Design method for improving catalytic efficiency and thermal stability of citrobacter vinegaticus hyaluronidase and expression application of citrobacter vinegaticus hyaluronidase

Abstract

The invention discloses a molecular modification design method for improving the catalytic activity and the thermal stability of citrobacter vinegaticus-derived hyaluronidase and expression application of the citrobacter vinegaticus-derived hyaluronidase. The interaction of citrobacter vinegaticus hyaluronidase active pocket amino acid residues and hyaluronate tetrasaccharide molecules is visually explored through Pymol, conservative analysis of pocket amino acid evolution is carried out through Consurf, and rational design is carried out through policies of FoldX for calculating enzyme-substrate binding free energy, so that the hyaluronidase catalytic activity is improved. A mutant with significantly improved enzyme activity is obtained through screening, and the thermal stability of the enzyme is further improved through PROSS design and an analysis strategy of mutation site amino acid evolution conservative property and position. According to the invention, the sequence, structure and function of hyaluronidase are deeply studied, and hyaluronidase is mutated from two aspects of stabilizing a catalytic structure and promoting a catalytic reaction, so that a high-activity enzyme with higher application value in industrial production is obtained.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and specifically relates to a design method and expression application for improving the catalytic activity and stability of hyaluronidase. Background Art

[0002] Hyaluronic acid (HA), also known as hyaluronic acid, has a molecular formula of (C 14 H 21 NO 11 ) n It is a glycosaminoglycan composed of disaccharide units of D-glucuronic acid and N-acetylglucosamine. D-glucuronic acid and N-acetylglucosamine are linked by β-1,3 glycosidic bonds, and disaccharides are linked by β-1,4 glycosidic bonds. Hyaluronic acid has important physiological functions in the human body, such as regulating the chemical composition of extracellular fluid, lubrication and promoting wound healing; regulating adhesion, growth, movement and activation of specific cells, etc. It is an important component of extracellular matrix, synovial fluid, cartilage tissue, and umbilical cord. In recent years, studies have found that the effect and biological activity of HA are directly related to its relative molecular weight (Mw). Compared with high-molecular-weight HA, low-molecular-weight HA and oligomeric HA have a variety of biological functions, such as delivering proteins and peptides, promoting wound healing, promoting angiogenesis and bone cell formation, inhibiting tumor cell apoptosis, and enhancing immune regulation.

[0003] Low-molecular-weight HA is degraded from high-molecular-weight HA using physical, chemical, and enzymatic methods. Physical methods primarily include mechanical shearing, sonication, gamma-irradiation, and heat treatment. These degradation methods do not require the addition of any chemical reagents, resulting in products with a narrow molecular spectrum and excellent thermal stability. However, these methods are relatively inefficient. Chemical methods primarily use acidic, alkaline, or oxidative conditions to break the chemical bonds of high-molecular-weight HA, producing HA fragments of varying degrees. However, these methods involve harsh reaction conditions, making it difficult to control the molecular spectrum and potentially damaging or modifying the HA molecular structure, resulting in varying degrees of product activity. Due to various drawbacks associated with physical and chemical HA degradation, they have been gradually replaced by enzymatic degradation. Enzymatic degradation offers high specificity, is gentle and efficient, and maintains good substrate activity. HA fragments with varying molecular spectra and structures can be obtained by using hyaluronidases from different sources, controlling enzyme dosage, reaction time, and product concentration, as needed. Extracting hyaluronidases from animal tissues is costly and carries the potential risk of cross-contamination with animal viruses, making large-scale production difficult. Hyaluronidases derived from microorganisms are not restricted by their origin, do not contain animal viruses, and have high enzymatic activity, offering unique advantages over animal-derived hyaluronidases. Chinese patent CN103484513B discloses a method for preparing low-molecular-weight hyaluronates with a molecular weight of 10kDa-1000kDa using hyaluronidase derived from Bacillus aeruginosa, as well as the resulting low-molecular-weight hyaluronates and their uses.

[0004] Currently, animal-derived hyaluronidases are limited and expensive. Although microbial hyaluronidases (HAases) are unrestricted and possess excellent degradation capabilities, they are difficult to apply to industrial production due to factors such as low expression levels in host cells, resulting in low enzyme activity and poor stability. Therefore, it is urgent to obtain a HAase with high expression, high catalytic activity, and good thermal stability, which is of great significance for the large-scale industrial production of small-molecule HA. Summary of the Invention

[0005] The present invention is to explore the interaction of Citrobacter rodentium hyaluronidase activity pocket amino acid residues with hyaluronic acid tetrasaccharide molecules, Consurf to pocket amino acid evolutionary conservation analysis and FoldX calculation enzyme-substrate binding free energy strategy by Pymol visualization and carry out rational design, to improve hyaluronidase catalytic activity.Screening obtains the mutant that enzyme activity is significantly improved, by PROSS design, mutation site amino acid evolutionary conservation and positional analysis strategy, further improves the thermostability of enzyme.The present invention is to carry out in-depth study on the sequence, structure and function of hyaluronidase, mutates hyaluronidase from two aspects of stabilizing catalytic structure and promoting catalytic reaction, obtains the high-activity enzyme that is more possessed of application value in industrial production.

[0006] Therefore, according to one aspect of the present invention, a hyaluronidase mutant is provided, which differs from a wild-type hyaluronidase of microbial origin in comprising the mutation T204R. The wild-type hyaluronidase of microbial origin may be derived from Citrobacter portucalensis, Group A Streptococcus, Staphylococcus aureus, Clostridium perfringens, or the like.

