Highly active beta-galactosidase mutants, methods for their production and use

Abstract

The application provides a high-activity beta-galactosidase mutant, a preparation method and application thereof. In the application, through analysis of a three-dimensional space structure of a complex of a wild-type beta-galactosidase, a substrate and metal ions, and site-directed mutation and combined mutation of amino acid sites related to beta-galactosidase activity and substrate binding, combined with random mutation, a series of beta-galactosidase mutants are successfully obtained, and the enzyme activity of the mutants is greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering, and more specifically, this invention relates to highly active β-galactosidase mutants, their preparation methods, and applications. Background Technology

[0002] β-galactosidase, or β-gal for short, is an exoglycosidase with galactosidase hydrolytic activity. It can specifically hydrolyze β-1,4-galactosidic bonds, catalyzing the hydrolysis of the disaccharide substrate lactose into monosaccharides: glucose and galactose, which is a key step in lactose metabolism. Under specific conditions in the feedback pathway (such as high concentrations of lactose), it can also transfer the galactosyl group in lactose to other sugar molecules to generate isolaxose. Isolaxose can bind to the lacZ repressor, relieving its inhibition of the operon, thereby initiating the expression of genes such as β-galactosidase, forming an induction regulatory pathway that regulates the expression of genes such as β-galactosidase in cells. At the same time, β-galactosidase can further hydrolyze isolaxose into glucose and galactose, completing the lactose metabolic cycle.

[0003] β-galactosidase, as a classic and highly efficient reporter molecule, is widely used in studies of spatiotemporal gene expression and regulation in various cell types. Using β-galactosidase to determine promoter and gene expression and regulatory mechanisms offers advantages such as simplicity, readily available and diverse substrates (e.g., chromogenic substrates X-gal, fluorescent substrates MUG), compatibility with various detection techniques (colorimetric, fluorescence, histochemical staining, etc.), and high stability. It can be applied to many areas, including gene expression and regulation mechanism analysis (promoter function analysis / transcriptional regulatory network research), subcellular localization labeling, in vivo / tissue in situ detection, tissue-specific expression, developmental regulation, mRNA stability, analysis of various organelle-targeting protein signal sequences, and aging-related research.

[0004] When detecting β-galactosidase activity, one of its substrates, o-nitrophenyl-β-D-galactopyranoside (ONPG), can be used. ONPG is decomposed to produce yellow o-nitrophenol (ONP), and enzyme activity is quantified by measuring the absorbance change at a wavelength of 410 nm. Alternatively, 4-methylumbelliferone-β-D-galactosidase (MUG) can be used, which hydrolyzes to produce the strongly fluorescent product 4-methylumbelliferone (MU), and enzyme activity is detected by a fluorescence spectrophotometer (excitation wavelength 365 nm, emission wavelength 445 nm). Another method is to use 5-bromo-4-chloro-3-indole-β-D-galactosidase (X-gal), which hydrolyzes to produce the insoluble blue precipitate 5-bromo-4-chloro-indigo, as used for blue-white screening of bacterial clones. Of course, enzyme activity detection methods are not limited to these methods. This invention primarily uses the first detection method for comparative detection of β-galactosidase activity in wild-type and a series of mutant strains.

[0005] With the advancement of biotechnology and the increasing demand for β-galactosidase in areas such as cell senescence and gene expression, the limited recombinant expression efficiency and original activity of E. coli-derived β-galactosidase have severely restricted its production and application in the field of biotechnology. Developing highly active β-galactosidase has become an urgent problem to be solved in the field of biotechnology.

[0006] Those skilled in the art have mutated β-galactosidases from Aspergillus oryzae, lactic acid bacteria, and other types of microorganisms, modifying them in various aspects such as pH tolerance, heat resistance, and catalytic activity, thereby improving enzyme activity or stability to some extent. For example, Martinez-Bilbao M et al. found that the Escherichia coli β-galactosidase G794D mutant significantly improved the catalytic activity against lactose, but at the same time, it also caused a significant decrease in heat stability and protease tolerance (Martinez-Bilbao M, Holdsworth RE, Edwards LA and Huber RE. J Biol Chem. 1991. 266(8):4979-86). Therefore, successful modifications to the enzyme properties of β-galactosidases in this field are still relatively few.

[0007] To further improve the substrate recognition, binding, and hydrolysis efficiency of β-galactosidase, there is an urgent need in this field to further improve the enzyme activity of β-galactosidase, thereby achieving the efficient production of highly active β-galactosidase for better application in various related technologies. Summary of the Invention

[0008] The purpose of this invention is to provide a highly active β-galactosidase mutant, its preparation method, and its application.

[0009] In a first aspect of the present invention, a method for improving the enzyme activity of β-galactosidase is provided, comprising: mutating β-galactosidase to form a β-galactosidase mutant; wherein the amino acid sequence of the mutant corresponds to the amino acid sequence shown in SEQ ID NO:1, M203 is mutated to an aromatic ring side chain amino acid, Q601 is mutated to an amino acid without a side chain, F602 is mutated to a hydrophobic side chain amino acid, and R801 is mutated to a negatively charged amino acid, wherein the mutation includes single-point mutation or combination mutation.

[0010] In one or more embodiments, the amino acid sequence of the mutant corresponds to the wild-type β-galactosidase amino acid sequence shown in SEQ ID NO:1, and includes the following mutations:

[0011] I. The following mutations or combinations thereof:

[0012] (i) The 203rd position is mutated from M to F, the 601st position is mutated from Q to G, and the 602nd position is mutated from F to L (M203F / Q601G / F602L);

[0013] (ii) The 203rd position is mutated from M to F, and the 601st position is mutated from Q to G (M203F / Q601G);

[0014] (iii) The 203rd position is mutated from M to F, and the 602nd position is mutated from F to L (M203F / F602L);

[0015] (iv) The 203rd position is mutated from M to F, the 601st position is mutated from Q to G, and the 801st position is mutated from R to E (M203F / Q601G / R801E);

[0016] (v) The 203rd position is mutated from M to F, and the 801st position is mutated from R to E (M203F / R801E);

[0017] (vi) The 203rd position is mutated from M to F (M203F);

[0018] (vii) The 601st position is mutated from Q to G (Q601G);

[0019] (viii) The 602nd position is mutated from F to L (F602L);

[0020] (ix) The 801st position is mutated from R to E (R801E);

[0021] II. A protein derived from I that has 80% or more (preferably 85% or more; more preferably 90% or more; even more preferably 95%, such as 98%, 99%) homology to the amino acid sequence of protein I and has the function of protein I, but the sites corresponding to (i)-(ix) in I are conserved (amino acid residues are unchanged).

