A beta-galactosidase, mutants thereof and uses thereof

Abstract

This invention provides a β-galactosidase, its mutants, and their applications, relating to the field of protein engineering technology. This invention is the first to discover that β-galactosidase M3 possesses superior fermentation activity and transglycosylation performance, representing a novel biological enzyme for lactose decomposition engineering. Furthermore, based on M3, this application analyzes its active site and identifies position 394 as a beneficial mutation site for β-galactosidase M3. This mutation further enhances the enzyme's fermentation activity and transglycosylation performance, significantly improving lactose decomposition efficiency and facilitating its application in industrial production.

Description

Technical Field

[0001] This application relates to the field of protein engineering technology, and in particular to a β-galactosidase, its mutants, and its applications. Background Technology

[0002] Lactose is the main carbohydrate in milk, and its digestion and absorption depend on lactase secreted by the brush border of the small intestine. Lactase is a glycoside hydrolase capable of hydrolyzing the disaccharide lactose into galactose and glucose monomers. The lactose hydrolytic activity of lactase can be referred to as lactase activity, β-galactosidase activity, or hydrolytic activity. However, in a significant proportion of the population, the activity of naturally occurring lactase in the intestine is insufficient, leading to the ineffective hydrolysis of ingested lactose, resulting in a series of adverse gastrointestinal reactions such as bloating and diarrhea. This phenomenon is known as "lactose intolerance."

[0003] To address the aforementioned issues, the food industry widely employs lactase, derived from Aspergillus or yeast fermentation, for pretreatment of dairy products. Through the catalytic action of this lactase, one molecule of lactose in milk can be hydrolyzed into one molecule of glucose and one molecule of galactose, resulting in low-lactose or lactose-free milk. However, due to the increased number of sugar molecules in the hydrolysis products and the enhanced sweetness per unit of each sugar component, lactose-free milk exhibits a significantly higher sweetness, thus affecting its palatability and consumer acceptance. Besides direct hydrolysis into glucose and galactose, some lactases exhibit β-galactosidase activity, which can also catalyze transglycosylation reactions to partially hydrolyze lactose and further convert it into galacto-oligosaccharides (GOS). This process not only effectively eliminates the triggers for lactose intolerance but also introduces galacto-oligosaccharides, a recognized dietary fiber, into the food matrix in situ. GOS can be selectively utilized by beneficial gut bacteria, thereby endowing the product with potential gut health regulatory functions.

[0004] In recent years, lactases such as Nurica from International Flavors & Fragrances, Inc., and Nola GOS from Kelley, Inc., have been used by some dairy companies to produce lactose-free milk containing natural prebiotics. Both of these lactases have high activity in converting lactose into galactooligosaccharides. However, the required dosage of these two lactases is more than ten times that of normal lactase (3 parts per thousand, compared to 3 parts per ten thousand for conventional lactase), resulting in higher usage costs and significantly increasing the production cost of lactose-free milk.

[0005] In conclusion, developing novel enzyme preparations capable of efficiently and selectively synthesizing galactooligosaccharides has become crucial for advancing the next generation of functional low-lactose dairy products. This urgently requires exploring novel microbial enzyme resources and systematically modifying existing enzyme molecules using comprehensive protein engineering strategies to obtain superior β-galactosidases with excellent catalytic performance that meet the needs of industrial production. Summary of the Invention

[0006] Therefore, this invention has obtained novel β-galactosidases and their mutants through bioinformatics mining and genetic engineering. Compared with known β-galactosidase molecules, the β-galactosidase molecules constructed in this invention exhibit significantly increased fermentation activity and improved oligogalactose synthesis capacity, thus possessing promising prospects for industrial production.

[0007] On the one hand, this application provides a β-galactosidase comprising an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence having at least 98% identity with SEQ ID NO.1.

[0008] The β-galactosidase comprises an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence having 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO.1.

[0009] In this application, a novel protein with β-galactosidase activity (WP_270266043.1) was discovered through screening. Based on this, the protein was truncated, and its active fragment was searched, ultimately yielding a new β-galactosidase. The amino acid sequence of the β-galactosidase is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. Experimental verification shows that it has better fermentation activity and transglycosylation performance, and has broad prospects for industrial applications.

[0010] On the other hand, this application also provides a β-galactosidase mutant, wherein the amino acid sequence of the β-galactosidase mutant contains an asparagine residue at position 394 mutated to a methionine residue, wherein the amino acid position corresponds to the position in SEQ ID NO.1, the mutant has at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO.1 or its mature polypeptide, and the mutant has β-galactosidase activity.

[0011] Furthermore, the amino acid sequence of the β-galactosidase mutant comprises the amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence having at least 98% identity with SEQ ID NO.3.

[0012] Preferably, the amino acid sequence of the β-galactosidase mutant comprises the amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence having 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO.3.

