Beta-galactosidase encoding genes and uses thereof

Abstract

The present application relates to the technical field of bioengineering, and particularly relates to a kind of divalent ion adjustable activity β-galactosidase coding gene and application thereof.The nucleotide sequence of the β-galactosidase coding gene described in the present application is as shown in SEQ ID NO:3, which is transferred into Pichia pastoris, and high-activity, high-yield, high-stability β-galactosidase can be obtained.The gene sequence as shown in SEQ ID NO:3 is subjected to point mutation in the present application, and the nucleotide sequence of the obtained gene sequence is as shown in SEQ ID NO:4, and the β-galactosidase with better heat-resistant stability is obtained by expression, and the amino acid sequence thereof is as shown in SEQ ID NO:5.The present application provides conditions for the development of industrial β-galactosidase source in the aspects of deep processing of lactose, bioenergy, biological medicine and the like.

Description

TECHNICAL FIELD

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

[0002] Beta-galactosidase (beta-galactosidase, EC3.2.1.23), full name beta-D-galactoside galacto hydrolase, commonly known as lactase, has the functions of catalyzing beta-1, 4 glycosidic bond hydrolysis and transgalactosidase. The source of beta-galactosidase is very wide, animals, plants and microorganisms can produce beta-galactosidase. Among them, the production of microbial beta-galactosidase is the highest, the gene operation is simple, and it is more stable than animal and plant beta-galactosidase. And microbial beta-galactosidase has the advantages of high enzyme activity, strong selectivity and wide substrate, which has been greatly popularized in industrial application. As a biological catalyst, beta-galactosidase can catalyze hydrolysis and synthesis reactions from different substrates, and the reaction does not require coenzyme, the reaction condition is mild, and the byproduct is less. Therefore, beta-galactosidase is widely used in many fields, such as food industry, medicine and health, chemical industry, environmental protection, energy development, etc., and 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 interface enzyme catalysis technology, the research on beta-galactosidase has been further deepened, especially the mutation breeding and molecular modification of beta-galactosidase producing strains have made great progress. However, beta-galactosidase needs to overcome several bottlenecks in application: high enzyme cost, low activity, low stability and low reaction yield. Therefore, in order to further improve the application of beta-galactosidase in industrial field, screening and developing microbial beta-galactosidase with new catalytic activity and high stability will become the research focus and key.

[0004] Therefore, the present application is proposed. SUMMARY

[0005] To solve the above technical problems, the present application provides a beta-galactosidase encoding gene and application thereof.

[0006] Specifically, the technical scheme of the present application is as follows:

[0007] In a first aspect, the present application provides a beta-galactosidase encoding gene, the nucleotide sequence of which is shown in SEQ ID NO: 3.

[0008] By using the gene engineering technology, the present application utilizes the beta-galactosidase encoding gene of Penicillium sp. (Penicillium sp.) to construct a recombinant strain. Penicillium oxalicum) and the Pichia pastoris codon bias, the low frequency codons are replaced by high frequency codons, the amino acid sequence of the mature protein is kept unchanged, and finally the beta-galactosidase coding gene as shown in SEQ ID NO: 3 is synthesized. The beta-galactosidase coding gene is introduced into Pichia pastoris, and high-activity, high-yield and high-stability beta-galactosidase can be obtained. It is verified that the activity of the engineering strain constructed by the coding gene provided by the present application can reach 128.7 U / mL at the level of a shaking table, and the optimal catalytic temperature of the obtained beta-galactosidase is 60℃, and the optimal pH is 5.

[0009] In a second aspect, the present application provides a biological material comprising the beta-galactosidase coding gene and having the function of expressing the beta-galactosidase coding gene.

[0010] Preferably, the biological material is a recombinant vector or an engineering strain.

[0011] More preferably, the engineering strain is Pichia pastoris.

[0012] In a more specific optional embodiment provided by the present application, the engineering strain is transformed with the beta-galactosidase coding gene, which is obtained by constructing a recombinant expression plasmid with the beta-galactosidase coding gene and then transforming Pichia pastoris.

[0013] In a third aspect, the present application provides the beta-galactosidase coding gene or the biological material for use in the industrial production of beta-galactosidase.

[0014] In a fourth aspect, the present application further provides a new beta-galactosidase, and the amino acid sequence thereof is shown in SEQ ID NO: 5.

[0015] The beta-galactosidase with the amino acid sequence shown in SEQ ID NO: 5 provided by the present application is obtained by pair mutation based on 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 site of the pair mutation is 61M-397V.

[0016] In a fifth aspect, the present application provides a coding gene having the function of coding the beta-galactosidase described in the fourth aspect.

