Heat-resistant alkaline protease mutant and application thereof in degrading prolamine

Abstract

This invention belongs to the field of enzyme engineering technology and discloses a thermostable alkaline protease mutant and its application in the degradation of zein. The thermostable alkaline protease mutant was obtained by site-directed mutagenesis screening of the alkaline protease gene hapR from *Bacillus belysae*. The alanine at position 122 of HapR was mutated to lysine, and the asparagine at position 132 was mutated to glycine. Compared with the wild-type alkaline protease HapR, the enzyme activity of the alkaline protease mutant was increased by 1.18 times. The half-life of this mutant at 60°C reached 30.03 min, and its thermostableness was improved by 1.65 times compared to the wild type, significantly enhancing its ability to degrade zein.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a heat-resistant alkaline protease mutant and its application in the hydrolysis of zein. Background Technology

[0002] Proteases have significant applications in the feed and food industries, one of their core functions being the efficient hydrolysis of various protein substrates to enhance the nutritional value and utilization efficiency of raw materials. However, the practical application effectiveness of proteases is severely limited by their thermal stability. Improving the heat resistance of proteases is crucial for reducing production costs and adapting to high-temperature processing environments.

[0003] In the production and preparation stage, heat resistance directly affects cost. Industrial production of enzyme preparations often employs spray drying for solidification. Conventional proteases are easily denatured and inactivated under high-temperature drying conditions. To preserve enzyme activity, expensive protective agents are often added, or milder processes are used, leading to high costs and low efficiency. If the protease itself possesses excellent heat resistance, it can withstand more intense drying conditions, thereby simplifying the process, reducing energy consumption and auxiliary material costs, and significantly improving economic efficiency. In the application and processing stage, heat resistance determines process adaptability. Especially in the feed industry, high-temperature pelleting is the mainstream process, but it causes common proteases to rapidly inactivate, forcing the industry to adopt complex post-coating technologies, increasing equipment investment and process instability. Heat-resistant proteases, on the other hand, can be added directly before pelleting and remain viable in the final product, thus simplifying the process and ensuring uniform activity and distribution.

[0004] The effectiveness of proteases depends not only on their heat resistance but also on their ability to hydrolyze specific recalcitrant substrates. For example, proteases, a class of storage proteins widely found in plant materials such as corn and wheat, have a dense molecular structure, are rich in hydrophobic amino acids, and have strong intramolecular forces, resulting in extremely poor solubility. They are difficult to degrade effectively by endogenous animal proteases or common proteases during conventional feed processing and deep food processing. This severely limits the release of the nutritional value and industrial utilization efficiency of these high-quality protein resources.

[0005] Currently, the efficient degradation of proteases mainly relies on screening or modifying proteases with specific hydrolytic capabilities. Therefore, developing proteases with excellent heat resistance and efficient proteolytic ability is a key research direction for improving the technological level of the feed and food industries. This not only overcomes the heat stability bottleneck in production and processing, achieving cost reduction and efficiency improvement, but also directly targets and solves the resource utilization problem of recalcitrant plant proteins such as proteases, fully releasing their nutritional potential. This invention develops a highly heat-resistant, highly active alkaline protease mutant and evaluates its application in the degradation of corn proteases. Summary of the Invention

[0006] The present invention aims to obtain an alkaline protease mutant with significantly improved thermal stability and enzyme activity, thereby greatly enhancing its degradation efficiency of zein.

[0007] To achieve the above objectives, the present invention employs the following technical measures; The thermostable alkaline protease HapR mutant is obtained by mutating alanine at position 122 to lysine and asparagine at position 132 to glycine in the alkaline protease HapR derived from Bacillus belysinus, resulting in a protein with the amino acid sequence shown in SEQ ID NO.2.

[0008] A polynucleotide encoding the thermostable alkaline protease HapR mutant, the nucleotide sequence of which is shown in SEQ ID NO.3. An expression vector or host cell containing the polynucleotide.

[0009] The genetically engineered bacteria expressing the thermostable alkaline protease mutant are preferably constructed using Bacillus amyloliquefaciens HZ-12 as the starting strain to express the thermostable alkaline protease HapR mutant.

[0010] Application of the thermostable alkaline protease HapR mutant in the degradation of alcohol-soluble proteins: In a specific embodiment of the present invention, the expression plasmids pHY-hapR and pHY-hapR are used. A122K / N132G (The mutants were mutated at position 122 to lysine and at position 132 to glycine.) These were electroporated into Bacillus amyloliquefaciens HZ-12 competent cells. After fermentation, the thermostable properties and activity of the alkaline protease in the fermentation supernatant were measured. The results showed that, compared with the wild-type alkaline protease HapR, the mutant had a half-life of 30.03 min at 60℃, exhibiting a 1.65-fold increase in thermostable properties and a 1.18-fold increase in alkaline protease activity, demonstrating a good degradation effect on zein. Attached Figure Description

[0011] Figure 1 Wild-type recombinant strain HZ-12 / pHYhapR and double mutant recombinant strain HZ-12 / pHYhapR A122K / N132G Comparison of the activities of expressed alkaline proteases. CK represents wild-type alkaline protease HapR, and A122K / N132G represents the alkaline protease HapR double mutant.