[0022] In another aspect of the invention, a β-galactosidase mutant is provided, the amino acid sequence of which corresponds to the amino acid sequence shown in SEQ ID NO:1, wherein M203 is mutated to an aromatic ring side chain amino acid, Q601 is mutated to an amino acid without a side chain, F602 is mutated to a hydrophobic side chain amino acid, and R801 is mutated to a negatively charged amino acid, the mutations including single-point mutations or combination mutations.

[0023] In one or more embodiments, the amino acid sequence of the mutant is as defined above.

[0024] In another aspect of the invention, a polynucleotide is provided that encodes the β-galactosidase mutant described above.

[0025] In one or more embodiments, the β-galactosidase (or the gene encoding it) is a β-galactosidase (or the gene encoding it) derived from Escherichia coli.

[0026] In another aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotides described above.

[0027] In another aspect of the invention, a genetically engineered cell containing the expression vector described above is provided, wherein the genetically engineered cell contains the expression vector or the polynucleotide integrated into its genome.

[0028] In one or more embodiments, the host cell includes: prokaryotic cells or eukaryotic cells; preferably, the eukaryotic host cell includes: yeast cells, fungal cells, insect cells, mammalian cells, etc.; the prokaryotic host cell includes Escherichia coli, Bacillus subtilis, etc.

[0029] In one or more embodiments, the genetically engineered cell is a prokaryotic cell, such as an Escherichia coli cell.

[0030] In one or more embodiments, the 5' end of the polynucleotide in the vector further includes a signal peptide, a tag peptide, a fluorescent protein, and / or a promoter. The 3' end of the polynucleotide further includes a tag peptide, a fluorescent protein, and / or a terminator.

[0031] In one or more embodiments, compared with the β-galactosidase of the amino acid sequence shown in SEQ ID NO:1, the enzyme activity of the modified β-galactosidase is increased by more than 5% or 8%, preferably by more than 10%, 20%, or 30%, and more preferably by more than 40%.

[0032] In another aspect of the invention, a method for producing the mutant of the β-galactosidase is provided, comprising the steps of: (a) culturing the host cells; (b) collecting a culture containing the mutant of the β-galactosidase; and (c) isolating the mutant of the β-galactosidase from the culture.

[0033] In another aspect of the invention, the use of the β-galactosidase mutant, a host cell expressing the mutant, or a lysis product thereof is provided for hydrolyzing galactosidic bonds.

[0034] In one or more embodiments, the β-galactosidase mutant, the host cell expressing the mutant, or its cleavage product are used to catalyze the hydrolysis of substrates containing galactosidic bonds.

[0035] In one or more embodiments, the substrate containing a galactosidic bond includes (but is not limited to): lactose (which can be broken down into glucose and galactose), galactose, o-nitrophenyl-β-D-galactopyranoside (ONPG), X-gal, or analogues thereof.

[0036] In another aspect of the invention, a method for hydrolyzing galactosidic bonds is provided, comprising: using the β-galactosidase mutant, a host cell expressing the mutant, or its lysis product to hydrolyze the galactosidic bonds; preferably, catalyzing the hydrolysis of a substrate containing galactosidic bonds.

[0037] In another aspect of the invention, a kit for hydrolyzing galactosidic bonds is provided, comprising: any of the β-galactosidase mutants described above; or the expression vectors described above; or the genetically engineered cells described above.

[0038] In one or more embodiments, the kit is also used to include (but is not limited to) the following detections: β-galactosidase reporter gene detection, in situ staining detection of β-galactosidase in cells or tissues, lysosomal β-galactosidase staining detection, and β-galactosidase staining detection in senescent cells or tissues.

[0039] Other aspects of the invention will be apparent to those skilled in the art from the disclosure of this invention. Attached Figure Description

[0040] Figure 1 A diagram showing the overall structure of the wild-type Escherichia coli β-galactosidase tetramer and its substrate complex.

[0041] Figure 2 Structural details of the two substrate-binding regions of wild-type β-galactosidase (AB).

[0042] Figure 3 A bar chart comparing the crude enzyme activity of wild-type β-galactosidase and each single-point mutant.

[0043] Figure 4 Electrophoresis diagrams and relative activity bar charts of nine β-galactosidase mutants and wild-type β-galactosidases with significantly enhanced enzyme activity are shown. A is the protein electrophoresis diagram with a loading amount of 10 μg for each β-galactosidase sample. B is the relative activity bar chart comparing wild-type β-galactosidase with the nine β-galactosidase mutants. ** indicates significant difference (p < 0.01).

[0044] Figure 5 Three-dimensional structural diagram of each mutant site of β-galactosidase with significantly improved enzyme activity. Detailed Implementation

[0045] Based on their previous research experience with various β-galactosidase reporter gene system products, the inventors analyzed the three-dimensional structure of the complex of wild-type β-galactosidase with substrates and metal ions. By rationally designing, site-directed mutagenesis, combined mutagenesis, and random mutagenesis targeting amino acid sites related to β-galactosidase activity and substrate binding, they successfully screened a series of β-galactosidase mutants with high enzyme activity, which showed a very significant improvement in enzyme activity.

[0046] the term

[0047] As used in this invention, unless otherwise stated, "β-galactosidase mutant" and "mutant β-galactosidase" are used interchangeably, referring to an enzyme (peptide / protein) formed by mutation at certain sites related to enzyme activity as determined by the inventors, corresponding to the β-galactosidase before mutation. Preferably, the mutation corresponds to the amino acid sequence shown in SEQ ID NO:1, and the mutation is selected from the following sites or combinations thereof: position 203, position 601, position 602, or position 801; preferably, it also includes some combined mutations, wherein the preferred multi-point mutant M81 (M203F / Q601G) has an enzyme activity that is increased to about 1.3 times that of wild-type β-galactosidase, and the optimal multi-point mutant M84 (M203F / Q601G / F602L) has an enzyme activity that is increased to about 1.4 times that of wild-type β-galactosidase.

[0048] If you need to represent the β-galactosidase before the mutation, you can refer to the enzyme with the amino acid sequence shown in SEQ ID NO:1.

[0049] Unless otherwise stated, at the protein level, the mutation sites of the mutants in this invention are based on the sequence shown in SEQ ID NO:1.

[0050] In this invention, unless otherwise stated, the identification of β-galactosidase mutants is to use "the amino acid that was replaced at the original amino acid position" to indicate the mutated amino acid in the β-galactosidase mutant, such as M203F, which means that the amino acid at position 203 is replaced by F by the starting enzyme.

[0051] As used in this invention, "aromatic ring side chain amino acids" include three types: phenylalanine (Phe, F), tryptophan (Trp, W), and tyrosine (Tyr, Y), which have similar benzene ring structures.

[0052] As used in this invention, “side-chain-free amino acids” include glycine (Gly, G) and alanine (Ala, A).

