清洗3次，最后用5mL pH7.0 Tris-HCl缓冲液悬浮细胞。超声波破碎处理制备粗酶液。
配制0.2M底物L-精氨酸(pH9.0，0.2M碳酸盐缓冲液)，取0.9ml底物溶液，加入0.1ml酶液，40℃反应10min。将酶反应液稀释相应的倍数，取1ml稀释后的反应液，用Chinard比色法测定反应液中L-鸟氨酸的含量。酶活定义单位为1min催化1umol L-精氨酸转化成L-鸟氨酸所需的酶量。
Chinard比色法：1ml标准液中依次加入1ml的冰醋酸，1ml混合酸(茚三酮溶液)，于沸水浴中反应1h，测定515nm下的吸光度值。
结果表明重组菌B.subtilis168/pMA5-argI表达的精氨酸酶比酶活为21.9U/mg，比出发菌株B.subtilis 168精氨酸酶酶活提高了26.7倍。
实施例6 全细胞转化法生产L-鸟氨酸最佳转化体系的构建
重组菌B.subtilis168/pMA5-argI接种于50ml LB培养基中培养12h(OD600≈3.5)，离心获得重组菌细胞，取0.2g重组菌细胞重悬于50ml缓冲液中，以100g/L的L-精氨酸为底物，转化生产L-鸟氨酸。
关于转化温度：分别在20-60℃下进行转化产L-鸟氨酸，测定在不同转化温度下转化液中L-鸟氨酸的含量。结果表明，温度在低于40℃时，转化速率随着温度的升高而升高，高于40℃时转化速率急剧下降。
关于缓冲液：分别考察磷酸盐缓冲液、碳酸盐缓冲液、硼砂缓冲液、Tris-HCl缓冲液、柠檬酸盐缓冲液和磷酸氢二钠-柠檬酸缓冲液对转化速率的影响，同时确定最佳的缓冲液浓度。结果如表1所示，碳酸盐缓冲液由于其在碱性条件下的pH缓冲能力较强，与其他几种缓冲液相比，应用碳酸盐缓冲液进行转化产L-鸟氨酸的转化速率最快。
关于转化体系中的金属离子：在转化液中添加0.1-5mM的Mn2+，考察对转化率的影响，结果表明转化体系中Mn2+的浓度在0-0.5mM时，转化率随着Mn2+浓度的升高而升高，继续升高Mn2+的浓度并不会促进转化率的升高，反而由于Mn2+在碱性条件(pH 9.0)下产生Mn(OH)2，不利于转化的进行。
表1：不同缓冲液L-鸟氨酸产率的影响
缓冲液组成 L-鸟氨酸产率(g/(L·h))
Na2HPO4-NaH2PO4 47.7±1.01
Na2HPO4-柠檬酸 37.9±0.57
柠檬酸钠-柠檬酸 34.8±0.65
Tris-HCl 45.8±0.72
NaHCO3-Na2CO3 51.1±0.92
硼酸钠-硼酸 46.1±0.77
实施例7 分批补加底物转化L-精氨酸生产L-鸟氨酸
重组菌B.subtilis 168/pMA5-argI接种于2L发酵培养基中培养16h(OD600≈14.6-15.2)，离心获得细胞，pH 7.0Tris-HCl洗涤两次后，重悬于2L底物缓冲液(0.25M碳酸盐缓冲液，200g/L L-精氨酸，0.5mM Mn2+，pH 9.0)，40℃下进行转化。转化过程中补加底物L-精氨 酸，使得底物浓度维持在120-200g/L之间。
结果表明，在12h内，共补加500g/L的底物L-精氨酸，L-鸟氨酸的产量为378.9g/L，底物L-精氨酸的摩尔转化率达到99.9％。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2015094424-appb-000001
Figure PCTCN2015094424-appb-000002

Claims (7)