[0007] In a preferred embodiment, the wild-type hyaluronidase is derived from Citrobacter portucalensis. Preferably, the amino acid sequence of the wild-type hyaluronidase is shown in SEQ ID No. 1, and more preferably, the encoding nucleotide sequence of the wild-type hyaluronidase is shown in SEQ ID No. 2.

[0008] In another preferred embodiment, the hyaluronidase mutant further comprises a mutation selected from the group consisting of: N206H, A408L, Q250E, T248Y, or a combination thereof. In a particularly preferred embodiment, the hyaluronidase mutant comprises the following mutation combinations relative to wild-type hyaluronidase: T204R+A408L, T204R+T248Y, T204R+K119N, T204R+Q250E, or T204R+N206H.

[0009] In another preferred embodiment, the amino acid sequence of the hyaluronidase mutant is shown as SEQ ID No. 3 or SEQ ID No. 5.

[0010] According to one aspect of the present invention, the present invention provides a gene encoding the hyaluronidase mutant according to the present invention.

[0011] In a preferred embodiment, the gene sequence is shown as SEQ ID No. 4 or SEQ ID No. 6.

[0012] According to one aspect of the present invention, the present invention provides a recombinant expression vector comprising and / or expressing the gene according to the present invention.

[0013] In another preferred embodiment, the recombinant expression vector is a pET series expression vector. The pET vector uses a strong T7 phage promoter to regulate the expression of the target gene and is induced by the T7 RNA polymerase provided by the host cell. Its characteristics include: low basal expression level, which can be precisely regulated by inducers (such as IPTG); providing a variety of fusion tags (such as 6×His, GST) and secretion signal peptides; and a variety of host bacteria (more than 15 species), such as BL21 (DE3), Rosetta, etc. Particularly preferably, the pET series expression vectors can include pET-28a (+), pET-22b, pTriEx series, and pGEX derivatives.

[0014] According to one aspect of the present invention, the present invention provides a microbial host cell comprising the recombinant expression vector according to the present invention or a gene expressing the hyaluronidase mutant according to the present invention, preferably an Escherichia coli, Bacillus subtilis, or yeast cell. Particularly preferably, the Escherichia coli host cell comprises BL21(DE3).

[0015] According to one aspect of the present invention, the present invention provides the use of a hyaluronidase mutant, gene, recombinant expression vector or microbial host cell according to the present invention in the preparation of low molecular weight hyaluronic acid or oligomeric hyaluronic acid. Low molecular weight hyaluronic acid generally refers to HA with a molecular weight between 10kDa and 1000kDa (such as 500kDa), wherein oligomeric hyaluronic acid generally refers to HA with a molecular weight <10kDa.

[0016] According to one aspect of the present invention, the present invention provides a molecular modification design method for improving the catalytic activity and / or thermal stability of hyaluronidase (HAase), comprising the following steps:

[0017] (1) Based on the amino acid sequence of HAase, the structure of HAase was predicted using Alphafold3;

[0018] (2) The amino acid sequence of HAase was uploaded to the NCBI and PDB databases respectively, and the same family enzyme-tetrasaccharide molecule (HA4) complex with the highest amino acid sequence similarity and the most similar three-dimensional structure as the target enzyme and the experimental X-ray diffraction crystal was searched as a reference for molecular docking. Based on the three-dimensional conformation of HAase obtained in step (1), molecular docking was performed to determine the binding range of the substrate molecule in the active pocket of HAase;

[0019] (3) Visualize the best docked complex three-dimensional structure in step (2) using Pymol software and find the distance from the substrate All amino acids within the distance were used to input the HAase amino acid sequence into the online server ConSurf for homologous evolution analysis of amino acids in the same family;

[0020] (4) Based on the Consurf online database in step (3), a conservation analysis of each amino acid residue of the enzyme was performed. Among the amino acid residues within the range, amino acid residues with a conservation score of less than 5 and a frequency of less than 50% were selected as mutation targets. FoldX was used in conjunction to calculate the free energy of substrate-enzyme binding for further screening and design, and mutants were constructed. The enzyme with the best catalytic activity was obtained through experimental screening; and

[0021] (5) Optionally, based on the result of step (4), the enzyme with the best catalytic activity is selected as the starting enzyme, and the enzyme sequence is submitted to the PROSS online database to obtain the optimal design scheme of the database. The position of the design site is visualized by Pymol, and the site located in the flexible loop of the enzyme is screened out. Subsequently, the ConSurf database is used to analyze the evolutionary conservation of the amino acids of the screened flexible loop sites, and sites with a score of less than 5 are selected to construct mutants, and mutants with enhanced thermal stability are screened out through experiments.

[0022] In a preferred embodiment of the method, the hyaluronidase is derived from Citrobacter portucalensis, and preferably the amino acid sequence of the wild-type hyaluronidase is shown in SEQ ID No. 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The above features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0024] Figure 1 The predicted spatial structure diagram of wild HAase from Citrobacter rodentium from Portugal by Alphafold3 modeling;

[0025] Figure 2 This is the molecular docking diagram of HAase and HA tetrasaccharide molecule;

[0026] Figure 3 is the binding free energy of T204R, Y97R and wild-type enzymes with substrates during molecular dynamics simulations; and

[0027] Figure 4 This is the thermal stability test result diagram.

