Beta-N-acetylglucosaminidase mutant and preparation method thereof

Abstract

The invention discloses a beta-N-acetylglucosaminidase mutant obtained based on random mutation and a preparation method of the beta-N-acetylglucosaminidase mutant. On the basis of an error-prone PCR (Polymerase Chain Reaction) technology, a mutant BsNagZ-14 (C17R / K209T / T436S) and a mutant BsNagZ-17 (E358G) of the beta-N-acetylglucosaminidase of the bacillus subtilis are obtained in the invention. The two mutants keep high enzyme activity within the pH range of 3-10, the thermal stability of BsNagZ-14 is better, 98% of enzyme activity (90% of original enzyme) is kept after the temperature is kept at 40 DEG C for 3 hours, 83% of enzyme activity is kept after the temperature is kept at 60 DEG C for 3 hours, and the parent is attenuated to 23% of the initial enzyme activity. The mutants BsNagZ-14 and BsNagZ-17 are connected with a carrier pET20b (+) and are transferred into escherichia coli BL21 (DE3), the extracellular highest enzyme activities of the two recombinant bacteria are 1.140 U / mL and 1.136 U / mL respectively, and the extracellular highest enzyme activities of the two recombinant bacteria are improved by 16.32% and 15.91% respectively compared with those of control group bacteria expressing NagZ. The beta-N-acetylglucosaminidase can be externally tangent to the chitosan oligosaccharide, so that the hydrolysis effect of the chitinase on the chitin is favorably improved. The beta-N-acetylglucosaminidase mutant has wide pH and temperature adaptability, can be adapted to chitinase with different enzymatic characteristics, and provides convenience for carrying out double-enzyme hydrolysis on chitin.

Description

Technical Field

[0001] The present invention relates to a beta-N-acetylglucosaminidase mutant based on random mutation modification and a preparation method thereof, belonging to the technical fields of protein engineering and enzyme engineering. Background Art

[0002] Chitin is formed by the polymerization of β-N-acetylglucosamine (GlcNAc) through covalent β-1,4 bonds. This long-chain polymer is mainly found in the exoskeletons of arthropods and insects, such as crabs, lobsters and locusts, and accounts for up to 75% of the dry weight of the exoskeleton. Chitin is also found in the cell walls of fungi and in the beaks and inner shells of cephalopods. As a biosphere, chitin is produced in an annual amount of about 10 12 –10 14 Tons of rich biomass. Chitin is second only to lignocellulose and, as a nitrogen-containing organic compound, its content is second only to protein.

[0003] β-N-acetylglucosaminidase hydrolyzes chito-oligosaccharides into the final product, β-N-acetylglucosamine. Adding β-N-acetylglucosamine to chitin hydrolysis helps reduce the inhibitory effect of N-acetylglucosaccharides on endochitinase, thereby improving hydrolysis efficiency. β-N-acetylglucosaminidase (NagZ) from Bacillus subtilis belongs to the glycoside hydrolase family and specifically recognizes and cleaves β-1,4-glycosidic bonds, progressively hydrolyzing chitobiose and its polymers and derivatives from the termini to produce N-acetylglucosamine. Therefore, it can be used in conjunction with chitinase to hydrolyze chitin, ultimately yielding N-acetylglucosamine.

[0004] Extracellularly expressed proteins are directly secreted into the culture medium and can be collected directly from the culture medium, avoiding cell disruption and complex intracellular extraction steps, significantly simplifying the purification process and reducing production costs. Extracellular expression also reduces the potential interference of the protein's large intracellular accumulation with the host cell's internal environment. Directed evolution is a non-rational design that simulates Darwin's natural evolutionary process in the laboratory, targeting the gene of a specific protein enzyme by inducing gene-level mutations and constructing a library, and then screening for excellent mutants. The present invention constructs a recombinant Escherichia coli BL21 (DE3) that secretively expresses NagZ derived from Bacillus subtilis, and obtains mutants BsNagZ-14 (C17R / K209T / T436S) and BsNagZ-17 (E358G) through error-prone PCR technology and library screening. Compared with NagZ, both BsNagZ-14 and BsNagZ-17 show better acid-base tolerance and thermal stability, and have higher extracellular enzyme activity when expressed in Escherichia coli BL21 (DE3). Summary of the Invention

