Chitosanase mutant with improved enzyme activity and preparation method and application thereof

Abstract

The application discloses a chitosanase mutant with improved enzyme activity, a preparation method and application thereof, and belongs to the technical field of enzyme engineering. The mutant has amino acid substitution at one or more of positions 5, 68, 193, 120, 171, 87, 86, 119 or 135 relative to a parent chitosanase (SEQ ID NO. 1) of Aspergillus fumigatus GH75 family. Based on AlphaFold3 modeling and molecular dynamics simulation guidance design, nine single-point mutants (N5E, S68R, T193A, H120Y, H171R, D87I, S86E, S119E, Q135R) with significantly improved enzyme activity are obtained, wherein the relative enzyme activity of N5E, S68R and T193A reaches 561%, 452% and 356% of the wild type, respectively. The application also provides nucleic acid molecules, recombinant expression vectors and strains encoding the mutant, and a preparation method and application thereof in the enzymatic preparation of chitooligosaccharide, and the product has high safety and good application prospect in the production of food and medical grade chitooligosaccharide.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a chitosanase mutant with enhanced enzyme activity, its encoding gene, recombinant expression vector, recombinant expression bacteria, preparation method, and application. Background Technology

[0002] Chitosanase (EC 3.2.1.132) is a specific glycoside hydrolase that catalyzes the hydrolysis of β-1,4-glycosidic bonds in chitosan molecules to generate chitooligosaccharides (COS) with various biological activities. Due to their antibacterial, antioxidant, immunomodulatory, and plant growth-promoting physiological functions, COS has broad application prospects in biomedicine, food industry, modern agriculture, and cosmetics. Compared with chemical and physical methods, enzymatic preparation of chitooligosaccharides has significant advantages such as mild reaction conditions, easy process control, controllable degree of polymerization of products, and environmental friendliness. Therefore, the development of highly efficient chitosanases has always been a research hotspot in this field.

[0003] Aspergillus fumigatus ( Aspergillus fumigatus Chitosanases from the GH75 family have been reported to have good chitosan degradation capabilities and product specificity. Cheng et al. first cloned and characterized the Aspergillus fumigatus GH75 chitosanase (GenBank accession number AY190324), whose full-length protein containing a signal peptide consists of 238 amino acids and hydrolyzes chitosan via endo-glucanase to produce oligosaccharide products such as chitobiose to chitohexaose. The team further conducted site-directed mutagenesis studies on 10 conserved carboxyl amino acids in the GH75 family, identifying Asp160 and Glu169 as essential residues for the enzyme's catalysis (Cheng CY, Chang CH, Wu YJ, Li YK. Exploration of glycosyl hydrolase family 75, a chitosanase from Aspergillus fumigatus. J Biol Chem. 2006 Feb 10;281(6):3137-44. doi: 10.1074 / jbc.M512506200. Epub 2005 Dec 5. PMID: 16330537.). However, the above studies only focused on the elucidation of the catalytic mechanism and did not involve the targeted modification of enzyme activity. Furthermore, the enzyme activity of wild-type chitosanase still needs to be further improved under industrial application conditions (such as high substrate concentration and continuous production), resulting in a relatively high production cost for enzymatic preparation of chitosan oligosaccharides.

[0004] Regarding chitosanase expression systems, current technologies mostly employ Escherichia coli (E. coli) Escherichia coliRecombinant chitosanase expression was performed using an expression system. However, exogenous proteins are often expressed in E. coli as inclusion bodies, requiring a complex denaturation-renaturation process to obtain active proteins. This process is not only cumbersome and yields low results, but also makes it difficult to guarantee refolding efficiency. Furthermore, impurities such as endotoxins (lipopolysaccharides) produced by E. coli during fermentation may remain in the product, affecting the safety of chitosan oligosaccharide products in the food and pharmaceutical fields.

