GDSL LIPASE, GENETICALLY-ENGINEERED BACTERIA AND APPLICATION THEREOF

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

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° 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.

[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° 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.

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° 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.

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° C., 200 r for 8˜12 h to a fermentation medium in an inoculum size of 5%.

[0043] (2) Ferment them for 8˜12 h, add IPTG till a final concentration of 0.4˜1 mmol/L, and ferment and culture at 37° C., 200 r for 18˜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 (250×4.6 mm, 4.5 μm); mobile phase: 100% methanol; detector: Shimadzu 10A ultraviolet detector; detection wavelength: 327 nm; flow rate: 1 mL/min.

[0050] Calculation formula of conversion rate:

[00001]$\mathrm{Conversion}\mathrm{rate}=\frac{\mathrm{Vitamin}A\mathrm{palmitate}\left(g/L\right)}{\left(\mathrm{Vitamin}A\left(g\right)+\mathrm{palmitic}\mathrm{acid}\left(g\right)\right)/n-\mathrm{hexane}\left(L\right)}\times 100\%.$