WO2024050928A1 - 一种吡咯烷酮的制备方法 - Google Patents

一种吡咯烷酮的制备方法 Download PDF

Info

Publication number
WO2024050928A1
WO2024050928A1 PCT/CN2022/126215 CN2022126215W WO2024050928A1 WO 2024050928 A1 WO2024050928 A1 WO 2024050928A1 CN 2022126215 W CN2022126215 W CN 2022126215W WO 2024050928 A1 WO2024050928 A1 WO 2024050928A1
Authority
WO
WIPO (PCT)
Prior art keywords
pyrrolidone
caic
carnitine
coa ligase
preparation
Prior art date
Application number
PCT/CN2022/126215
Other languages
English (en)
French (fr)
Inventor
吴静
姜旭玲
刘立明
宋伟
周怡雯
陈修来
刘佳
高聪
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
Priority to US17/998,217 priority Critical patent/US20240132925A1/en
Application filed by 江南大学 filed Critical 江南大学
Publication of WO2024050928A1 publication Critical patent/WO2024050928A1/zh

Links

Images

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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • 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
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y602/00—Ligases forming carbon-sulfur bonds (6.2)
    • C12Y602/01—Acid-Thiol Ligases (6.2.1)

Definitions

  • the invention relates to a preparation method of pyrrolidone and belongs to the technical field of bioengineering.
  • Pyrrolidone also known as butyrolactam and ⁇ -pyrrolidone
  • the synthesis of pyrrolidone is mainly based on chemical methods.
  • the target product is obtained with a yield of 94% by reacting ⁇ -butyrolactone with ammonia at high temperature and high pressure.
  • the chemical method has a high yield, it consumes a lot of energy and has a toxic effect on the environment, which does not meet the requirements of green production, safe production and sustainable development.
  • the preparation of pyrrolidone through biological methods has the characteristics of stable and safe product quality, mild process conditions, and environmental protection. It can reduce pressure on the environment and resources. Therefore, there is an urgent need for an effective biological method to prepare pyrrolidone efficiently.
  • the present invention provides a method for preparing pyrrolidone. Specifically, the present invention provides a method for preparing pyrrolidone by using carnitine-CoA ligase CaiC to catalyze ⁇ -aminobutyric acid, or using the carnitine-CoA ligase to catalyze ⁇ -aminobutyric acid to prepare pyrrolidone.
  • the enzyme CaiC is used to construct recombinant bacteria and perform whole-cell transformation of ⁇ -aminobutyric acid to produce pyrrolidone. It has the advantages of low environmental damage, short production cycle, and reduced by-products during transformation, which greatly improves the industrial production efficiency.
  • the first object of the present invention is to provide a method for preparing pyrrolidone, which method uses carnitine-CoA ligase CaiC or whole cells expressing carnitine-CoA ligase CaiC as a catalyst to catalyze the preparation of ⁇ -aminobutyric acid. Pyrrolidone.
  • amino acid sequence of the carnitine-CoA ligase CaiC is shown in SEQ ID NO.1.
  • nucleotide sequence of the carnitine-CoA ligase CaiC is shown in SEQ ID NO. 2.
  • the recombinant strain uses Escherichia coli as the host and PET-28a as the expression vector to express carnitine-CoA ligase CaiC.
  • Escherichia coli is Escherichia coli BL21 (DE3).
  • reaction system of the catalytic reaction contains ⁇ -aminobutyric acid, ATP and Mg 2+ .
  • the final concentration of whole cells is 15-25g/L.
  • the final concentration of ⁇ -aminobutyric acid is 5 to 15 g/L.
  • reaction system contains 40-60mM ATP and 20-40mM Mg 2+ .
  • the pH of the reaction system is 7.4-7.6, and the reaction temperature is 35-38°C.