[0005] Therefore, developing a chitosanase mutant of Aspergillus fumigatus GH75 with significantly enhanced enzyme activity and establishing a recombinant expression system suitable for food-grade production are of great practical significance for reducing the cost of chitosan oligosaccharide preparation and promoting the industrial application of chitosanase. Summary of the Invention

[0006] The purpose of this invention is to provide a chitosanase mutant with enhanced enzyme activity. This mutant has amino acid substitutions at selected amino acid sites corresponding to SEQ ID NO.1, relative to the parental chitosanase with an amino acid sequence as shown in SEQ ID NO.1, thereby achieving a significant increase in enzyme activity.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a chitosanase mutant with enhanced enzyme activity, wherein the mutant has amino acid substitutions at one or more sites corresponding to the parental chitosanase with the amino acid sequence as shown in SEQ ID NO.1, at positions 5, 68, 193, 120, 171, 87, 86, 119, or 135 of SEQ ID NO.1.

[0008] Preferably, the amino acid sequence of the parental chitosanase is shown in SEQ ID NO.1. The parental chitosanase is derived from *Aspergillus fumigatus* (…). Aspergillus fumigatus ), belongs to the GH75 glycoside hydrolase family, which is composed of 238 amino acids.

[0009] Preferably, the chitosanase mutant has any one of the amino acid substitutions selected from the group consisting of: N5E: The 5th position of asparagine (Asn, N) is replaced by glutamic acid (Glu, E); S68R: Serine at position 68 (Ser, S) is replaced by arginine (Arg, R); T193A: Threonine at position 193 (Thr, T) is replaced by alanine (Ala, A); H120Y: Histidine at position 120 (His, H) is replaced by tyrosine (Tyr, Y); H171R: Histidine at position 171 (His, H) is replaced by arginine (Arg, R); D87I: The 87th position of aspartic acid (Asp, D) is replaced by isoleucine (Ile, I); S86E: Serine at position 86 (Ser, S) is replaced by glutamic acid (Glu, E); S119E: Serine at position 119 (Ser, S) is replaced by glutamic acid (Glu, E); Q135R: Glutamine (Gln, Q) at position 135 is replaced by arginine (Arg, R).

[0010] More preferably, the chitosanase mutant is selected from N5E, S68R, and T193A. With the wild-type parental chitosanase, whose amino acid sequence is as shown in SEQ ID NO. 1, having an enzyme activity of 100%, the relative enzyme activity of mutant N5E is 561%, the relative enzyme activity of mutant S68R is 452%, and the relative enzyme activity of mutant T193A is 356%.

[0011] In a second aspect, the present invention provides a nucleic acid molecule that encodes a chitosanase mutant as described in any of the first aspects.

[0012] Preferably, the nucleotide sequence of the nucleic acid molecule is targeted at Pichia pastoris (Pichia pastoris). Pichia pastoris The codon bias of X33 was optimized to improve the expression level of exogenous proteins in Pichia pastoris.

[0013] Thirdly, the present invention provides a recombinant expression vector containing the nucleic acid molecule described in the second aspect.

[0014] Preferably, the expression plasmid of the recombinant expression vector is pPICZαA. The pPICZαA plasmid contains a methanol-inducible AOX1 promoter and an α-factor secretion signal peptide sequence, enabling efficient secretory expression of the chitosanase mutant.

[0015] Fourthly, the present invention provides a variety of recombinant expression bacteria, wherein the recombinant expression bacteria contain the recombinant expression vector described in the third aspect.

[0016] Preferably, the host strain of the recombinant expression strain is Pichia pastoris (Pichia pastoris). Pichia pastoris Pichia pastoris, as a eukaryotic expression system, has advantages such as being safe and endotoxin-free, capable of post-translational modification, and able to achieve high-density fermentation and efficient secretory expression of exogenous proteins. It is especially suitable for the production of food and pharmaceutical grade recombinant proteins.

[0017] Fifthly, the present invention provides a method for preparing any of the chitosanase mutants described in the first aspect, comprising the following steps: (1) Cultivate the recombinant expression bacteria described in the fourth aspect; (2) Induce the recombinant expression bacteria to express chitosanase mutant.

[0018] Preferably, in step (1), recombinant Pichia pastoris X33 is inoculated into BMGY medium and cultured at 28–30°C with shaking at 200–250 rpm until OD. 600 Reaching 2 to 6; In step (2), the bacterial cells were collected and resuspended in BMMY medium, and methanol was added to a final concentration of 0.5% to 1.0% (v / v) for induction expression. Methanol was added every 24 h. After induction expression for 72 to 120 h, the fermentation supernatant was collected and purified by nickel column affinity chromatography or ion exchange chromatography to obtain the chitosanase mutant.