[0013] Preferably, the β-galactosidase mutant is obtained by mutating the asparagine residue (N) at position 394 of the wild-type β-galactosidase to a methionine residue (M), with the amino acid sequence shown in SEQ ID NO.3 and the nucleotide sequence shown in SEQ ID NO.4.

[0014] This application is the first to discover that position 394 is a beneficial mutation site for β-galactosidase M3. After mutation, the enzyme’s fermentation activity and transglycosylation performance can be improved, providing a new engineered enzyme for lactose decomposition.

[0015] On the other hand, this application also provides biological materials, said biological materials comprising any one of the following A1)-A4):

[0016] A1) A nucleic acid molecule, said nucleic acid molecule containing a nucleic acid molecule encoding the β-galactosidase or a mutant of the β-galactosidase;

[0017] A2) An expression cassette, wherein the expression cassette contains the nucleic acid molecule described in A1);

[0018] A3) A recombinant vector containing the nucleic acid molecule described in A1) and / or the expression cassette described in A2);

[0019] A4) Host cell, wherein the host cell contains the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3).

[0020] The expression cassette described herein may also include functional elements such as promoters, terminators, and marker genes. Those skilled in the art can make conventional selections according to the actual situation, as long as the expression of A1 nucleic acid molecules can be completed. No further restrictions are placed on the structure and composition of the expression cassette here.

[0021] Preferably, the expression cassette further includes a protein expression signal peptide; more preferably, the protein expression signal peptide is derived from Bacillus clausti alkaline protease AprE.

[0022] Preferably, the promoter is the amyE gene promoter derived from the ATCC6051a strain.

[0023] The vectors described herein refer to those capable of delivering exogenous DNA or target genes into host cells for amplification and expression. These vectors can be any vector (e.g., plasmids or viruses) that facilitates recombinant DNA manipulation and the expression of nucleic acid sequences. The choice of vector typically depends on its compatibility with the host cell to which it will be introduced. Vectors can be linear or closed-circular plasmids. Vectors can be self-replicating vectors (i.e., complete structures existing outside the chromosome that can replicate independently of the chromosome), such as plasmids, extrachromosomal elements, microchromosomes, or artificial chromosomes. Vectors can contain any mechanism that ensures self-replication. Alternatively, a vector is one that, upon introduction into a host cell, integrates into the genome and replicates along with the integrated chromosome. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, or transposons, may be used, as those skilled in the art can choose according to the specific circumstances; no excessive limitations are imposed here.

[0024] Preferably, the plasmid is pUC57-BsaI-free-amyE plasmid.

[0025] Furthermore, the host cell is one or more of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, and Saccharomyces cerevisiae.

[0026] Preferably, the host cell is Bacillus subtilis (Bacillus subtilis). Bacillus subtilis ).

[0027] In a preferred embodiment, the Bacillus subtilis is ATCC6051a.

[0028] Preferably, the host cell further includes the following modifications:

[0029] B1) Suppression or non-expression aprE, nprE, spo and amyE Gene;

[0030] B2) Integration at the aprE site controlled by the xylose-inducible promoter comK Gene.

[0031] Those skilled in the art will understand that conventional fermentation strains or any known industrial strain can be used as the starting strain, as long as they can complete the expression of the mutant described in this application. No specific strain is limited here.

[0032] Preferably, the suppression or non-expression is achieved by gene knockout.

[0033] In one optional implementation, the recombinant vector and the host cell contain an resistance selection marker gene. Those skilled in the art can choose according to the actual situation. This application does not impose mandatory limitations on the resistance selection marker gene.

[0034] A resistance selection marker gene is a gene whose product confers resistance to biocides or viruses, resistance to heavy metals, or a protrophic auxotrophic phenotype. Examples of bacterial selection markers include the dal gene in Bacillus subtilis or Bacillus licheniformis, or resistance markers for antibiotics such as ampicillin, kanamycin, chloramphenicol, or tetracycline.

[0035] Further, the nucleic acid molecule includes a nucleotide sequence as shown in SEQ ID NO.2 or a nucleotide sequence having at least 95% or more identity with SEQ ID NO.2, or a nucleotide sequence as shown in SEQ ID NO.4 or a nucleotide sequence having at least 95% or more identity with SEQ ID NO.4.

[0036] Preferably, the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO.2 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO.2, or a nucleotide sequence as shown in SEQ ID NO.4 or a nucleotide sequence with SEQ ID NO.2. NO.4 has nucleotide sequences with 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, and 99.9% identity.

[0037] On the other hand, this application also provides the use of the aforementioned biomaterials in the preparation of β-galactosidase and / or β-galactosidase mutants.