  • the invention provides a method for preparing pyrrolidone by utilizing carnitine coenzyme A ligase CaiC to catalyze ⁇ -aminobutyric acid.
  • the amino acid sequence of the carnitine-CoA ligase CaiC is shown in SEQ ID NO: 1. This ligase has the activity of catalyzing the cyclization of ⁇ -aminobutyric acid to generate pyrrolidone.
  • the output of pyrrolidone in 24 hours is 3.26g/L, and the molar yield can reach 39.53%, which reduces the production cycle, increases the output of pyrrolidone, and speeds up the production of pyrrolidone.
  • the industrialization process of producing pyrrolidone by enzymatic conversion method is 3.26g/L, and the molar yield can reach 39.53%, which reduces the production cycle, increases the output of pyrrolidone, and speeds up the production of pyrrolidone.
  • Figure 1 is an SDS-PAGE diagram of the induced expression of carnitine-coenzyme A ligase CaiC of the present invention
  • lane M refers to the low molecular weight protein marker
  • lanes 1 to 3 are respectively the supernatant and precipitation after induction of expression at 0.2mM IPTG concentration at 25°C. and the size of the target protein band in whole cells.
  • Figure 2 is an enzyme activity verification diagram, which performs deletion control on each component in the transformation system and compares the production of pyrrolidone.
  • Figure 3 is a graph showing the relationship between the pH of the conversion buffer and the production of pyrrolidone.
  • Figure 4 is a graph showing the relationship between Mg 2+ concentration and pyrrolidone production.
  • Figure 5 is a graph showing the relationship between conversion temperature and pyrrolidone production.
  • Figure 6 is a graph showing the relationship between substrate concentration and pyrrolidone production.
  • the pET-28a(+) plasmid involved in the following examples was purchased from Novagen (Madison, WI, U.S.A.), and the restriction enzyme, primeSTAR, homologous recombinase, etc. were purchased from TaKaRa (Dalian, China).
  • the standard products ⁇ -aminobutyric acid and pyrrolidone were purchased from Sigma-Aldrich Company in the United States, and the other reagents were purchased from the market.
  • LB liquid culture medium peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, sterilized at 121°C for 20 minutes.
  • LB solid medium On the basis of LB liquid medium, add 2% agar.
  • TB liquid medium KH 2 PO 4 2.31g/L, K 2 HPO 4 ⁇ 3H 2 O 16.42g/L, yeast powder 24g/L, peptone 12g/L, glycerol 4g/L.
  • F1 agcaaatgggtcgcggatccgaattcATGGATATCATTGGCGGACAACATCTAC(SEQ ID NO.3);
  • R1 tggtgctcgagtgcggccgcaagcttTTTCAGATTCTTTCTAATTATTTTCCCCGAGCAAT(SEQ ID NO.4).
  • the cDNA sequence of the coding region of the carnitine-CoA ligase CaiC gene was obtained. After the PCR product was recovered, it was connected with the pET-28a(+) plasmid vector that had been digested by the same double enzyme through homologous recombination to obtain the recombinant expression plasmid pET-28a(+)- CaiC, the recombinant plasmid pET-28a(+)-CaiC was transformed into E.coli BL21(DE3). After PCR identification, the positive engineering bacterium was named E.coli BL21/pET-28a(+)-CaiC.
  • the engineering strain E.coli BL21/pET-28a(+)-CaiC was inserted into the LB liquid medium, and after culturing for 12 hours, the seed liquid was obtained.
  • the seed liquid was inoculated into fresh TB at an inoculum volume of 5% (v/v).
  • Liquid culture medium after culturing for 2 hours, add IPTG with a final concentration of 0.2mM, and culture at 25°C for 14 hours to induce the expression of the recombinant target protein. Take 150mL of induced fermentation broth and centrifuge at 6000r/min to collect the bacteria.