[0028] Description of the sequence listing (preferred sequences of the present invention and sequences used in the examples)

[0029] SEQ ID No. 1 - wild HAase from Citrobacter portucalensis (excluding the signal peptide of the N-terminal 20 amino acid sequence)

[0030] QIATENVNLPVVKSTTTTSTQQQHIIERMRDTWRQNFVPSGPAAPELSAEYVASLNKTANIFWKGIDKNTPAGQLWADTVLDSESTSGRLKLGTTLYTVYQRLFTLAKAWATPGTDLYKNAQLNTVLKSALINLNQDYYNDQTPEWGNWWNWELGISRSVNNTLVILYDDLPSTLIDKYNLATRHFVRDPRYLAEGSGAPYSTTKNAFTSTGGNRIDSAMVVFVRGLLANDPGEISAAVTSVPEVLNTVQSGDGFYKDGSFIQHKDLPYSGTYGQVLLNGLGLIKNSVAGTPWDFSVEDNRRIYDVIRQAFLPLLHEGKMPDAVNGRSISRKNGQDQDVGASVMNAIALFVNGAPPEEKRHIEQVLKAQLNSKTTEYYHTHLPENLTSWQVITRIQQHSHLPPAPRTAGGKLYADMDRLIYQGTNYLAVVAMHSNRTGSYECINNENLKGQRTSDGMTWLYLPNDDQYRDYWPVVDSRFLPGTTSAGEQGWCDEQYRVTQLGRANIAWAGGNTLNKWASASMHLKVPTYSLKAKKSWFMAPHEMIMLGSQISSSSPAVTTIANQKISGSAKVLVDGIVLLPGEERKATQSVVLNDKGNNIIWKPLAGSSAQVSVKQRQGNWADIGTSSGKVSAQFLTIIQPHSAESDNHYAWVVFPSGSASPSVNADITLLANDAKVQAVSLPGQQVIYANFWRSATVGGIHALTPMSLIMTPTTQGYQIAVSSPRRDSRVSFQLPDNAIPFHISSDPDKRVSLNGEIVSVNMTNLRGSSYSFELSKNK

[0031] SEQ ID No. 2 - Coding nucleotide sequence of wild-type HAase

[0032]

[0033] SEQ ID No. 3 - Amino acid sequence of hyaluronidase mutant

[0034] QIATENVNLPVVKSTTTTSTQQQHIIERMRDTWRQNFVPSGPAAPELSAEYVASLNKTANIFWKGIDKNTPAGQLWADTVLDSESTSGRLKLGTTLYTVYQRLFTLAKAWATPGTDLYKNAQLNTVLKSALINLNQDYYNDQTPEWGNWWNWELGISRSVNNTLVILYDDLPSTLIDKYNLATRHFVRDPRYLAEGSGAPYSTRKNAFTSTGGNRIDSAMVVFVRGLLANDPGEISAAVTSVPEVLNTVQSGDGFYKDGSFIQHKDLPYSGTYGQVLLNGLGLIKNSVAGTPWDFSVEDNRRIYDVIRQAFLPLLHEGKMPDAVNGRSISRKNGQDQDVGASVMNAIALFVNGAPPEEKRHIEQVLKAQLNSKTTEYYHTHLPENLTSWQVITRIQQHSHLPPAPRTAGGKLYADMDRLIYQGTNYLAVVAMHSNRTGSYECINNENLKGQRTSDGMTWLYLPNDDQYRDYWPVVDSRFLPGTTSAGEQGWCDEQYRVTQLGRANIAWAGGNTLNKWASASMHLKVPTYSLKAKKSWFMAPHEMIMLGSQISSSSPAVTTIANQKISGSAKVLVDGIVLLPGEERKATQSVVLNDKGNNIIWKPLAGSSAQVSVKQRQGNWADIGTSSGKVSAQFLTIIQPHSAESDNHYAWVVFPSGSASPSVNADITLLANDAKVQAVSLPGQQVIYANFWRSATVGGIHALTPMSLIMTPTTQGYQIAVSSPRRDSRVSFQLPDNAIPFHISSDPDKRVSLNGEIVSVNMTNLRGSSYSFELSKNK

[0035] SEQ ID No. 4 - Gene sequence of hyaluronidase mutant

[0036]

[0037] Amino acid sequence of hyaluronidase mutant of SEQ ID No. 5

[0038] QIATENVNLPVVKSTTTTSTQQQHIIERMRDTWRQNFVPSGPAAPELSAEYVASLNKTANIFWKGIDKNTPAGQLWADTVLDSESTSGRLKLGTTLYTVYQRLFTLAKAWATPGTDLYKNAQLNTVLKSALINLNQDYYNDQTPEWGNWWNWELGISRSVNNTLVILYDDLPSTLIDKYNLATRHFVRDPRYLAEGSGAPYSTRKHAFTSTGGNRIDSAMVVFVRGLLANDPGEISAAVTSVPEVLNTVQSGDGFYKDGSFIQHKDLPYSGTYGQVLLNGLGLIKNSVAGTPWDFSVEDNRRIYDVIRQAFLPLLHEGKMPDAVNGRSISRKNGQDQDVGASVMNAIALFVNGAPPEEKRHIEQVLKAQLNSKTTEYYHTHLPENLTSWQVITRIQQHSHLPPAPRTAGGKLYADMDRLIYQGTNYLAVVAMHSNRTGSYECINNENLKGQRTSDGMTWLYLPNDDQYRDYWPVVDSRFLPGTTSAGEQGWCDEQYRVTQLGRANIAWAGGNTLNKWASASMHLKVPTYSLKAKKSWFMAPHEMIMLGSQISSSSPAVTTIANQKISGSAKVLVDGIVLLPGEERKATQSVVLNDKGNNIIWKPLAGSSAQVSVKQRQGNWADIGTSSGKVSAQFLTIIQPHSAESDNHYAWVVFPSGSASPSVNADITLLANDAKVQAVSLPGQQVIYANFWRSATVGGIHALTPMSLIMTPTTQGYQIAVSSPRRDSRVSFQLPDNAIPFHISSDPDKRVSLNGEIVSVNMTNLRGSSYSFELSKNK

[0039] Gene sequence of hyaluronidase mutant of SEQ ID No. 6

[0040] DETAILED DESCRIPTION

[0041] Unless otherwise indicated, the terms used herein have their ordinary technical meanings as understood by those skilled in the art. For definitions and terms in the art, the skilled artisan is particularly referred to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0042] In the present invention, the singular articles "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. All references cited herein are hereby incorporated by reference in their entirety.

