Patent application title: GDSL LIPASE, GENETICALLY-ENGINEERED BACTERIA AND APPLICATION THEREOF
Inventors:
IPC8 Class: AC12N920FI
USPC Class:
Class name:
Publication date: 2022-02-03
Patent application number: 20220033789
Abstract:
The invention relates to a GDSL lipase, genetically-engineered bacteria
and an application thereof. The GDSL lipase is derived from Streptomyces
diastaticus CS1801 and its amino acid sequence is as shown in SEQ ID
NO.2. After construction of a genetically-engineered bacterium strain, a
GDSL lipase is generated through fermentation. Through this enzyme,
vitamin A and palmitic acid are converted to produce vitamin A palmitate.
The content of the vitamin A palmitate obtained from the conversion is
16.35 mg/L at most. The conversion efficiency is 81.75% at most. This
lipase provides a new path to synthesize vitamin A palmitate by the
enzymatic method and has an important application prospect.Claims:
1. A GDSL lipase, wherein its amino acid sequence is as shown in SEQ ID
NO.2.
2. A gene encoding the GDSL lipase as in claim 1.
3. The gene according to claim 2, wherein its nucleotide sequence is as shown in SEQ ID NO.1
4. A recombinant vector containing the gene as in claim 3.
5. The recombinant vector according to claim 4, wherein its expression vector is pET 32a(+).
6. An engineered bacterium containing the recombinant vector as in claim 5.
7. The engineered bacterium according to claim 6, wherein the host cell is E. coli BL21(DE3).
8. An application of the engineered bacterium as in claim 6 in the production of vitamin A palmitate, including: (1) inoculating the engineered bacterium to an LB medium for seed culture; (2) transferring the seed solution to a fermentation medium for fermentation culture, and then adding an inducer to induce expression of enzymes; (3) centrifuging the fermentation broth to obtain a supernate, and obtaining enzyme powder through precipitation by ammonium sulfate and lyophilization; and (4) adding the enzyme powder to an organic phase system containing vitamin A and palmitic acid to produce vitamin A palmitate.
9. The application according to claim 8, wherein the fermentation medium comprises: tryptone 10 g/L, yeast powder 5 g/L, NaCl 10 g/L, MgSO.sub.4.7H.sub.2O 1 g/L, KH.sub.2PO.sub.4 0.5 g/L, K.sub.2HPO.sub.4 0.5 g/L, olive oil emulsion 12 mL/L, and distilled water added till volume 1 L.
10. The application according to claim 9, wherein 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.
Description:
TECHNICAL FIELD
[0001] 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.
BACKGROUND ART
[0002] 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.
[0003] 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.
SUMMARY OF THE INVENTION
[0004] 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.
[0005] In order to achieve the above object, the present invention adopts the following technical solution:
[0006] A GDSL lipase, wherein its amino acid sequence is as shown in SEQ ID NO.2.
[0007] The present invention further provides a gene encoding the foregoing GDSL lipase.
[0008] Specifically, the nucleotide sequence of the gene encoding the GDSL lipase is as shown in SEQ ID NO.1.
[0009] The foregoing GDSL lipase is derived from Streptomyces diastaticus CS1801, which has been disclosed in the applicant's prior application CN109337843A.
[0010] 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 (+).
[0011] The present invention further provides a genetically-engineered bacterium containing the foregoing recombinant vector.
[0012] Further, the host cell of the engineered bacterium is E. coli BL21(DE3).
[0013] The present invention further provides an application of the foregoing engineered bacterium in the production of vitamin A palmitate by the enzymatic method, including:
[0014] (1) inoculating the engineered bacterium to an LB medium for seed culture;
[0015] (2) transferring the seed solution to a fermentation medium for fermentation culture, and then adding an inducer to induce expression of enzymes;
[0016] (3) centrifuging the fermentation broth to obtain a supernate, and obtaining enzyme powder through precipitation by ammonium sulfate and lyophilization; and
[0017] (4) adding the enzyme powder to an organic phase system containing vitamin A and palmitic acid to produce vitamin A palmitate.