[0005] The present invention aims to provide a mutant of β-N-acetylglucosaminidase and a preparation method thereof. The β-N-acetylglucosaminidase mutants BsNagZ-14 and BsNagZ-17 are shown in SEQ ID NO.1 and SEQ ID NO.2 with amino acid sequences. Compared with the parent Bacillus subtilis β-N-acetylglucosaminidase (NagZ), the mutant BsNagZ-14 mutates the 17th cysteine of the parent β-N-acetylglucosaminidase to arginine, the 209th lysine to threonine, and the 436th threonine to serine. The mutant BsNagZ-17 mutates the 358th glutamic acid of the parent β-N-acetylglucosaminidase to glycine.

[0006] A mutant library of the NagZ gene was constructed by error-prone PCR of the parental NagZ gene. The NagZ mutant library was ligated into the pET20b(+) vector using overlap extension PCR and transformed into Escherichia coli BL21(DE3) for gene expression. Library screening and sequencing analysis yielded mutants BsNagZ-14 and BsNagZ-17. Compared with NagZ, BsNagZ-14 and BsNagZ-17 exhibited a broader pH range, maintaining high enzyme activity within the pH range of 3.0 to 10.0. BsNagZ-14 exhibited superior thermostability compared to the control. After incubation at 40°C for 3 hours, the enzyme activity of BsNagZ-14 remained approximately 96% of its initial value, while that of NagZ decreased to 90%. After incubation at 60°C for 3 hours, the enzyme activity of BsNagZ-14 remained approximately 89% of its initial value, while that of NagZ decreased to 59%.

[0007] E. coli BL21 / pET-20b(+)-bsnagZ-14 and E. coli BL21 / pET-20b(+)-bsnagZ-17 carry pET20b(+) plasmids connected to the DNA fragments encoding SEQ ID NO.3 and SEQ ID NO.4, respectively, and can be used for extracellular expression of BsNagZ-14 and BsNagZ-17. The recombinant bacteria were inoculated into a fermentation medium (50-90 mM glycine, 11.8 g / L peptone, 23.6 g / L yeast extract, 9.4 g / L K2HPO4, 2.2 g / L KH2PO4, 5 g / L glycerol), cultured at 25°C and 220 rpm for 2 hours, 0.5 mM IPTG was added, and the culture was continued for 12-60 hours. The supernatant was collected to obtain the β-N-acetylglucosaminidase mutant. The maximum extracellular enzyme activities of E.coli BL21 / pET-20b(+)-bsnagZ-14 and E.coli BL21 / pET-20b(+)-bsnagZ-17 were 1.140 and 1.136 U / mL, respectively, which were increased by 16.32% and 15.91% compared with the control group expressing NagZ.

[0008] The beneficial effects of the present invention are:

[0009] The present invention aims to provide β-N-acetylglucosaminidase mutants and preparation methods thereof. Based on error-prone PCR technology, the present invention uses Bacillus subtilis β-N-acetylglucosaminidase to develop β-N-acetylglucosaminidase mutants BsNagZ-14 (C17R / K209T / T436S) and BsNagZ-17 (E358G). The two mutants can be expressed in Escherichia coli BL21 (DE3). The maximum extracellular enzyme activity of the two recombinant bacteria is increased by 16.32% and 15.91% respectively compared with the control bacteria expressing NagZ. The extracellular secretion expression of the recombinant protein can avoid the steps of cell disruption and intracellular protein removal, greatly reducing costs. The two mutants maintain high enzyme activity in the pH range of 3.0 to 10.0, and the thermal stability of BsNagZ-14 is better than that of the parent. Chitinases from different sources have widely varying optimal pH values. β-N-acetylglucosaminidase mutants with broad pH and temperature adaptability can adapt to chitinases with different enzymatic properties, facilitating dual-enzyme hydrolysis of chitin. This invention provides a beneficial method for β-N-acetylglucosaminidase production research and offers a valuable solution for promoting the comprehensive utilization of chitin. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Comparison of extracellular enzyme activities of β-N-acetylglucosaminidase mutants expressed in recombinant Escherichia coli;

