Beta-galactosidase coding gene and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to a beta-galactosidase coding gene with divalent ion adjustable activity and application of the beta-galactosidase coding gene. The nucleotide sequence of the beta-galactosidase coding gene is shown as SEQ ID NO: 3, and the beta-galactosidase with high activity, high yield and high stability can be obtained by transferring the beta-galactosidase coding gene into pichia pastoris. The gene sequence as shown in SEQ ID NO: 3 is subjected to point mutation to obtain a gene sequence as shown in SEQ ID NO: 4, the beta-galactosidase with better heat-resistant stability is obtained through expression, and the amino acid sequence of the beta-galactosidase is as shown in SEQ ID NO: 5. The method provides conditions for development of industrial beta-galactosidase sources in the aspects of lactose deep processing, biological energy sources, biological medicines and the like.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a beta-galactosidase encoding gene and application thereof. Background Art

[0002] β-galactosidase (EC3.2.1.23), full name β-D-galactoside galacto hydrolase, commonly known as lactase, has the function of catalyzing the hydrolysis of β-1,4 glycosidic bonds and transgalactosylation. β-galactosidase has a wide range of sources, and can be produced by animals, plants and microorganisms. Among them, microbial β-galactosidase has the highest yield, simple genetic manipulation, and is more stable than animal and plant β-galactosidase. Moreover, microbial β-galactosidase has been greatly promoted in industrial applications due to its advantages such as high enzyme activity, strong selectivity and wide substrate range. As a biocatalyst, β-galactosidase can catalyze hydrolysis and synthesis reactions starting from different substrates, and the reaction does not require coenzymes, the reaction conditions are mild, and there are few by-products. Therefore, β-galactosidase is widely used, such as in the fields of food industry, medicine and health, chemistry and chemical engineering, environmental protection, energy development, etc. It has become the most widely used enzyme in biotechnology and organic synthesis.

[0003] With the rapid development of cell engineering, genetic engineering, immobilized enzyme technology, and interfacial enzymatic reactions, the research on β-galactosidase has been further deepened, especially in the mutagenesis breeding and molecular modification of β-galactosidase-producing strains. However, β-galactosidase needs to overcome several bottlenecks in its application: high enzyme cost, low activity, low stability, and low reaction yield. Therefore, in order to further improve the application of β-galactosidase in the industrial field, screening and developing microbial β-galactosidase with new catalytic activity and high stability will become a hot spot and focus of research.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a β-galactosidase encoding gene and application thereof.

[0006] Specifically, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a β-galactosidase encoding gene, the nucleotide sequence of which is shown in SEQ ID NO:3.

[0007] The present invention uses genetic engineering technology to utilize Penicillium ( Penicillium oxalicum) of the β-galactosidase gene sequence (Genebank: EPS25349.1, the specific nucleotide sequence is shown in SEQ ID NO: 1; the corresponding amino acid sequence is shown in SEQ ID NO: 2) and the codon preference of Pichia pastoris, and the codons with high usage frequency are used to replace the codons with low usage frequency, keeping the amino acid sequence of the mature protein unchanged, and finally synthesizing the β-galactosidase encoding gene shown in SEQ ID NO: 3. The present invention transfers it into Pichia pastoris to obtain high-activity, high-yield and high-stability β-galactosidase. It has been verified that the engineering strain constructed by the encoding gene provided by the present invention can reach 128.7U / mL at the shaking table level, and the optimal catalytic temperature of the obtained β-galactosidase is 60°C and the optimal pH is 5.

[0008] In a second aspect, the present invention provides a biomaterial, which comprises the β-galactosidase encoding gene and has the function of expressing the β-galactosidase encoding gene.

[0009] Preferably, the biological material is a recombinant vector or an engineered strain.

[0010] More preferably, the engineered strain is Pichia pastoris.

[0011] In a more specific optional embodiment provided by the present invention, the engineered strain is transformed with the β-galactosidase encoding gene, which is obtained by constructing a recombinant expression plasmid with the β-galactosidase encoding gene and then transforming Pichia pastoris.

[0012] In a third aspect, the present invention provides the use of the β-galactosidase encoding gene or the biomaterial in the industrial production of β-galactosidase.