[0018] Specifically, the method includes: inoculating the engineered bacteria cultured in an LB medium at 37.degree. C., 200 r for 8.about.12 h to a fermentation medium in an inoculum size of 5%; fermenting them for 8.about.12 h, then adding IPTG till a final concentration of 0.4.about.1 mmol/L and fermenting and culturing at 37.degree. C., 200 r for 18.about.24 h; centrifuging at 4000 r for 10 min to obtain a supernate of the fermentation broth; precipitating zymoprotein by 50% ammonium sulfate and lyophilizing the precipitate for 48 h to obtain enzyme powder; and adding the enzyme powder to an organic phase system (vitamin A: palmitic acid=10 g: 10 g, dissolved in 1 L of normal hexane) at a ratio of 5%, and determining the content of vitamin A palmitate and calculating the conversion rate after a specific time of conversion.
[0019] Further, the fermentation medium comprises:
[0020] tryptone 10 g/L, yeast powder 5 g/L, NaCl 10 g/L, MgSO.sub.4.7H.sub.2O 1 g/L, KH.sub.2PO.sub.4 0.5 g/L, K.sub.2HPO.sub.4 0.5 g/L, and olive oil emulsion 12 mL/L.
[0021] Further, 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.
[0022] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 shows target strips of PCR amplification of GDSL lipase;
[0024] FIG. 2 is an SDS-PAGE electrophoretogram of E. coli;
[0025] FIG. 3 shows the conversion time of GDSL lipase in the production of vitamin A palmitate.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Embodiment 1
[0026] This embodiment describes a method for PCR amplification of GDSL lipase derived from Streptomyces diastaticus.
[0027] 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.degree. C. for 2.about.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.
[0028] The primers for GDSL lipase gene amplification are designed as follows:
TABLE-US-00001 GDSL2-up: 5' -GTGGCCGGGCTCACGTCCTC-3' GDSL2-down: 5' -TCATTCCGGCAGGCTCCG-3'
[0029] 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.
[0030] The specific amplification procedure is as follows: pre-denature at 95.degree. C. for 10 min and add Taq enzyme; denature at 95.degree. C. for 1 min, anneal at 55.degree. C. for 30 s, extend at 72.degree. C. for 1 min, repeat this process for 29 cycles and lastly extend at 72.degree. 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.
Embodiment 2
[0031] This embodiment describes the PCR amplification method of GDSL lipase gene with restriction enzyme cutting sites.
[0032] The primers for amplification of GDSL lipase gene with restriction enzyme cutting sites are designed as follows:
TABLE-US-00002 GDSL2-up: 5' -CCGGAATTCGTGGCCGGGCTCACGTCCTC-3' GDSL2-down: 5' -CCGCTCGAGTCATTCCGGCAGGCTCCG-3'
[0033] 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.
[0034] The specific amplification procedure is as follows: pre-denature at 95.degree. C. for 2 min; denature at 95.degree. C. for 1 min, anneal at 55.degree. C. for 30 s, extend at 72.degree. C. for 1 min, repeat this process for 29 cycles and lastly extend at 72.degree. 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.
Embodiment 3
[0035] This embodiment describes a method for constructing a recombinant cloning vector of GDSL lipase.
[0036] 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.
Embodiment 4
[0037] This embodiment describes a method for constructing a recombinant expression vector of GDSL lipase.
[0038] 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.
Embodiment 5
[0039] This embodiment describes a method for constructing genetically-engineered bacteria of GDSL lipase.
[0040] 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. Compared with other lanes, obvious strips are found at molecular weight 44k Da. After removal of the fusion expression protein on the plasmid, it is consistent with the predicted target protein in size, suggesting that GDSL lipase is successfully expressed in recombinant bacteria.
Embodiment 6
[0041] This embodiment describes an application of genetically-engineered bacteria of GDSL lipase in vitamin A palmitate.
[0042] (1) Inoculate the genetically-engineered bacteria cultured in an LB medium at 37.degree. C., 200 r for 8.about.12 h to a fermentation medium in an inoculum size of 5%.