[0043] In the present invention, the terms "comprising" or "including" are open-ended expressions, referring to the specific components or steps being described, while not excluding other components or steps that have no substantial effect. When describing a protein or nucleotide sequence, the sequence may constitute the target molecule in isolation, or may have additional amino acids or nucleotides appended to one or both ends, or undergo protein engineering, while retaining the functional activity described herein.

[0044] The term "and / or" as used herein includes all combinations of the items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and / or B" includes "A," "A and B," and "B." For another example, "A, B, and / or C" includes "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0045] In the present invention, the term "expression" may refer to "overexpression", which is defined as a gene expression level higher than the natural state, which may be achieved by increasing the transcription level (producing more messenger mRNA) or improving the translation efficiency (generating more functional proteins). In a specific embodiment, preferably, the expression or overexpression of the exogenous gene can be achieved by inserting the target gene into the genomic non-translation site, or by plasmid overexpression. Preferably, the CRISPR / Cas9 method is used for gene insertion. Preferably, the plasmid overexpression is introduced into the chassis strain by electrotransformation, chemical transformation or conjugation transformation.

[0046] The main purpose of this application is to provide a design method and expression application for improving the catalytic activity and stability of HAase from microorganisms, selecting key amino acids for purposeful rational mutation, and aiming to solve the problems existing in the prior art identified in the background technology.

[0047] In order to achieve the above objectives, this application provides the following technologies:

[0048] The present invention provides an HAase derived from Citrobacter rodentium, whose amino acid sequence is shown in SEQ ID No. 1.

[0049] The gene encoding the above hyaluronidase has a sequence shown in SEQ ID No. 2.

[0050] A HAase mutant derived from Citrobacter rodentium, whose amino acid sequence is shown as SEQ ID No. 3 or SEQ ID No. 5.

[0051] The gene encoding the above-mentioned hyaluronidase mutant has a sequence as shown in SEQ ID No. 4 or SEQ ID No. 6.

[0052] A recombinant expression vector comprising the above gene nucleotide sequence.

[0053] A host cell comprising the above recombinant expression vector.

[0054] The present invention also provides the use of the HAase gene for expressing HAase in chassis cells.

[0055] The rationally designed new HAase gene is used to construct a recombinant expression vector to induce HAase expression.

[0056] In the above application, the chassis cells may be Escherichia coli BL21 (DE3).

[0057] The present invention also provides the use of the above-mentioned HAase, HAase mutant, gene, vector or cell in the preparation of low molecular weight hyaluronic acid or oligomeric hyaluronic acid. The application can be achieved by enzymatic hydrolysis of high molecular weight hyaluronic acid (usually with a molecular weight higher than 2×10 6 Da) to achieve it.

[0058] The present invention also provides a method for molecular modification of HAase, characterized by comprising the following steps:

[0059] Using Pymol visualization to explore the interaction between the active pocket amino acid residues of the hyaluronidase and the hyaluronan tetrasaccharide molecule, Consurf analysis of the evolutionary conservation of pocket amino acids, and FoldX calculation of enzyme-substrate binding free energy, a rational mutant was constructed. Experimental verification screened for a mutant enzyme with optimal catalytic efficiency. Using a mutant enzyme with further improved catalytic efficiency as a starting point, PROSS design and analysis of the evolutionary conservation and position of amino acids at the mutation site were used to construct thermostable mutants. Experimental verification screened for a mutant enzyme with further improved catalytic activity and thermostability.

[0060] In a specific embodiment, a molecular engineering design method for improving the catalytic activity and thermal stability of hyaluronidase (HAase) derived from Citrobacter rodentium is provided, comprising the following steps:

[0061] (1) The amino acid sequence of HAase (SEQ ID No. 1) was submitted to the protein tertiary structure prediction website Alphafold3 for structure prediction;

[0062] (2) The amino acid sequence of HAase was uploaded to the NCBI and PDB databases respectively, and the same family enzyme-tetrasaccharide molecule (HA4) complex (PDB accession number: 1LXK) with the highest similarity (>30%) to the amino acid sequence of the target enzyme, the most similar three-dimensional structure, and the experimental X-ray diffraction crystal was found as a reference for molecular docking to determine the binding range of the substrate molecule in the HAase active pocket. Based on the three-dimensional conformation of the target enzyme obtained in step (1), the PDB database obtained the spatial structure file of the HA4 molecule, pre-processed the HA4 molecule and the target HAase tertiary structure file, and used AutoDock vina for molecular docking. The screening principle of the docking results is that the reducing end of the HA4 molecule must be located at the narrow end of the HAase active pocket; secondly, the docking position of the substrate must overlap with the reference substrate as much as possible, and the binding energy must be minimized;

[0063] (3) Visualize the best docked complex three-dimensional structure in step (2) using Pymol software and find the distance from the substrate All amino acids within the distance were used to input the HAase amino acid sequence into the online server ConSurf for homologous evolution analysis of amino acids in the same family and to understand the catalytic mechanism of the enzyme;

[0064] (4) Based on the Consurf online database in step (3), a conservation analysis of each amino acid residue of the enzyme was performed. Among the amino acid residues within the range, the amino acid residues with a conservation score of less than 5 and a usage frequency of less than 50% were selected as mutation targets. Less than Among the amino acid residues within 5, those with a conservation score of less than 5 were selected as mutation targets. FoldX was used in parallel to calculate the free energy of substrate-enzyme binding for further screening and design. Eleven mutants were constructed: E384R, D338R, Y97R, N338R, T204R, S627K, N444G, T204D, T94D, N148G, and S342Q.