[0038] On the other hand, this application also provides a method for preparing the β-galactosidase or the β-galactosidase mutant, the method comprising: culturing the host cells to obtain the β-galactosidase or the β-galactosidase mutant.

[0039] A method for preparing β-galactosidase or a β-galactosidase mutant, the method comprising:

[0040] Step 1: Construct the host cell expressing β-galactosidase or a β-galactosidase mutant;

[0041] Step 2: Cultivate the host cells.

[0042] Those skilled in the art can culture the host cells using conventional methods.

[0043] In a preferred embodiment, the method includes:

[0044] Step 1: Construct the host cell expressing β-galactosidase or a β-galactosidase mutant;

[0045] Step 2: Inoculate the host cells into the fermentation medium and culture at 25℃-40℃ and 100-300 rpm for 50-100 hours.

[0046] On the other hand, this application also provides the application of the β-galactosidase or the β-galactosidase mutant or the β-galactosidase prepared by the biomaterial or the preparation method described above in reducing lactose content, catalyzing lactose decomposition and / or preparing galactooligosaccharides.

[0047] On the other hand, this application also provides the application of the β-galactosidase mutant or the biomaterials described herein in improving the fermentation activity of β-galactosidase.

[0048] The β-galactosidase mutant of this application has a fermentation activity of β-galactosidase greater than or equal to 60 U / mL.

[0049] On the other hand, this application also provides a method for improving the fermentation activity of β-galactosidase, comprising: mutating an asparagine residue at position 394 of β-galactosidase to a methionine residue, wherein the β-galactosidase comprises the amino acid sequence shown in SEQ ID NO.1.

[0050] On the other hand, this application also provides the application of the β-galactosidase mutant or the biomaterials described herein in improving the transglycosylation ability of β-galactosidase or the ability to synthesize galactooligosaccharides.

[0051] The transglycosylation ability coefficient of the β-galactosidase mutant in this application is greater than or equal to 2.69.

[0052] On the other hand, this application also provides a method for improving the transglycosylation ability of β-galactosidase or the ability to synthesize galactooligosaccharides, comprising: mutating the asparagine residue at position 394 of β-galactosidase to a methionine residue, wherein the β-galactosidase comprises the amino acid sequence shown in SEQ ID NO.1.

[0053] On the other hand, this application also provides compositions containing the β-galactosidase and / or the β-galactosidase mutant.

[0054] The compositions of this application also include physiologically acceptable excipients.

[0055] As used herein, the term "physiologically acceptable" means a molecular entity and composition that is physiologically tolerable and, when administered to humans, generally does not produce toxicity or sensitization or similar adverse reactions (such as stomach upset, dizziness, etc.). Optionally, as used herein, the term "physiologically acceptable" means approved by a regulatory agency or approved in a pharmacopoeia or other generally accepted manner for use in animals or humans.

[0056] The composition of this application may also contain excipients, which may be suitable solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc.

[0057] This application provides the use of the β-galactosidase or the β-galactosidase mutant or the biomaterial or the preparation method of the β-galactosidase or the β-galactosidase mutant in reducing lactose content, catalyzing lactose decomposition and / or preparing galactooligosaccharides.

[0058] The compositions of this application can be prepared by common methods, wherein one or more diluents or carriers can be added to form dosage forms commonly used in the food or pharmaceutical fields.

[0059] One aspect of this application relates to compositions, which are any compositions capable of achieving the effects described in this application. The compositions include, but are not limited to, the simultaneous or sequential use of the components. "Simultaneous use" includes using them together in the same formulation or separately in different formulations. "Sequential use" includes using them sequentially in different formulations, with no restriction on the order of sequential use.

[0060] On the other hand, this application also provides a method for producing food, the method comprising treating a lactose-containing substrate with the β-galactosidase and / or the β-galactosidase mutant.

[0061] Preferably, the food is a low-lactose or lactose-free food.

[0062] Preferably, the food is a food containing galactooligosaccharides.

[0063] The present invention has the following beneficial effects:

[0064] This invention marks the first time that β-galactosidase M3 has been obtained, exhibiting superior fermentation activity and transglycosylation performance compared to existing technologies such as Nurica, providing a novel bioenzyme for lactose decomposition engineering. Furthermore, based on β-galactosidase M3, this application analyzed its active site and discovered that position 394 is a beneficial mutation site for β-galactosidase M3. This mutation further enhances the enzyme's fermentation activity and transglycosylation performance, significantly improving lactose decomposition efficiency and facilitating its application in industrial production. Attached Figure Description

[0065] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0066] Figure 1 This is a graph showing the SDS-PAGE results of β-galactosidase molecules.

