Protein used as lipase as well as coding gene and application thereof

Abstract

The invention discloses a protein used as lipase as well as a coding gene and application thereof. The invention firstly discloses a protein used as lipase. The protein is a protein with an amino acid sequence as shown in the 21st-321st sites of SEQ ID NO.1 or a protein obtained by connecting a signal peptide to the N terminal or / and the C terminal of the amino acid sequence as shown in the 21st-321st sites of SEQ ID NO.1. The invention further discloses application of the protein as lipase. As lipase, the protein has the characteristics of being psychrophilic, neutral and resistant to organic solvents, and can meet various applications of the lipase.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a protein used as lipase, a coding gene and application thereof. Background Art

[0002] Lipase (Lipase, EC3.1.1.3), as a class of ester bond hydrolase, can catalyze the hydrolysis of triacylglycerol to generate glycerol and free fatty acids. The natural substrate of lipase is long-chain fatty acid ester, which can work in a two-phase system (oil, water interface) or in an aqueous phase. Lipase has position specificity and stereospecificity and can carry out multiple reactions such as esterification, transesterification, alcoholysis, acidolysis and aminolysis. Therefore, the application field of lipase is very extensive, such as including the fields of food, medicine, detergent, cosmetics, chemical industry, environmental protection, feed, energy development, leather, etc. In addition, lipase has also become the most widely used class enzyme in biotechnology and organic synthesis.

[0003] Different application fields require lipase to stably catalyze reactions under industrial production and practical application conditions such as high temperature, alkalinity, and the presence of organic solvents and surfactants. The diversification of application environments requires the development of diversified lipases that can meet various needs. Lipases are widely found in animals, plants, and microorganisms. At present, lipases from microorganisms have the advantages of rich variety and excellent enzymatic properties, and have become the main source of lipases for industrial production.

[0004] Existing lipases generally have only one or two good properties, such as low temperature, alkali resistance or organic solvent resistance, which limits the application of lipases. Since the actual application environment of lipases places stringent requirements on the various properties of lipases, and the discovery of new lipase genes is the basis of lipase application and genetic engineering modification, the development of new enzymes has always been the goal of lipase research. Summary of the invention

[0005] One object of the present invention is to provide a new protein which can be used as a lipase and has the characteristics of being psychrophilic, neutral and resistant to organic solvents, and can meet the various applications of lipase.

[0006] Another object of the present invention is to provide a preparation method and application of the above lipase.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention first provides a protein used as a lipase, wherein the protein is shown in A1) or A2) or A3) or A4):

[0009] A1) the protein whose amino acid sequence is shown in positions 21 to 321 of SEQ ID NO.1;

[0010] A2) a protein obtained by connecting a signal peptide to the N-terminus or / and the C-terminus of the amino acid sequence shown in positions 21-321 of SEQ ID NO.1;

[0011] A3) a fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of A1) or A2);

[0012] A4) A protein obtained by replacing and / or deleting and / or adding one or more amino acid residues of any of the proteins shown in A1) to A3) and having the same function.

[0013] Furthermore, the protein may be a protein whose amino acid sequence is shown in SEQ ID NO.1, including 321 amino acids, wherein the N-terminal 20 amino acids are its signal peptide sequence, and the sequence is shown in positions 1 to 20 of SEQ ID NO.1.

[0014] Furthermore, the coding gene of the above protein is also within the protection scope of the present invention, and the coding gene is as shown in a1) or a2) or a3) or a4):

[0015] a1) a DNA molecule whose nucleotide sequence is shown as positions 61-966 of SEQ ID NO.2;

[0016] a2) a DNA molecule having a nucleotide sequence as shown in SEQ ID NO.2 (wherein the coding sequence of the signal peptide is shown in positions 1 to 60 of SEQ ID NO.2);

[0017] a3) a DNA molecule that has 75% or more identity with the nucleotide sequence defined in a1) or a2) and encodes the above protein;

[0018] a4) A DNA molecule which hybridizes with the nucleotide sequence defined in a1) or a2) under stringent conditions and encodes the above-mentioned protein.