[0019] In a sixth aspect, the present invention provides the use of any of the chitosanase mutants described in the first aspect or the recombinant expression bacteria described in the fourth aspect in the preparation of chitosan oligosaccharides.

[0020] Preferably, the application includes: using chitosan as a substrate, and under conditions of pH 5.0–6.5 and temperature 40–55°C, using the chitosanase mutant to catalyze the hydrolysis of chitosan to prepare chitobiose to chitohexaose and other chitosan oligosaccharide products.

[0021] In a seventh aspect, the present invention provides a method for improving chitosanase activity, comprising the following steps: site-directed mutagenesis of the parental chitosanase with an amino acid sequence as shown in SEQ ID NO.1, wherein the site-directed mutagenesis site is selected from any one of the following groups: replacing N with E at position 5, replacing S with R at position 68, replacing T with A at position 193, replacing H with Y at position 120, replacing H with R at position 171, replacing D with I at position 87, replacing S with E at position 86, replacing S with E at position 119, or replacing Q with R at position 135.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention successfully obtained nine single-point mutants with significantly enhanced enzyme activity by systematically screening the non-catalytic active sites of chitosanase from Aspergillus fumigatus GH75 through site-directed mutagenesis. With the enzyme activity of the wild-type parental chitosanase (SEQ ID NO.1) as 100%, the relative enzyme activity of each mutant increased to 172%–561%. Among them, the preferred mutants N5E reached a relative enzyme activity of 561%, S68R reached 452%, and T193A reached 356%, which are significantly higher than the enzyme activity enhancement levels of single-point mutants of chitosanase from Aspergillus fumigatus GH75 reported in the prior art.

[0023] (2) The present invention uses the Pichia pastoris X33 / pPICZαA eukaryotic expression system to recombinantly express chitosanase mutants, avoiding the inclusion body refolding problem and endotoxin residue risk of traditional Escherichia coli expression system, realizing efficient soluble secretory expression of chitosanase mutants, with higher product safety, and is especially suitable for enzymatic preparation of food and pharmaceutical grade chitosan oligosaccharides.

[0024] (3) The nine mutation sites provided by this invention cover multiple different structural regions in the primary sequence of Aspergillus fumigatus GH75 chitosanase. The identification of these sites provides key site information for further enhancing enzyme activity through combined mutations (such as double mutations or multiple mutations), and also provides structural information reference for the rational design and modification of other GH75 family chitosanases. Attached Figure Description

[0025] Figure 1 The predicted three-dimensional structure of wild-type chitosanase Csn75 using AlphaFold3.

[0026] Figure 2 The root mean square fluctuation (RMSF) analysis plots are obtained from 50 ns molecular dynamics simulations of wild-type chitosanase Csn75 at 300 K, 350 K, and 400 K.

[0027] Figure 3 Map of the wild-type chitosanase Csn75 expression plasmid pPICZαA-Csn75.

[0028] Figure 4 The image shows the expression plasmid pPICZαA-Csn75 for wild-type chitosanase Csn75. Lane M is the molecular weight standard for nucleic acids. Lanes 1 and 2 are the products of pPICZαA-Csn75 after double digestion with EcoRI and NotI. Lane 3 is the product of PCR amplification using the recombinant plasmid as a template and Csn75-specific primers.

[0029] Figure 5 The results show the specific activity of wild-type chitosanase Csn75 and its mutants. Detailed Implementation

[0030] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0031] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0032] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0033] Example 1 Construction and preparation of chitosanase mutant This invention uses chitosanase derived from Aspergillus fumigatus as the parent chitosanase, the amino acid sequence of which is shown in SEQ ID NO.1 of this invention. This parent chitosanase belongs to the GH75 family and has the ability to catalyze the hydrolysis of β-1,4 glycosidic bonds in chitosan to generate chitosan oligosaccharides.

