A gene encoding a broad spectrum acid-base tolerant chitinase

Abstract

The present application relates to the field of enzyme engineering, and in particular to a broad-spectrum acid-base resistant chitinase. The existing chitinase has problems such as low activity, poor thermal stability, narrow pH range, and the like, which limit the application range of the enzyme. The present application provides a chitinase gene that can be efficiently expressed in Pichia pastoris. The enzyme not only has high thermal stability, but also has good pH stability in the pH range of 3.0-11.0. These two characteristics make it have good production application prospect.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering, specifically to the encoding gene of a chitinase with broad-spectrum acid and alkali tolerance. Characterization of its enzymatic properties revealed that the enzyme possesses both good thermal stability and broad-spectrum pH tolerance. Background Technology

[0002] Chitin, also known as chitin, is a biopolymer composed of N-acetylglucosamine (GlcNac, or N-acetylglucosamine) linked by β-1,4-glycosidic bonds. It is an abundant biopolymer on Earth. [1-2] It is mainly distributed in the exoskeletons of crustaceans (such as shrimp, crabs, and lobsters) and insects (such as ants and beetles), as well as in the cell walls of fungi (such as yeasts and mushrooms). [3-4] The total amount of chitin produced annually in some natural ecosystems is approximately 10. 10 Up to 10 12 With the rapid development of shrimp and crayfish farming in China, the output of chitin waste has continued to rise. However, chitin is relatively stable and insoluble in water, dilute acids, dilute alkalis, and most organic solvents, thus hindering resource recycling and utilization. [4-6] .

[0003] The degradation products of chitin include some water-soluble substances, such as chitosan oligosaccharides, chitin oligosaccharides, and glucosamine (GlcN) monomers. [7-9] These degradation products show promising development prospects in fields such as medicine, food, industry, and agriculture. [8] For example, chitosan oligosaccharides have antibacterial, antimicrobial, food preservation, plant growth regulation, antioxidant, and antitumor effects. [9] For example, chitosan oligosaccharides possess a variety of biological activities, including but not limited to antibacterial, antifungal, antitumor, anti-inflammatory, and immune-enhancing activities. [5] Therefore, degrading chitin can not only alleviate environmental pressure and improve resource utilization, but also yield derivatives with wide-ranging applications.

[0004] Traditional methods for chitin degradation include chemical, physical, and biological methods. Chemical methods include acid-base methods, EDTA methods, ionic liquid methods (IL methods), and DES methods. However, acid-base and EDTA methods require strong acid or alkali solutions, which can easily pollute the environment and increase wastewater treatment costs. IL and DES methods are less toxic to the environment than the former two, but their costs are very high, making them unsuitable for large-scale industrial use. [8,10-11]Physical methods, including photodegradation, microwave degradation, and ultrasonic degradation, produce less pollution compared to chemical methods, but their yields are also low and the products are non-uniform. Therefore, they are typically used only as supplementary methods. [5,8,10] Compared to chemical methods, biological methods are more environmentally friendly, operate under milder conditions, and can reduce subsequent wastewater treatment costs. Biological methods include microbial fermentation and enzymatic hydrolysis. Although microbial fermentation has a simple process and is easy to operate, it suffers from low efficiency due to issues such as long growth times and unstable growth states of microbial strains. Therefore, enzymatic hydrolysis has become a superior solution.

[10] .

[0005] The enzymes used in the enzymatic degradation of chitin include chitinase, chitosanase, chitin deacetylase, polysaccharide monooxygenase, and N-acetylaminohexosidase. [5,11] Chitinase has many functions, including degrading chitin, antifungal activity, pest and disease control, preparation of fungal protoplasts, and food preservation. [7,12-13] For example, chitinase Chit33 can inhibit the growth of Rhizoctonia solani, and chitinase Chit36 has an inhibitory effect on Fusarium oxysporum and Sclerotium rolfsii.

[14] For example, chitinase produced by the *Serratia marcescens* strain can kill female parasitic mites of the Western honeybee, and chitinase AO-492 has a strong degrading effect on *Caenorhabditis elegans* and its eggs. [13-15] .

[0006] The application prospects of chitinase are very promising. First, chitinase can increase the degradation rate of shrimp and crab shells, which helps reduce the accumulation of these wastes in the environment. [16-17] Secondly, the derivatives produced by chitinase from the degradation of chitin, such as chitosan oligosaccharides and chitosan oligosaccharides, have antibacterial, antitumor, and immune-regulating biological activities, and can be applied in the fields of medicine and food. [7] Third, chitinase itself has shown great development potential in fields such as medicine, agriculture, industry, and environmental protection.

[18] However, existing chitinases have relatively low activity, poor thermal stability, and most are acidic, making it difficult to meet practical application requirements and limiting large-scale production and industrial application.