[0067] Figure 2 For β-galactosidase M3, Ca 2+ A schematic diagram of the N394M mutant near the binding site. Detailed Implementation

[0068] Technical terms:

[0069] Sequence identity: The term "sequence identity" describes the correlation between two amino acid sequences or two nucleotide sequences. Sequence identity refers to the percentage of sequence similarity determined by performing optimal alignment (maximum sequence identity) of two sequences within a comparison window. Optimal alignment can be achieved through additions or deletions (i.e., gaps). The percentage of sequence identity can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears in the optimal alignment mode to generate the number of matching positions, dividing the number of matching positions by the total number of positions compared, and then multiplying the result by 100 to obtain the percentage of sequence identity. Sequence identity between two amino acid sequences can be determined using available local alignment tools (e.g., BLAST) or global alignment tools (e.g., the Needleman-Wunsch algorithm). For example, a global alignment tool could be the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. 48:443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 5.0.0 or later) to determine sequence identity between two amino acid sequences. Default parameters such as a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 substitution matrix (the EMBOSS version of BLOSUM62) can be used. Alternatively, the parameters used could be a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL substitution matrix (the EMBOSS version of NCBI NUC4.4). In some implementations, sequence identity comparison does not include signal peptides and / or leader peptides; that is, sequence alignment is performed within the range of amino acid sequences that do not contain signal peptides and / or leader peptides.

[0070] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, *Journal of Molecular Biology* 48:443-453), preferably version 6.6.0 or later, implemented in the Needle program within the EMBOSS software package, is used to determine the sequence identity between two amino acid sequences as the output result of "longest identity". The parameters used are: a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. To have the Needle program report the longest identity, the -nobrief option is specified in the command line. The Needle program output marked as "longest identity" is calculated as follows:

[0071] (Number of identical residues × 100) / (Alignment length - Total number of vacancies in the alignment).

[0072] "Corresponding to...", "reference position", "position", or similar terms refer to the position in the reference amino acid sequence that corresponds to a specific position in the reference amino acid sequence when the queried amino acid sequence is best aligned with the reference amino acid sequence (e.g., the best alignment as described above).

[0073] Recombination: In a broad sense, any gene exchange process that causes a change in genotype is called recombination.

[0074] Expression cassette: An expression cassette is a set of DNA sequences that consists of promoters, target genes, and reporter genes, and can be expressed in specific tissues and is easily detected.

[0075] Recombinant vectors: Recombinant vectors are vectors into which the target gene is transferred based on the basic framework of a cloning vector, thereby enabling the target gene to be expressed.

[0076] Host cell: The term "host cell" refers to any cell type that can be transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "host cell" also encompasses any parental cell progeny that is not completely identical to the parental cell due to mutations that occur during replication.

[0077] Whole-cell catalysts: Whole-cell biocatalysis refers to the process of using a complete biological organism (i.e., whole cell, tissue, or even individual) as a catalyst for chemical transformation. The complete biological organism that participates in this catalytic process is called a whole-cell catalyst.

[0078] Free expression: Free expression is the expression of target genes using free plasmids. Free plasmids are independent DNA molecules that exist in cells and have the ability to replicate and be transmitted independently. They are widely used in genetic engineering and molecular biology research.

[0079] Integrated expression: Expression that occurs when a gene is integrated into the genome.

[0080] β-Galactosidase catalyzes the hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides, such as lactose (1,4-O-β-D-galactopyranose-D-glucose), oligosaccharides, glycolipids, and glycoproteins. In a preferred embodiment, the activity of β-galactosidase can be determined using o-nitrobenzene-β-D-galactoside as a substrate at pH 6.5. Its transglycosylation performance is evaluated by changes in β-galactosidase activity in substrate solutions containing cellobiose.

[0081] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0082] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0083] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0084] Unless otherwise specified, all reagents or instruments used in the following embodiments, unless otherwise indicated by the manufacturer, are commercially available products. Where specific conditions are not specified in the embodiments, they are performed under standard conditions or conditions recommended by the manufacturer.

[0085] The plasmids, restriction enzymes, PCR enzymes, column DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products. The specific operations were performed according to the kit instructions.

[0086] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Specifically, they can be performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0087] In this specification, the amino acids at the corresponding sites are represented by the recognized IUPAC single-letter abbreviations, where each amino acid and its abbreviation are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0088] In this specification, mutations of amino acids are referred to as "original amino acid, site, substituted amino acid". For example, the mutation of asparagine N to methionine M at position 394, starting from the N (nitrogen) end of the sequence and counting sequentially from the C (carbon) end, is represented as N394M.