[0034] To improve the enzymatic activity of the parental chitosanase, the inventors used AlphaFold3 modeling based on the amino acid sequence of SEQ ID NO.1, and its protein structure is as follows: Figure 1 As shown, molecular dynamics simulations of this structure were performed using Gromacs at 300 K, 350 K, and 400 K for 50 ns (see [link to Gromacs simulation]). Figure 2 After analyzing regions with large fluctuations, nine mutants were designed as shown in Table 1. Each mutant in the design contains a substitution of one amino acid relative to the parental chitosanase. These mutants were named N5E, S68R, T193A, H120Y, H171R, D87I, S86E, S119E, and Q135R, respectively.

[0035] Table 1 Chitosanase variants

[0036] The nucleic acid sequence (SEQ ID NO.2) of the parental chitosanase expressed in Pichia pastoris was synthesized by General Biosystems (Anhui) Co., Ltd. This sequence was optimized for the codon bias of Pichia pastoris. The gene was cloned into a Pichia pastoris vector, and the chitosanase expression plasmid pPICZαA-Csn75 (e.g., ...) was used. Figure 3 (As shown).

[0037] The mutant plasmids were named pPICZαA-Csn75-N5E to pPICZαA-Csn75-Q135R according to the variant names in Table 1. To express the parental chitosanase and its variants, the Pichia expression kit (Invitrogen) was used, and the *Pichia pastoris* X33 strain and plasmids were manipulated according to its instructions. Specifically, *Pichia pastoris* X33 strain was cultured on YPD medium (1% yeast extract, 2% peptone, 2% glucose, and 1.5% agar) plates at 30°C for 48 h. Single colonies were then picked and cultured in 4 mL of YPD liquid medium (1% yeast extract, 2% peptone, 2% glucose) at 30°C and 200 rpm for 12 h. Subsequently, they were transferred to Erlenmeyer flasks containing 30 mL of YPD liquid medium and cultured at 30°C and 220 rpm for 4-5 h, and OD was detected. 600 After the bacterial concentration reached the range of 1.1-1.3, the culture medium was centrifuged at 4℃ and 9000 rpm for 10 min. 4 mL of bacterial cells were collected into sterile EP tubes, the supernatant was gently discarded, and the remaining supernatant was blotted dry with sterile filter paper. The bacterial cells were then resuspended in 1 mL of pre-chilled sorbitol and centrifuged at 4℃ and 9000 rpm for 2 min, and the supernatant was discarded. The above steps were repeated. Finally, 100-150 μL of pre-chilled sorbitol (1 mol / L) was added to resuspend the bacterial cells. The competent cells were now prepared. The expression plasmid pPICZαA-Csn75 and nine other variants were linearized using Sac I (e.g., ...). Figure 4 (As shown), the linearized fragment was purified and recovered, and then transformed into the above-mentioned Pichia pastoris X33 competent cells by electroporation. The mixture was then uniformly spread on a substrate containing 100 μg / mL Zeocin. TM The Pichia pastoris was incubated upside down on YPD plates at 30°C for 2-3 days. Recombinant Pichia pastoris strains were screened on the plates and named Csn75, Csn75-N5E to Csn75-Q135R.

[0038] The clones obtained from the above screening were transferred to BMGY medium and cultured in a shaking incubator at 30℃ and 200 rpm for 24 h. Then they were transferred to BMMY medium and cultured at 30℃ and 200 rpm. Methanol was added every 24 h to a final concentration of 0.5% (v / v) and expression was induced for 96 h. The cells were removed by centrifugation at 9000-12000 rpm for 10 min to obtain fermentation supernatant containing chitosanase Csn75 and 9 other variants.

[0039] Example 2: Determination of the properties of chitosanase (1) Determination of optimal temperature and enzyme specific activity Chitosanase activity was determined using the DNS method. 1% (w / v) chitosan (deacetylation ≥95%, dissolved in acetate-sodium acetate buffer) was used as the substrate. 500 μL of the substrate solution was preheated in a water bath at different temperatures for 3 min, followed by the addition of 100 μL of appropriately diluted enzyme solution and a reaction time of 30 min. The reaction was terminated by adding 500 μL of DNS reagent, followed by boiling in a water bath for 5 min for color development. After cooling, 200 μL was taken and the absorbance was measured at 540 nm. A standard curve was plotted using D-glucosamine hydrochloride as a standard. The enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute under optimal conditions.