[0011] Figure 2 To analyze the enzymatic properties of β-N-acetylglucosaminidase mutants BsNagZ-14 and BsNagZ-17;

[0012] Figure 3 The amino acid sequence alignment of β-N-acetylglucosaminidase mutants BsNagZ-14 and BsNagZ-17 is shown;

[0013] Figure 4 To simulate and analyze the protein structure of β-N-acetylglucosaminidase mutants BsNagZ-14 and BsNagZ-17;

[0014] Figure 5 Chitinase (ChiA) and β-N-acetylglucosaminidase mutant BsNagZ-14 were combined to hydrolyze chitin. DETAILED DESCRIPTION

[0015] The present invention will be further described below with reference to specific examples so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention.

[0016] Example 1: Construction and screening of β-N-acetylglucosaminidase mutant library

[0017] Increase the change of Mn 2+ A low-fidelity DNA polymerase (0.2 mM) and a low-fidelity DNA polymerase can increase the frequency of base mutations in the amplified product, allowing incorrect bases to be randomly incorporated into the amplified gene at a certain frequency, increasing the diversity of the mutation spectrum. This results in a randomly mutated DNA sequence, allowing the selection of proteins that meet the needs. Using the Bacillus subtilis genome as a template, the β-N-acetylglucosaminidase gene bsnagZ was amplified using primers nagZbs(Nde I)+: ATACATATGAGACCTG TCTTTCCGCTTATC and NagZbs(Xho I)-: tatctcgagAAGCGGTCTTCCCGTTTTG. The plasmid pET-20b(+)-bsnagZ was constructed by one-step cloning and ligation into pET-20b(+). Error-prone PCR was performed using pET-20b(+)-bsnagZ as a template using primers mutation-F: TTGTTTAACTTTAAGAAGGAGATATACATATG and mutation-R: GGTGGTGGTGGTG CTCGAG, as shown in Table 1. The resulting product was digested with Dpn I inhibitory endonuclease to remove the plasmid template. The reaction was performed for 1 hour, and the resulting product was recovered by gel recovery. The resulting product was the mutant library gene.

[0018] Overlap extension PCR was used to connect the mutant library gene to the vector. The pET-20b(+) plasmid was first linearized using primers pET-20b(+)-F: CATATGTATATCTCCTTC TTAAAGTTAAACAA and pET-20b(+)-R: CTCGAGCACCACCACCACC. The PCR amplification system is shown in Table 2, and the reaction conditions are the same as in Table 1. Overlap extension PCR was then performed using the mutant product and the linearized plasmid as templates and primers to generate the mutant recombinant plasmid. The overlap extension PCR amplification system and reaction conditions are the same as in Table 2 and Table 1, respectively. The overlapping extension PCR product was transferred into E. coli BL21 (DE3) and a mutant library was obtained by colony PCR screening. The recombinant bacteria were inoculated into a fermentation medium (50-90 mM glycine, peptone, yeast powder), cultured at 25°C and 220 rpm for 2 hours, 0.5 mM IPTG was added, and the culture was continued for 12-60 hours. The supernatant was collected to obtain the β-N-acetylglucosaminidase mutant.

[0019] Table 1 Parameters of error-prone PCR amplification system

[0020]

[0021] Table 2 PCR amplification system

[0022]

[0023] β-N-acetylglucosaminidase activity assay: Enzyme activity is determined using 4-nitro-N-acetyl-β-D-glucosamine (pNP-β-GlcNAc) as a substrate. 10 μL of crude enzyme solution is added to 90 μL of 1 nmol / L pNP-β-GlcNAc. The reaction is allowed to proceed for 1 hour under appropriate conditions. The reaction is then terminated by adding 200 μL of 0.2 mol / L Na2CO3. The absorbance of the reactants is measured at 405 nm. One unit (U) of NagZ enzyme activity is defined as the amount of enzyme required to hydrolyze the substrate to produce 1 μmol of pNP per hour under appropriate conditions.