[0089] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0090] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0091] Example 1: Construction and screening of β-galactosidase expression strains derived from Bifidobacterium bifidum

[0092] In this embodiment, the lactase Nurica sequence was used as a probe to perform a search in Blast (BLAST: Basic Local Alignment Search Tool), and a sequence from Bifidobacterium bifidum was found. Bifidobacterium bifidumThe protein, with NCBI ID WP_270266043.1, was predicted to have β-galactosidase activity and was labeled as a wild-type β-galactosidase. The full-length sequence of this wild-type β-galactosidase is 1935 AA. After identifying a potentially preferred wild-type β-galactosidase, this application further truncated it to obtain a novel β-galactosidase M3 with the amino acid sequence shown in SEQ ID NO.1. The nucleotide sequence of the novel β-galactosidase M3 is based on Bacillus subtilis (…). Bacillus subtilis Codon bias was optimized (at https: / / www.genscript.com.cn / gensmart-free-gene-codon-optimization.html), and the nucleotide sequence is shown in SEQ ID NO.2. The gene was then synthesized and the plasmid was constructed by Nanjing GenScript.

[0093] The recombinant plasmid and strain expressing novel β-galactosidase M3 were constructed according to the description in Chinese Patent Publication No. CN115247166A. The specific method is as follows:

[0094] The protein expression signal peptide was derived from the alkaline protease AprE of Bacillus clavatum, as shown in SEQ ID NO. 5. The novel β-galactosidase M3 gene was expressed using the amyE gene promoter from strain ATCC6051a. The amyE gene promoter sequence is shown in SEQ ID NO. 6, and this promoter is contained at the end of the amyE-5' fragment, directly linked to the aprE signal peptide. The terminator was selected from the terminator sequence of the Bacillus amyloliquefaciens α-amylase gene (amyQ), as shown in SEQ ID NO. 7. A novel β-galactosidase M3 expression cassette, containing the amyE gene promoter, protein expression signal peptide, novel β-galactosidase M3, and terminator, was synthesized by Nanjing GenScript Biotech Co., Ltd.

[0095] The pUC57-BsaI-free plasmid was obtained by point mutation of the pUC57 plasmid to destroy the BsaI restriction site, and this plasmid was used as the backbone for construction. The pUC57-BsaI-free plasmid was linearized using BamHI-HindIII, and the 2674 bp fragment was recovered by gel electrophoresis. Using Bacillus subtilis ATCC6051a genomic DNA as a template, the upstream homologous arm of amyE (abbreviated as amyE-5') was amplified using primers amyE-5′-F and amyE-5′-R, with a PCR product size of 668 bp. The downstream homologous arm of amyE (abbreviated as amyE-3') was amplified using primers amyE-3′-F and amyE-3′-R, with a PCR product size of 671 bp. The three fragments of 2674 bp, 668 bp, and 671 bp were then recombinated using CloneZ recombinase (manufactured by Genscript), inserting two nucleotides between the amyE-5' and amy-3' fragments. BsaI The site was used for subsequent plasmid construction, and the resulting plasmid was named pUC57-BsaI-free-amyE. Primer sequences are shown in Table 1.

[0096] The pUC57-BsaI-free-amyE plasmid was used BsaI Linearization was performed to obtain a 3876 bp fragment. Using Bacillus licheniformis ATCC14580 genomic DNA as a template, the chloramphenicol resistance gene expression cassette was amplified using Cm-F and Cm-R primers, with a PCR product size of 1264 bp. The above three fragments—pUC57-BsaI-free-amyE linearized plasmid, novel β-galactosidase M3 expression cassette, and chloramphenicol resistance gene expression cassette—were recombined using CloneZ recombinase (manufactured by Genscript). The resulting plasmid was called the β-galactosidase expression plasmid, and the primer sequences are shown in Table 1.

[0097] The constructed β-galactosidase expression plasmid was introduced into competent cells of Bacillus subtilis A164Δ4-comK to construct a β-galactosidase expression strain. The experimental host, A164Δ4-comK, was obtained by modifying Bacillus subtilis strain ATCC6051a, and the modification included... aprE , nprE , spo and amyE Gene inactivation, and aprE The site integrates a mechanism for enhancing competent valence. comK Genes are manipulated by xylose-inducible promoters.

[0098] The β-galactosidase expression strains obtained above were subjected to shake-flask fermentation. The experimental method is as follows: Appropriate amounts of bacterial cells were inoculated into 250 mL Erlenmeyer flasks containing 50 mL of LB liquid medium (5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, with the remainder being water). The flasks were incubated overnight at 37℃ and 220 rpm. Then, 1% of the inoculum was inoculated into 250 mL Erlenmeyer flasks containing 50 mL of AKP fermentation medium (formulation: 10% glucose, 6% soybean meal, 1% anhydrous disodium hydrogen phosphate, with the remainder being water). The flasks were incubated at 37℃ and 220 rpm for 96 h. 2 mL of the fermentation broth was then centrifuged at 12000 rpm for 2 min. The supernatant was used to detect β-galactosidase activity and transglycosylation performance (galacto-oligosaccharide synthesis performance) and SDS-PAGE was performed. The β-galactosidase activity and transglycosylation performance are shown in Table 2. The SDS-PAGE results are as follows: Figure 1 As shown. Furthermore, Nurica was used as a control for evaluating enzymatic performance. Its construction and expression methods are described in the section on the construction of recombinant plasmids and strains of novel β-galactosidase M3. The amino acid sequence of Nurica is shown in SEQ ID NO:8.