[0007] This invention relates to a broad-spectrum acid-base tolerant chitinase. Although the optimal pH of this enzyme is 6.0, it retains over 80% of its catalytic activity after treatment with 50mM buffer at 30°C and pH 3.0-11.0 for 24 h. Compared with other existing chitinases, this tolerance and stability over a wider pH range allows it to adapt to more complex catalytic environments and has special value in the study of catalytic mechanisms. Furthermore, after treatment at 60°C for 20 min, the enzyme retains 62.04% of its residual enzyme activity. Compared with other existing chitinases, it exhibits stronger thermostability and can meet the needs of more industrial applications.

[0008] References

[0009] [1] Zhang Jie, Xie Chen, Guo Xiaohong, et al. Cloning, expression and activity of thermostable chitinases from Thermoascus aurantiacus var. aurantiacus [J]. Acta Mycologica Sinica, 2010, 29(5):691-697.

[0010] [2]Micocci KC, Moreira AC, Sanchez AD, et al. Identification, cloning, and characterization of a novel chitinase from leaf-cutting ant Attasexdens: An enzyme with antifungal and insecticidal activity[J]. Biochimicaet Biophysica Acta (BBA)-General Subjects, 2023, 1867(1): 130249.

[0011] [3]Mahajan G, Sharma N, Kaur M, et al. Chitinases: Key players inplant defense mechanisms against fungal pathogens[J]. Physiological andMolecular Plant Pathology, 2025, 138: 102664.

[0012] [4]Deng JJ, Shi D, Mao HH, et al. Heterologous expression and characterization of an antifungal chitinase (Chit46) from Trichodermaharzianum GIM 3.442 and its application in colloidal chitin conversion[J]. International Journal of Biological Macromolecules, 2019, 134: 113-121.

[0013] [5] Yuan Yuan. Study on the efficient expression and chitin degradation of chitinases from different sources in Bacillus subtilis [D]. Jiangnan University, 2023.

[0014] [6]Itoh T. Structures and functions of carbohydrate-active enzymes ofchitinolytic bacteria Paenibacillus sp. str. FPU-7[J]. Bioscience,Biotechnology, and Biochemistry, 2021, 85(6): 1314-1323.

[0015] [7] Liu, Lirui; Pan, Jie; Li, Meng. Research progress, application and prospect of microbial chitinases [J]. Bioresources, 2020, 42(5): 494-504.

[0016] [8] Lu Chunlan, Wang Bei. Preparation methods of chitosan oligosaccharide and chitin oligosaccharide and their application in aquaculture [J]. Guangdong Agricultural Sciences, 2023, 50(2): 136-146.

[0017] [9] Xue Pinghong, Deng Yunhong, Tian Xueqin, et al. Research progress on the preparation of chitosan oligosaccharides by biological method [J]. Acta Microbiologica Sinica, 2025, 65(2): 453-466.

[0018]

[10] Yin Kaibo, Zheng Zilu, Jin Jiayue, et al. Progress in the preparation and application of chitin from shrimp and crab shell waste [J]. Food Industry Technology, 2024, (20): 407-414.

[0019]

[11] Zhang Alei, Wei Guoguang, Zhang Chi, et al. Research progress on biodegradation and high-value transformation of chitin resources [J]. Synthetic Biology, 2024, 5(6): 1279-1299.

[0020]

[12] Lü Mengyuan, Shi Jiaxian, Xia Xiang, et al. Enzymatic properties of chitinase expressed in Escherichia coli on different substrates and analysis of its degradation products [J]. Food and Fermentation Industries, 2015, (3): 26-32.

[0021]

[13] Ma Saimai, Li Tongyuan, Ma Yanjun, et al. Research progress of chitinase in biological control of crop diseases and pests [J]. Biotechnology Bulletin, 2023, 39(10): 29-40.

[0022]

[14] Gajera H, Domadiya R, Patel S, et al. Molecular mechanism of Trichoderma as bio-control agents against phytopathogen system–a review[J]. Current Research in Microbiology and Biotechnology, 2013, 1(4): 133-142.

[0023]

[15] Zhang Jiahua, Zhang Huimei, Ma Xixi, et al. Study on the degradation effect of chitinase AO-492 of Oligosporium argentis on nematodes [J]. Biotechnology Bulletin, 2024, 40(5): 261-268.

[0024]

[16] Mi Yanxia, ​​Ren Hui, Zhang Chang, et al. Research progress on chitinase [J]. Life Science Research, 2015, 19(5): 437-443.

[0025]

[17] Sharma S, Kaur R, et al. A review on valorization of chitinouswaste[J]. Journal of Polymer Research, 2021, 28(11): 1-20.

[0026]

[18] Wang Lin, Chen Yaru, Cheng Meijie, et al. Research progress and application of microbial chitinases [J]. China Biotechnology Journal, 2022, 42(12): 101-110. Summary of the Invention

[0027] The purpose of this invention is to provide a gene encoding a chitinase with broad-spectrum acid-base tolerance.

[0028] Metagenomic DNA was extracted from soil, degenerate primers were designed and synthesized, and the gene was amplified by PCR. The amplified DNA was cloned into pPICZ-αA, and a recombinant plasmid pPICZ-αA-Tam-chit1 containing the chitinase gene Tam-chit1 was obtained by sequencing. After transforming Pichia pastoris with this plasmid, activity verification confirmed that the Tam-chit1 gene can encode a chitinase with broad-spectrum acid-base tolerance and thermostability.