  • Lanes 1 to 3 are the band sizes of the proteins contained in the supernatant, pellet and whole cells respectively. It can be seen that the target protein is expressed in both whole cells, supernatant and pellet, and The strips are the same size.
  • the seed liquid was obtained after culturing at 200 rpm and 37°C for 12 hours at a constant temperature.
  • the seed liquid was inoculated into fresh TB liquid culture medium at an inoculation volume of 5% (v/v). After 2 hours of culture, the final concentration was added. 0.2mM IPTG was used for induction and culture at 25°C for 14h, and the cells were collected after completion.
  • P represents the final molar concentration of pyrrolidone
  • S 0 represents the initial molar concentration of ⁇ -aminobutyric acid
  • the pyrrolidone production is 2.72g/L, and the molar yield is 32.96%. Subsequently, the cyclization activity decreased with further increase in pH. This shows that a neutral environment is more favorable for carnitine-CoA ligase CaiC to catalyze the cyclization reaction, and the whole cell has better cyclization activity at pH 7.5.
  • Example 4 Optimal Mg 2+ concentration for whole cells
  • Example 3 For specific implementation details, see Example 3. The difference is that the conversion of carnitine-CoA ligase CaiC at different Mg 2+ concentrations (10, 20, 30, 40, 50, 60mM) was measured for 24 h under the condition that the buffer pH was 7.5. The yield of pyrrolidone and calculate the molar yield. The results showed that the cyclization activity of carnitine-CoA ligase CaiC increased with the increase of Mg 2+ concentration in the range of 10-30mM, reaching a peak at 30mM. The pyrrolidone production was 2.80g/L, and the molar yield was 33.81%. However, there is almost no change in the cyclization activity in the range of 30-60mM.
  • carnitine-CoA ligase CaiC it is more favorable for carnitine-CoA ligase CaiC to catalyze the cyclization reaction at a Mg 2+ concentration of 30 mM, and carnitine-CoA ligase CaiC has better cyclization activity at this Mg 2+ concentration.
  • Example 3 For specific implementation details, see Example 3. The difference is that the carnitine-CoA ligase CaiC was measured under the conditions of buffer pH 7.5 and 30mM MgSO 4 under different temperature conditions (16, 20, 25, 30, 37, 44 °C) to convert the pyrrolidone production for 24 h, and calculate the molar yield.
  • the results show that the cyclization activity of carnitine-CoA ligase CaiC increases with the increase of temperature in the range of 16-37°C, while the cyclization activity decreases with the increase of temperature in the range of 37-44°C. °C reached the peak, the pyrrolidone production was 3.26g/L, and the molar yield was 39.53%. Therefore, the conversion temperature of 37°C is more favorable for carnitine-CoA ligase CaiC to catalyze the cyclization reaction, and carnitine-CoA ligase CaiC has better cyclization activity at this temperature.
  • Example 6 Optimal substrate concentration for whole cells
  • Example 3 For specific implementation details, see Example 3. The difference is that the carnitine- CoA ligase CaiC was measured at different substrate concentrations (5, 10, 20, 30 , 40, 50g/L) to convert the pyrrolidone yield in 24h, and calculate the molar yield.
  • the results show that the cyclization activity of CaiC enzyme increases with the increase of temperature in the range of 5-10g/L, while the cyclization activity decreases with the increase of temperature in the range of 10-50g/L, reaching 10g/L.
  • pyrrolidone production was 3.26g/L, and the molar yield was 39.53%. Therefore, at a substrate concentration of 10 g/L, it is more favorable for carnitine-CoA ligase CaiC to catalyze the cyclization reaction, and an increase in substrate concentration will cause inhibition.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