[0099] β-galactosidase activity was determined according to section 6.1.1 of GB / T 33409-2016, "Spectrophotometric Method for the Determination of β-galactosidase Activity," using the phosphate buffer method at pH 6.5. β-galactosidase fermentation activity is defined as U / mL, where U is defined as the amount of enzyme that catalyzes the conversion of one micromolar of o-nitrobenzene-β-D-galactoside per minute under specified reaction conditions. The principle of enzyme activity detection is that β-galactosidase catalyzes the formation of o-nitrobenzene from ONPG (o-nitrobenzene-β-D-galactoside) at pH 6.5, and the absorbance of the solution is measured at a wavelength of 420 nm using a spectrophotometer. Enzyme activity is proportional to absorbance.

[0100] Transglycosylation performance (transgalactosylation activity) was evaluated by changes in the enzyme activity of β-galactosidase in a substrate solution containing cellobiose. 0.25 g of ONPG and 0.685 g of cellobiose (20 mM) were weighed and dissolved in 80 mL of pH 6.5 phosphate buffer in a beaker. The solution was stirred on a magnetic stirrer until completely dissolved (20 min), then transferred to a 100 mL volumetric flask and brought to volume to prepare the transglycosylation performance assay substrate solution. Using the same method as for β-galactosidase activity assay, the above transglycosylation performance assay substrate solution was used to replace the original substrate (ONPG substrate) solution to measure the A produced by β-galactosidase hydrolysis of ONPG. 420 .

[0101] The formula for calculating the transglycosylation capacity coefficient is: A measured by the substrate in the transglycosylation performance assay. 420A measured from the original substrate 420 If the transglycosylation capacity coefficient is greater than 1, the sample is considered to have transglycosylation capacity.

[0102] Table 1: Primer sequences

[0103]

[0104] Table 2: Fermentation activity and transglycosylation ability of the new β-galactosidase sequence

[0105]

[0106] As shown in Table 2, compared with the known β-galactosidase Nurica, the novel β-galactosidase M3 obtained in this embodiment has higher fermentation activity in Bacillus subtilis and its transglycosylation performance is comparable to that of Nurica. Compared with the existing technology, it can achieve significant advantages in fermentation cost and has good application scenarios.

[0107] Example 2: Construction of a β-galactosidase mutant with improved fermentation activity

[0108] This embodiment analyzes the enzyme structure of the novel β-galactosidase M3 from Example 1. For example... Figure 2 As shown, several amino acids (blue sticks) near the calcium ion (pink ball) binding site of the novel β-galactosidase M3 were selected and mutated to obtain β-galactosidase M3 mutants with improved enzyme activity. Analysis results showed that position 394 may be a potentially important site for improving enzyme activity, especially the mutation of the asparagine residue at position 394 to a methionine residue.

[0109] In this embodiment, two mutants, N394M (amino acid sequence as shown in SEQ ID NO.3, nucleotide sequence as shown in SEQ ID NO.4) and N394T, were selected. Strains expressing the β-galactosidase M3 mutants N394M and N394T were constructed using the same method as in Example 1, and the fermentation activity and transglycosylation performance of β-galactosidase were detected using the same method. The results are shown in Table 3.

[0110] Table 3: β-galactosidase mutants and performance test results

[0111]

[0112] The results are shown in Table 3. The mutation of the M3 mutant N394M can increase the fermentation activity of β-galactosidase M3 from 60 U / mL to 76 U / mL while maintaining comparable transglycosylation performance. Compared with Nurica, the M3 mutant N394M has significantly enhanced fermentation enzyme activity and comparable galacto-oligosaccharide synthesis energy, showing broad application prospects.

[0113] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

[0114] sequence list

[0115] >SEQ ID NO.1 (Novel β-galactosidase M3 amino acid sequence)