[0024] The mutants BsNagZ-14 and BsNagZ-17 with the highest β-N-acetylglucosaminidase activity were screened and inoculated into a fermentation medium (50-90 mM glycine, 11.8 g / L peptone, 23.6 g / L yeast powder, 9.4 g / L K2HPO4, 2.2 g / L KH2PO4, 5 g / L glycerol), cultured at 25 ° C, 220 rpm for 2 hours, added 0.5 mM IPTG, and continued to culture for 12-60 hours. The supernatant was collected to obtain the β-N-acetylglucosaminidase mutant. The highest enzyme activities of the mutants were 1.14 U / mL and 1.136 U / mL, respectively, which were increased by 16.32% and 15.91% respectively compared with the enzyme activities of the control group expressing NagZ. The results are shown in FIG. Figure 1 .

[0025] Example 2: Analysis of Enzymatic Properties of Mutants BsNagZ-14 and BsNagZ-17

[0026] In order to further understand the enzymatic properties of β-N-acetylglucosaminidase, the optimal temperature, pH and thermal stability of the enzyme before and after mutation were analyzed and compared. The enzymatic activity of β-N-acetylglucosaminidase was measured at different temperatures (30-70°C). The temperature corresponding to the peak of the enzyme activity was determined as the optimal reaction temperature of the recombinant enzyme. The enzymatic activity of β-N-acetylglucosaminidase was measured in 10mM buffer with a pH of 3.0-10.0 and at the optimal temperature, and the pH corresponding to the peak of the enzyme activity was determined as the optimal reaction pH of the enzyme. The recombinant enzyme was placed in an environment of 40°C and 60°C, and after heat treatment for different periods of time, its enzymatic activity was measured. Taking the enzyme activity without treatment as the benchmark (set as 100%), the relative residual enzyme activity of the recombinant enzyme after different heat treatment periods was compared to understand the thermal stability of the enzyme. The results are shown in Figure 2 .like Figure 2As shown in A, recombinant NagZ is active within the range of 30-60°C, with the highest activity at 30-50°C. Since some enzymes reach peak activity at 40°C and 60°C, their temperature stability was analyzed at 40°C and 60°C, respectively. Figure 2 As shown in B, when kept at 40℃ for 180min, the enzyme activity of the recombinant enzyme exceeded 90%. Figure 2 As shown in Figure C, after incubation at 60°C for 20 minutes, the enzyme activity began to decline continuously. In contrast, BL21 / pET-20b(+)-bsnagZ-14 retained 83% of its activity after incubation at 60°C for 180 minutes, while the control enzyme, BL21 / pET-20b(+)-bsnagZ, retained only 23% of its activity under the same conditions. Overall, BL21 / pET-20b(+)-bsnagZ-17 and BL21 / pET-20b(+)-bsnagZ-14 exhibited strong temperature tolerance, with BL21 / pET-20b(+)-bsnagZ-17 showing particularly strong activity and temperature tolerance. Therefore, the optimal temperature for recombinant NagZ is either 40°C or 60°C. Mutant recombinant NagZ exhibited high activity across the pH range of 4.0 to 9.0, with the lowest activity at pH 6.0, demonstrating its broad adaptability. The enzyme activity of BL21 / pET-20b(+)-bsnagZ was highest at pH 6.0, and decreased significantly when the pH value deviated from this value, indicating that its stability was poor.

[0027] Example 3: Comparison of amino acid sequences of mutant BsNagZ-14 and mutant BsNagZ-17 and partial structural analysis of mutation sites

[0028] The inserted sequence of plasmid BL21 / pET-20b(+)-bsnagZ-14 was analyzed by Sanger sequencing to obtain the DNA and protein sequences of mutants BsNagZ-14 and BsNagZ-17.