[0017] Alternatively, the nucleotide sequence thereof is shown in SEQ ID NO: 4.

[0018] In a sixth aspect, the present invention provides a biomaterial comprising the coding gene described in the fifth aspect above, and having the function of expressing the coding gene and producing the corresponding β-galactosidase.

[0019] Preferably, the biomaterial is a recombinant vector or an engineered bacterial strain.

[0020] In a seventh aspect, the present invention provides the application of the encoding gene or the biological material in the industrial production of β-galactosidase.

[0021] Preferably, the application includes: expressing the engineered strain containing the coding gene described in the fifth aspect above in a shaker under the conditions of 28±2℃ and 220±rpm.

[0022] Beneficial effects:

[0023] This invention provides a β-galactosidase encoding gene, the nucleotide sequence of which is shown in SEQ ID NO: 3. This invention utilizes Penicillium (…) through genetic engineering technology. Penicillium oxalicum Based on 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, high-frequency codons were replaced with high-frequency codons while maintaining the amino acid sequence of the mature protein, ultimately synthesizing the β-galactosidase encoding gene as shown in SEQ ID NO: 3. This invention transforms this gene into Pichia pastoris, yielding β-galactosidase with high activity, high yield, and high stability. Verification showed that the engineered strain constructed using the nucleotide sequence SEQ ID NO: 3 provided by this invention achieved a fermentation activity of 128.7 U / mL at the shaker level, with the optimal catalytic temperature of 60℃ and the optimal pH of 5 for the obtained β-galactosidase. Furthermore, this invention also performed point mutations on the amino acids of the β-galactosidase, obtaining a β-galactosidase with better thermostability, an optimal temperature of 65℃, and an enzyme activity of 58.6 U / 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. This invention provides conditions for the development of industrial β-galactosidase sources in the fields of lactose deep processing, bioenergy, and biomedicine. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described below.

[0025] Figure 1Optimum pH analysis chart of the beta-galactosidase in Example 1 of the present application.

[0026] Figure 2 Optimum catalytic temperature analysis chart of the beta-galactosidase in Example 1 of the present application. DETAILED DESCRIPTION

[0027] The in-depth development of the novel beta-galactosidase can be realized by taking new biotechnological means. According to the codon bias of Pichia pastoris, the gene sequence of the Penicillium beta-galactosidase is optimized in the present application, so as to obtain the beta-galactosidase with high activity, high yield and high stability, thereby providing conditions for the development of industrial lipase sources in the aspects of deep processing of lactose, biological energy and biological medicine.

[0028] Specifically, the nucleotide sequence of the beta-galactosidase coding gene provided in the present application is shown in SEQ ID NO: 3. The beta-galactosidase with high activity, high yield and high stability can be obtained by transferring the gene into Pichia pastoris. It has been verified that the enzyme activity of the engineering strain constructed by the coding gene provided in the present application can reach 128.7 U / mL at the level of a shaking table, and the optimum catalytic temperature of the obtained beta-galactosidase is 60℃, and the optimum pH is 5.

[0029] Further, the amino acid of the beta-galactosidase is subjected to point mutation in the present application, and the beta-galactosidase with better heat-resistant stability is obtained.

[0030] Specifically, the amino acid sequence of the beta-galactosidase provided in the present application is shown in SEQ ID NO: 5 (i.e., mutant 61).

[0031] The present application further uses the divalent metal ions Zn 2+ , Mn 2+ , Ca 2+ to adjust the activity of the beta-galactosidase.

[0032] Specifically, in order to improve the activity of the beta-galactosidase of the mutant 61, the mutant 61 is bathed with 0.014 mg / mL Zn 2+ , 0.5 mg / mL Mn 2+ , 5 mg / mL Ca 2+ at room temperature for 10 minutes, and then the activity is measured again, and the enzyme activity is improved by 228%, which is 133.7 U / mL.

[0033] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0034] The endpoints of the ranges and any values disclosed in the specification are not limited to the precise values stated. The ranges or values should be construed to be approximations that allow for significant variation. Various ranges of values are stated in terms of being between two specific ends. When these ranges are approximations, the teaching also should be understood to allow for a range of values to include these two specific ends and also to include values outside of that range.

[0035] In the description of the present application, the description of the terms "one embodiment", "some embodiments", "specific embodiments", or "some specific embodiments" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the embodiments of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples without contradiction.

[0036] In the embodiments provided in the present specification, the specific techniques or conditions not specified are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments used without specifying the manufacturer are conventional products that can be purchased through regular channels.

[0037] In the following examples, the definition of beta-galactosidase activity unit (enzyme activity) is as follows: under the specified reaction conditions, the enzyme catalyzing the conversion of one micromole of o-nitrophenyl-beta-D-galactoside per minute.

