Gene for efficiently expressing CALB lipase and application thereof

Abstract

The invention discloses a gene for efficiently expressing CALB lipase and application of the gene, and belongs to the technical field of bioengineering, the gene is CALB-S and has a nucleotide sequence as shown in SEQ ID NO.1. Gene optimization is performed on the basis of wild-type candida antarctica, and the CALB-S has a nucleotide sequence as shown in SEQ ID NO.2. The CALB-S has a nucleotide sequence as shown in SEQ ID NO.1. The constructed pichia pastoris engineering bacterium has the advantages of simple culture conditions and simple and convenient operation process, has a remarkable effect of improving the esterification function of the CALB lipase, has utilization potential, and provides important experimental data and theoretical support for efficient expression of the CALB lipase; by constructing the recombinant engineering bacteria, the yield of ethyl hexanoate is increased, the cost is low, the operation is simple, and the application in industrial production is facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a gene for efficiently expressing CALB lipase and an application thereof. Background Art

[0002] Chinese liquor is one of the world's six famous distilled liquors. After a long history of development, it has formed four basic flavors: light fragrance, sauce fragrance, strong fragrance, and rice fragrance. Among them, strong-flavor liquor accounts for more than 70% of the market share. It has the flavor characteristics of mellow body, long aftertaste, and lingering fragrance in the empty cup. Among them, ethyl hexanoate has fruity, floral and sweet aromas, and is recognized as the main aroma component of strong-flavor Daqu liquor.

[0003] The synthesis of ethyl caproate in Luzhou-aroma Daqu is mainly based on the enzymatic reaction of esterifying enzymes such as lipase. In the current liquor production, the method of adding lipase as a catalyst is often used to catalyze the synthesis of ester substances such as ethyl caproate, which has the advantages of mild reaction conditions, high conversion rate, few by-products, and easy extraction.

[0004] At present, Antarctic Candida lipase B (abbreviated as "CALB") derived from Antarctic Candida isolated from the Antarctic continent is widely used. It shows strong catalytic activity in various catalytic reactions such as hydrolysis, esterification, and transesterification in water-soluble and water-insoluble substances. Therefore, it is used in many fields such as food processing, organic synthesis, and biodiesel.

[0005] However, the low yield of the enzyme in the original strain of Candida antarctica, the long fermentation cycle and the high separation cost are not conducive to industrial utilization. Therefore, it is crucial to explore methods for efficiently expressing CALB. Summary of the invention

[0006] One of the purposes of the present invention is to provide a gene for efficiently expressing CALB lipase to solve the above problems.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows: a gene for efficiently expressing CALB lipase, the gene is CALB-S, which has a nucleotide sequence as shown in SEQ ID NO.1.

[0008] The inventors of the present invention have proved through a large number of experiments that by replacing rare codons with optimal codons, adjusting the GC content and other methods to optimize codons, a gene fragment capable of efficiently expressing CALB lipase is obtained.

[0009] The second object of the present invention is to provide an application of the CALB-S gene in the efficient expression of CALB lipase.

[0010] As a preferred technical solution, the CALB-S gene is used to construct a recombinant engineered bacterium that efficiently expresses CALB lipase.

[0011] As a further preferred technical solution, the method for constructing the recombinant engineered bacteria comprises the following steps: cloning the CALB-S gene, constructing a recombinant vector pPIC9K- CALB-S , transfer the vector into Pichia pastoris for induced expression.

[0012] Pichia pastoris is a unicellular eukaryotic organism with the advantages of fast growth, easy cultivation, simple genetic manipulation, easy fermentation control, and no endotoxin production. Therefore, the recombinant strain constructed in this application has good application potential in the industrial application of CALB lipase.

[0013] As a further preferred technical solution, the Pichia pastoris is Pichia pastoris GS115 The third object of the present invention is to provide an application of the above-mentioned recombinant engineering bacteria for flavor enhancement of Luzhou-flavor liquor. The recombinant engineering bacteria can produce ethyl caproate in high yield, and thus can be used for flavor enhancement of Luzhou-flavor liquor.

[0014] The present invention utilizes gene CALB-S Construction of recombinant engineering bacteria with high expression of CALB lipase, gene CALB-S From the strain purchased from CGMCC - Antarctic Candida ( Candida Antarctica ) and optimized using optipyzer. CALB-S Gene, construction of recombinant vector pPIC9K- CALB-S , transfer the vector into Pichia pastoris The expression was induced in GS115 and verified by SDS-PAGE. CALB-S The engineered strain of the recombinant vector was distilled in a caproic acid and ethanol reaction system to obtain 28.68 g / L of ethyl caproate, which greatly improved the caproic acid conversion rate in the production of Luzhou-flavor liquor. The present invention provides important data for further studying the efficient expression of CALB lipase and its ability to esterify caproic acid and ethanol.