[0029] The protein sequence of BsNagZ-14 is shown in SEQ ID NO.1:

[0030] MRPVFPLILSAVLFLSRFFGARQTEASASKRAIDANQIVNRMSLDEKLGQ

[0031] MLMPDFRNWQKEGESSPQALTKMNDEVASLVKKYQFGGIILFAENVKTTKQT

[0032] VQLTDDYQKASPKIPLMLSIDQEGGIVTRLGEGTNFPGNMALGAARSRINAYQ

[0033] TGSIIGKELSALGINTDFSPVVDINNNPDNPVIGVRSFSSNRELTSRLGLYTMTG

[0034] LQRQDIASALKHFPGHGDTDVDSHYGLPLVSHGQERLREVELYPFQKAIDAG

[0035] ADMVMTAHVQFPAFDDTTYKSKLDGSDILVPATLSKKVMTGLLRQEMGFNG

[0036] VIVTDALNMKAIADHFGQEEAVVMAVKAGVDIALMPASVTSLKEEQKFARVI

[0037] QALKEAVKNGDIPEQQINNSVERIISLKIKRGMYPARNSDSTKEKIAKAKKIVG

[0038] SKQHLKAEKKLAEKAVSVLKNEQHTLPFKPKKGSRILIVAPYEEQTASIEQTIH

[0039] DLIKRKKIKPVSLSKMNFASQVFKTEHEKQVKEADYIITGSYVVKNDPVVND

[0040] GVIDDTISDSSKWATVFPRAVMKAALQHNKPFVLMSLRNPYDAANFEEAKAL

[0041] IAVYGFKGYANGRYLQPNIPAGVMAIFGQAKPKGTLPVDIPSVTKPGNTLYPLGYGLNIKTGRPLLEHHHHHH(SEQ ID NO.1)

[0042] The protein sequence of BsNagZ-17, as shown in SEQ ID NO.2:

[0043] MRPVFPLILSAVLFLSCFFGARQTEASASKRAIDANQIVNRMSLDEKLGQ

[0044] MLMPDFRNWQKEGESSPQALTKMNDEVASLVKKYQFGGIILFAENVKTTKQT

[0045] VQLTDDYQKASPKIPLMLSIDQEGGIVTRLGEGTNFPGNMALGAARSRINAYQ

[0046] TGSIIGKELSALGINTDFSPVVDINNNPDNPVIGVRSFSSNRELTSRLGLYTMKG

[0047] LQRQDIASALKHFPGHGDTDVDSHYGLPLVSHGQERLREVELYPFQKAIDAG

[0048] ADMVMTAHVQFPAFDDTTYKSKLDGSDILVPATLSKKVMTGLLRQEMGFNG

[0049] VIVTDALNMKAIADHFGQEEAVVMAVKAGVDIALMPASVTSLKEGQKFARVI

[0050] QALKEAVKNGDIPEQQINNSVERIISLKIKRGMYPARNSDSTKEKIAKAKKIVG

[0051] SKQHLKAEKKLAEKAVTVLKNEQHTLPFKPKKGSRILIVAPYEEQTASIEQTIH

[0052] DLIKRKKIKPVSLSKMNFASQVFKTEHEKQVKEADYIITGSYVVKNDPVVND

[0053] GVIDDTISDSSKWATVFPRAVMKAALQHNKPFVLMSLRNPYDAANFEEAKAL

[0054] IAVYGFKGYANGRYLQPNIPAGVMAIFGQAKPKGTLPVDIPSVTKPGNTLYPLGYGLNIKTGRPLLEHHHHHH(SEQ ID NO.2)

[0055] The protein sequence of BsNagZ-14, as SEQ ID NO.3:

[0056]

[0057] The protein sequence of BsNagZ-17 is shown in SEQ ID NO.4:

[0058]

[0059] Multiple sequence alignments were performed between the mutant sequences and the wild-type gene to identify the sites of base and corresponding amino acid changes. To elucidate the effects of the mutations on protein structure, a three-dimensional structural model of the mutant was constructed using a deep learning algorithm. A molecular visualization system was then used to analyze the topological differences between the mutant and wild-type proteins, focusing on the molecular conformational changes caused by the mutations and their effects on the spatial arrangement of key functional domains, such as the active center or ligand binding site. The potential mechanisms by which the three mutations (C17R, K209T, and T436S) in the mutant BsNagZ-14 enhance its function and activity are as follows: The C17R mutation is located in the signal peptide region. The positive charge of arginine (R) may enhance the interaction between the signal peptide and components of the secretion machinery, such as SecA or SRP, thereby improving protein secretion efficiency. The K209T mutation is located in the N-terminal catalytic domain, close to the catalytic center, and may optimize the catalytic center conformation, stabilize the transition state, and thus enhance catalytic efficiency. The T436S mutation is located in the C-terminal domain. The shorter side chain of serine (S) may reduce steric hindrance, increase local structural flexibility or stability, promote the binding of the enzyme active site to the substrate, or optimize the conformational conversion efficiency of the enzyme. The mutant BsNagZ-17 mutated at the E358G site in the N-terminal domain. The potential mechanism is as follows: The E358G mutation eliminates the large side chain of glutamic acid (E), reduces steric hindrance, and makes it easier for the substrate to approach the active center. The high flexibility of glycine (G) may make it easier for the catalytic residues to adjust their positions, promote the "induced fit" mechanism, thereby accelerating the catalytic step and improving enzyme activity. The results are shown in Figure 3 and Figure 4 .