本发明公开了一种吡咯烷酮的制备方法，本发明提供一种利用肉碱辅酶A连接酶CaiC催化γ-氨基丁酸制备吡咯烷酮的方法。所述肉碱辅酶A连接酶CaiC的氨基酸序列如SEQ ID NO:1所示。该连接酶具有催化γ-氨基丁酸环化生成吡咯烷酮的活性。本发明提供的肉碱辅酶A连接酶以γ-氨基丁酸为底物时24h吡咯烷酮的产量为3.26g/L，摩尔产率可达39.53％，减少了生产周期，提高了吡咯烷酮的产量，加快了酶转化法生产吡咯烷酮的工业化进程。

Description

一种吡咯烷酮的制备方法 技术领域
本发明涉及一种吡咯烷酮的制备方法，属于生物工程技术领域。
背景技术
吡咯烷酮又称丁内酰胺、α-吡咯烷酮，是一种无色结晶，可用作溶剂及有机合成中间体，以及用来制造尼龙4和乙烯基吡咯烷酮等多种化合物的前体，在工业上具有许多重要的应用。吡咯烷酮及其衍生物作为五元氮杂环分子，在生物活性方面具有某些独特的性质，许多天然产物中都有此类杂环化合物的分子骨架存在。
目前吡咯烷酮的合成以化学法为主，由γ-丁内酯与氨气在高温、高压下反应，以94％的收率得到目标产物。但是化学法虽然得率高，能耗却较大，同时会对环境产生毒害作用，不符合绿色生产、安全生产和可持续发展的要求。相对传统的化学法，通过生物法制备吡咯烷酮具有产品质量稳定安全、工艺条件温和、环保等特点，可以减轻环境和资源压力，因此迫切需要一种有效的生物法高效制备吡咯烷酮。
近几年国内外对吡咯烷酮的生物法合成进行了一些研究，目前生物法合成吡咯烷酮主要采用微生物发酵法和酶转化法。但是微生物发酵法，发酵周期比较长，生产强度低，并不适用于工业生产。而现有的酶转化法催化效率低、产量极低，因此迫切需要一种有效的酶转化法高效制备吡咯烷酮。
发明内容
为解决上述技术问题，本发明提供一种吡咯烷酮的制备方法，本发明具体是提供一种利用肉碱辅酶A连接酶CaiC催化γ-氨基丁酸制备吡咯烷酮的方法，或利用该肉碱辅酶A连接酶CaiC构建重组菌，进行全细胞法转化γ-氨基丁酸生产吡咯烷酮的方法，具有低环境损害、生产周期短、减少了转化中的副产物等优点，极大提高了工业化的生产效率。
本发明的第一个目的是提供一种吡咯烷酮的制备方法，所述方法是以肉碱辅酶A连接酶CaiC或表达肉碱辅酶A连接酶CaiC的全细胞为催化剂，催化γ-氨基丁酸制备 吡咯烷酮。
进一步地，所述的肉碱辅酶A连接酶CaiC的氨基酸序列如SEQ ID NO.1所示。
进一步地，所述的肉碱辅酶A连接酶CaiC的核苷酸序列如SEQ ID NO.2所示。
进一步地，所述的全细胞是将表达肉碱辅酶A连接酶CaiC的重组菌株经过IPTG诱导12-16h后收集得到。
进一步地，所述的重组菌株是以大肠杆菌为宿主，以PET-28a为表达载体表达肉碱辅酶A连接酶CaiC。
进一步地，所述的大肠杆菌为Escherichia coli BL21(DE3)。
进一步地，催化反应的反应体系中含有γ-氨基丁酸、ATP和Mg 2+。
进一步地，所述的反应体系中，全细胞的终浓度为15～25g/L。
进一步地，所述的反应体系中，γ-氨基丁酸的终浓度为5～15g/L。
进一步地，所述的反应体系中，含有40～60mM ATP以及20～40mM Mg 2+。
进一步地，反应体系的pH为7.4-7.6，反应温度为35～38℃。
本发明的有益效果是：
本发明提供一种利用肉碱辅酶A连接酶CaiC催化γ-氨基丁酸制备吡咯烷酮的方法。所述肉碱辅酶A连接酶CaiC的氨基酸序列如SEQ ID NO:1所示。该连接酶具有催化γ-氨基丁酸环化生成吡咯烷酮的活性。本发明提供的肉碱辅酶A连接酶以γ-氨基丁酸为底物时24h吡咯烷酮的产量为3.26g/L，摩尔产率可达39.53％，减少了生产周期，提高了吡咯烷酮的产量，加快了酶转化法生产吡咯烷酮的工业化进程。
附图说明
图1为本发明肉碱辅酶A连接酶CaiC诱导表达的SDS-PAGE图；泳道M是指低分子量蛋白Marker；泳道1～3分别是25℃下0.2mM IPTG浓度诱导表达后上清液、沉淀和全细胞中所目的蛋白条带大小。
