US20220033789A1 - Gdsl lipase, genetically-engineered bacteria and application thereof - Google Patents

Gdsl lipase, genetically-engineered bacteria and application thereof Download PDF

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US20220033789A1
US20220033789A1 US17/279,733 US201917279733A US2022033789A1 US 20220033789 A1 US20220033789 A1 US 20220033789A1 US 201917279733 A US201917279733 A US 201917279733A US 2022033789 A1 US2022033789 A1 US 2022033789A1
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vitamin
lipase
palmitate
gdsl
olive oil
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Limei Wang
Tiantian XU
Yamei JI
Bin Qi
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Changshu Institute of Technology
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    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • 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
    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • 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

Definitions

  • the present invention relates to the fields of genetic engineering and protein engineering and to a method for gene cloning and expression of a new type of GDSL lipase and an application in the production of vitamin A palmitate by the enzymatic method and pertains to a technology in the industrial microorganism field.
  • Lipase also known as triacylglycerol lipase, is a type of enzymes that can degrade natural oils into glycerol and free fatty acids. It is widely found in animals, plants and microorganisms. Microorganisms are an important source of lipase, mainly including Rhizopus, Aspergillus and Candida . According to the analysis of the amino acid sequences of different lipases and their basic biological properties, lipases can be divided into eight families, of which the second family is also known as a GDSL family.
  • GDSL lipase (lipase, EC 3.1.1.3) is a type of hydrolase that can hydrolyze various substrates such as thioesters, aryl esters, phospholipids and amino acids. As GDSL lipase is a new type of lipase, little research has been done on its expression and function. Because GDSL lipase has ester hydrolysis activity, people are deepening the research on it.
  • Vitamin A palmitate can help maintain normal visual function and participate in various metabolic activities to maintain the health of the organisms. It is currently the most commonly used vitamin A derivative and is widely used in various industries such as food, cosmetics and medicine. At present, vitamin A palmitate is synthesized mainly by the chemical method and the enzymatic method. The chemical method for the synthesis of vitamin A palmitate has problems such as environmental pollution and cost, while the enzymatic method features less pollution, high space-time yield and low cost.
  • the enzyme that is used to produce vitamin A palmitate is a lipase with ester hydrolysis activity.
  • the object of the present invention is to provide a GDSL lipase, genetically-engineered bacteria and an application thereof.
  • the lipase has high activity and can be used in the production of vitamin A palmitate.
  • the present invention adopts the following technical solution:
  • a GDSL lipase wherein its amino acid sequence is as shown in SEQ ID NO.2.
  • the present invention further provides a gene encoding the foregoing GDSL lipase.
  • nucleotide sequence of the gene encoding the GDSL lipase is as shown in SEQ ID NO.1.
  • GDSL lipase is derived from Streptomyces diastaticus CS1801, which has been disclosed in the applicant's prior application CN109337843A.
  • the present invention further provides a recombinant vector, comprising the gene encoding the GDSL lipase and an expression vector, and the nucleotide sequence of the gene is as shown in SEQ ID NO.1.
  • the expression vector is pET-32a (+).
  • the foregoing gene is inserted between multiple cloning sites of the expression vector pET-32a (+).
  • the present invention further provides a genetically-engineered bacterium containing the foregoing recombinant vector.
  • the host cell of the engineered bacterium is E. coli BL21(DE3).
  • the present invention further provides an application of the foregoing engineered bacterium in the production of vitamin A palmitate by the enzymatic method, including:
  • the fermentation medium comprises:
  • tryptone 10 g/L tryptone 10 g/L, yeast powder 5 g/L, NaCl 10 g/L, MgSO 4 .7H 2 O 1 g/L, KH 2 PO 4 0.5 g/L, K 2 HPO 4 0.5 g/L, and olive oil emulsion 12 mL/L.
  • an olive oil emulsifier is prepared by the following method: mixing olive oil emulsifier PVA with olive oil at a volume ratio of 3:1 and emulsifying the mixture by ultrasound.
  • the genetically-engineered bacterium constructed by the enzyme in the present invention is used to produce vitamin A palmitate by the enzymatic method.
  • the content of the vitamin A palmitate obtained from conversion is 16.35 mg/L at most.
  • the maximum conversion rate is 81.75%.
  • This lipase provides a new path to synthesize vitamin A palmitate by the enzymatic method and has an important application prospect.
  • FIG. 1 shows target strips of PCR amplification of GDSL lipase
  • FIG. 2 is an SDS-PAGE electrophoretogram of E. coli
  • FIG. 3 shows the conversion time of GDSL lipase in the production of vitamin A palmitate.
  • This embodiment describes a method for PCR amplification of GDSL lipase derived from Streptomyces diastaticus.
  • Streptomyces diastaticus CS1801 stored on a test tube slant is used for plate activation, and a single colony is inoculated to an LB liquid medium and cultured at 30° C. for 2 ⁇ 3 days.