[0065] (5) Based on the results of step (4), the enzyme with the best catalytic activity was selected as the T204R starting enzyme. The enzyme sequence was submitted to the PROSS online database to obtain the optimal design scheme of the database. The position of the design site was visualized by Pymol, and the site located in the flexible loop of the enzyme was screened. The amino acid evolutionary conservation of the screened flexible loop sites was then analyzed using the ConSurf database, and sites with a score of less than 5 were selected. Structural analysis of the enzyme activity pocket showed that the catalytic region of the enzyme is mainly composed of two major regions: the α-domain and the random coil (Link region) between the α-helix and the β-sheet. Therefore, the enhancement of the thermal stability of the Link region and the α-domain may be more important. From the ConSurf analysis results, the sites located in the Link region and the α-helix were further screened as the final mutation sites. Five mutants were constructed, T204R / A408L, T204R / T248Y, T204R / K119N, T204R / Q250E, and T204R / N206H.

[0066] Compared with the existing technology, this application can bring the following technical effects:

[0067] 1. Based on the embodiment of this application, the present invention characterized the wild HAase from Citrobacter rodentium from Portugal using Escherichia coli BL21 (DE3) as the host. The specific enzyme activity of the wild enzyme under the optimal reaction conditions can reach (1.29×10 6 )U / mg;

[0068] 2. The present invention conducted in-depth research on the sequence, three-dimensional structure, catalytic mechanism, and amino acid evolutionary conservation of wild HAase from Citrobacter rodentium, Portugal. By using computer simulation of enzyme-substrate binding energy, the wild HAase was mutated from two aspects: stabilizing the catalytic structure and promoting the catalytic reaction. The final T204R was screened and the specific enzyme activity was 1.21 times that of the wild-type enzyme (1.56×10 6) U / mg. The amino acid sequence of this enzyme is shown in SEQ ID No. 3, and the nucleotide sequence is shown in SEQ ID No. 4. Using T204R as the starting enzyme for thermostability modification, the preferred T204R / N206H enzyme exhibits an inactivation half-life of 75 minutes, 1.4 times that of the wild-type enzyme. The amino acid sequence of this enzyme is shown in SEQ ID No. 5, and the nucleotide sequence is shown in SEQ ID No. 6. This results in a stable and highly active enzyme with greater application value in the industrial production of low-molecular-weight HA.

[0069] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0070] Example 1 Molecular docking and catalytic rational design of HAase from Citrobacter rodentium

[0071] (1) The amino acid sequence of HAase (SEQ ID No. 1) was submitted to the protein tertiary spatial structure prediction website Alphafold3 (https: / / alphafoldserver.com) for structure prediction. Figure 1 The spatial structure of the enzyme was observed using Pymol software (DeLano Scientific LLC).

[0072] (2) The amino acid sequence of HAase (SEQ ID No. 1) was uploaded to the NCBI and PDB databases respectively, and the same family enzyme-tetrasaccharide molecule (HA4) complex (PDB accession number: 1LXK) with the highest similarity (>30%) to the amino acid sequence of the target enzyme, the most similar three-dimensional structure, and the experimental X-ray diffraction crystal was found as a reference for molecular docking to determine the binding range of the substrate molecule in the HAase active pocket. Based on the three-dimensional conformation of the target enzyme obtained in step (1), the spatial structure file of the HA4 molecule was obtained from the PDB database, and the HA4 molecule and the target HAase tertiary structure file were preprocessed, and molecular docking was performed using AutoDock vina (https: / / autodock.scripps.edu). The screening principle of the docking results is that the reducing end of the HA4 molecule (N-acetylglucosamine C1 is a reducing aldehyde group) must be located at the narrow end of the HAase active pocket; secondly, the docking position of the substrate must overlap with the reference substrate as much as possible, and the binding energy must be minimized. The results are as follows: Figure 2 As shown (red is the reference complex, green is the target complex).

[0073] (3) Visualize the best docked complex three-dimensional structure in step (2) using Pymol software and find the distance from the substrate All amino acids within the distance were analyzed, and the HAase amino acid sequence was input into the online server ConSurf (https: / / consurf.tau.ac.il / consurf_index.php) for homologous evolution analysis of amino acids in the same family and to understand the catalytic mechanism of the enzyme.

[0074] (4) Based on the Consurf online database in step (3), the conservation analysis of each amino acid residue of the enzyme was carried out. Among the amino acid residues within the range, we selected amino acid residues with a conservation score of less than 5 and a frequency of less than 50% as mutation targets. Less than Among the amino acid residues within 5, we selected amino acid residues with a conservation score of less than 5 as mutation targets. Under physiological conditions, HA is negatively charged, and the interaction between HAase and its main substrate HA depends to a certain extent on ionic interactions and involves basic amino acid clusters. Therefore, salt bridges and hydrogen bonds play a key role in this interaction. The positive charge in the active pocket is conducive to the binding of HAase molecules to negatively charged HA. Therefore, the non-conserved amino acid residues in the enzyme active pocket binding region are mainly mutated to basic amino acids and polar amino acids to increase the formation of salt bridges and hydrogen bonds between the enzyme and the substrate, thereby increasing the affinity between the two. FoldX is used in parallel to calculate the free energy of substrate-enzyme binding for further screening and design.