[0013] In a fourth aspect, the present invention further provides a novel β-galactosidase, whose amino acid sequence is shown in SEQ ID NO:5.

[0014] The β-galactosidase provided by the present invention, whose amino acid sequence is shown in SEQ ID NO: 5, is obtained by paired mutation on the basis of the amino acid sequence shown in SEQ ID NO: 2 (the sequence shown in SEQ ID NO: 5 does not contain the signal peptide at the front end of the sequence shown in SEQ ID NO: 2); the sites of the paired mutation are 61M-397V.

[0015] In a fifth aspect, the present invention provides a coding gene having the function of encoding the β-galactosidase described in the fourth aspect.

[0016] Optionally, the nucleotide sequence is shown in SEQ ID NO:4.

[0017] In a sixth aspect, the present invention provides a biomaterial, which comprises the coding gene described in the fifth aspect and has the function of expressing the coding gene and producing the corresponding β-galactosidase.

[0018] Preferably, the biological material is a recombinant vector or an engineered strain.

[0019] In a seventh aspect, the present invention provides the use of the encoding gene or the biological material in the industrial production of β-galactosidase.

[0020] Preferably, the application comprises: performing shaking expression on the engineered strain comprising the coding gene described in the fifth aspect, and the expression conditions are 28±2°C and 220±rpm.

[0021] Beneficial effects: The present invention provides a β-galactosidase encoding gene, the nucleotide sequence of which is shown in SEQ ID NO: 3. The present invention uses genetic engineering technology to utilize Penicillium ( Penicillium oxalicum ) of the β-galactosidase gene sequence (Genebank: EPS25349.1, the specific nucleotide sequence is shown in SEQ ID NO: 1; the corresponding amino acid sequence is shown in SEQ ID NO: 2) and the codon preference of Pichia pastoris, and the codons with high usage frequency are used to replace the codons with low usage frequency, keeping the amino acid sequence of the mature protein unchanged, and finally synthesizing the β-galactosidase encoding gene shown in SEQ ID NO: 3. The present invention transfers it into Pichia pastoris to obtain β-galactosidase with high activity, high yield and high stability. It has been verified that the fermentation activity of the engineering strain constructed by the nucleotide sequence SEQ ID NO: 3 encoding gene provided by the present invention can reach 128.7 U / mL at the shaking table level, and the optimal catalytic temperature of the obtained β-galactosidase is 60°C and the optimal pH is 5. In addition, the present invention also performs point mutation on the amino acid of β-galactosidase to obtain a β-galactosidase with better heat resistance and stability, with an optimal temperature of 65°C and an enzyme activity of 58.6U / mL. After Zn 2+ , Mn 2+ , Ca 2+ After co-bathing at room temperature, the enzyme activity increased by 228% to 133.7 U / mL. The invention provides conditions for the development of industrial β-galactosidase sources in the aspects of lactose deep processing, bioenergy, biomedicine, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0023] Figure 1This is the optimum pH analysis diagram of β-galactosidase in Example 1 of the present invention.

[0024] Figure 2 This is an analysis diagram of the optimum catalytic temperature of β-galactosidase in Example 1 of the present invention. DETAILED DESCRIPTION

[0025] The in-depth development and research of new β-galactosidase can be achieved by adopting new biotechnology means. According to the codon preference of Pichia pastoris, the present invention optimizes the gene sequence of Penicillium β-galactosidase to obtain high-activity, high-yield and high-stability β-galactosidase, thereby providing conditions for the development of industrial lipase sources in the aspects of lactose deep processing, bioenergy, biomedicine, etc.

[0026] Specifically, the nucleotide sequence of the β-galactosidase encoding gene provided by the present invention is shown in SEQ ID NO: 3. The present invention transfers it into Pichia pastoris to obtain β-galactosidase with high activity, high yield and high stability. It has been verified that the enzyme activity of the engineering strain constructed by the encoding gene provided by the present invention can reach 128.7U / mL at the shaking table level, and the optimal catalytic temperature of the obtained β-galactosidase is 60°C and the optimal pH is 5.

[0027] Furthermore, the present invention also performs point mutation on the amino acids of β-galactosidase to obtain β-galactosidase with better heat resistance and stability.