图2为酶活性验证图，对转化体系内各组分进行缺失对照，对比吡咯烷酮的生成情况图。
图3为转化缓冲液pH和吡咯烷酮生产量的关系图。
图4为Mg 2+浓度和吡咯烷酮生产量的关系图。
图5为转化温度和吡咯烷酮生产量的关系图。
图6为底物浓度和吡咯烷酮生产量的关系图。
具体实施方式
下面结合具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
下述实施例中所涉及的pET-28a(+)质粒购自Novagen(Madison,WI,U.S.A.)，限制性内切酶、primeSTAR、同源重组酶等购自TaKaRa(Dalian,China)。标品γ-氨基丁酸、吡咯烷酮均购自美国Sigma-Aldrich公司，其余试剂均为市场购买所得。
下述实施例中所涉及的培养基如下：
LB液体培养基：蛋白胨10g/L，酵母粉5g/L，氯化钠10g/L，在121℃灭菌20min。
LB固体培养基：在LB液体培养基基础上，添加2％的琼脂。
TB液体培养基：KH 2PO 4 2.31g/L，K 2HPO 4·3H 2O 16.42g/L，酵母粉24g/L，蛋白胨12g/L，甘油4g/L。
实施例1：肉碱辅酶A连接酶CaiC的表达与纯化
基因工程菌的构建及蛋白的表达：
以大肠杆菌Escherichia coli(strain K12)中目的蛋白编码基因的核苷酸序列(SEQ ID NO.2所示)为模板，利用F1和R1为引物(下划线分别是EcoR I和HindⅢ限制性酶切位点)PCR扩增，扩增条件为：
95℃5min，29个循环(98℃10s，55℃15s，72℃1.5min)，72℃5min。
F1：agcaaatgggtcgcggatccgaattcATGGATATCATTGGCGGACAACATCTAC(SEQ ID NO.3)；
R1：tggtgctcgagtgcggccgcaagcttTTTCAGATTCTTTCTAATTATTTTCCCCGAGCAAT(SEQ ID NO.4)。
获得肉碱辅酶A连接酶CaiC基因编码区cDNA序列，PCR产物回收后与经同样双酶切的pET-28a(+)质粒载体通过同源重组连接，获得重组表达质粒pET-28a(+)-CaiC，将重组质粒pET-28a(+)-CaiC转化入E.coli BL21(DE3)，经过PCR鉴定，获得阳性的工程菌命名为E.coli BL21/pET-28a(+)-CaiC。
将工程菌E.coli BL21/pET-28a(+)-CaiC接入LB液体培养基，培养12h后得到种子液，将种子液按照5％(v/v)的接种量，接种至新鲜的TB液体培养基，培养2h后，加入终浓度为0.2mM的IPTG，25℃培养14h，诱导表达重组目的蛋白。取150mL诱导发酵液经6000r/min离心收集菌体。
结果如图1：泳道1～3分别是上清液、沉淀和全细胞中所含蛋白的条带大小，可见，不论是全细胞还是上清液和沉淀中，目的蛋白都得到了表达，并且条带大小相同。
实施例2：肉碱辅酶A连接酶CaiC活性验证
具体步骤如下：
将从甘油管中保存的菌株E.coli BL21/pET-28a(+)-CaiC涂布于LB固体培养基上， 在37℃下恒温培养至长出单克隆，挑取单克隆至新鲜的LB液体培养基中，以200rpm、37℃下恒温培养12h后得到种子液，将种子液按照5％(v/v)的接种量，接种至新鲜的TB液体培养基，培养2h后，加入终浓度为0.2mM的IPTG，于25℃下诱导培养14h，结束后收集细胞。
在100mL锥形瓶中分别加入0.2g诱导培养后表达肉碱辅酶A连接酶CaiC蛋白的全细胞、0.1gγ-氨基丁酸(C4H9NO2，GABA)、500μL 1M ATP、500μL 1M MgSO 4和9mL PBS缓冲液(pH7.4)，30℃反应24h后12000r/min离心10min，吸取上上清液，过0.22μm水膜，进行高效液相色谱法HPLC分析。