[0075] Example 2 Experimental Verification Screening of Mutant Enzymes with Improved Catalytic Activity

[0076] 1. Construction and transformation of recombinant vectors

[0077] (1) SnapGene 6.02 software was used to construct the recombinant vector. pET-28a(+) was used as a blank plasmid. Based on the amino acid sequence of SEQ ID No. 1, codon optimization was performed (SEQ ID No. 2). 6×His tags were added to the N-terminus and C-terminus of the enzyme, respectively, and finally a recombinant expression plasmid was constructed. By designing point mutation primers and using PCR amplification, 11 mutants were constructed, including E384R, D338R, Y97R, N338R, T204R, S627K, N444G, T204D, T94D, N148G, and S342Q (completed by Beijing Qingke Biotechnology Co., Ltd.).

[0078] (2) Streak the recombinant plasmid E. coli Top10 culture liquid on a resistant (containing 100 μg / mL Kana) LB plate and culture it in a constant temperature incubator at 37°C overnight. Then pick a single colony of the recombinant plasmid E. coli Top10 and place it in 10 ml of liquid LB medium (containing 100 μg / mL Kana). Incubate it in a shaker at 37°C and 200 rpm for 12 to 16 hours. Then, perform plasmid extraction according to the instructions of the plasmid DNA small-scale extraction kit.

[0079] (3) The target plasmid was transformed into Escherichia coli BL21 (DE3) by heat shock transformation. The coated LB plates were placed in a 37°C incubator for 24 h. Positive transformants were selected from the plates and verified by colony PCR. The PCR products were detected by 1.0% agarose gel electrophoresis, and the corresponding remaining products were sequenced. The sequencing results were compared with the template gene sequence for final verification. The above method was used for both the wild-type enzyme and each mutant.

[0080] 2. Effects of different IPTG concentrations on HAase expression

[0081] (1) Streak the previously stored positive glycerol bacteria (BL21) containing the recombinant plasmid onto an LB plate (containing 100 μg / mL Kana) and culture at 37°C overnight.

[0082] (2) Pick a single colony from the LB plate and inoculate it into 10 mL of liquid culture medium (containing 100 μg / mL Kana) and culture it at 37°C with shaking at 200 rpm for 12 to 16 h (the appropriate culture time is selected based on the growth of the bacteria).

[0083] (3) Inoculate 1% of the inoculum into 50 mL of liquid culture medium (containing 100 μg / mL Kana) and culture at 37°C and 200 rpm for 3-4 h until the OD value reaches between 0.6 and 0.8.

[0084] (4) Add an appropriate amount of IPTG stock solution (concentration of 0.21 mol / L) to make the final IPTG concentration in the culture medium 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mM. Prepare three bottles for each IPTG concentration gradient and culture at 16°C for 20 h. After induction, collect the bacteria and obtain the recombinant protein, and then analyze the target protein by SDS-PAGE.

[0085] 3. Isolation and Purification of Recombinant Protein

[0086] When constructing the recombinant vector, the present invention adds 6×His tags to the N-terminus and C-terminus of the target protein, and utilizes its affinity with the nickel column to achieve the purpose of separating and purifying the target protein. Histidine, as a basic amino acid, can bind to nickel ions, and then the purified protein is obtained by elution with different concentrations of imidazole.

[0087] (1) Select the most appropriate concentration of IPTG among the above inducers and obtain the bacterial solution after crushing and centrifugation.

[0088] (2) Place the collected bacterial lysate on ice and take 20 μL of the supernatant for subsequent testing.

[0089] (3) Take 1mL of 50% BeyoGold TM Prepare His-Tag Purification Resin (reduction-resistant chelating type) by centrifugation at 4°C (1000g × 60s) and discard the storage solution. Add 0.5mL of non-denaturing lysis buffer to the gel and mix thoroughly to equilibrate the gel. Centrifuge at 4°C (1000g × 60s) and discard the liquid. Repeat equilibration 1-2 times and discard the liquid. Add 5mL of bacterial lysate supernatant and supplement with 2-3mL of non-denaturing lysis buffer. Place on ice and shake slowly on a rocking platform for 2 hours to allow binding.

[0090] (4) After the combination is completed, BeyoGold TM The mixture of His-Tag Purification Resin and lysate is loaded into the empty column tube of the affinity chromatography column.

[0091] (5) Open the lid at the bottom of the purification column and allow the liquid in the column to flow out under the action of gravity. Collect about 20 μL of the flow-through liquid for subsequent analysis.

[0092] (6) Wash the column five times, adding 0.5-1 mL of non-denaturing wash buffer each time, and collect approximately 20 μL of the wash buffer for subsequent analysis and testing. The column washing and elution process combined with the Bradford assay can quickly and easily detect the protein content in each wash buffer and eluate, thereby determining whether to increase or decrease the number of washes and elutions.

[0093] (7) Elute the target protein 6 to 10 times, using 0.5 mL of non-denaturing eluent each time. Collect the eluent from each time into separate centrifuge tubes. The collected eluent is the purified target protein.

[0094] (8) The washing solution and the eluate to be tested are subjected to SDS-PAGE gel electrophoresis to detect the protein purity.