[0053] As used in this invention, "hydrophobic side chain amino acid" includes leucine (Leu, L), isoleucine (Ile, I), and methionine (Met, M).

[0054] As used in this invention, “negatively charged amino acids” include glutamic acid (Glu, E) and aspartic acid (Asp, D).

[0055] As used in this invention, "isolated β-galactosidase" refers to a β-galactosidase mutant that is substantially free of other naturally occurring proteins, lipids, carbohydrates, or other substances associated with it. Those skilled in the art can purify β-galactosidase mutants using standard protein purification techniques. Substantially pure protein produces a single master band on a non-reducing polyacrylamide gel.

[0056] As used in this invention, "improved enzyme activity" refers to a statistically significant increase, or a marked increase, in the enzyme activity of the mutated β-galactosidase compared to the unmodified β-galactosidase. For example, under the same reaction conditions / environment, after a certain reaction time, the mutant β-galactosidase with improved enzyme activity shows a significant increase of 5% or more, or 8% or more, preferably 10%, 20%, or 30% or more, and even more than 40% or more, compared to the unmodified enzyme.

[0057] As used in this invention, "a library of β-galactosidase mutants or polynucleotides encoding them" refers to a collection of polypeptides or polynucleotides containing a series of mutant β-galactosidases provided by this invention. The assembly of multiple β-galactosidase mutants or polynucleotides encoding them with different enzyme activities into a library facilitates the selection of appropriate β-galactosidases or their encoding nucleic acids by those skilled in the art based on their required reaction conditions.

[0058] As used in this invention, the term "effective amount" refers to the amount that produces the function or enzyme activity of the reaction of interest in this invention, achieving the desired effect (accurate detection results).

[0059] As used in this invention, the terms "containing" or "comprising" include "comprising," "consistently made of," and "made of." The term "consistently made of" means that, in addition to containing the essential ingredients or components, the composition / reaction system / kit may contain small amounts of minor components and / or impurities that do not affect the active ingredient.

[0060] β-galactosidase and its modification

[0061] This invention uses a protein three-dimensional spatial structure visualization and analysis tool to analyze the published structures of wild-type β-galactosidase complexes containing substrates and metal ions. It considers a large number of key amino acid sites related to enzyme activity and, combined with site-directed mutagenesis, designs and screens a series of site-directed and random mutants of β-galactosidase (verifying 80 single-point mutants and 5 multi-point mutants of β-galactosidase). Furthermore, the mutants with higher enzyme activity are expressed, purified, and their enzyme activity is measured and compared.

[0062] In a specific embodiment of the present invention, after the β-galactosidase mutant plasmid was confirmed by sequencing, it was simultaneously transformed into E. coli competent cells along with the wild-type plasmid. After culture and induction, E. coli bacterial cultures expressing β-galactosidase were obtained. Cells were lysed to release β-galactosidase, and the enzyme activities of each mutant were detected and compared. For mutants with significantly increased enzyme activity, further large-scale culture and induction expression were performed on the wild-type control, and the β-galactosidase proteins were purified to compare the differences in enzyme activity between the mutants and the wild-type. Using the above strategy, β-galactosidase mutants with high activity were successfully screened. In the amino acid sequence of the mutants, M203 was mutated to an aromatic ring side chain amino acid, Q601 was mutated to an amino acid without a side chain, F602 was mutated to a hydrophobic side chain amino acid, and R801 was mutated to a negatively charged amino acid. Furthermore, the superior mutant M81 (M203F / Q601G) showed a 30% increase in activity compared to the wild-type control β-galactosidase, while the optimal mutant M84 (M203F / Q601G / F602L) showed a 40% increase in activity compared to the wild-type control β-galactosidase, achieving highly efficient expression of the highly active β-galactosidase. Consequently, these enzyme mutants can be widely used in kits related to cell / tissue senescence detection, cell / tissue section staining, and gene spatiotemporal expression and regulation.

[0063] The β-galactosidase mutant of the present invention can be a chemically synthesized product or produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology.

[0064] This invention also includes fragments, derivatives, and analogs of the β-galactosidase mutant. As used herein, the terms "fragment," "derivative," and "analyte" refer to proteins that substantially retain the same biological function or enzymatic activity as the native β-galactosidase mutant of this invention. The protein fragments, derivatives, or analogs of this invention may be (i) proteins with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) proteins having substituent groups in one or more amino acid residues; or (iii) proteins formed by fusing an additional amino acid sequence to this protein sequence (such as a leader sequence, secretory sequence, or sequence used to purify this protein, or a proteogenic sequence, or a fusion protein). According to the definition of this invention, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art. However, a condition must be met: the amino acid sequence of the β-galactosidase mutant and its fragments, derivatives, and analogs must necessarily contain at least one mutation specifically pointed out above in this invention.

[0065] In this invention, the term "β-galactosidase mutant" also includes (but is not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-20, more preferably 1-10, and even more preferably 1-8, 1-5, 1-3, or 1-2), and the addition or deletion of one or more amino acids (typically up to 20, preferably up to 10, and more preferably up to 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition or deletion of one or more amino acids at the C-terminus and / or N-terminus generally does not alter the function of the protein. The term also includes enzyme-active fragments and enzyme-active derivatives of β-galactosidase mutants. However, at least one mutation described above in this invention is certainly present in these variant forms.

[0066] In this invention, the term "β-galactosidase mutant" also includes (but is not limited to): derived proteins that retain the protease activity of the β-galactosidase mutant and have at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, such as at least 98% or 99%, sequence identity with the amino acid sequence of the β-galactosidase mutant. Similarly, these derived proteins certainly contain at least one mutation as described above in this invention.

[0067] The invention also provides analogs of the β-galactosidase mutant. These analogs may differ from the β-galactosidase mutant in that they may be differences in amino acid sequence, differences in modifications that do not affect the sequence, or both.

[0068] The present invention also provides a multinucleotide sequence encoding the β-galactosidase mutant of the present invention or its conserved variant protein.

[0069] The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.

[0070] The polynucleotide encoding the mature protein of the mutant includes: a coding sequence that encodes only the mature protein; a coding sequence for the mature protein and various additional coding sequences; a coding sequence for the mature protein (and optional additional coding sequences) and a non-coding sequence.

[0071] "A polynucleotide encoding a protein" can be a polynucleotide that includes the protein itself, or it can include polynucleotides that also include additional coding and / or non-coding sequences.

[0072] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered using the vectors of the present invention or β-galactosidase mutant coding sequences, and methods for generating the mutant enzymes of the present invention via recombination technology.