[0043] (2) Ferment them for 8.about.12 h, add IPTG till a final concentration of 0.4.about.1 mmol/L, and ferment and culture at 37.degree. C., 200 r for 18.about.24 h.
[0044] (3) Centrifuge at 4000 r for 10 min to get a supernate of the fermentation broth; use a 50% ammonium sulfate solution to precipitate zymoprotein, lyophilize it for 48 h to obtain enzyme powder, and determine the enzyme activity of GDSL lipase according to the national standard GBT23535-2009, which is 1.53 U/mg.
[0045] (4) Add 5% (w/v) enzyme powder to an organic phase system (vitamin A: palmitic acid=10 g: 10 g, dissolved in 1 L of normal hexane), determine the content of vitamin A palmitate after 2, 4, 6, 8 and 10 h of conversion and calculate the conversion rates. As shown in FIG. 3, after 8 h of conversion, the content of vitamin A palmitate is 16.35 mg/L at most and the conversion rate is 81.75%.
[0046] The fermentation medium comprises:
[0047] tryptone 10 g/L, yeast powder 5 g/L, NaCl 10 g/L, MgSO.sub.4.7H.sub.2O 1 g/L, KH.sub.2PO.sub.4 0.5 g/L, K.sub.2HPO.sub.4 0.5 g/L, olive oil emulsion 12 mL/L, and distilled water added till volume 1 L.
[0048] 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.
[0049] The vitamin A palmitate is determined by HPLC and quantitatively determined by the external standard method. Chromatographic conditions: chromatographic column: Alltech C18 (250.times.4.6 mm, 4.5 .mu.m); mobile phase: 100% methanol; detector: Shimadzu 10A ultraviolet detector; detection wavelength: 327 nm; flow rate: 1 mL/min.
[0050] Calculation formula of conversion rate:
Conversion .times. .times. rate = Vitamin .times. .times. A .times. .times. palmitate .times. .times. ( g / L ) ( Vitamin .times. .times. A .function. ( g ) + palmitic .times. .times. acid .times. .times. ( g ) ) / n - hexane .times. .times. ( L ) .times. 100 .times. % . ##EQU00001##
Sequence CWU
1
1
21741DNAArtificial SequenceCDS(1)..(741)The sequence is synthesized 1gtg
gcc ggg ctc acg tcc tcg gtg ccg cgc cgc tgg gag atg atg ctg 48Val
Ala Gly Leu Thr Ser Ser Val Pro Arg Arg Trp Glu Met Met Leu1
5 10 15ccc atg cgc ttc ctc ttc gtc
ggc gac tcc atg acg gtc ggc cgg gcc 96Pro Met Arg Phe Leu Phe Val
Gly Asp Ser Met Thr Val Gly Arg Ala 20 25
30ggg gac ttc acc tgg cgc cac cgc atg tgg cag cac ctg gag
acg acc 144Gly Asp Phe Thr Trp Arg His Arg Met Trp Gln His Leu Glu
Thr Thr 35 40 45ctc ggc ccc ggc
gcg tac acc atc acc ggc ccc cgc acc ggc ctg tac 192Leu Gly Pro Gly
Ala Tyr Thr Ile Thr Gly Pro Arg Thr Gly Leu Tyr 50 55
60gcg ggc gac ggc gcc gac gcc tcc gag gcg tac gcg gac
ccc gcc ttc 240Ala Gly Asp Gly Ala Asp Ala Ser Glu Ala Tyr Ala Asp
Pro Ala Phe65 70 75
80ccg ccc gcc gcg cgc cgc cac ctc gcg ggc tgg ggc gag ggg tgg cgg