[0019] Furthermore, a recombinant vector comprising the above-mentioned coding gene is also within the protection scope of the present invention. The recombinant vector is the above-mentioned coding gene connected between the EcoRI and NotI restriction enzyme sites of pPIC9K, and the other sequences of pPIC9K are kept unchanged to obtain the recombinant vector pPIC9K-Pclip7.

[0020] Furthermore, a recombinant strain comprising the above-mentioned recombinant vector is also within the protection scope of the present invention, and the host cell of the recombinant strain is a Pichia pastoris strain, preferably a Pichia pastoris strain GS115.

[0021] The use of the above protein as lipase is also within the protection scope of the present invention.

[0022] The present invention further provides a method for preparing the above lipase, comprising the following steps:

[0023] (1) transforming a host cell with the above-mentioned recombinant vector to obtain a recombinant strain;

[0024] (2) culturing the recombinant strain and inducing lipase expression;

[0025] (3) Purify to obtain lipase.

[0026] The present invention further provides the application of the lipase in the fields of biotechnology, industrial production and food processing.

[0027] The beneficial effects of the present invention are as follows:

[0028] The protein of the present invention can be used as a lipase, which has the characteristics of being cold-loving, neutral and resistant to DSMO (DSMO is an important solvent for reactions such as ester and sugar ester synthesis, which is beneficial for the enzyme to be used in reactions such as ester synthesis and ester exchange), and has good stability at 50°C, which can meet the various applications of lipase in the fields of biotechnology, industrial production and food processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0030] Figure 1 This is a purification diagram of lipase Pclip7, where M: low molecular weight standard protein; 1: crude enzyme solution; 2: pure enzyme solution.

[0031] Figure 2 This is the optimal pH screening diagram for lipase Pclip7.

[0032] Figure 3 This is the pH stability diagram of lipase Pclip7.

[0033] Figure 4 This is the optimal temperature screening diagram for lipase Pclip7.

[0034] Figure 5 This is the temperature stability diagram of lipase Pclip7. DETAILED DESCRIPTION

[0035] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and accompanying drawings. Similar components in the accompanying drawings are represented by the same reference numerals. It should be understood by those skilled in the art that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0036] Example 1 Discovery and analysis of lipase Pclip7

[0037] 1. Discovery of the new enzyme Pclip7

[0038] A new enzyme Pclip7 was discovered through transcriptome sequencing analysis of Phanerochaete carnosa. The amino acid sequence of the new enzyme Pclip7 is shown in positions 21-321 of SEQ ID NO.1.

[0039] 2. Amino acid sequence analysis of the new enzyme Pclip7

[0040] The new enzyme Pclip7 shown in SEQ ID NO.1 at positions 21-321 was analyzed, and it was determined that Pclip7 belongs to the family III of lipid hydrolase. The highest consistency between the enzyme and the sequence of the registered protein in GenBank (Phanerochaete sordida, GJE98300.1) is 84.8%, so the enzyme is a new gene. It is predicted to be a lipase and named lipase Pclip7. At present, the functional characteristics and application research have not been carried out.

[0041] Example 2 Cloning and expression of lipase Pclip7

[0042] 1. Cloning of the gene encoding lipase Pclip7

[0043] The mRNA of Phanerochaete carnosa was extracted and reverse transcribed into cDNA. The cDNA was used as a template for PCR amplification and a PCR amplification product was obtained. The coding gene of lipase Pclip7 (shown in positions 61-966 of SEQ ID NO.2) was purified.

[0044] 2. Construction of recombinant vector

[0045] The purified PCR amplification product (i.e., the coding gene of lipase Pclip7) and pPIC9k plasmid were double-digested with EcoRⅠ and NotⅠ, respectively, and then ligated and transformed into TransI-T1 competent cells, and the transformants were selected for sequencing verification. The transformants that were correctly verified by sequencing were the recombinant yeast expression plasmid pPIC9K-Pclip7. The recombinant vector pPIC9K-Pclip7 was linearized with restriction endonuclease SacI and then electroporated into Pichia pastoris GS115 (Invitrogen, USA, catalog number C18100) to form the recombinant bacteria GS115 / pPIC9K-Pclip7.