[0095] 4. Test of enzyme degradation products

[0096] (1) Mix 5 mL of 0.5 mg / mL enzyme solution with 0.5 L of 5% (w / v) high molecular weight sodium hyaluronate (Sodium Hyaluronate, SH, average M W =100wDa, Fufeng Biotechnology Co., Ltd., pH = 7) were uniformly mixed at 37 ° C and placed on a horizontal shaker at 220 rpm for 1 hour. The sample was taken and boiled for 5 minutes to terminate the reaction. The molecular weight of the hyaluronic acid product was determined using the agarose gel method. The molecular weight and distribution of the hyaluronic acid product were determined using gel permeation chromatography (GPC), and the Mp (peak molecular weight), weight average molar mass (Mw), number average molecular weight (Mn) and polydispersity index (PD, PD = Mw / Mn) of the product fragments were calculated.

[0097] 5. Determination of enzyme kinetic parameters

[0098] Kinetic parameters of the wild-type enzyme and various mutant enzymes were determined using SH as a substrate. SH substrate concentrations ranged from 0.25% to 3% were prepared in a citric acid-sodium hydrogen phosphate buffer solution. The catalytic activity of the enzyme at various substrate concentrations was determined within a first-order reaction time. The data were imported into GraphPad Prism, and nonlinear regression analysis was performed using the Michaelis-Menten model.

[0099] 6. Enzyme properties detection

[0100] (1) The enzyme activity unit U of HAase is defined as the amount of enzyme required to produce 1 μg of N-acetylglucosamine (NAG) reducing equivalent reducing sugar from HA in 1 h. The purified wild-type (WT) and mutant enzymes were used for subsequent experiments. The optimal pH of the enzyme reaction was determined by placing the diluted enzyme solution in different pH substrates for reaction at 37°C for 30 min, and the enzyme activity was determined by the DNS method. The substrate was composed of high molecular weight sodium hyaluronate (SH, average M W =100 wDa, Fufeng Biotechnology Co., Ltd.) and different pH buffers: 0.1 mol / L citric acid-disodium hydrogen phosphate (pH 3.0-8.0) and 0.1 mol / L glycine-sodium hydroxide (pH 9.0-12.0). The maximum enzyme activity was defined as 100%. The pH stability of the enzyme reaction was determined by mixing the enzyme solution with 0.1 mol / L pH 3.0-11.0 buffer at room temperature for 2 hours. The enzymatic reaction was then carried out at the optimal pH and temperature for 30 minutes. The enzyme activity of the untreated enzyme solution was used as a control.

[0101] (2) Determination of the optimal temperature for enzyme reaction: Under the optimal pH conditions, the enzyme catalyzed reaction was carried out at 4°C to 70°C for 30 minutes, and the enzyme activity was measured. The highest enzyme activity was defined as 100%. Determination of thermal stability: The enzyme solution was placed in a water bath at 40°C, 45°C, and 50°C, and allowed to stand for 0 to 60 minutes. After that, the enzyme activity was measured by reacting with 0.5% (w / v) SH at the optimal pH and temperature for 30 minutes. The enzyme activity of the enzyme solution that had not been heat-treated was used as a control.

[0102] (3) 5 mL of 0.5 mg / mL enzyme solution was mixed with 0.5 L of 5% (w / v) high molecular weight SH (M W =100 wDa) were uniformly mixed under the optimal conditions of each enzyme, and placed on a horizontal shaker at 220 rpm for 1 h. The enzyme activity was measured using the DNS method.

[0103] 7. Molecular dynamics simulation

[0104] Molecular dynamics (MD) simulations of WT and each mutant were performed using the software YASARA 16.3.5 (default force field: YAMBER, NaCl 0.9%, water solvent density 0.997 g / ml), with the temperature set to 298 K, the pH set to 7, and the dynamics simulations were performed for 30 ns without restrictions.

[0105] Through the above experimental screening, it was found that among all the predicted mutants, T204R and Y97R had increased enzyme activities compared to the wild type, with T204R performing the best. The test results of mutants T204R and Y97R are as follows (Table 1):

[0106] Table 1

[0107] Wild enzyme T204R Y97R Mp(Da) 11335 9127 9658 Mn(Da) 8630 7076 7336 Mw(Da) 13061 10185 10662 Km(g / L) 11.32±0.19 9.98±0.34 10.32±0.39 <![CDATA[Kcat(*10 2 s -1 )]]> 6.97±0.08 9.04±0.09 8.11±0.15 Kcat / Km(L / (g·s)) 61.62±0.95 90.56±2.27 78.67±1.83 <![CDATA[Specific enzyme activity (*10 6 U / mg)]]> 1.29±0.006 1.56±0.01 1.43±0.009

[0108] The molecular dynamics simulation results are as follows Figure 3 shown.

[0109] Example 3 Experimental Verification and Screening of Mutant Enzymes with Improved Thermal Stability

[0110] 1. PROSS design of thermostable mutants

[0111] T204R with the best catalytic activity was selected as the starting enzyme. First, the enzyme sequence was submitted to the PROSS online database (https: / / pross.weizmann.ac.il / step / pross-terms / ) to obtain the optimal design scheme of the database. The position of the design site was visualized by Pymol, and the site located in the flexible loop of the enzyme was screened. The ConSurf database was then used to analyze the evolutionary conservation of the amino acids in the screened flexible loop sites, and sites with a score of less than 5 were selected. Structural analysis of the enzyme activity pocket showed that the catalytic region of the enzyme is mainly composed of two major regions: the α-domain and the random coil (Link region) between the α-helix and the β-fold. Therefore, the enhancement of the thermal stability of the Link region and the α-domain may be more important. From the ConSurf analysis results, the sites located in the Link region and the α-helix were further screened as the final mutation sites. By designing point mutation primers and using PCR amplification, five double-point mutation mutants were constructed, namely T204R / A408L, T204R / T248Y, T204R / K119N, T204R / Q250E and T204R / N206H (completed by Beijing Qingke Biotechnology Co., Ltd.).