[0015] Compared with the prior art, the advantages of the present invention are as follows: the present invention adopts optimized genes to construct Pichia pastoris engineered bacteria, which has the advantages of simple culture conditions and simple operation procedures, and has a significant effect on improving the esterification function of CALB lipase, has utilization potential, and provides important experimental data and theoretical support for the efficient expression of CALB lipase; by constructing recombinant engineered bacteria, the yield of ethyl hexanoate is increased, the cost is low, the operation is simple, and it can be used in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 for CALB-S Gene amplification map, Figure 1 Middle: M is the DNA molecular weight standard (DL10000); 1 and 2 are CALB-S PCR amplification products of genes; Figure 2 For pPIC9K- CALB-S Recombinant plasmid map; Figure 3 For pPIC9K- CALB-S Recombinant plasmid Sac I single enzyme digestion map, Figure 3 Middle: M is DNA molecular weight standard (DL15000); 1 is pPIC9K- CALB-S Linearization product; Figure 4 for CALB-S Expression profile of the coded protein, Figure 4 In the figure, M is a protein marker, and 1-2 are the expression products induced by the recombinant plasmid transformed bacteria; Figure 5 For wild-type CALB lipase and CALB-S Comparison of lipase esterification capabilities. DETAILED DESCRIPTION

[0017] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0018] In the following examples, the materials and reagents used are: Candida antarctica ( Candida Antarctica ) were purchased from CGMCC, vector pPIC9K and expression strain Pichia pastoris GS115 were purchased from Chengdu Qingkezixi Biotechnology Co., Ltd.

[0019] YPD liquid medium: yeast powder 10g / L, peptone 20g / L, glucose 20g / L; YPD solid medium: yeast powder 10g / L, peptone 20g / L, glucose 20g / L, agar 20g / L; BMGY liquid medium: yeast powder 10g / L, peptone 20g / L, glycerol 10mL / L, potassium phosphate buffer 100mL / L, add 500×D-biotin 2mL / L, 10×YNB 100mL / L before use; BMMY liquid medium: yeast powder 10 g / L, peptone 20 g / L, potassium phosphate buffer 100 mL / L, add 500×D-biotin 2 mL / L, 10×YNB 100 mL / L, anhydrous methanol 50 mL / L before use; MD solid medium: glucose 20 g / L, agar 15 g / L, add 500×D-biotin 2 mL / L, 10×YNB 100 mL / L before use. Example

[0020] A gene for efficiently expressing CALB lipase, having a nucleotide sequence as shown in SEQ ID NO.1, The method of obtaining and verifying it: (1) Based on CALB The original gene sequence (GenBank accession No. Z30645.1) was optimized using optipyzer without changing the protein amino acid sequence. Eco R Ⅰ and Not I restriction endonuclease site, and obtain the nucleotide sequence shown in SEQ ID NO.1; (2) Based on CALB-S Gene sequence synthesis primers; pPIC9K universal primers AOX1 and AOX2 were used, and the primer sequences are as follows: Upstream primer AOX1: 5′-GACTGGTTCCAATTGACAAGC-3′ Downstream primer AOX2: 5′-GGCAAATGGCATTCTGACATCC-3′ The above were synthesized by Chengdu Qingke Zixi Biotechnology Co., Ltd.; (3) CALB-S Gene cloning Chengdu Qingke Zixi Biotechnology Co., Ltd. was commissioned to artificially synthesize the corresponding sequence of the target gene and connect it with the vector pPIC9K, and then transferred into E. coli DH5α competent cells for plasmid amplification. 100 μL of the transformation solution was spread on an LB plate containing 100 μg / mL ampicillin sodium, cultured at 37°C overnight, and a single colony was picked and inoculated into LB liquid culture medium containing 100 μg / mL ampicillin sodium, and cultured at 37°C and 180 rpm for 10-12 hours. The plasmid was extracted for PCR verification: PCR reaction system 25uL: 2×Rapid Taq Master Mix 12.5 μL, sterile ddH2O 9.5 μL, CALB-F 1 μL, CALB-R 1 μL, genomic DNA 1 μL. PCR reaction conditions: 95 ℃ pre-denaturation for 3 min, enter the cycle; 95 ℃ denaturation for 15 s; 53 ℃ annealing for 15 s; 72 ℃ extension for 23 s; 35 cycles; 72 ℃ extension for 5 min; pPIC9K- CALB-S Recombinant plasmid map Figure 2 As shown; (4) CALB-S Inducible gene expression; The verified pPIC9K- CALB-S Recombinant plasmid use Sac After linearization, electroporation Pichia pastoris GS115, screened using MD solid medium, cultured at 30°C for 2-3 days until clearly visible colonies appeared. Pick a single colony and inoculate it into a 5 mL YPD liquid medium test tube, cultured at 30°C, 180 rpm / min for 18-24 h, inoculated the bacterial solution into 50 mL BMGY liquid medium at a ratio of 1:50, and cultured at 30°C, 180 rpm / min until OD 600 =4-6, centrifuged at 4000×g for 10 min at 4°C, the cells were resuspended in 50 mL BMMY liquid medium, 1% volume of anhydrous methanol was added to the culture medium every 24 h, and the expression was induced at 30°C for 120 h; Collect all bacterial liquid in a 50 mL centrifuge tube, centrifuge at 4000×g for 10 min at 4°C, take 400 μL of supernatant in a 1.5 mL EP tube, add 100 μL of 5x loading buffer and mix evenly, boil in a water bath for 3-5 min, and centrifuge at 12000 rpm / min for 5 min. Wash the sample wells with ddH2O, pour off the ddH2O, and dry with absorbent paper; take 20 μL of the above sample solution for SDS-PAGE analysis; stain the SDS-PAGE gel with the prepared Coomassie Brilliant Blue staining solution, and then decolorize with decolorizing solution. Example