[0060] Example 4: Chitinase (ChiA) and mutant BsNagZ-14 complex hydrolyze chitin

[0061] The chitinase used is derived from Bacillus thuringiensis. The optimum temperature of the enzyme is 40°C and the optimum pH is 4.0. When 1.00g / L colloidal chitin is used as the substrate, chitinase and β-N-acetylglucosaminidase are compounded to hydrolyze chitin. The ratio of chitinase: β-N-acetylglucosaminidase is 1U:1U. The control group is the supernatant of the enzyme-free culture medium. The reaction is carried out for 10 hours, and samples are taken every 2 hours. After centrifuging the sampled solution, the supernatant is heated in a 100°C metal bath for 5 minutes to inactivate the enzyme. The results are shown in FIG. Figure 5 .like Figure 5 As shown, when chitinase hydrolyzed chitin alone, the yield of GlcNAc reached a maximum of 0.13 g / L; when chitinase:β-N-acetylglucosaminidase was combined to hydrolyze chitin, the yield of GlcNAc reached a maximum of 0.33 g / L.

[0062] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A β-N-acetylglucosaminidase mutant, characterized in that The amino acid sequences of the β-N-acetylglucosaminidase mutants BsNagZ-14 and BsNagZ-17 are shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. The β-N-acetylglucosaminidase mutant according to claim 1, characterized in that Bacillus subtilis β-N-acetylglucosaminidase (NagZ) was used as the parent and obtained by error-prone PCR. The mutant, BsNagZ-14, mutated the cysteine at position 17 of the parent β-N-acetylglucosaminidase to arginine, the lysine at position 209 to threonine, and the threonine at position 436 to serine. The mutant, BsNagZ-17, mutated the glutamic acid at position 358 of the parent β-N-acetylglucosaminidase to glycine.

3. The gene fragment of the mutant according to claim 1, characterized in that The nucleotide sequence of the mutant BsNagZ-14 gene fragment is shown in SEQ ID NO: 3, and the nucleotide sequence of the mutant BsNagZ-17 gene fragment is shown in SEQ ID NO:

4.

4. A recombinant expression vector containing the coding gene according to claim 3.

5. A recombinant host cell containing the recombinant expression vector according to claim 3.

6. The method for preparing the β-N-acetylglucosaminidase mutant according to claim 1, characterized in that The following steps are involved: (1) Recombinant Escherichia coli BL21 (DE3) containing the pET20b(+) plasmid linked to the DNA fragments described in SEQ ID NO: 2 and SEQ ID NO: 3 were constructed, respectively, and named BL21 / pET-20b(+)-bsnagZ-14 and BL21 / pET-20b(+)-bsnagZ-17, which can be used for the extracellular expression of BsNagZ-14 and BsNagZ-17. (2) The recombinant bacteria BL21 / pET-20b(+)-bsnagZ-14 and BL21 / pET-20b(+)-bsnagZ-17 were inoculated into a fermentation medium (50-90 mM glycine, 11.8 g / L peptone, 23.6 g / L yeast powder, 9.4 g / L K2HPO4, 2.2 g / L KH2PO4, 5 g / L glycerol), cultured at 25°C and 220 rpm for 2 h, 0.5 mM IPTG was added, and the culture was continued for 12-60 h. The supernatant was collected to obtain the β-N-acetylglucosaminidase mutant.

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