上述具体的高效液相色谱法HPLC分析方法为：
以Agilent ZORBAX SB-C18(5μm，250×4.6mm)作为色谱柱，以经过抽滤、超声脱气的甲醇/乙腈/水(5/5/90，v/v/v)为流动相，进样量为10μL，柱温为30℃，紫外检测器的波长为205nm，流速为0.5mL/min，样品处理时间为10min。在此检测条件下，吡咯烷酮的保留时间为8.078min。
吡咯烷酮摩尔产率＝(P/S 0)×100％；
其中：P代表吡咯烷酮的最终摩尔浓度，S 0代表γ-氨基丁酸的初始摩尔浓度。
具体的结果如图2所示，由图2可知全细胞的催化效果为29.52％摩尔产率。从结果可以看出肉碱辅酶A连接酶CaiC具有明显的环化活性。相反无菌泥和无底物的对照组均没有相应的催化效果，在没有ATP、以及没有MgSO 4的反应体系中，摩尔产率都显著降低。
实施例3：全细胞最适反应pH
在100mL锥形瓶中加入20g/L全细胞，10g/Lγ-氨基丁酸，50mM的ATP以及50mM的MgSO 4，分别于pH 6.0、pH 6.5、pH 7.0、pH 7.5、pH 8.0和pH 8.5的PBS缓冲液组成10mL反应体系。于30℃、200rpm恒温摇床中反应24h。按照上述检测方法测定吡咯烷酮的产量，并计算摩尔产率。结果显示，肉碱辅酶A连接酶CaiC在pH 6.0-pH 7.5内环化活性随着pH的上升而增长，在pH 7.5左右达到峰值，吡咯烷酮产量为2.72g/L，摩尔产率为32.96％，随后环化活性随pH的进一步升高而下降。这说明中性环境对肉碱辅酶A连接酶CaiC催化环化反应更有利，全细胞在pH 7.5具有更好的环化活性。
实施例4：全细胞最适Mg 2+浓度
具体实施方式参见实施例3，区别在于，在缓冲液pH为7.5的条件下测定肉碱辅酶A连接酶CaiC在不同Mg 2+浓度下(10、20、30、40、50、60mM)转化24h的吡咯烷酮产量，并计算摩尔产率。结果显示，肉碱辅酶A连接酶CaiC在10-30mM范围内环化活性随着 Mg 2+浓度的上升而上升，在30mM达到峰值，吡咯烷酮产量为2.80g/L，摩尔产率为33.81％。而在30-60mM范围内环化活性几乎没有变化。因而，在30mM的Mg 2+浓度下对肉碱辅酶A连接酶CaiC催化环化反应更有利，肉碱辅酶A连接酶CaiC在该Mg 2+浓度下具有更好的环化活性。
实施例5：全细胞最适反应温度
具体实施方式参见实施例3，区别在于，在缓冲液pH为7.5以及30mM的MgSO 4的条件下测定肉碱辅酶A连接酶CaiC在不同温度条件下(16、20、25、30、37、44℃)转化24h的吡咯烷酮产量，并计算摩尔产率。结果显示，肉碱辅酶A连接酶CaiC在16-37℃范围内环化活性随着温度的上升而上升，而在37-44℃范围内环化活性却随着温度的上升而下降，在37℃达到峰值，吡咯烷酮产量为3.26g/L，摩尔产率为39.53％。因而，在37℃的转化温度对肉碱辅酶A连接酶CaiC催化环化反应更有利，肉碱辅酶A连接酶CaiC在该温度下具有更好的环化活性。
实施例6：全细胞最适底物浓度
具体实施方式参见实施例3，区别在于，在缓冲液pH为7.5、30mM的MgSO 4以及37℃的条件下测定肉碱辅酶A连接酶CaiC在不同底物浓度下(5、10、20、30、40、50g/L)转化24h的吡咯烷酮产量，并计算摩尔产率。结果显示，CaiC酶在5-10g/L范围内环化活性随着温度的上升而上升，而在10-50g/L范围内环化活性却随着温度的上升而下降，在10g/L达到峰值，吡咯烷酮产量为3.26g/L，摩尔产率为39.53％。因而，在10g/L的底物浓度时肉碱辅酶A连接酶CaiC催化环化反应更有利，底物浓度升高会产生抑制。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (10)