[0021] The preparation of the recombinant engineering strain and its functional verification include the following steps: (1) CALB-S Cloning of genes; Using the sequence-optimized genomic DNA as a template, PCR amplification was performed using the upstream primer AOX1 and the downstream primer AOX2 shown in step (2) of Example 1. After purification, a fragment of about 1500 bp was obtained. The electrophoresis of the amplified product is shown in FIG. Figure 1 As shown; (2) CALB-S Inducible expression of proteins; Extract the recombinant plasmid containing CALB-S The recombinant plasmid was verified by enzyme digestion and PCR. The results are as follows Figure 3 As shown, the sequencing results show that the fragment size is the same as the target gene size, proving that it contains CALB-S Gene expression vector pPIC9K- CALB-S The construction was successful. After electroporation and methanol induction, CALB-S Gene expression vector pPIC9K- CALB-S exist Pichia pastoris It was successfully expressed in GS115 with a molecular weight of approximately 33 kDa, which is consistent with the expected target protein size (34.64 kDa). Figure 4 As shown, it shows that under the induction of methanol, CALB-S Able to be heterologously expressed in Pichia pastoris; (3) CALB-S Esterification Functional Studies The original Antarctic Candida and the recombinant engineered bacteria were induced with methanol for 120 h, 100 mL of hexanoic acid, 300 mL of ethanol and 200 mL of distilled water were taken, 100 mL of crude enzyme solution was added for distillation, and 500 g of sample was collected for gas chromatography analysis. Gas chromatography detection conditions: n-pentyl acetate was used as the internal standard, the chromatographic column was Pheromon ZB-WAXPLUS (30m*0.25mm, 0.25um), the initial temperature was 35 ℃, maintained for 4 min, and the temperature was increased to 70, 106, and 226 ℃, respectively, maintained for 0.5, 2.5, and 8 min, the detector temperature was 250 ℃, and the injector temperature was 250 ℃.

[0022] The results are as follows Figure 5 As shown, from Figure 5 As can be seen from the figure, wild-type CALB lipase catalyzes the production of 2.3 g / L ethyl hexanoate, with a hexanoic acid conversion rate of 0.61%. CALB-S The gene-induced lipase esterification produced 28.68 g / L ethyl hexanoate, and the hexanoic acid conversion rate was 16.4%. The results showed that the constructed Pichia pastoris containing the recombinant vector pPIC9K-CALB-S had a high efficiency in esterification of hexanoic acid and ethanol, and was much higher than the wild-type CALB.

[0023] The above embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

[0024] CALB-S gene sequence (1002bp):

Claims

1. A gene for efficiently expressing CALB lipase, characterized in that: The gene is CALB-S, which has a nucleotide sequence as shown in SEQ ID NO.

1.

2. Use of the CALB-S gene according to claim 1 in the efficient expression of CALB lipase.

3. The use according to claim 2, characterized in that: The CALB-S gene is used to construct a recombinant engineering bacterium that efficiently expresses CALB lipase.

4. The use according to claim 3, characterized in that: The method for constructing the recombinant engineering bacteria comprises the following steps: cloning the CALB-S gene, constructing a recombinant vector pPIC9K- CALB-S , transfer the vector into Pichia pastoris for induced expression.

5. The use according to claim 4, characterized in that: The Pichia yeast is Pichia pastoris GS115.

6. The use of the recombinant engineered bacteria according to claim 3, characterized in that: Used to enhance the flavor of Luzhou-flavor liquor.

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