[0028] Specifically, the amino acid sequence of β-galactosidase provided by the present invention is shown in SEQ ID NO: 5 (ie, mutant mutation 61).

[0029] The present invention further uses divalent metal ions Zn 2+ , Mn 2+ , Ca 2+ Regulates β-galactosidase activity.

[0030] Specifically, in order to improve the β-galactosidase activity of mutant mutant 61, mutant 61 was treated with 0.014 mg / mL Zn 2+ , 0.5mg / mL Mn 2+ , 5mg / mL Ca 2+ After 10 minutes of bathing at room temperature, the activity was measured again and the enzyme activity increased by 228% to 133.7 U / mL.

[0031] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0033] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0034] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0035] In the following examples, a β-galactosidase activity unit (i.e., enzyme activity) is defined as the enzyme that catalyzes the conversion of one micromole of o-nitrophenyl-β-D-galactoside per minute under specified reaction conditions.

[0036] The sequence involved in the present invention is as follows: SEQ ID NO: 01

[0037] SEQ ID NO: 02——

[0038] SEQ ID NO:03——

[0039] SEQ ID NO: 04 (mutation 61)

[0040] SEQ ID NO: 05 (mutation 61) 。

[0041] Example 1 (1) Optimize the design of β-galactosidase gene sequence.

[0042] According to the Penicillium β-galactosidase gene sequence reported in GenBank and the codon preference of Pichia pastoris, the Penicillium β-galactosidase gene was redesigned to replace the low-frequency codons with high-frequency codons, keeping the amino acid sequence of the mature protein unchanged. The designed sequence was fully synthesized by Beijing Qingke Biotechnology Co., Ltd. In order to facilitate cloning, EcoRI and XbaI restriction sites were designed at the 5' and 3' ends of the sequence, respectively.

[0043] (2) Construction and transformation of Pichia pastoris expression vector.

[0044] The previously synthesized gene was double-digested with restriction endonucleases EcoRI and XbaI, and then ligated into the downstream of the α factor sequence in the vector pPICZαA (Invitrogen, USA) that had been digested with the same enzymes. The ligation product was transformed into Escherichia coli Top10 competent cells, and positive clones were screened using LB resistance plates containing 25 μg / mL of the antibiotic Zeocin. The plasmid was extracted to obtain the Pichia pastoris expression vector for β-galactosidase.

[0045] Take 10 μL of Pichia expression vector and digest it with restriction endonuclease Sac I at 37℃ for 24 h. Add 80 μL of Pichia X-33 competent cells, mix well, place in a 0.2 cm electric shock cup, ice bath for 5 min, shock at 2000 volts (25μF), quickly add 1 mL 1 mol / L sorbitol, and place it at 28℃ for 3 h. Then spread the culture on a YPDS (1% yeast extract, 2% peptone, 2% glucose, 2% agar, and the rest is water) plate containing the antibiotic Zeocin (100 μg / mL) and culture at 28℃ for 2-4 days until clear colonies grow.

[0046] (3) Screening of Pichia pastoris engineered strains that efficiently secrete and express β-galactosidase.

[0047] Positive recombinants were picked and inoculated into 50 mL BMGY (1% yeast extract, 2% peptone, 1.34% YNB, 4×10 -5 The cells were cultured in a 500 mL shake flask containing 1% biotin, 1% glycerol, 100 mM pH 6.0 phosphate buffer, and the rest water at 28°C and 220 rpm until the OD600 was 2-6. The cells were then collected by centrifugation at room temperature and washed with BMMY (1% yeast extract, 2% peptone, 1.34% YNB, 4×10-5 % Zeocin, 1% glycerol, 100 mM pH 6.0 phosphate buffer, and the rest water) to resuspend the bacteria to an OD600 of 1.0, and then use methanol with a final concentration of 0.5% (V / V) as the only carbon source for induction culture. As a result, a recombinant strain with high enzyme activity was screened and named B-Gal.