[0073] Recombinant β-galactosidase mutants can be expressed or produced using the polynucleotide sequences of this invention through conventional recombinant DNA technology. Generally, the following steps are involved:

[0074] (1). Transform or transduce suitable host cells with the polynucleotide (or variant) encoding the β-galactosidase mutant of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0075] (2) Host cells cultured in a suitable culture medium;

[0076] (3) Isolate and purify proteins from culture media or cells.

[0077] In this invention, the β-galactosidase mutant polynucleotide sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well-known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0078] Methods well known to those skilled in the art can be used to construct expression vectors containing a β-galactosidase mutant encoding DNA sequence and suitable transcription / translation control signals. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Preferably, the expression vector contains one or more selective marker genes to provide phenotypic traits for selecting transformed host cells.

[0079] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform suitable host cells to enable them to express proteins. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.

[0080] In this invention, the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a mold cell or a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples include: *Escherichia coli*, *Bacillus subtilis*, *Streptomyces*, and *Agrobacterium*; eukaryotic cells such as yeast and plant cells. In a specific embodiment of this invention, *Escherichia coli* is used as the host cell.

[0081] application

[0082] Under suitable reaction conditions, β-galactosidase can hydrolyze galactosidic bonds; that is, the β-galactosidase of the present invention can be used as a catalyst to catalyze the formation of degradation products (monosaccharides) (such as glucose, galactose, o-nitrophenyl-β-D-galactopyranoside (ONPG), X-gal, etc.) from substrates containing galactosidic bonds, and can be applied in a variety of scenarios in which this reaction occurs, including some detection methods.

[0083] It should be understood that the β-galactosidase mutant of the present invention, which has higher enzyme activity, is applicable to a wide variety of technologies known or under development in the art and has broad applicability.

[0084] The present invention also provides a composition, or reaction system, or detection system, comprising an effective amount of the β-galactosidase mutant of the present invention, and other components required for cell or tissue hydrolysis (enzymatic digestion), or other components required for the reaction or detection, such as solutions that can form the reaction system. Those skilled in the art can determine the effective amount of the β-galactosidase mutant in the composition according to the actual use of the composition. Other substances may also be added to the composition to further regulate the enzyme activity of the β-galactosidase mutant of the present invention or to promote the reaction process.

[0085] To facilitate expanded or commercial applications, this invention also provides a kit comprising: the β-galactosidase mutant of this invention, or the aforementioned composition, or a cell / tissue hydrolysis solution, or a reaction system, or a detection system. Each reagent can be placed independently in a separate container, or two or more of them can be mixed in the same container.

[0086] In addition, the test kit may also include an instruction manual to guide people on how to use the test kit of the present invention correctly.

[0087] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the manufacturer's recommendations.

[0088] 1. Experimental materials, reagents and instruments

[0089] All materials and reagents, including Escherichia coli DH5α, BL21(DE3), site-directed mutagenesis kits, random mutagenesis kits, endonucleases DpnⅠ, NdeⅠ, XhoⅠ, LB medium-related reagents, antibiotics, inducers, plasmid mini-extraction kits, Ni affinity chromatography column packing, β-galactosidase assay reagents, protein concentration assay reagents, and 96-well plates, are sourced from our company.

[0090] The PCR instrument was purchased from Bio-Rad.

[0091] The ultrasonic cell disruptor was purchased from Ningbo Xinzhi Biotechnology Co., Ltd.

[0092] The high-pressure cell disruptor was purchased from Antos Nanotechnology (Suzhou) Co., Ltd.

[0093] The Clinx gel imaging system was purchased from Shanghai Qinxiang Scientific Instruments Co., Ltd.

[0094] The Varioskan LUX multi-functional microplate reader was purchased from ThermoFisher.

[0095] 2. Structural analysis of wild-type β-galactosidase complexes with substrates and metal ions

[0096] The three-dimensional complex structure of an enzyme-substrate binding enzyme can visually demonstrate the regions of protein-substrate interaction, the modes of substrate binding and release, and thus analyze the possible mechanisms of substrate catalysis, and screen for key amino acid sites involved in substrate-specific recognition. Escherichia coli β-galactosidase is a tetramer composed of four identical polypeptide subunits, each containing 1024 amino acids.

[0097] Amino acid sequence (SEQ ID NO:1) of β-galactosidase (wild type) from Escherichia coli:

[0098] MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLN

[0099] GEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNVTYPITVNP

[0100] PFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVNSAFHLWCNGRWVGYGQDSRL

[0101] PSEFDLSAFLRAGENRLAVMVLRWSDGSYLEDQD M WRMSGIFRDVSLLHKPTTQIS

[0102] DFHVATRFNDDFSRAVLEAEVQMCGELRDYLRVTVSLWQGETQVASGTAPFGGEII

[0103] DERGGYADRVTLRLNVENPKLWSAEIPNLYRAVVELHTADGTLIEAEACDVGFREV

[0104] RIENGLLLLNGKPLLIRGVNRHEHHPLHGQVMDEQTMVQDILLMKQNNFNAVRCSH

[0105] YPNHPLWYTLCDRYGLYVVDEANIETHGMVPMNRLTDDPRWLPAMSERVTRMVQ

[0106] RDRNHPSVIIWSLGNESGHGANHDALYRWIKSVDPSRPVQYEGGGADTTATDIICPM

[0107] YARVDEDQPFPAVPKWSIKKWLSLPGETRPLILCEYAHAMGNSLGGFAKYWQAFRQ

[0108] YPRLQGGFVWDWVDQSLIKYDENGNPWSAYGGDFGDTPNDR QF CMNGLVFADRT

[0109] PHPALTEAKHQQQFFQFRLSGQTIEVTSEYLFRHSDNELLHWMVALDGKPLASGEVP

[0110] LDVAPQGKQLIELPELPQPESAGQLWLTVRVVQPNATAWSEAGHISAWQQWRLAE

[0111] NLSVTLPAASHAIPHLTTSEMDFCIELGNKRWQFNRQSGFLSQMWIGDKKQLLTPLR

[0112] DQFTRAPLDNDIGVSEAT R IDPNAWVERWKAAGHYQAEAALLQCTADTLADAVLI

[0113] TTAHAWQHQGKTLFISRKTYRIDGSGQMAITVDVEVASDTPHPARIGLNCQLAQVA

[0114] ERVNWLGLGPQENYPDRLTAACFDRWDLPLSDMYTPYVFPSENGLRCGTRELNYGP

[0115] HQWRGDFQFNISRYSQQQLMETSHRHLLHAEEGTWLNIDGFHMGIGGDDSWSPSVS

[0116] AEFQLSAGRYHYQLVWCQK

[0117] Using three-dimensional spatial structure analysis tools, the overall structure and substrate-binding active sites of β-galactosidase were analyzed. It was found that the catalytic hydrolysis of the substrate by β-galactosidase mainly proceeds through two steps: The first catalytic step, called galactosylation, involves β-galactosidase anchoring the substrate to the substrate-binding pocket through hydrogen bonds and hydrophobic interactions, enabling the enzyme's active site to recognize and specifically bind the substrate. Subsequently, through conformational changes at the top of the β-galactosidase active site and interactions between the substrate and nearby amino acid side chains, the substrate is propelled from the top of the active site towards the catalytic center. This step is initiated by a proton donation from Glu461 and completed by the nucleophilic translocation of Glu537, which covalently interacts with the galactose substrate. The second catalytic step, called degalactosylation, is initiated by a proton acquisition from Glu461, propelling a second translocation of water and glucose. Based on the molecular mechanism of the β-galactosidase catalytic reaction, the spatial structure of the β-galactosidase-substrate and metal ion complex was analyzed in depth, leading to the deduction of key amino acid sites involved in substrate recognition and binding, and influencing catalytic activity.