[0116] VEDATRSDSTQMSSTPEVVYSSAVDSKQNRTSDFDANWKFMLSDSVQAQDPTFDDSAWQQVDLPHDYSITQKYSQSNEAESAYLPGGTGWYRKSFTIDRDLAGKRIAINFDGVYMNATVWFNGVKLGTHPYGYSPFSFDLTGNAKFGEENTIVVKVENRLPSSRWYSGSGIYRDVTLTVTDGVHVGNNGVAIKTPSLATQNGGNVTMNLTTKVANDTKAANITLKQTVFPKGGKTDAAIGTVTTTASKSIAAGASADVTSTITAASPKLWSIKNPNLYTVRTEVLNGGKVLDTYDTEYGFRWTGFDATSGFSLNGEKVKLKGVSMHHDQGSLGAVANRRAIERQVEILQKMGVNSIRTTHNPAAKALIDVCNEKGVLVVEEVFDMWNRSKNGNTEDYGKWFGQTIAGDNAVLGGDKDETWAKFDLTSTINRDRNAPSVIMWS LGNEMMEGISGSVSDFPATSAKLVAWTKAADSTRPMTHGDNKIKANWNESNTMGDNLTANGGVVGTNYSNGANYDQIRTGHPSWAIYGSETASAINSRGIYNRTTGGAQSSDKQLTSYDNSAVGWGAVASSAYYDVVQRDFVAGTYVWTGFDYLGEPTPWNGTGGSGAVGSWPSPKNSYFGIVDTAGFPKDTYYFYQSQWNDDVHTLHILPAWNENVVAKDSSNKVPVVVYTDAAKVKLYFTPKGSTEKRLIGEKSFTKKTTAAGYTYQVYEGTDKDSTAHKNMYLTWNVPWAEGTISAEAYDENNRLIPEGSTEGNASVTTTGKAAKLKADADRKTITADGKDLSYIEVDVTDANGHIVPDAANRVTFDVKGAGKLVGVDNGSSPDHDSYQADNRKAFSGKVLAIVQSTKEAGEITVTAKADGLQSFTVKIATTAVGTSTEKT

[0117] >SEQ ID NO. 2

[0118]

[0119] >SEQ ID NO. 3

[0120] VEDATRSDSTTQMSSTPEVVYSSAVDSKQNRTSDFDANWKFMLSDSVQAQDPTFDDSAWQQVDLPHDYSITQKYSQSNEAESAYLPGGTGWYRKSFTIDRDLAGKRIAINFDGVYMNATVWFNGVKLGTHPYGYSPFSFDLTGNAKFGEENTIVVKVENRLPSSRWYSGSGIYRDVTLTVTDGVHVGNNGVAIKTPSLATQNGGNVTMNLTTKVANDTKAA ANITLKQTVFPKGGKTDAAIGTVTTASKSIAAGASADVTSTITAASPKLWSIKNPNLYTVRTEVLNGGKVLDTYDTEYGFRWTGFDATSGFSLNGEKVKLKGVSMHHDQGSLGAVANRRAIERQVEILQKMGVNSIRTTHNPAAKALIDVCNEKGVLVVEEVFDMWNRSKNGMTEDYGKWFGQTIAGDNAVLGGDKDETWAKFDLTSTINRDRNAPSVIMWS LGNEMMEGISGSVSDFPATSAKLVAWTKAADSTRPMTHGDNKIKANWNESNTMGDNLTANGGVVGTNYSNGANYDQIRTGHPSWAIYGSETASAINSRGIYNRTTGGAQSSDKQLTSYDNSAVGWGAVASSAWYDVVQRDFVAGTYVWTGFDYLGEPTPWNGTGSGAVGSWPSPKNSYFGIVDTAGFPKDTYYFYQSQWNDDVHTLHILPAWNEVVAKDSS NKVPVVVYTDAAKVKLYFTPKGSTEKRLIGEKSFTKKTTAAGYTYQVYEGTDKDSTAHKNMYLTWNVPWAEGTISAEAYDENNRLIPEGSTEGNASVTTTGKAAKLKADADRKTITADGKDLSYIEVDVTDANGHIVPDAANRVTFDVKGAGKLVGVDNGSSPDHDSYQADNRKAFSGKVLAIVQSTKEAGEITVTAKADGLQSFTVKIATTAVPGTSTEKT

[0121] >SEQ ID NO.4 (nucleotide sequence of novel β-galactosidase M3 mutant N394M)

[0122]

[0123] >SEQ ID NO.5 (Signal Peptide)

[0124] MKKPLGKIVASTALLISVAFSSSIASA

[0125] >SEQ ID NO.6 (amyE gene promoter sequence of ATCC6051a strain)

[0126] gtaagcgttaacaaaattctccagtcttcacatcggtttgaaaggaggaagcggaagaatgaagtaagaggggatttttgactccgaagtaagtcttcaaa aaatcaaata aggagtgtcaaga

[0127] >SEQ ID NO.7 (Terminator)

[0128] TAATCAATAAAAAAACGCTGTGCGGTTAAAGGGCACAGCGTTTTTTTGTGTGT

[0129] >SEQ ID NO.8 (Nurica amino acid sequence of lactase)