  • the culture solution is centrifuged at 8000 r for 2 min, thalli are collected and a bacterial genome extraction kit is used for total genome extraction.
  • the extraction steps are described in the bacterial genome extraction kit manual of Sangon Biotech (Shanghai) Co., Ltd.
  • the primers for GDSL lipase gene amplification are designed as follows:
  • GDSL2-up 5′ -GTGGCCGGGCTCACGTCCTC-3′
  • GDSL2-down 5′ -TCATTCCGGCAGGCTCCG-3′
  • the extracted Streptomyces diastaticus genome is used as a template, and the above primers and a PCR amplification kit with high GC content from Sangon Biotech (Shanghai) Co., Ltd. are used for amplification, but no Taq enzyme is added.
  • the specific amplification procedure is as follows: pre-denature at 95° C. for 10 min and add Taq enzyme; denature at 95° C. for 1 min, anneal at 55° C. for 30 s, extend at 72° C. for 1 min, repeat this process for 29 cycles and lastly extend at 72° C. for 30 min; and take the product to perform agarose gel electrophoresis (AGE), cut gel and extract and store target strips.
  • the gel extraction kit is purchased from Sangon Biotech (Shanghai) Co., Ltd.
  • This embodiment describes the PCR amplification method of GDSL lipase gene with restriction enzyme cutting sites.
  • the primers for amplification of GDSL lipase gene with restriction enzyme cutting sites are designed as follows:
  • GDSL2-up 5′ -CCGGAATTCGTGGCCGGGCTCACGTCCTC-3′
  • GDSL2-down 5′ -CCGCTCGAGTCATTCCGGCAGGCTCCG-3′
  • Embodiment 1 The gel extraction product in Embodiment 1 is used as a template, and the above primers and a PCR amplification kit with high GC content from Sangon Biotech (Shanghai) Co., Ltd. are used for amplification.
  • the specific amplification procedure is as follows: pre-denature at 95° C. for 2 min; denature at 95° C. for 1 min, anneal at 55° C. for 30 s, extend at 72° C. for 1 min, repeat this process for 29 cycles and lastly extend at 72° C. for 30 min; and take the product to perform agarose gel electrophoresis (AGE), and cut gel, extract the target strips as shown in FIG. 1 and send them to Sangon Biotech (Shanghai) Co., Ltd. for sequence measurement to obtain sequence SEQ ID NO.1.
  • AGE agarose gel electrophoresis
  • This embodiment describes a method for constructing a recombinant cloning vector of GDSL lipase.
  • the gel extraction product in Embodiment 2 is linked to a T vector. After conversion to DH-5a, positive clones are picked for verification. After extraction of plasmid, the sequence is measured for verification.
  • This embodiment describes a method for constructing a recombinant expression vector of GDSL lipase.
  • XhoI and EcoRI are used to perform double digestion of the plasmid in Embodiment 3 and extract target strips, meanwhile XhoI and EcoRI are used to perform double digestion of pET32a(+) vector and extract large fragments in the vector, the extracted target gene fragments are linked to vector fragments and they are imported to host cell E. coli DH5a. After resistance screening, positive clones are picked to measure the sequence for verification.
  • This embodiment describes a method for constructing genetically-engineered bacteria of GDSL lipase.
  • the plasmid of the positive clones with a correct sequence in Embodiment 4 is extracted and directly converted and imported to host cell E. coli BL21 (DE3). Genetically-engineered bacteria of GDSL lipase are successfully constructed. In the fermentation process, an inducer like IPTG needs to be added to efficiently express GDSL lipase protein. Through SDS-PAGE, it is verified that the fusion protein is successfully expressed. SDS-PAGE electrophoretogram is as shown in FIG. 2 , lane 1 is E. coli pET32a-GDSL not induced, and lanes 2, 3 and 4 are recombinant E. coli pET32a-GDSL that has been induced by IPTG for 4, 8 and 16 h, respectively.
  • This embodiment describes an application of genetically-engineered bacteria of GDSL lipase in vitamin A palmitate.
  • the fermentation medium comprises:
  • tryptone 10 g/L tryptone 10 g/L, yeast powder 5 g/L, NaCl 10 g/L, MgSO 4 .7H 2 O 1 g/L, KH 2 PO 4 0.5 g/L, K 2 HPO 4 0.5 g/L, olive oil emulsion 12 mL/L, and distilled water added till volume 1 L.
  • the olive oil emulsion is prepared by the following method: mixing olive oil emulsifier PVA with olive oil at a volume ratio of 3:1 and emulsifying the mixture by ultrasound.
  • the vitamin A palmitate is determined by HPLC and quantitatively determined by the external standard method. Chromatographic conditions: chromatographic column: Alltech C18 (250 ⁇ 4.6 mm, 4.5 ⁇ m); mobile phase: 100% methanol; detector: Shimadzu 10A ultraviolet detector; detection wavelength: 327 nm; flow rate: 1 mL/min.

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US17/279,733 2019-03-15 2019-10-31 Gdsl lipase, genetically-engineered bacteria and application thereof Abandoned US20220033789A1 (en)

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CN201910199413.XA CN109777793B (zh) 2019-03-15 2019-03-15 一种gdsl脂肪酶、基因工程菌及其应用
CN201910199413.X 2019-03-15
PCT/CN2019/114789 WO2020186768A1 (zh) 2019-03-15 2019-10-31 一种gdsl脂肪酶、基因工程菌及其应用

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CN110564649B (zh) * 2019-09-27 2021-05-18 常熟理工学院 一株产脂肪酶菌株及其应用
CN113481186B (zh) * 2021-06-15 2023-01-03 常熟理工学院 一种GH18几丁质酶ChiA及其应用
CN114854717B (zh) * 2022-05-07 2023-08-11 万华化学集团股份有限公司 一种脂肪酶及其编码基因与应用

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JP2022505573A (ja) 2022-01-14
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JP7162374B2 (ja) 2022-10-28
CN109777793A (zh) 2019-05-21
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