[0118] Subsequently, based on the results of structural analysis, a series of site-directed mutants were constructed on the basis of wild-type β-galactosidase. Enzyme activity was tested on these site-directed mutants in the hope of screening out mutants that can significantly improve β-galactosidase activity.

[0119] 3. Construction of β-galactosidase site-directed mutants

[0120] a. Construction of wild-type β-galactosidase expression vector: The codon-optimized wild-type β-galactosidase gene (SEQ ID NO:1) was obtained by gene synthesis and cloned into the pET22b expression vector through NdeⅠ and XhoⅠ restriction sites. A 6XHis purification tag and a stop codon were added to the N-terminus of the protein to obtain the pET22b-β-galactosidase (wild-type) plasmid.

[0121] b. Design and synthesis of site-directed mutagenesis primers: Using pET22b-β-galactosidase (wild-type) plasmid as a template, site-directed mutagenesis PCR primers were designed and synthesized.

[0122] c. Site-directed mutation: using QuickMutation TM Site-directed mutagenesis PCR was performed using a gene site-directed mutagenesis kit (Beyotime product, D0206). The 50 μl reaction mixture included: 29 μl nuclease-free water and 5 μl 10X BeyoFusion. TM Buffer (with Mg2+), 10 μl dNTP mix (2.5 mM each), 2 μl forward primer (10 μM), 2 μl reverse primer (10 μM), 1 μl pET22b-β-galactosidase plasmid (200 ng), and 1 μl BeyoFusion TM DNA Polymerase

[0123] d. The PCR program was as follows: pre-denaturation at 95℃ for 3 min; 20 cycles: 95℃ for 30 s, 55℃ for 30 s, 68℃ for 3 min (30 s / kb); extension at 68℃ for 15 min; temporary storage at 4℃.

[0124] e. After the PCR reaction, add 1 μl of restriction enzyme DpnⅠ (Beyotime product, D6257) to the PCR reaction system, mix well, and incubate at 37°C for 1 hour to digest the template plasmid. Take 10 μl of each digested PCR product and add it to 100 μl of DH5α supercompetent cells (Beyotime product, D1031), mix well, place on ice for 30 min, heat shock at 42°C for 90 s, and incubate on ice for 2 min. Add 900 μl of antibiotic-free LB medium and shake at 37°C for 1 hour. Centrifuge to collect the bacterial cells, resuspend in 100 μl of antibiotic-free LB medium, and evenly spread the bacterial solution on LB agar plates containing 100 μg / ml ampicillin (Beyotime product, ST007). Invert the plates in a 37°C incubator and incubate overnight. The next day, pick single colonies and transfer them to 10 ml of LB liquid medium containing 100 μg / ml ampicillin, and incubate overnight.

[0125] f. Plasmid extraction and sequencing verification: Plasmids were extracted from the bacterial cultures of each mutant strain using a plasmid mini-extraction kit (Beyotime product, D0003), and the plasmid concentration was determined. The target gene was sequenced using universal primers T7 and T7-Terminator. The sequencing results were compared with the wild-type β-galactosidase sequence to identify the mutation site and mutation type.

[0126] g. Multiple-point mutation: For mutants that require the introduction of multiple mutation sites, multiple rounds of site-directed mutagenesis are required, with one mutation introduced in each round. Each round must be verified by sequencing to ensure success before proceeding to the next round.

[0127] h. Enzyme activity assay: The activity of different β-galactosidase site-directed mutants was detected using a β-galactosidase reporter gene assay kit (Beyotime product, RG0036) (substrate: ONPG).

[0128] 4. Construction of random mutants of β-galactosidase

[0129] To maximize the performance of β-galactosidase mutants and screen for more and better high-activity mutants, site-directed mutagenesis was performed, using the pET22b-β-galactosidase (wild-type) plasmid as a template, and QuickMutation was employed. TM A random mutation kit (Beyotime product, D0219) was used to perform random mutation PCR on E. coli β-galactosidase, construct a random mutant library, and detect and screen the enzyme activity of the random mutants. The main steps for constructing random mutants of β-galactosidase are as follows:

[0130] The 50 μl reaction system for random mutagenesis PCR includes: 31 μl PCR Grade Water, 5 μl 10x RandomMut buffer, 5 μl Mutation enhancer, 5 μl dNTPs (2.5 mM each), 1 μl template pET22b-β-galactosidase (wild-type) plasmid (1 ng), 1 μl forward primer (10 μM), 1 μl reverse primer (10 μM), and 1 μl RandomMut DNA polymerase.

[0131] The PCR program was as follows: 94℃ pre-denaturation for 30s; 25 cycles: 94℃ for 30s, 55℃ for 30s, 72℃ for 6min; extension at 72℃ for 10min; temporary storage at 4℃.

[0132] The PCR product was double-digested with NdeI and XhoI: The 50 μl double digestion reaction system consisted of: PCR Grade Water, 5 μl 10x Buffer R, 1 μl NdeI (Beyotime product, D6485), 1 μl XhoI (Beyotime, D6721), and PCR product (2 μg). Digestion was performed overnight at 37°C.

[0133] The overnight digested PCR product was ligated with the pET22b vector, which was linearized by digestion with NdeⅠ and XhoⅠ, and the ligation product was transformed into BL31(DE3) competent cells. The transformation process was as follows: 10 μl of the ligation product was added to 100 μl of DH5α supercompetent cells (Beyotime product, D1031), mixed well, and incubated on ice for 30 min. Then, the cells were heat-shocked at 42℃ for 90 s and incubated on ice for 2 min. 900 μl of antibiotic-free LB medium was added, and the cells were shaken at 37℃ for 1 hour. The cells were collected by centrifugation, resuspended in 100 μl of antibiotic-free LB medium, and the bacterial culture was evenly spread on LB agar plates containing 100 μg / ml ampicillin (Beyotime product, ST007). The plates were inverted and incubated overnight at 37℃.