[0130] VEDATRSDSTQMSSTPEVVYSSAVDSKQNRTSDFDANWKFMLSDSVQAQDPAFDDSAWQQVDLPHDYSITQKYSQSNEAESAYLPGGTGWYRKSFTIDRDLAGKRIAINFDGVYMNATVWFNGVKLGTHPYGYSPFSFDLTGNAKFGGENTIVVKVENRLPSSRWYSGSGIYRDVTLTTVTGVHVGNNGVAIKTPSLATQNGGDVTMNLTTKVANDTEAAANITLKQTVFPKGGKTDAAIGTVTTASKSIAAGASADVTSTITAASPKLWSIKNPNLYTVRTEVRNGGKVLDTYDTEYGFRWTGFDATSGFSLNGEKVKLKGVSMHHDQGSLGAVANRRAIERQVEILQKMGVNSIRTTHNPAAKALIDVCNEKGVLVVEEVFDMWNRSKNGNTEDYGKWFGQAIAGDNAVLGGDKDETWAKFDLTSTINRDRNAPSVIMWS LGNEMMEGISGSVSGFPATSAKLVAWTKAADSTRPMTYGDNKIKANWNESNTMGDNLTANGGVVGTNYSDGANYDKIRTTHPSWAIYGSETASAINSRGIYNRTTGGAQSSDKQLTSYDNSAVGWGAVASSAYDVVQRDFVAGTYVWTGFDYLGEPTPWNGTGGSGAVGSWPSPKNSYFGIVDTAGFPKDTYYFYQSQWNDDVHTLHILPAWNENVVAKGSGNNVPVVVYTDAAKVKLYFTPKGSTEKRLIGEKSFTKKTTAAGYTYQVYEGSDKDSTAHKNMYLTWNVPWAEGTISAEAYDENNRLIPEGSTEGNASVTTTGKAAKLKADADRKTITADGKDLSYIEVDVTDANGHIVPDAANRVTFDVKGAGKLVGVDNGSSPDHDSYQADNRKAFSGKVLAIVQSTKEAGEITVTAKADGLQSSTVKIATTAVGTSTEKT

[0131] >SEQ ID NO.9

[0132] tcggtacctcgcgaatgcatctagatatcggatccgcggcattatgtttgaatttccgtttaaag

[0133] >SEQ ID NO.10

[0134] ctTgagacctttgagcttccgagactggtctcatcttgacactccttatttgattttttgaagac

[0135] >SEQ ID NO.11

[0136] aagatgagaccagtctcggaagctcaaaggtctcaagcgcccaagtgcccggtcagaatc

[0137] >SEQ ID NO.12

[0138] acaggaaacagctatgaccatgattacgccaagcttcttcactaacgatgcctttgaaaatcttc

[0139] >SEQ ID NO.13

[0140] aacgtcgtgactgggaaaacc

[0141] >SEQ ID NO.14

[0142] agccaggctgattctgaccgggcacttgggcgctttattggtatgactggttttaagcgc

Claims

1. A β-galactosidase mutant, characterized in that, The amino acid sequence of the β-galactosidase mutant was obtained by mutating the asparagine residue at position 394 of the β-galactosidase to a methionine residue. The amino acid sequence of the β-galactosidase is shown in SEQ ID NO.

1. The β-galactosidase mutant has β-galactosidase activity.

2. The β-galactosidase mutant according to claim 1, characterized in that, The amino acid sequence of the β-galactosidase mutant is shown in SEQ ID NO.

3.

3. A biomaterial, characterized in that, The biomaterial includes any one of the following A1)-A4): A1) A nucleic acid molecule, wherein the nucleic acid molecule is a nucleic acid molecule encoding the β-galactosidase mutant of claim 1 or 2; A2) An expression cassette, wherein the expression cassette contains the nucleic acid molecule described in A1); A3) A recombinant vector containing the nucleic acid molecule described in A1) or the expression cassette described in A2); A4) Host cell, wherein the host cell contains the nucleic acid molecule described in A1), the expression cassette described in A2), or the recombinant vector described in A3).

4. The biomaterial according to claim 3, characterized in that, The host cell is one or more of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, and Saccharomyces cerevisiae.

5. The biomaterial according to claim 3, characterized in that, The nucleic acid molecule is shown in SEQ ID NO.

4.

6. A method for preparing the β-galactosidase mutant as described in claim 1 or 2, characterized in that, The method includes: culturing the host cells as described in any one of claims 3-5 to obtain the β-galactosidase mutant.

7. The application of the β-galactosidase mutant as described in claim 1 or 2, or the biomaterial as described in any one of claims 3-5, or the β-galactosidase mutant prepared by the method as described in claim 6, in reducing lactose content, catalyzing lactose decomposition, or preparing galactooligosaccharides.

8. A composition, characterized in that, The composition contains the β-galactosidase mutant as described in claim 1 or 2.

9. A method for producing food, characterized in that, The method includes treating a lactose-containing substrate with a β-galactosidase mutant as described in claim 1 or 2.

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