  1. 一株产精氨酸酶的重组枯草芽孢杆菌，是将精氨酸酶基因argI，以pMA5为表达载体，以Bacillus subtilis 168为宿主，构建了基因工程菌株B.subtilis 168/pMA5-argI。
  2. 根据权利要求1所述的重组枯草芽孢杆菌，其特征在于，所述精氨酸酶基因来源于Bacillus cereus，序列如SEQ ID NO.1所示。
  3. 一种应用权利要求1所述的重组枯草芽孢杆菌全细胞转化L-精氨酸生产L-鸟氨酸的方法，其特征在于，是以重组菌全细胞作为生物催化剂，构建了转化法生产L-鸟氨酸的转化体系；所述转化体系采用pH为9.0的0.25-0.3 M碳酸盐缓冲液，含有0-0.5 mM的Mn2+、100-200g/L的L-精氨酸，转化温度为35-40℃；转化过程中适时补加底物L-精氨酸，使得底物浓度维持在120-200 g/L之间。
  4. 根据权利要求3所述的方法，其特征在于，所述转化体系采用pH为9.0的0.25 M碳酸盐缓冲液。
  5. 根据权利要求3所述的方法，其特征在于，所述转化体系含有0.5 mM的Mn2+。
  6. 根据权利要求3所述的方法，其特征在于，所述转化温度为40℃。
  7. 根据权利要求3所述的方法，其特征在于，所述转化体系中全细胞用量为3-5 g/L。
PCT/CN2015/094424 2015-08-25 2015-11-12 一种利用重组枯草芽孢杆菌全细胞转化生产l-鸟氨酸的方法 WO2017031838A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510526416.1 2015-08-25
CN201510526416.1A CN105062941B (zh) 2015-08-25 2015-08-25 一种利用重组枯草芽孢杆菌全细胞转化生产l-鸟氨酸的方法

Publications (1)

Publication Number Publication Date
WO2017031838A1 true WO2017031838A1 (zh) 2017-03-02

Family

ID=54492461

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/094424 WO2017031838A1 (zh) 2015-08-25 2015-11-12 一种利用重组枯草芽孢杆菌全细胞转化生产l-鸟氨酸的方法

Country Status (2)

Country Link
CN (1) CN105062941B (zh)
WO (1) WO2017031838A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023102816A1 (zh) * 2021-12-09 2023-06-15 武汉远大弘元股份有限公司 一种基因工程菌及用其制备l-鸟氨酸的方法

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105886491A (zh) * 2016-04-11 2016-08-24 湖北大学 一种实现人源精氨酸酶-1在大肠杆菌表面展示的方法
CN105886566A (zh) * 2016-05-10 2016-08-24 宁波市镇海海德生化科技有限责任公司 一种微生物酶法转化生产l-鸟氨酸的方法
CN105950697B (zh) * 2016-07-20 2019-10-15 江南大学 一种双菌混合发酵生产l-鸟氨酸的方法
CN106434611A (zh) * 2016-10-14 2017-02-22 江南大学 一种以l‑精氨酸为原料的l‑鸟氨酸双酶耦合制备方法
CN107354142A (zh) * 2017-09-14 2017-11-17 精晶药业股份有限公司 一种L‑天门冬氨酸‑α‑脱羧酶酶液处理方法
CN110964683B (zh) * 2019-12-02 2021-08-13 天津科技大学 生产l-精氨酸的基因工程菌及其构建方法与应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1918298A (zh) * 2002-06-20 2007-02-21 康达医药科技有限公司 利用精氨酸剥夺治疗人恶性肿瘤的药物组合物及方法
CN102191291A (zh) * 2011-04-25 2011-09-21 武汉远大弘元股份有限公司 一种利用基因工程菌生产l-鸟氨酸盐酸盐的方法
CN102703370A (zh) * 2012-06-01 2012-10-03 南开大学 一株组成型表达精氨酸酶的基因工程菌株及其应用
CN103923936A (zh) * 2013-12-05 2014-07-16 江南大学 一种产精氨酸酶工程菌的构建及应用该菌生产l-鸟氨酸

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1918298A (zh) * 2002-06-20 2007-02-21 康达医药科技有限公司 利用精氨酸剥夺治疗人恶性肿瘤的药物组合物及方法
CN102191291A (zh) * 2011-04-25 2011-09-21 武汉远大弘元股份有限公司 一种利用基因工程菌生产l-鸟氨酸盐酸盐的方法
CN102703370A (zh) * 2012-06-01 2012-10-03 南开大学 一株组成型表达精氨酸酶的基因工程菌株及其应用
CN103923936A (zh) * 2013-12-05 2014-07-16 江南大学 一种产精氨酸酶工程菌的构建及应用该菌生产l-鸟氨酸