[0134] The following day, single colonies were picked from the plate and transferred to 10 ml of LB liquid medium containing 100 μg / ml ampicillin. The culture was carried out at 37°C and protein expression was induced. Subsequently, the enzyme activity of different random mutants of β-galactosidase was detected using a β-galactosidase reporter gene assay kit (Beyotime product, RG0036).

[0135] Based on the enzyme activity assay results, plasmid extraction and sequencing were performed on the random mutants of β-galactosidase with significantly increased activity. Plasmids for each mutant were extracted using a small-scale plasmid extraction kit (Beyotime product, D0003), and the plasmid concentration was determined. The target gene was sequenced using universal primers T7 and T7-Terminator. The sequencing results were compared with the wild-type β-galactosidase sequence to identify the mutation site and mutation type.

[0136] 5. Enzyme activity detection of crude enzyme from β-galactosidase mutant

[0137] a. Wild-type pET22b-β-galactosidase and β-galactosidase site-directed / random mutant plasmids were transformed into Escherichia coli BL21(DE3) competent cells (Beyotime product, D1013S) and cultured overnight at 37°C on LB plates containing ampicillin.

[0138] b. The next day, pick a single colony and put it into 1 ml of LB liquid medium containing ampicillin. Incubate at 37°C and 220 rpm in a shaker until the OD600 value is between 0.6 and 0.8. Add IPTG (Beyotime product, ST098) to a final concentration of 1 mM and induce protein expression at 37°C.

[0139] c. Centrifuge 1 ml of the cultured bacterial solution at room temperature, 8000 rpm, for 5 minutes to collect the bacterial cells; remove the supernatant after centrifugation, and add 500 μl of BeyoLytic acid to the bacterial cells. TM Bacterial active protein extraction reagent (Beyotime product, P0013Q): Mix thoroughly by pipetting and let stand at room temperature for 1 hour to allow for complete lysis and protein extraction.

[0140] d. After complete lysis, the culture was centrifuged at 4°C and 12,000 rpm for 10 minutes, and the supernatant was transferred to a new 1.5 ml centrifuge tube for preliminary screening of crude enzyme activity.

[0141] e. Collect the crude β-galactosidase wild-type and mutant enzymes after induction expression and preliminary lysis extraction. Analyze their activity using a β-galactosidase reporter gene assay kit (Beyotime product, RG0036), and compare the enzyme activity differences between different β-galactosidase mutants and the control wild-type β-galactosidase. First, remove the β-galactosidase reporter gene assay kit from the -20℃ freezer and allow it to fully dissolve and equilibrate to room temperature. To avoid excessively high signal values ​​exceeding the linear range of the microplate reader, dilute the lysed crude enzyme sample 10-fold with 1X PBS, then take 50 μl to BeyoGold... TMIn a pure white 96-well cell culture plate (flat-bottomed with a lid, individually packaged) (Beyotime product, FCP968), use 50 μl of 1XPBS as a blank control. Then add 50 μl of β-galactosidase assay reagent (Beyotime product, RG0036-2) to each well, mix thoroughly by pipetting, and cover the 96-well plate. Alternatively, you can seal the 96-well plate with parafilm or plastic wrap to prevent evaporation. Incubate at 37°C for approximately 5 minutes or until a light yellow color appears in the sample wells. Typically, the absorbance plateaus after about 3 hours; longer incubation will not significantly increase the absorbance. Finally, add 150 μl of β-galactosidase stop solution (Beyotime product, RG0036-3) to terminate the reaction, mix well, and avoid air bubbles as much as possible. Use a microplate reader or spectrophotometer, set the measurement wavelength to 420 nm, and measure the absorbance. The absorbance value roughly reflects the activity level of different mutants.

[0142] 6. Purification of high-activity β-galactosidase mutants

[0143] a. Based on the preliminary results of crude enzyme activity detection, single-point mutants of β-galactosidase with significantly increased enzyme activity were screened for stacked combination mutations. The construction process of multi-point stacked combination mutants of β-galactosidase was carried out in the manner described above.

[0144] b. The successfully constructed high-activity β-galactosidase single-point mutant, multi-point combination mutant and control wild-type β-galactosidase plasmid were transformed into competent cells of BL21(DE3) strain.

[0145] c. The next day, a single colony was picked and inoculated into 10 ml of LB liquid medium containing ampicillin, and cultured overnight at 37°C and 220 rpm in a shaker. Then, 10 ml of the bacterial culture was transferred to 1000 ml of LB liquid medium containing ampicillin at a 1:100 ratio, and cultured at 37°C and 220 rpm in a shaker until the OD600 value reached approximately 0.6-0.8. The culture temperature was then lowered to 18°C, and 0.1 mM IPTG (Beyotime product, ST098) was added. The culture was continued for 16 hours with shaking to induce protein expression. 10 ml of the bacterial culture before and after induction was collected by centrifugation and resuspended in 10 ml of 1X PBS. Then, 50 μl of the bacterial culture samples before and after induction were added to 10 μl of 6X SDS-PAGE protein loading buffer (Beyotime product, P0015F), heated in a boiling water bath at 100°C for 10 min, and centrifuged at 12000 rpm for 10 min. Take 20 μl of each sample for SDS-PAGE electrophoresis to observe whether β-galactosidase protein is expressed in the induced bacterial cells.

[0146] d. The following purification steps were all performed at 4℃: Induced bacterial cells were collected and resuspended in 100ml of 1X PBS (10mM Na2HPO4, 2mM NaH2PO4, 135mM NaCl, 4.7mM KCl, pH 7.3±0.1 (25℃)) (Beyotime product, C0221A). The cells were then cyclically disrupted three times in a high-pressure cell disruptor at 850 bar. After centrifugation at 12000rpm for 30min, the supernatant was collected and purified using BeyoGold... TM His-tag Purification Resin (reduction-resistant chelating type) (Beyotime product, P2218) affinity purification column was used to purify the target protein, and the purified β-galactosidase was dialyzed into 1X PBS.

[0147] e. Protein concentration was determined using the Bradford Protein Concentration Assay Kit (detergent compatible) (Beyotime product, P0006) and detected by SDS-PAGE electrophoresis to further confirm the consistency of protein content in each β-galactosidase sample for subsequent enzyme activity detection and comparison. The aliquoted proteins were flash-frozen in liquid nitrogen and stored at -80°C.

[0148] 7. Comparison of the enzyme activities of purified wild-type β-galactosidase and various highly active β-galactosidase mutants.