288Pro Pro Ala Ala Arg Arg His Leu Ala Gly Trp Gly Glu Gly Trp Arg
85 90 95cac atg gcc ccg ctg atc
cag ccg gtc gtc gcc acc acc cgc gcc gac 336His Met Ala Pro Leu Ile
Gln Pro Val Val Ala Thr Thr Arg Ala Asp 100
105 110gtg ctg ctg gtc gcc ctc ggc ctg atc gac ctc ggc
ttc tac gcc cac 384Val Leu Leu Val Ala Leu Gly Leu Ile Asp Leu Gly
Phe Tyr Ala His 115 120 125gcc gag
gag acc gcc gag cac gcc cgg acc ttc ctg tcc cgg gcc cgc 432Ala Glu
Glu Thr Ala Glu His Ala Arg Thr Phe Leu Ser Arg Ala Arg 130
135 140gcc gcc aag ccg gac gta cgc gcc gtc atc ctc
ccg gtc gtc ccc aac 480Ala Ala Lys Pro Asp Val Arg Ala Val Ile Leu
Pro Val Val Pro Asn145 150 155
160gtc cgc gcc cgc acc gac ccc ttc ttc gcc gac gac tgc gcc cgc ttc
528Val Arg Ala Arg Thr Asp Pro Phe Phe Ala Asp Asp Cys Ala Arg Phe
165 170 175aac acg ctc ctc gcc
aag acc gtc gcc gag ctg gac cgc ccc ggc tcc 576Asn Thr Leu Leu Ala
Lys Thr Val Ala Glu Leu Asp Arg Pro Gly Ser 180
185 190ccg ctc ctg ctc gcc tcc cac ccg ccc ggc tac acc
ctg gac gcc gac 624Pro Leu Leu Leu Ala Ser His Pro Pro Gly Tyr Thr
Leu Asp Ala Asp 195 200 205acc tac
gac ggc acc cat ccc ggt ccc tcc ggc gaa cac cgc atc gcc 672Thr Tyr
Asp Gly Thr His Pro Gly Pro Ser Gly Glu His Arg Ile Ala 210
215 220gcc gcc ttc gcc gac gcg ctg cac cag ggc tgg
ggc gtc ggc ggg ccg 720Ala Ala Phe Ala Asp Ala Leu His Gln Gly Trp
Gly Val Gly Gly Pro225 230 235
240tac cgg agc ctg ccg gaa tga
741Tyr Arg Ser Leu Pro Glu 2452246PRTArtificial
SequenceThe sequence is synthesized 2Val Ala Gly Leu Thr Ser Ser Val Pro
Arg Arg Trp Glu Met Met Leu1 5 10
15Pro Met Arg Phe Leu Phe Val Gly Asp Ser Met Thr Val Gly Arg
Ala 20 25 30Gly Asp Phe Thr
Trp Arg His Arg Met Trp Gln His Leu Glu Thr Thr 35
40 45Leu Gly Pro Gly Ala Tyr Thr Ile Thr Gly Pro Arg
Thr Gly Leu Tyr 50 55 60Ala Gly Asp
Gly Ala Asp Ala Ser Glu Ala Tyr Ala Asp Pro Ala Phe65 70
75 80Pro Pro Ala Ala Arg Arg His Leu
Ala Gly Trp Gly Glu Gly Trp Arg 85 90
95His Met Ala Pro Leu Ile Gln Pro Val Val Ala Thr Thr Arg
Ala Asp 100 105 110Val Leu Leu
Val Ala Leu Gly Leu Ile Asp Leu Gly Phe Tyr Ala His 115
120 125Ala Glu Glu Thr Ala Glu His Ala Arg Thr Phe
Leu Ser Arg Ala Arg 130 135 140Ala Ala
Lys Pro Asp Val Arg Ala Val Ile Leu Pro Val Val Pro Asn145
150 155 160Val Arg Ala Arg Thr Asp Pro
Phe Phe Ala Asp Asp Cys Ala Arg Phe 165
170 175Asn Thr Leu Leu Ala Lys Thr Val Ala Glu Leu Asp
Arg Pro Gly Ser 180 185 190Pro
Leu Leu Leu Ala Ser His Pro Pro Gly Tyr Thr Leu Asp Ala Asp 195
200 205Thr Tyr Asp Gly Thr His Pro Gly Pro
Ser Gly Glu His Arg Ile Ala 210 215
220Ala Ala Phe Ala Asp Ala Leu His Gln Gly Trp Gly Val Gly Gly Pro225
230 235 240Tyr Arg Ser Leu
Pro Glu 245
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