[0012] Figure 2 Half-life analysis of wild-type alkaline protease HapR and thermostable alkaline protease mutant A122K / N132G at 60℃ for different treatment times (10, 20, 30, 40, 50, 60 min).

[0013] Figure 3SDS-PAGE analysis results of zein degradation by A122K / N132G mutant and wild-type HapR protease. Lane 1: CK group, untreated; Lane 2: Zein degradation by untreated HapR protease; Lane 3: Zein degradation by untreated A122K / N132G mutant; Lane 4: Zein degradation by HapR protease after treatment at 60℃ for 10 min; Lane 5: Zein degradation by A122K / N132G mutant after treatment at 60℃ for 10 min; Lane 6: Zein degradation by HapR protease after treatment at 80℃ for 90 s; Lane 7: Distribution of zein degradation by A122K / N132G mutant after treatment at 80℃ for 90 s; Lane 8: Protein Marker. Detailed Implementation

[0014] The present invention will be described in detail below through examples.

[0015] Biological material source description: Bacillus amyloliquefaciens ( B. amyloliquefaciens HZ-12 has been disclosed in the paper "Analysis of Spermine Metabolism and Key Gene Mining in Bacillus Amyloliquefaciens" (DOI:10.27158 / d.cnki.ghznu.2020.001154.), while Bacillus belyssus WH-7 and plasmid pHY-hapR have been disclosed in the paper "Genetic identification and expression optimization of a novel protease HapR from Bacillus velezensi The pHY-hapR plasmid was constructed by fusing the P43 promoter, hapR gene and TamyL terminator via SOE-PCR and then inserting them into the SmaI and XbaI sites of the pHY300PLK plasmid. The microorganisms and plasmids mentioned above are currently stored in the Microbial Engineering Laboratory of Huazhong Agricultural University.

[0016] Example 1: Construction of mutant expression vector Using the laboratory-preserved plasmid pHY-hapR as a template, the nucleotide sequence of its hapR gene is shown in SEQ ID NO.1. Two mutant bases were simultaneously introduced at the optimal combination mutation sites (A122K and N132G) of the alkaline protease HapR, and forward and reverse mutation primers containing these mutant bases were designed. Amplification was performed using site-directed mutagenesis PCR. The amplification system is shown in Table 1, and the amplification primers are as follows: Upstream primer A122K-F: 5'-GGTGAAGCCTTGAGAGTGCAGCTTAGGGGCTTTAATCTGTGATAC-3' Downstream primer A122K-R: 5'-GTATCACAGATTAAAGCCCCTAAGCTGCACTCTCAAGGCTTCACC-3' Upstream primer N132G-F: 5'-AACCGCTACTTTAACACCTGATCCGGTGAAGCCTTGAGAG-3' Downstream primer N132G-R: 5'CTCTCAAGGCTTCACCGGATCAGGTGTTAAAGTAGCGGTT-3'.

[0017] Table 1 Amplification reaction system First, using plasmid pHY-hapR as a template, PCR amplification was performed using primers containing the A122K mutation site. The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 90 s, 30 cycles; 72℃ extension for 5 min, 10℃ hold for 5 min. The obtained PCR amplification product was subjected to 0.8% agarose gel electrophoresis, yielding a 1149 bp band. The PCR product was recovered using a small-volume DNA recovery kit, yielding the mutant alkaline protease gene hapR. After PCR amplification, DpnI enzyme was added and digested at 37℃ for 1 h to remove methylated template plasmid DNA, retaining only the newly synthesized mutant product. The PCR product was transformed into E. coli DH5α competent cells, and sequencing confirmed the presence of the recombinant plasmid pHY-hapR containing the A122K mutation. A122K Subsequently, pHY-hapR A122K Using a template, a second PCR amplification was performed using primers containing the N132G mutation site, under the same PCR conditions as above. After PCR amplification, DpnI enzyme was added and the cells were digested at 37°C for 1 h, then transformed into DH5α competent cells. Sequencing confirmed successful mutation, indicating that both the A122K and N132G mutations in the hapR gene were successfully introduced. The mutated gene sequence is shown in SEQ ID NO.3, and the recombinant plasmid pHY-hapR was finally obtained. A122K / N132G .