[0040] Following the chitosanase activity assay method, the wild-type chitosanase and its mutants obtained in Example 1 were subjected to enzyme activity detection. The wild-type chitosanase exhibited activity over a wide pH range, reaching maximum activity between pH 4.5 and 7.0, with optimal activity at pH 6.0. Given that this enzymatic reaction is typically carried out at pH 6.0 in industrial production, subsequent determinations of the enzymatic properties of the wild-type and all mutants were conducted uniformly at pH 6.0.

[0041] First, the reaction system was subjected to different temperature conditions: 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, and 75℃. The optimal reaction temperature for each chitosanase mutant was determined and is shown in Table 2.

[0042] The nine variants obtained in Example 1 and the parental chitosanase Csn75 were diluted with pure water to appropriate concentrations (controlling the absorbance OD value at 540 nm wavelength between 0.2 and 0.8), and then the enzyme activity of each sample was measured. Using the enzyme activity of the parental chitosanase Csn75 as 100% as a reference, the relative enzyme activity of each variant was calculated, and the results are shown in Table 2. Figure 5 As shown.

[0043] Table 2. Relative enzyme activities of each mutant

[0044] The results showed that the relative enzyme activities of mutants N5E increased to 561%, S68R to 452%, T193A to 356%, H120Y to 317%, H171R to 232%, D87I to 212%, S86E to 194%, S119E to 191%, and Q135R to 172%. These results indicate that the chitosanase variants provided by this invention have significantly improved enzyme activities compared to the parental chitosanase, with variants N5E, S68R, and T193A showing particularly significant effects. These variants have potential application value in the industrial production and application of chitosanase.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chitosanase mutant with enhanced enzyme activity, characterized in that, The mutant has an amino acid substitution at one or more sites corresponding to the parental chitosanase with the amino acid sequence as shown in SEQ ID NO.1, at positions 5, 68, 193, 120, 171, 87, 86, 119, or 135 of SEQ ID NO.

1.

2. The chitosanase mutant according to claim 1, characterized in that, The mutant has any one of the amino acid substitutions selected from the group consisting of: N5E: Asparagine at position 5 is replaced by glutamic acid; S68R: Serine at position 68 is replaced by arginine; T193A: Threonine at position 193 is replaced by alanine; H120Y: Histidine at position 120 is replaced by tyrosine; H171R: Histidine at position 171 is replaced by arginine; D87I: Aspartic acid at position 87 is replaced by isoleucine; S86E: Serine at position 86 is replaced by glutamic acid; S119E: Serine at position 119 is replaced by glutamic acid; Q135R: Glutamine at position 135 is replaced by arginine.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the chitosanase mutant according to any one of claims 1-2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid molecule as described in claim 3.

5. The recombinant expression vector according to claim 4, characterized in that, The expression plasmid of the recombinant expression vector is pPICZαA.

6. A recombinant expression bacterium, characterized in that, The recombinant expression bacteria contains the recombinant expression vector as described in claim 4 or 5.

7. The recombinant expression bacterium according to claim 6, characterized in that, The host strain of the recombinant expression strain is Pichia pastoris X33.

8. A method for preparing the chitosanase mutant according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Cultivating the recombinant expression bacteria as described in claim 6 or 7; (2) Induce the recombinant expression bacteria to express the chitosanase mutant.

9. The use of the chitosanase mutant according to any one of claims 1-2 or the recombinant expression bacteria according to claim 6 or 7 in the preparation of chitosan oligosaccharides.

10. A method for improving chitosanase activity, characterized in that, The method includes the following steps: site-directed mutagenesis of the parental chitosanase with an amino acid sequence as shown in SEQ ID NO.1, wherein the site-directed mutagenesis site is selected from any one of the following groups: replacing N with E at position 5, replacing S with R at position 68, replacing T with A at position 193, replacing H with Y at position 120, replacing H with R at position 171, replacing D with I at position 87, replacing S with E at position 86, replacing S with E at position 119, or replacing Q with R at position 135.

Images