[0112] 2. Thermal stability test of each mutant

[0113] Equal amounts of each mutant enzyme (0.5 mg / mL) were placed in a 45°C water bath and incubated for 2 h. Every 10 min, 100 μL of the sample was evenly mixed with 1 mL of 3% SH solution (pH = 5). The mixture was reacted at 37°C for 30 min. The enzyme activity was measured using DNS, and the enzyme activity of the untreated enzyme solution was defined as 100%. The inactivation rate constant (k d ) determination: For a first-order reaction, the rate equation InA = -K d t can be used to obtain K d , where A is the enzyme activity retention rate (%); t is the inactivation time. Half-life (t 1 / 2 ) determination: the time required for the enzyme activity to decrease by half at a specific temperature. For a first-order reaction, t 1 / 2 =0.693 / k d .

[0114] Through the above experimental screening, the test results are as follows (Table 2):

[0115] Table 2

[0116] enzymes <![CDATA[Kd(min -1 )]]> <![CDATA[Half-life (t 1 / 2 = 0.693 / Kd)]]> T204R 0.0133 52min T204R / A408L 0.0115 60min T204R / Q250E 0.0098 71 minutes T204R / T248Y 0.0112 62 minutes T204R / N206H 0.0092 75min

[0117] From the table above, we can see that the inactivation half-life of T204R is 52 min, and the half-lives of T204R / A408L, T204R / Q250E, T204R / T248Y, and T204R / N206H are 60, 71, 62, and 75 min, respectively, which are 1.2, 1.4, 1.2, and 1.4 times that of the control T204R. Figure 4 shown.

[0118] Those skilled in the art will appreciate that, although the present invention has been specifically described with reference to the above embodiments, the present invention is not limited to these specific embodiments. Based on the methods and technical solutions taught by the present invention, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present invention, and the equivalent embodiments obtained therefrom are within the scope of the present invention.

Claims

1. A hyaluronidase mutant, which differs from a wild-type hyaluronidase derived from a microorganism in that it comprises the mutation T204R.

2. The hyaluronidase mutant according to claim 1, wherein the wild-type hyaluronidase is derived from Citrobacter portucalensis, preferably the amino acid sequence of the wild-type hyaluronidase is shown in SEQ ID No. 1, more preferably the encoding nucleotide sequence of the wild-type hyaluronidase is shown in SEQ ID No.

2.

3. The hyaluronidase mutant according to claim 1 or 2, wherein the hyaluronidase mutant further comprises a mutation selected from the group consisting of N206H, A408L, Q250E, T248Y or a combination thereof. 4 . The hyaluronidase mutant according to claim 1 , wherein the amino acid sequence of the hyaluronidase mutant is shown as SEQ ID No. 3 or SEQ ID No.

5.

5. A gene encoding the hyaluronidase mutant according to any one of claims 1 to 4, preferably the gene sequence is shown as SEQ ID No. 4 or SEQ ID No.

6.

6. A recombinant expression vector comprising and / or expressing the gene according to claim 5, preferably the recombinant expression vector is a pET series expression vector.

7. A microbial host cell comprising the recombinant expression vector according to claim 6 or expressing the gene according to claim 5, preferably an Escherichia coli, Bacillus subtilis or yeast cell.

8. Use of the hyaluronidase mutant according to any one of claims 1 to 4, the gene according to claim 5, the recombinant expression vector according to claim 6, or the microbial host cell according to claim 7 in the preparation of low molecular weight hyaluronic acid or oligomeric hyaluronic acid.

9. A molecular engineering design method for improving the catalytic activity and / or thermal stability of hyaluronidase (HAase), comprising the following steps: (1) Based on the amino acid sequence of HAase, the structure of HAase was predicted using Alphafold3; (2) The amino acid sequence of HAase was uploaded to the NCBI and PDB databases respectively, and the same family enzyme-tetrasaccharide molecule (HA4) complex with the highest amino acid sequence similarity and the most similar three-dimensional structure as the target enzyme and the experimental X-ray diffraction crystal was searched as a reference for molecular docking. Based on the three-dimensional conformation of HAase obtained in step (1), molecular docking was performed to determine the binding range of the substrate molecule in the active pocket of HAase; (3) Visualize the best docked complex three-dimensional structure in step (2) using Pymol software and find the distance from the substrate All amino acids within the distance were used to input the HAase amino acid sequence into the online server ConSurf for homologous evolution analysis of amino acids in the same family; (4) Based on the Consurf online database in step (3), a conservation analysis of each amino acid residue of the enzyme was performed. Among the amino acid residues within the range, amino acid residues with a conservation score of less than 5 and a frequency of less than 50% were selected as mutation targets. FoldX was used in conjunction to calculate the free energy of substrate-enzyme binding for further screening and design, and mutants were constructed. The enzyme with the best catalytic activity was obtained through experimental screening; and (5) Optionally, based on the result of step (4), the enzyme with the best catalytic activity is selected as the starting enzyme, and the enzyme sequence is submitted to the PROSS online database to obtain the optimal design scheme of the database. The position of the design site is visualized by Pymol, and the site located in the flexible loop of the enzyme is screened out. Subsequently, the ConSurf database is used to analyze the evolutionary conservation of the amino acids of the screened flexible loop sites, and sites with a score of less than 5 are selected to construct mutants, and mutants with enhanced thermal stability are screened out through experiments.

10. The method of claim 9, wherein the hyaluronidase is derived from Citrobacter portucalensis.

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