[0046] 3. Expression of recombinant bacteria

[0047] The recombinant bacteria GS115 / pPIC9K-Pclip7 were coated on MD plates (2% glucose, 1.34% YNB, 4×10 -5 % biotin), and after culturing at 30℃ for 3-5 days, the transformants were picked and plated in BMGY medium (1% yeast extract, 2% tryptone, 100mM phosphate buffer at pH 7.5, 1.34% YNB, 4×10 -5 The cells were cultured in BMMY medium (1% yeast extract, 2% tryptone, 100 mM phosphate buffer at pH 7.5, 1.34% YNB, 4 × 10 -5 % biotin, 0.5% methanol) and added methanol every 12 h to a final concentration of 1%. The culture was induced at 220 rpm and 30°C to obtain the fermentation supernatant (i.e., crude enzyme solution).

[0048] 4. Purification of lipase PcLip7

[0049] The fermentation supernatant was dialyzed for 24 hours with 20mM Tris-HCl buffer (buffer A) at pH 7.5, and then separated and purified by HiTrapQ FF (purchased from Cytiva) strong anion exchange chromatography, with a sample loading flow rate of 1mL / min. After loading, the sample was eluted with buffer A until the eluate OD280 was <0.05 (flow rate of 1.0mL / min), and then linearly eluted with buffer A containing 0-500mM NaCl for 10 column volumes, and the enzyme solution (i.e., pure enzyme solution) was collected to obtain the purified lipase Pclip7 (sequence as shown in SEQ ID NO.1, 21-321), and the purity was detected by SDS-PAGE. The results are shown in Figure 1 As shown, the molecular weight of lipase Pclip7 is 31.5 kDa, which is consistent with the predicted molecular weight of 31.5. Lipase Pclip7 is an electrophoresis grade pure enzyme.

[0050] Example 3 Determination of enzyme activity and enzymatic properties of recombinant lipase Pclip7

[0051] Enzyme activity determination method: add 50μL of appropriately diluted enzyme solution to 400μL 50mM pH 7.5 Tris-HCl buffer, preheat in a 50℃ water bath for 3min, add 50μL p-nitrophenyl palmitate substrate (pNPP, 10mM, dissolved in isopropanol), react at 50℃ for 10min, add 500μL 95% ethanol to terminate the reaction, centrifuge at 12000rpm for 3min, take the supernatant and measure the absorbance at 410nm. Enzyme activity unit definition: under the above conditions, the amount of enzyme required to generate 1μmol p-nitrophenol per minute is one enzyme activity unit (U).

[0052] 1. Optimum pH and pH stability

[0053] Prepare 50 mM buffer solutions with different pH values ​​(Citrate-Na 2 HPO 4 , pH 5.5-7.0; Tris-HCl, pH 7.0-8.5; Gly-NaOH, pH 8.5-9.5), and the activity of lipase PcLip7 in different pH buffer solutions was determined by the enzyme activity determination method to determine the optimal pH of Pclip7; the enzyme solution was treated at 37°C for 1h at different pH values ​​(pH 2.0-10.0), and the activity of lipase Pclip7 was determined by the enzyme activity determination method in pH 7.5 50mM Tris-HCl as buffer solution to determine the pH stability of lipase Pclip7.

[0054] The results are as follows Figure 2 and 3 As shown, the lipase Pclip7 has the highest activity in 50 mM Tris-HCl buffer at pH 7.5 ( Figure 2 In terms of pH stability, when the pH value is in the range of 4.0 to 9.0, the relative residual enzyme of lipase Pclip7 is maintained above 60% ( Figure 3 ). Therefore, the optimum pH value of Pclip7 is 7.5, and the enzyme belongs to neutral lipase. Neutral lipase has important application value in improving food quality, flavor and extending the shelf life of food. Neutral lipase can improve the baking quality of flour products, improve the texture of bread, and extend the shelf life of products. In addition, neutral lipase can increase the content of acid and ester volatile flavor substances in cheese and improve the flavor; neutral lipase helps to quickly form the flavor of cured meat products and accelerate the maturation of cured meat products.