  1. 一种吡咯烷酮的制备方法，其特征在于，所述方法是以肉碱辅酶A连接酶CaiC或表达肉碱辅酶A连接酶CaiC的全细胞为催化剂，催化γ-氨基丁酸制备吡咯烷酮。
  2. 根据权利要求1所述的制备方法，其特征在于，所述的肉碱辅酶A连接酶CaiC的氨基酸序列如SEQ ID NO.1所示。
  3. 根据权利要求1所述的制备方法，其特征在于，所述的肉碱辅酶A连接酶CaiC的核苷酸序列如SEQ ID NO.2所示。
  4. 根据权利要求1所述的制备方法，其特征在于，所述的全细胞是将表达肉碱辅酶A连接酶CaiC的重组菌株经过IPTG诱导12-16h后收集得到。
  5. 根据权利要求4所述的制备方法，其特征在于，所述的重组菌株是以大肠杆菌为宿主，以PET-28a为表达载体表达肉碱辅酶A连接酶CaiC。
  6. 根据权利要求5所述的制备方法，其特征在于，所述的大肠杆菌为Escherichia coli BL21(DE3)。
  7. 根据权利要求1所述的制备方法，其特征在于，催化反应的反应体系中含有γ-氨基丁酸、ATP和Mg 2+。
  8. 根据权利要求7所述的制备方法，其特征在于，所述的反应体系中，全细胞的终浓度为15～25g/L，γ-氨基丁酸的终浓度为5～15g/L。
  9. 根据权利要求7所述的制备方法，其特征在于，所述的反应体系中，含有40～60mM ATP以及20～40mM Mg 2+。
  10. 根据权利要求7所述的制备方法，其特征在于，反应体系的pH为7.4-7.6，反应温度为35～38℃。
PCT/CN2022/126215 2022-09-05 2022-10-19 一种吡咯烷酮的制备方法 WO2024050928A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/998,217 US20240132925A1 (en) 2022-09-05 2022-10-18 Method for preparing pyrrolidone