[0018] Example 2: Expression and preparation of alkaline protease HapR mutant in Bacillus amyloliquefaciens HZ-12 The wild-type recombinant plasmid pHYhapR and the mutant plasmid pHYhapR of alkaline protease HapR were used. A122K / N132GThe bacteria were electroporated into Bacillus amyloliquefaciens HZ-12, and after selection for tetracycline (T) resistance and enzyme digestion verification, wild-type recombinant strain HZ-12 / pHYhapR and mutant recombinant strain HZ-12 / pHYhapR were obtained. A122K / N132G The recombinant strain HZ-12 / pHYhapR of Bacillus amyloliquefaciens mutant was used. A122K / N132G The wild-type recombinant strain HZ-12 / pHYhapR was inoculated separately into 50 mL of fermentation medium (containing 50 μg / mL kanamycin) and cultured overnight at 37°C and 220 r / min. Then, it was transferred to 50 mL of fermentation medium at a 3% inoculation rate and cultured for another 48 h at 37°C and 220 r / min. The fermentation medium consisted of: 80 g / L tryptone, 25 g / L yeast extract, 5 g / L KH₂PO₄, and 6 g / L NH₄Cl, and was sterilized at 121°C for 30 min. The supernatant (crude enzyme solution) of the fermentation broth was collected by centrifugation, and its alkaline protease activity was tested using the Folin-phenol method. The results are as follows: Figure 1 As shown, the alkaline protease HapR expressed by the wild-type recombinant strain HZ-12 / pHYhapR has an enzyme activity of 207.73 U / mL, while that of the mutant recombinant strain HZ-12 / pHYhapR is significantly lower. A122K / N132G The expressed alkaline protease HapR double mutant A122K / N132G exhibited an enzyme activity of 452.85 U / mL, which was 1.18 times higher than that of the wild-type. Further thermostability assessments were performed on the alkaline protease HapR double mutant and wild-type alkaline protease HapR. Figure 2 As shown, the double mutant A122K / N132G retained over 40% activity after incubation at 60°C for 30 min, while the wild type only retained 12.22%. Half-life analysis revealed that the half-life of the double mutant A122K / N132G at 60°C was extended to 30.03 min, a 1.65-fold increase compared to the wild type (11.33 min). Therefore, this invention successfully yielded the alkaline protease HapR double mutant A122K / N132G, which possesses both high thermostability and enzymatic activity.

[0019] Example 3: Application of the alkaline protease HapR mutant in corn alcohol hydrolysis experiment Enzymatic hydrolysis of zein was performed using the alkaline protease HapR double mutant A122K / N132G. The specific steps were as follows: 1 g of zein was weighed and added to 20 mL of 70% (v / v) ethanol solution, and stirred thoroughly until completely dissolved. Then, 60 mL of deionized water was added to adjust the ethanol concentration to a suitable final level to avoid the inhibitory effect of high ethanol concentration on protease activity. 2 mL of wild-type alkaline protease HapR or its double mutant protease solution was added to the above system. The protease solution was divided into three groups: no heat treatment group, 60℃ for 10 min group, and 80℃ for 90 s group. No protease solution was added as a control group. The enzymatic hydrolysis reaction was then carried out at 37℃. After the reaction, 100 μL of the reaction solution sample was taken, mixed with 400 μL of deionized water, and centrifuged at low temperature to collect the precipitate. After drying the precipitate, 30 μL of 8 M urea solution, 20 μL of 3 M thiourea solution, and 50 μL of SDS-PAGE loading buffer were added sequentially. The mixture was thoroughly mixed and then heated to boiling for 10 min to fully dissolve the protein. Subsequently, the mixture was centrifuged at 12,000 rpm for 10 min, and 15 μL of the supernatant was used for SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis to evaluate the enzymatic digestion effect of zein.

[0020] SDS-PAGE analysis results ( Figure 3 The results showed that the control group (without enzyme) exhibited a clear high-molecular-weight protein band throughout the incubation period. The zeaxanthin supplemented with wild-type HapR or the A122K / N132G mutant showed fewer intact bands compared to the control group, indicating that HapR and its double mutants can effectively enzymatically hydrolyze zeaxanthin. Compared to wild-type HapR, the A122K / N132G mutant showed significantly fewer intact zeaxanthin bands under non-heat-treated conditions, indicating that the enzyme activity of the A122K / N132G mutant was significantly higher than that of the wild-type alkaline protease HapR. Although the degradation efficiency of zeaxanthin by both wild-type and A122K / N132G mutants decreased under heat treatment conditions of 60℃ and 80℃, the degradation efficiency of zeaxanthin by the A122K / N132G mutant was still significantly higher than that of the wild-type, indicating that the double mutation enhanced the residual functional activity of the alkaline protease HapR under heat stress conditions. The above results confirm that zein undergoes time-dependent and continuous degradation under the action of the alkaline protease HapR double mutant A122K / N132G.

Claims

1. A thermostable alkaline protease HapR mutant, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.

2.

2. A polynucleotide encoding the thermostable alkaline protease HapR mutant of claim 1, characterized in that, The sequence of the polynucleotide is shown in SEQ ID NO.

3.

3. An expression vector containing the polynucleotide of claim 2.

4. Expressing the thermostable alkaline protease HapR mutant of claim 1 or a host cell containing the expression vector of claim 3.

5. Genetically engineered bacteria expressing the thermostable alkaline protease HapR mutant of claim 1.

6. The genetically engineered bacterium according to claim 5, characterized in that, An engineered bacterium expressing the thermostable alkaline protease HapR mutant of claim 1 was constructed using Bacillus amyloliquefaciens HZ-12 as the starting bacterium.

7. The use of the genetically engineered bacteria according to claim 5 or 6 in the production of heat-resistant alkaline protease.

8. The application of the thermostable alkaline protease HapR mutant of claim 1 in the degradation of zein.

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