[0055] 2. Optimum temperature and temperature stability

[0056] The enzyme solution was appropriately diluted with 50 mM Tris-HCl buffer at pH 7.5, and the enzyme activity was determined at 10-70°C according to the enzyme activity determination method; the lipase Pclip7 was treated in a water bath at 40-70°C for 120 minutes, and the residual activity of the lipase Pclip7 was determined at 20°C.

[0057] The results of enzyme activity determination at 10-70°C are as follows Figure 4 As shown in Figure 2, the optimal reaction temperature of lipase Pclip7 is 20°C. The temperature stability results of lipase Pclip7 are shown in Figure 2 Figure 5As shown, after treatment at 50℃ for 60min, the remaining enzyme activity was 77.3%; after treatment at 60℃ for 60min, the remaining enzyme activity was 49.4%; after treatment at 70℃, the enzyme activity dropped rapidly and was almost completely inactivated after 30min. In the detergent industry, low-temperature lipase maintains a high catalytic activity at 10-40℃, which helps to improve the cleaning ability of detergents. In the production process of leather products, low-temperature lipase can be used to treat leather to improve its texture and appearance. In the papermaking industry, low-temperature lipase can be used to optimize the quality and performance of paper. In addition, the temperature stability of the enzyme meets the synthesis conditions of structural lipids or sugar esters, such as the synthesis temperature of sugar esters is generally 30-50℃, and the reaction time is generally less than 10h; the synthesis temperature of sugar esters is generally 30-55℃, and the reaction time is generally less than 24h.

[0058] Example 4 Substrate specificity of lipase Pclip7

[0059] The substrate specificity of the recombinant lipase Pclip7 to p-nitrophenyl ester was determined according to the enzyme activity determination method in Example 3. When the substrate specificity was determined using p-nitrophenyl ester as the substrate, the substrate concentration was 10 mM, and the reaction conditions were 20°C, 50 mM pH 7.5 Tris-HCl buffer for 10 min. Lipase Pclip7 can catalyze the degradation of p-nitrophenyl ester containing C2, C8, C10, C12, C14, C16 and C18 fatty chains. The enzyme has the highest hydrolysis activity for p-nitrophenyl decanoate, with a specific enzyme activity of 3.35 U / mg (Table 1). Lipase Pclip7 has high activity for medium and long carbon chain substrates and is a typical lipase.

[0060] Table 1 Substrate specificity of lipase Pclip7

[0061] Substrate Specific enzyme activity (U / mg) p-Nitrophenyl acetate (C2) 0.71±0.2 p-Nitrophenylbutyrate (C4) NA p-Nitrophenylhexanoate (C6) NA p-Nitrophenyl octanoate (C8) 0.2±0.14 p-Nitrophenyldecanoate (C10) 3.35±0.36 p-Nitrophenyl Laurate (C12) 0.54±0.02 p-Nitrophenyl myristate (C14) 0.72±0.03 p-Nitrophenyl palmitate (C16) 1.38±0.39 p-Nitrophenyl Stearate (C18) 1.29±0.2

[0062] Example 5 Effect of metal ions on the activity of lipase Pclip7

[0063] Different metal ion solutions were prepared with 50 mM Tris-HCl, pH 7.5 as solvent, and the final concentration of metal ions was 3 mM. The activity of lipase Pclip7 was determined according to the enzyme activity determination method in Example 3, and no metal ions were added as a control. 2+ 、Mo 2+ , K + , Mn 2+ , Cu 2+ 、Zn 2+ 、Co 2+ and Ni +The relative enzyme activities of Fe and Fe-2+ were 89.54%, 73.49%, 66.37%, 40.22%, 34.81%, 28.22%, 24.89% and 16.77%, respectively. 3+ The enzyme activity was completely inhibited (Table 2). 2+ The catalytic activity of Pclip7 was strongly promoted in the presence of 3, 5, 10, 20 and 30 mM Mg 2+ In the reaction system, the relative activities reached 288.55%, 343.91%, 371.30%, 313.49% and 287.04% of the initial enzyme activity, respectively. Different metal ions have obvious differences in the effects on the enzyme activity, so the performance of lipase Pclip7 can be regulated by selecting and regulating metal ions. This feature can improve the application efficiency and effect of Pclip7 in industrial production, biocatalysis and other fields. In addition, when lipase is used as a feed additive, the metal ions contained in the feed will affect the catalytic activity of lipase, thereby affecting the feeding effect of lipase.