[0149] a. Based on the previous protein concentration determination results and SDS-PAGE electrophoresis results, the protein concentrations of various β-galactosidase mutants and wild-type β-galactosidases were diluted to 1 mg / ml using enzyme storage solution 1X PBS, and then diluted again by 200 times.

[0150] b. Enzyme activity detection and comparison using redissolved β-galactosidase samples. Using a β-galactosidase reporter gene assay kit (Beyotime product, RG0036), add 50 μl of redissolved wild-type and mutant β-galactosidase samples to each well of a 96-well plate. Then add 50 μl of β-galactosidase assay reagent (Beyotime product, RG0036-2) to each well, mix thoroughly by pipetting, and then cover the 96-well plate. Alternatively, use parafilm or plastic wrap to seal the 96-well plate to prevent evaporation. Incubate at 37°C for approximately 5 minutes or until a light yellow color appears in the sample wells. The absorbance typically plateaus after about 3 hours; longer incubation will not significantly increase the absorbance. Finally, add 150 μl of β-galactosidase reaction stop solution (Beyotime product, RG0036-3) to terminate the reaction, mix well, and avoid air bubbles affecting the absorbance measurement. Using an ELISA reader or spectrophotometer, the measurement wavelength was set to 420 nm to measure the absorbance. The absorbance value roughly reflects the activity levels of different mutants. Based on the absorbance measurement results, the differences in enzyme activity between different β-galactosidase mutants and the control wild-type β-galactosidase were compared and analyzed.

[0151] c. Comparison of the predicted three-dimensional structures of each mutant site of β-galactosidase with the structure of wild-type β-galactosidase

[0152] After screening for highly active β-galactosidase mutants, protein structure prediction tools were used to predict the structures of four mutation sites involved in nine β-galactosidase mutants with significantly enhanced enzyme activity. Protein three-dimensional structure visualization and analysis tools were used to compare and analyze the structures of β-galactosidase mutants and wild-type β-galactosidase, elucidating the mechanism by which mutation sites improve enzyme activity.

[0153] Example 1: Structural analysis of β-galactosidase and acquisition of single-point mutants

[0154] The overall structure of the β-galactosidase tetramer, substrate, and metal ions is shown below. Figure 1 The image shows the overall three-dimensional structure of the β-galactosidase tetramer, substrate, and metal ion complex from two different perspectives. Escherichia coli β-galactosidase exerts its catalytic activity as a homotetramer composed of four identical polypeptide subunits.

[0155] The structural details of the two substrate-binding regions and the catalytically active region of β-galactosidase are shown in the figure below. Figure 2 As shown: Figure 2 A shows a detailed diagram of the interaction between the substrate-binding and catalytically active regions, primarily located at the N-terminus of β-galactosidase. Figure 2Figure B shows a detailed view of the second substrate-binding region, which is mainly located at the C-terminus of β-galactosidase and adjacent to the catalytically active region.

[0156] Construction of single-point mutants: PCR products from various β-galactosidase site-directed mutations were digested with DpnI and transformed into *E. coli* DH5α competent cells. Three single clones were selected from each mutant for small-scale culture, and plasmids were extracted. Sequencing verification showed that most mutants had at least one correct sequence among the three clones sent for sequencing. Ultimately, all 80 designed single-point mutants yielded positive clones, and the plasmid sequences were all verified by sequencing to ensure their correctness.

[0157] The bar chart comparing the activity assays of wild-type β-galactosidase and the crude enzymes of each single mutant is shown below. Figure 3 As shown: Among the 80 successfully constructed β-galactosidase single-point mutants, four mutants, M13 (M203F), M65 (Q601G), M68 (F602L), and M79 (R801E), exhibited significantly increased enzyme activity compared to wild-type β-galactosidase. Specifically, mutants M13 (M203F) and M65 (Q601G) increased enzyme activity by 20% and 15%, respectively, while M68 (F602L) and M79 (R801E) increased it by 10%. (Details are as follows...) Figure 3 As shown.

[0158] The amino acid sequence of the single-point mutant is as follows:

[0159] Amino acid sequence of mutant M13 (M203F):

[0160] Based on SEQ ID NO:1, position 203 is mutated from M to F.

[0161] Amino acid sequence of mutant M65(Q601G):

[0162] Based on SEQ ID NO:1, position 601 is mutated from Q to G.

[0163] Amino acid sequence of mutant M68(F602L):

[0164] Based on SEQ ID NO:1, position 602 is mutated from F to L.

[0165] The amino acid sequence of mutant M79(R801E):

[0166] Based on SEQ ID NO:1, position 801 is mutated from R to E.

[0167] Example 2: Obtaining multi-point mutants

[0168] Construction of multi-point mutants: Based on the results of crude enzyme activity detection and comparison, four single-point mutation sites that significantly improved activity were combined with mutants to construct multi-point superimposed mutants of β-galactosidase, further enhancing enzyme activity. The PCR products of various β-galactosidase multi-point mutants were digested with DpnI and transformed into *E. coli* DH5α competent cells. Three single clones were selected from each mutant for small-scale culture, and plasmids were extracted. Sequencing verification showed that most mutants had at least one correct sequence among the three single clones sent for sequencing. Ultimately, all five designed multi-point combined mutants yielded positive clones, and the plasmid sequences were all verified by sequencing to ensure their correctness.

[0169] The results of purification and enzyme activity assays of high-activity β-galactosidase mutants and wild-type β-galactosidase are as follows: Figure 4 As shown: By detecting the enzyme activity of crude β-galactosidase mutants, four single-point mutants of β-galactosidase with high enzyme activity, five constructed multi-point combination mutants, and wild-type β-galactosidase were subjected to large-scale induction expression, purification, protein concentration quantification, and activity detection for comparison. The protein quantitative electrophoresis results are shown below. Figure 4 As shown in Figure A, the loading volume of each sample was 10 μg. The SDS-PAGE electrophoresis results showed that the bands were similar, indicating that the protein content of each β-galactosidase sample was basically the same.

[0170] Enzyme activity was measured and analyzed using samples after protein quantification. The differences in enzyme activity between wild-type β-galactosidase and various high-activity β-galactosidase mutants were further compared. The results are as follows: Figure 4 As shown in Figure B, compared with the wild-type β-galactosidase control, the enzyme activities of the remaining nine β-galactosidase mutants were significantly increased, approximately 1.1-1.4 times that of the control. Among them, the activity of the superior activity mutant M81 (M203F / Q601G) was about 30% higher than that of the wild-type β-galactosidase control; the activity of the optimal activity mutant M84 (M203F / Q601G / F602L) was about 40% higher than that of the wild-type β-galactosidase control.

[0171] The amino acid sequence of the combined mutant is as follows:

[0172] Amino acid sequence of mutant M81 (M203F / Q601G):

[0173] Based on SEQ ID NO:1, position 203 is mutated from M to F; position 601 is mutated from Q to G.