[0038] The sequences involved in the present application are as follows:

[0039] SEQ ID NO: 01

[0040]

[0041] SEQ ID NO: 02 —

[0042]

[0043] SEQ ID NO: 03 —

[0044]

[0045] SEQ ID NO: 04 (thul 61) —

[0046]

[0047] SEQ ID NO: 05 (tuf61) ——

[0048] AAISHKLDGFTLREHADASKRELLQKYVTWDKHSIFINGERLCMFSGEVHPYRLPVASLYIDVFEKIKALGFNCVSFYVDWALLEGKPGHYTAEGIFDLQPFFDAAKEAGIYLLARPGPYINAEVSGGGFPGWLQRVNGTLRTSIGDYLKSTDNYASHIAKTIAKAQITNGGPIILYQPENEYSGACCGYEDFPDGSYMQYIEDHARDAGIVVPFISNDAYAGGHNAPGTGKGAVDIYGHDGYPLGFDCANPSVWPDGNLPTNYHTLHEEQSPTTPFSIVEFQGGAFDPWGGVGFAKCAHLLNHEFERVFYKNDFSFGVTFFNLYMIFGGTNWGNLGHPGGYTSYDYGSAISESRNVTREKYSELKLLGNFAKVSPGYCVANPGNLTTSKYTKTADLTVTPLLGESSSAGSFFVIRHSNYNSQASVKYQLTLPTSAGELTIPQLGGELTLSGRDSKIHVTDYDVAGSNILYSTAEVFTWKKFDNGKVLILYGGPGEHHEFAVTGASASSVIEGSSSGITSKKIGDALVVAWDVSSKRRIIKIGDLKVFLLDRNSAYNYWVPHLPTKGKAPGYVSKKAIDSSIIVKAGYLVRSAFLSGKDLHIQADFNATTPIEIIGAPSSAKNLIINGKKTKTNVDDNGIWSASVSYSTPEIHLPSLKDLKWQSIDTLPEVKNSYDDSAWTSADQPHTLNTAHELQTPTTLFSSDYGYHTGTLLYRGHFVANGKESIFFIQTQGGSAYGHSVWVNETYVGSWEGSGSNDNYNATYTLPSLATGKKYVITVVIDNMGLDENWVIGQEGMKNPRGIIRYNLAGHDASAISWKLTGNLGGEDYRDTVRGPLNEGGLYAERQGFHQPKPSTKNWDSSSPFTGLTKPGIRFYSASFDLDLPSGYDIPLYFNFGNSTAPPDAYRVQLFVNGYQYGKYVNNVGPQTSFPVPEGILNYHGTNWIALSLWAQQESGAKLNSFELINTTPVLTSLDKVKSVDQPKYKSRKGAY

[0049] Example 1

[0050] (1) Optimize the design of β-galactosidase gene sequence.

[0051] According to the β-galactosidase gene sequence of Penicillium reported by GenBank and the codon bias of Pichia pastoris, the β-galactosidase gene of Penicillium was redesigned to replace the low frequency codon with high frequency codon, while keeping the amino acid sequence of mature protein unchanged. The designed sequence was synthesized by Beijing Genki 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.

[0052] (2) Construction and transformation of Pichia pastoris expression vector.

[0053] After double digestion of the previously synthesized gene with restriction enzymes EcoRI and XbaI, it was connected downstream of the α factor sequence of the vector pPICZαA (Invitrogen, USA) digested with the same enzymes. The ligation product was transformed into E. coli Top10 competent cells, and positive clones were selected on LB resistance plates containing 25 μg / mL of antibiotic Zeocin. The plasmid was extracted to obtain the Pichia pastoris expression vector of β-galactosidase.

[0054] Take 10 μL of Pichia pastoris expression vector, and digest it with restriction enzyme Sac I at 37°C for 24 h. Add 80 μL of Pichia pastoris X-33 competent cells, mix well, and place in a 0.2 cm electroporation cup on ice for 5 min. Electroporate at 2000 volts (25 μF), quickly add 1 mL of 1 mol / L sorbitol, and incubate at 28°C for 3 h. Then spread the culture on YPDS (1% yeast extract, 2% peptone, 2% glucose, 2% agar, and the rest water) plates containing antibiotic Zeocin (100 μg / mL), and incubate at 28°C for 2-4 d until clear colonies grow.

[0055] (3) Screening of high-efficiency secretory expression of β-galactosidase Pichia pastoris engineering strain.