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211097246.6A CN115725669A (zh) 2022-09-05 2022-09-05 一种吡咯烷酮的制备方法
CN202211097246.6 2022-09-05

Publications (1)

Publication Number Publication Date
WO2024050928A1 true WO2024050928A1 (zh) 2024-03-14

Family

ID=85293119

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/126215 WO2024050928A1 (zh) 2022-09-05 2022-10-19 一种吡咯烷酮的制备方法

Country Status (3)

Country Link
US (1) US20240132925A1 (zh)
CN (1) CN115725669A (zh)
WO (1) WO2024050928A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103189520A (zh) * 2010-09-30 2013-07-03 大象株式会社 利用生物物质的2-吡咯烷酮的制备方法
CN107438667A (zh) * 2015-04-13 2017-12-05 韩国科学技术院 内酰胺的制备方法
CN108191731A (zh) * 2017-12-25 2018-06-22 恒天生物基材料工程技术（宁波）有限公司 一种基于生物质原料氨基丁酸的新型丁内酰胺制备工艺

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10227622B2 (en) * 2015-04-14 2019-03-12 The Regents Of The University Of California Host cell modified to produce 2-pyrrolidone

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103189520A (zh) * 2010-09-30 2013-07-03 大象株式会社 利用生物物质的2-吡咯烷酮的制备方法
CN107438667A (zh) * 2015-04-13 2017-12-05 韩国科学技术院 内酰胺的制备方法
CN108191731A (zh) * 2017-12-25 2018-06-22 恒天生物基材料工程技术（宁波）有限公司 一种基于生物质原料氨基丁酸的新型丁内酰胺制备工艺

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BERNAL V., ARENSE P., BLATZ V., MANDRAND-BERTHELOT M.A., CÁNOVAS M., IBORRA J.L.: "Role of betaine:CoA ligase (CaiC) in the activation of betaines and the transfer of coenzyme A in Escherichia coli", JOURNAL OF APPLIED MICROBIOLOGY, WILEY-BLACKWELL PUBLISHING LTD., GB, vol. 105, no. 1, 1 July 2008 (2008-07-01), GB , pages 42 - 50, XP093146979, ISSN: 1364-5072, DOI: 10.1111/j.1365-2672.2008.03740.x *
DATABASE Protein 7 March 2022 (2022-03-07), ANONYMOUS : "MULTISPECIES: crotonobetaine/carnitine-CoA ligase [Escherichia] ", XP093146980, retrieved from NCBI Database accession no. WP_001350478.1 *

Also Published As

Publication number Publication date
US20240132925A1 (en) 2024-04-25
CN115725669A (zh) 2023-03-03

Similar Documents

Publication Publication Date Title
CN112961875B (zh) 一种生物法生产四氢嘧啶的工程菌株的构建方法
CN104388373A (zh) 一种共表达羰基还原酶和葡萄糖脱氢酶的大肠杆菌系统的构建
CN108070581B (zh) 一种酶活提高的L-天冬氨酸β-脱羧酶突变体及其应用
CN105624128B (zh) 一种固定化单胺氧化酶及其在合成手性氮杂双环化合物中的应用
CN112708589A (zh) 一种基因工程菌及其构建方法与在发酵产5-羟基色氨酸上的应用
CN108359626B (zh) 一种工程菌及其在制备(r)-3-羟基-5-己烯酸酯中的应用
WO2024050928A1 (zh) 一种吡咯烷酮的制备方法
CN111394289B (zh) 一种基因工程菌及其应用，生产前列腺素e2的方法
CN116814572A (zh) 一种羰基还原酶及其突变体及其在制备手性(r)-8-氯-6-羟基辛酸乙酯中的应用
WO2022088263A1 (zh) 一种高效生产琥珀酸的重组大肠杆菌及其构建方法
CN113502305B (zh) 一种利用重组酰亚胺酶合成(r)-异丁基戊二酸单酰胺的方法
CN112481320B (zh) 一种催化效率高的制备(-)γ-内酰胺的方法
CN101469318A (zh) (r)-羰基还原酶与甲酸脱氢酶偶联促进(r)-苯基乙二醇的合成
CN110804602B (zh) 一种L-天冬氨酸β-脱羧酶突变体及其应用
CN102140483A (zh) 一种固定化酶合成l-色氨酸的方法
CN113215120A (zh) 重组大肠杆菌转化生产反式-4-羟基-l-脯氨酸的方法
WO2023092632A1 (zh) 高效生产戊二酸的重组大肠杆菌及其构建方法与应用
CN112195202B (zh) 一种s-吲哚啉-2-羧酸的生物催化制备方法
CN115820610B (zh) 氨肽酶重组菌、氨肽酶及其制备方法和应用
CN111575334B (zh) 一种制备(s)-2-氯-1-(3,4-二氟苯基)乙醇的方法
CN110628849A (zh) 氧化态烟酰胺类辅因子再生的方法
CN111944776B (zh) 一种羰基还原酶及其应用
CN112442474B (zh) 一种(-)γ-内酰胺的制备方法
WO2021128432A1 (zh) 一种l-阿拉伯糖异构酶异构体及其应用
CN105200088A (zh) 一种酶法转化dl-2-氨基-△2-噻唑啉-4-羧酸合成l-半胱氨酸的方法

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: 22957894

Country of ref document: EP

Kind code of ref document: A1