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [O] 28 May 2015 (2015-05-28), JOHNSON, S.L. ET AL.: "Bacillus cereus G9241, complete genome", XP055365946, Database accession no. CP009590 *
SONG, WEI ET AL.: "Enzymatic production of L-ornithine from 1-arginine with recombinant thermophilic arginase", JOURNAL OF MOLECULAR CATALYSIS B: ENZYMATIC, vol. 110, 20 September 2014 (2014-09-20), pages 1 - 7, XP055365952, ISSN: 1381-1177 *
WANG, MEIZHOU ET AL.: "Construction of a highly efficient bacillus subtilis 168 whole- cell biocatalyst and its application in the production of L-ornithine", J IND MICROBIOL BIOTECHNOL, vol. 42, no. 11, 29 August 2015 (2015-08-29), pages 1427 - 1437, XP035602975, ISSN: 1367-5435 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023102816A1 (zh) * 2021-12-09 2023-06-15 武汉远大弘元股份有限公司 一种基因工程菌及用其制备l-鸟氨酸的方法

Also Published As

Publication number Publication date
CN105062941B (zh) 2019-03-05
CN105062941A (zh) 2015-11-18

Similar Documents

Publication Publication Date Title
WO2017031838A1 (zh) 一种利用重组枯草芽孢杆菌全细胞转化生产l-鸟氨酸的方法
US11225675B2 (en) D-lactate dehydrogenase, engineered strain containing D-lactate dehydrogenase and construction method and use of engineered strain
WO2019085445A1 (zh) 生产l-赖氨酸的重组菌、其构建方法以及l-赖氨酸的生产方法
WO2020133990A1 (zh) 草铵膦脱氢酶突变体及其合成l-草铵膦的应用
WO2022228169A1 (zh) 一种产乳酰-n-新四糖的基因工程菌及生产方法
WO2017080111A1 (zh) 一株生产戊二胺的基因工程菌及其制备戊二胺的方法
CN113481185B (zh) 一种耐盐β-半乳糖苷酶GalNC2-13及其制备方法和应用
WO2022174597A1 (zh) 一种用于l-肌氨酸生产的基因工程菌及构建方法与应用
CN113801833B (zh) 一种产d-阿洛酮糖的重组菌单细胞工厂及其构建与应用
WO2017031839A1 (zh) 一种酶活提高的l-天冬酰胺酶突变体及其构建方法
CN110951661B (zh) 一种高产l-谷氨酰胺的谷氨酸棒杆菌及其构建方法与应用
CN110540976B (zh) 一种异丙基苹果酸合成酶及其应用
CN113637660B (zh) 一种β-半乳糖苷酶GalNC3-89及其制备方法和应用
CN109468288A (zh) 一种高效降解组胺的新多铜氧化酶
WO2022217827A1 (zh) 一种用于制备β-烟酰胺单核苷酸的酶组合物及其应用
CN112391329B (zh) 一种抗酸胁迫能力提高的大肠杆菌工程菌及其应用
CN113549633A (zh) 一种l-半胱氨酸转运蛋白突变体及其在生产l半胱氨酸中的应用
WO2021037190A1 (zh) 一种2-异丙基苹果酸合成酶及其工程菌与应用
WO2017088094A1 (zh) 一种信号肽及其在利用魔芋粉合成l-谷氨酸及其高值化的应用
CN111057672B (zh) 重组菌株及其应用
WO2023115997A1 (zh) 一种用于生产异麦芽酮糖的重组谷氨酸棒杆菌及其应用
CN105112437A (zh) 一种利用重组钝齿棒杆菌一步发酵法生产l-鸟氨酸的方法
CN113881611A (zh) 一种提高谷氨酸棒杆菌合成l-谷氨酸产量的方法
WO2023102816A1 (zh) 一种基因工程菌及用其制备l-鸟氨酸的方法
WO2022133917A1 (zh) 改造的磷酸烯醇丙酮酸羧化酶及其在提高谷氨酸棒杆菌氨基酸产量中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15902107

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15902107

Country of ref document: EP

Kind code of ref document: A1