[0056] Pick positive recombinants and inoculate them in 50 mL of BMGY (1% yeast extract, 2% peptone, 1.34% YNB, 4×10 -5BMMY (1% yeast extract, 2% peptone, 1.34% YNB, 4x10 -5 BMMY (1% yeast extract, 2% peptone, 1.34% YNB, 4x10

[0057] D-galactopyranoside (ONPG) was dissolved in about 80 ml of the corresponding reaction buffer, and the reaction buffer was added to 100 mL, shaken well, and prepared within 2 h before use. 5 mL of the substrate was preheated at 60°C for 5 min, and 1 mL of the appropriately diluted enzyme solution and 1 mL of the boiled enzyme solution were added to the substrate and mixed well. The mixture was reacted at 60°C in a constant temperature water bath for 10 min, then 2 mL of Na2CO3 termination liquid was added and mixed well to terminate the enzyme reaction.

[0058] (5) Enzymatic property analysis of β-galactosidase.

[0059] ① Optimum pH

[0060] Determination of optimum pH: The substrate was prepared using trihydrogen dipotassium phosphate-citric acid monohydrate buffer with pH values of 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, and 8, respectively, and the enzyme activity was determined at a reaction temperature of 60°C to determine the optimum pH of the enzyme (see Figure 1 ).

[0061] ② Optimum temperature

[0062] Determination of optimum temperature: The substrate ONPG was prepared using pH 5.0 buffer, and the enzyme activity was determined at 25-75°C to determine the optimum reaction temperature (see Figure 2 ).

[0063] Example 2

[0064] Based on the sequence shown in SEQ ID NO: 2, site-directed gene mutation was carried out, the residue number was consistent with the arrangement number of SEQ ID NO: 2, and a total of 9 pairs of mutation sites were designed, and the pairs of mutations were cysteine. The specific mutation sites were: 61M-397V, 72L-348G, 89G-388N, 151W-846E, 150G-727H, 245A-290S, 352G-857N, 357PRO-753G, and 372S-950P.

[0065] The designed sequence was synthesized by Beijing Genki Biotechnology Co., Ltd. A total of 9 mutants were synthesized and named as Mut61, Mut72, Mut89, Mut151, Mut150, Mut245, Mut352, Mut357, and Mut372. In order to facilitate cloning, EcoRI and XbaI restriction sites were designed at the 5' and 3' ends of the sequence.

[0066] After the construction and transformation of the Pichia pastoris expression vector, the high-efficiency secretory expression of the heat-resistant β-galactosidase Pichia pastoris engineering strain was screened, and the enzymatic properties of each mutant were measured. It was found that only Mut61 had improved heat resistance, and the optimum temperature was 65℃, and the enzyme activity was 58.7 U / mL. Under the condition of 65℃, the activity of unmutated β-galactosidase was 52.1 U / mL; the activities of Mut72, Mut89, Mut151, Mut150, Mut245, Mut352, Mut357, and Mut372 were 7.7 U / mL, 6.4 U / mL, 5.6 U / mL, 0 U / mL, 18.9 U / mL, 0 U / mL, 0 U / mL, and 18.7 U / mL, respectively.

[0067] In this embodiment, divalent metal ions Zn 2+ , Mn 2+ , Ca 2+ were further used to adjust the activity of β-galactosidase.

[0068] To improve the activity of Mut61 β-galactosidase, Mut61 β-galactosidase was incubated with 0.014 mg / mL Zn 2+ , 0.5 mg / mL Mn 2+ , and 5 mg / mL Ca 2+ at room temperature for 10 minutes, and then the activity was measured again, and the enzyme activity was increased by 228%, to 133.7 U / mL.

[0069] It should be pointed out finally that the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit the same; and although the present application has been described in detail with reference to the foregoing embodiments, it should be appreciated by those skilled in the art that the technical solutions recorded in the foregoing embodiments can be modified, or some technical features thereof can be replaced equivalently; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A β-galactosidase characterized in that, The amino acid sequence is shown as SEQ ID NO:

5.

2. A coding gene, characterized in that, The coding gene encodes the β-galactosidase of claim 1.

3. The genetic code according to claim 2, wherein, The nucleotide sequence is shown as SEQ ID NO:

4.

4. Biomaterial, characterized in that, The biological material contains the coding gene of claim 2 or 3, and has the function of expressing the coding gene of claim 2 or 3 and producing the β-galactosidase of claim 1; the biological material is a recombinant vector or an engineered strain.

5. The coding gene of claim 2 or 3 or the biological material of claim 4 is applied to the industrial production of β-galactosidase.

6. Use according to claim 5, characterized in that, The engineered strain containing the coding gene of claim 3 is subjected to shaker expression, and the expression condition is 28±2℃, 220±rpm.

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