[0174] Amino acid sequence of mutant M82 (M203F / F602L):

[0175] Based on SEQ ID NO:1, position 203 is mutated from M to F; position 602 is mutated from F to L.

[0176] Amino acid sequence of mutant M83 (M203F / R801E):

[0177] Based on SEQ ID NO:1, position 203 is mutated from M to F; position 801 is mutated from R to E.

[0178] Amino acid sequence of mutant M84 (M203F / Q601G / F602L):

[0179] Based on SEQ ID NO:1, position 203 is mutated from M to F; position 601 is mutated from Q to G; and position 602 is mutated from F to L.

[0180] Amino acid sequence of mutant M85 (M203F / Q601G / R801E):

[0181] Based on SEQ ID NO:1, position 203 is mutated from M to F; position 601 is mutated from Q to G; and position 801 is mutated from R to E.

[0182] The mutant β-galactosidases share the same topological conformation as the wild-type β-galactosidase in their overall three-dimensional structure, exhibiting high structural similarity. Furthermore, the spatial regions responsible for substrate binding, metal ion anchoring, and enzyme catalysis are identical. Further detailed localization of the substrate / metal ion binding and catalytic domains of β-galactosidases reveals differences in amino acid side chains and deflection angles before and after mutations at each mutation site, providing a deeper and more specific explanation of the molecular mechanisms underlying the improved and enhanced enzyme activity of the series of highly active β-galactosidase mutants screened in this project. β-galactosidase requires binding to metal ions such as sodium, potassium, and magnesium to fully exert its activity; the participation of both monovalent and divalent cations is crucial for substrate binding and catalytic reactions. M203, Q601, and F602 are located in key active regions for substrate binding, cation anchoring, and catalysis, and can interact with 1-3 water molecules, playing a vital role in the recognition and immobilization of substrates, sodium ions, and magnesium ions. In this invention, M203 is mutated from a long-side-chain amino acid M to an aromatic ring side-chain amino acid such as F, causing a conformational change in the flexible region where the amino acid at position 203 is located. This enhances the hydrophobic interaction with neighboring amino acids and monovalent cations, promoting the binding and anchoring of sodium and magnesium ions. Q601 and F602 mainly participate in the stable binding of sodium and magnesium ions and also contribute to the binding of substrates. In particular, the π-electron cloud of the benzyl group in F602 can act as a "ligand" for monovalent cations, helping to neutralize the negative charge of the substrate's phosphate group and also interacting with sodium ions. Q601 is mutated from a long-side-chain amino acid to a side-chain-less amino acid (such as Q601G), and F602 is mutated from an aromatic ring side-chain amino acid to a hydrophobic side-chain amino acid (such as F602L). This can modify the hydrophobic properties of the substrate channel and reduce steric hindrance to more effectively allow substrates, sodium ions, and magnesium ions to enter and exit the active region, thereby improving enzyme activity. R801, located in another sodium ion-binding region adjacent to the active site, is mutated from a positively charged amino acid to a negatively charged amino acid such as E, enhancing the electrostatic interaction with sodium ions and stabilizing sodium ion binding. The conformational changes caused by mutations and modifications at these four key sites in the active region, leading to increased substrate affinity, are a key reason for the significantly enhanced enzyme activity in this mutant.

[0183] The effects of mutations at various sites on enzyme conformation / activity are summarized in Table 1.

[0184] Table 1

[0185]

[0186] The structures of various mutant sites of β-galactosidase with significantly improved enzyme activity are shown in the figure below. Figure 5 As shown.

[0187] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. A method for increasing the enzyme activity of β-galactosidase, comprising: β-galactosidase was mutated to form a β-galactosidase mutant; the amino acid sequence of the mutant was obtained by the following mutations based on the wild-type β-galactosidase amino acid sequence shown in SEQ ID NO: 1: (i) The 203rd position mutates from M to F, the 601st position mutates from Q to G, and the 602nd position mutates from F to L; (ii) The 203rd position is mutated from M to F, and the 601st position is mutated from Q to G; (iii) The 203rd position mutates from M to F, and the 602nd position mutates from F to L; (iv) The 203rd position is mutated from M to F, the 601st position is mutated from Q to G, and the 801st position is mutated from R to E; (v) The 203rd position mutates from M to F, and the 801st position mutates from R to E; (vi) The 203rd position is changed from M to F; (vii) The 601st position is mutated from Q to G; or (viii) The 801st position is mutated from R to E.

2. A β-galactosidase mutant, wherein the amino acid sequence of the mutant is based on the wild-type β-galactosidase amino acid sequence shown in SEQ ID NO: 1, by the following mutations: (i) The 203rd position mutates from M to F, the 601st position mutates from Q to G, and the 602nd position mutates from F to L; (ii) The 203rd position is mutated from M to F, and the 601st position is mutated from Q to G; (iii) The 203rd position mutates from M to F, and the 602nd position mutates from F to L; (iv) The 203rd position is mutated from M to F, the 601st position is mutated from Q to G, and the 801st position is mutated from R to E; (v) The 203rd position mutates from M to F, and the 801st position mutates from R to E; (vi) The 203rd position is changed from M to F; (vii) The 601st position is mutated from Q to G; or (viii) The 801st position is mutated from R to E.

3. A polynucleotide encoding the β-galactosidase mutant of claim 2.

4. An expression vector comprising the polynucleotide of claim 3.

5. A genetically engineered cell, said genetically engineered cell containing the expression vector of claim 4 or the genome thereof having the polynucleotide of claim 3 integrated therein.

6. A method for producing a mutant of β-galactosidase, comprising the steps of: (a) Culturing the host cells as described in claim 5; (b) Collect cultures containing the β-galactosidase mutant described above; (c) Isolate the β-galactosidase mutant from the culture.

7. Use of the β-galactosidase mutant of claim 2, a host cell expressing the mutant, or its lysis product thereof, for hydrolyzing galactosidic bonds.

8. The use as described in claim 7, characterized in that, The β-galactosidase mutant, the host cell expressing the mutant, or its lysis product are used to catalyze the hydrolysis of substrates containing galactosidic bonds.

9. A method for hydrolyzing galactosidic bonds, comprising: The β-galactosidase mutant of claim 2, the host cell expressing the mutant, or its cleavage product are used to hydrolyze galactosidic bonds.

10. The method as described in claim 9, characterized in that, The β-galactosidase mutant, the host cell expressing the mutant, or its cleavage products catalyze the hydrolysis of substrates containing galactosidic bonds.

11. A kit for hydrolyzing galactosidic bonds, comprising: The β-galactosidase mutant according to claim 2; or The expression vector according to claim 4; or The genetically engineered cell as described in claim 5.

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