[0064] Table 2 Effect of metal ions on enzyme activity

[0065]

[0066]

[0067] Example 6 Effect of organic solvents on the activity of lipase Pclip7.

[0068] 2% (v / v) methanol, ethanol, n-butanol, isopropanol and dimethyl sulfoxide (DMSO) were added to the enzyme activity assay system, and the enzyme activity was determined according to the enzyme activity assay method in Example 3 under the optimal conditions. The reaction without adding organic solvent was used as a control, and the enzyme activity of the reaction was 100% to determine the effect of organic solvents on the activity of lipase Pclip7. The results are shown in Table 3. Methanol, ethanol, and isopropanol have an inhibitory effect on the activity of lipase Pclip7, while n-butanol and DMSO have a promoting effect on the activity of Pclip7; lipase Pclip7 has good DSMO resistance, and the enzyme activity can still reach 117.61% in 50% DSMO.

[0069] Table 3 Effects of organic solvents on the activity of lipase Pclip7

[0070]

[0071] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A protein for use as a lipase, characterized in that The protein is shown in A1) or A2) or A3) or A4): A1) the protein whose amino acid sequence is shown in positions 21 to 321 of SEQ ID NO.1; A2) a protein obtained by connecting a signal peptide to the N-terminus or / and the C-terminus of the amino acid sequence shown in positions 21-321 of SEQ ID NO.1; A3) a fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of A1) or A2); A4) A protein obtained by replacing and / or deleting and / or adding one or more amino acid residues of any of the proteins shown in A1) to A3) and having the same function.

2. The protein according to claim 1, characterized in that The protein is a protein whose amino acid sequence is shown in SEQ ID NO.

1.

3. A gene encoding the protein according to claim 1 or 2.

4. The coding gene according to claim 3, characterized in that The coding gene is shown as a1) or a2) or a3) or a4): a1) a DNA molecule whose nucleotide sequence is shown as positions 61-966 of SEQ ID NO.2; a2) a DNA molecule with a nucleotide sequence as shown in SEQ ID NO.2; a3) a DNA molecule having 75% or more identity with the nucleotide sequence defined in a1) or a2) and encoding the protein of claim 1 or 2; a4) A DNA molecule which hybridizes with the nucleotide sequence defined in a1) or a2) under stringent conditions and encodes the protein according to claim 1 or 2.

5. A recombinant vector or recombinant strain comprising the encoding gene according to claim 3 or 4.

6. The recombinant vector according to claim 5, characterized in that The recombinant vector is obtained by connecting the coding gene described in claim 3 or 4 between the EcoRI and NotI restriction enzyme sites of pPIC9K, while keeping other sequences of pPIC9K unchanged, to obtain the recombinant vector pPIC9K-Pclip7.

7. The recombinant strain according to claim 5, characterized in that The host cell of the recombinant strain is a Pichia pastoris strain, preferably a Pichia pastoris strain GS115.

8. Use of the protein according to claim 1 or 2 as lipase.

9. A method for preparing lipase, characterized in that: The lipase comprises the protein according to claim 1 or claim 2, and the method comprises the following steps: (1) transforming a host cell with the recombinant vector according to claim 5 or 6 to obtain a recombinant strain; (2) culturing the recombinant strain and inducing lipase expression; (3) Purify to obtain lipase.

10. Use of the lipase prepared by the method according to claim 9 in the fields of biotechnology, industrial production and / or food processing.

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