[0048] Dissolve 250.0 mg of o-nitrophenyl-β-D-galactopyranoside (ONPG) in about 80 ml of the corresponding reaction buffer, dilute to 100 ml with the reaction buffer, shake well, and prepare within 2 hours before use. Preheat 5 ml of substrate at 60 °C for 5 min, pipette 1 ml of appropriately diluted enzyme solution and boiled enzyme solution into the substrate and mix thoroughly, react in a 60 °C constant temperature water bath for 10 min, then add 2 ml of Na2CO3 stop solution, shake and mix well, and terminate the enzyme reaction.

[0049] (5) Analysis of the enzymatic properties of β-galactosidase.

[0050] ①Optimum pH value Determination of the optimal pH value: Prepare substrates with potassium phosphate trihydrate-citrate monohydrate buffer at pH 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, and 8, respectively, and measure enzyme activity at a reaction temperature of 60°C to determine the optimal pH of the enzyme (see Figure 1 ).

[0051] ②Optimum temperature Determination of the optimal temperature: Prepare the substrate ONPG with pH 5.0 buffer, measure the enzyme activity at 25-75℃, and determine its optimal reaction temperature (see Figure 2 ).

[0052] Example 2 Site-directed gene mutation was performed based on the sequence shown in SEQ ID NO: 2, and the residue numbering was consistent with the arrangement numbering of SEQ ID NO: 2. A total of 9 paired mutation sites were designed, and the paired mutations were mutated into cysteine. The specific mutation sites were: 61M-397V, 72L-348G, 89G-388N, 151W-846E, 150G-727H, 245A-290S, 352G-857N, 357PRO-753G, 372S-950P.

[0053] The designed sequence was fully synthesized by Beijing Qingke Biotechnology Co., Ltd., and a total of 9 mutants were synthesized, named as mutation 61, mutation 72, mutation 89, mutation 151, mutation 150, mutation 245, mutation 352, mutation 357, and mutation 372. In order to facilitate cloning, EcoRI and XbaI restriction sites were designed at the 5' and 3' ends of the sequence, respectively.

[0054] The 9 mutants were constructed and transformed with Pichia pastoris expression vectors, and the Pichia pastoris engineering strains with efficient secretory expression of thermostable β-galactosidase were screened, and the enzymatic properties of each mutant were measured. The results showed that only the thermostable mutated 61 was improved, with an optimum temperature of 65℃ and an enzyme activity of 58.7U / mL. Under the condition of 65℃, the β-galactosidase activity of the unmutated ...

[0055] This example further uses divalent metal ions Zn 2+ , Mn 2+ , Ca 2+ Regulates β-galactosidase activity.

[0056] To improve the activity of mutant mutant β-galactosidase, mutant β-galactosidase was mixed with 0.014 mg / mL Zn 2+ , 0.5mg / mL 2+ , 5mg / mL Ca 2+ After 10 minutes of bathing at room temperature, the activity was measured again and the enzyme activity increased by 228% to 133.7 U / mL.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A β-galactosidase encoding gene, characterized in that The nucleotide sequence thereof is shown in SEQ ID NO:

3.

2. Biomaterial, characterized in that The biological material comprises the β-galactosidase encoding gene according to claim 1 and has the function of expressing the β-galactosidase encoding gene according to claim 1.

3. The biomaterial according to claim 2, characterized in that: The biological material is a recombinant vector or an engineered strain.

4. Use of the β-galactosidase encoding gene according to claim 1 or the biomaterial according to any one of claims 2 to 3 in the industrial production of β-galactosidase.

5. β-galactosidase, characterized in that Its amino acid sequence is shown in SEQ ID NO:

5.

6. A coding gene, characterized in that The coding gene has the function of encoding the β-galactosidase according to claim 5.

7. The coding gene according to claim 6, characterized in that The nucleotide sequence thereof is shown in SEQ ID NO:

4.

8. Biomaterial, characterized in that The biological material comprises the coding gene of claim 6 or 7, and has the function of expressing the coding gene of claim 6 or 7 and producing the β-galactosidase of claim 5; Preferably, the biological material is a recombinant vector or an engineered strain.

9. Use of the coding gene according to claim 6 or 7 or the biomaterial according to claim 8 in the industrial production of β-galactosidase.

10. The use according to claim 9, characterized in that: The engineered strain containing the coding gene of claim 7 is subjected to shaking expression, and the expression conditions are 28±2°C and 220±rpm.

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