[0029] The specific research plan is as follows:

[0030] 1) Metagenomic DNA was extracted from the soil and amplified by PCR using degenerate primers designed based on the chitinase gene. DNA fragments of about 1200 bp in length were obtained by gel recovery.

[0031] 2) Using the BBI plasmid extraction kit, the pPICZ-α-A plasmid was extracted and double-digested with EcoRI and KpnI (digestion system: 60 μL deionized water, 16 μL buffer, 80 μL plasmid, 3 μL EcoRI, 3 μL KpnI) at 37℃ for 12 h. The digestion products were subjected to agarose gel electrophoresis and purified using a gel extraction kit. The purified plasmid vector was obtained from the gel extraction.

[0032] 3) The target gene obtained in 1) and the plasmid vector obtained in 2) were mixed at a molar ratio of 3:1. 5 μL of Containerless Cloning Mix was added, and the mixture was incubated in a 50°C water bath for 20 min. The ligated target gene and plasmid vector were then electroporated into *E. coli* strain DB3.1. After recovery at 37°C for 1.5 h, the electroporated bacterial culture was plated onto LLB solid medium containing Zeocin. After incubation at 37°C for 12 h, colony PCR was performed to verify the colonies on the plates. Colonies with correct electrophoretic bands were inoculated into LLB liquid medium containing Zeocin to obtain the recombinant plasmid pPICZ-α-A-Tam-chit1 (e.g., ...). Figure 1 (as shown)

[0033] 4) Extract plasmids using the BBI plasmid extraction kit, digest them with EcoRI and KpnI, verify the digestion products and the original plasmids by agarose gel electrophoresis, and then verify them by DNA sequencing.

[0034] 5) The recombinant plasmid that was verified above was digested with SacI and then electroporated into Pichia pastoris. After successful electroporation, it was sequenced again for verification. The correct DNA sequence is shown in SEQUENCE LISTING.

[0035] 6) The above-mentioned recombinant Pichia pastoris strain was induced and expressed. Using colloidal chitin as a substrate, its enzyme activity was measured using the DNS method, and its enzymatic properties were characterized. Tam-chit1 was found to have high thermal stability and good pH stability within the pH range of 3.0–11.0. Specific analytical methods are detailed in the examples.

[0036] Compared with other chitinases, the broad-spectrum acid-base tolerant chitinase described in this invention has the following outstanding advantages:

[0037] 1) The chitinase described in this invention exhibits enhanced thermostability with broad-spectrum acid and alkali tolerance, retaining 62.04% of its enzyme activity after treatment at 60°C for 20 min. Therefore, this enzyme has promising application prospects.

[0038] 2) The chitinase with broad-spectrum acid and alkali tolerance described in this invention has an optimal pH of 6.0, but after treatment in an environment with pH 3.0 to 11.0 for 24 hours, it can retain more than 80% of its residual activity. This acid and alkali tolerance also makes this enzyme particularly valuable in the study of catalytic mechanisms. Attached Figure Description

[0039] Figure 1. Physical map of the plasmid pPICZ-α-A-Tam-chit1 constructed in this invention. Tam-chit1 is the encoding gene for a chitinase with broad-spectrum acid-base tolerance.

[0040] Figure 2. Detection of the optimal pH for the Tam-chit1 gene-encoded protein obtained in this invention. The average of three replicates for each reaction was taken, and the percentage ratio of sample activity to the highest activity was used as the relative activity.

[0041] Figure 3 The optimal temperature for detecting the Tam-chit1 gene-encoded protein obtained in this invention was determined. The average of three replicates for each reaction was taken, and the percentage ratio of sample activity to the highest activity was used as the relative activity.

[0042] Figure 4 The temperature stability of the Tam-chit1 gene-encoded protein obtained in this invention was determined. The average of three replicates for each reaction was taken, and the percentage of sample activity to the untreated group was used as the relative activity.

[0043] Figure 5The pH stability of the Tam-chit1 gene-encoded protein obtained in this invention was determined. Each reaction was calculated as the average of three replicates, and the percentage of sample activity relative to the untreated group was used as the relative activity.

[0044] Figure 6 The effects of high and low concentrations of metal ions on the chitinase encoded by the Tam-chit1 gene obtained in this invention were investigated. The average value of three replicates for each reaction was taken, and the percentage of sample activity compared to the untreated group was used as the relative activity.

[0045] Figure 7 This invention investigates the effects of high and low concentrations of organic matter on the chitinase encoded by the Tam-chit1 gene. The average value of three replicates for each reaction was used, and the percentage of activity of the sample compared to the untreated group represents the relative activity.

[0046] Figure 8 HPLC chromatograms of the hydrolysis products of the Tam-chit1 gene-encoded protein obtained in this invention after reaction with colloidal chitin. a is the HPLC result of the Tam-chit1 enzymatic hydrolysis product; b is the HPLC result of the blank control enzymatic hydrolysis product.

[0047] Table 1. Kinetic parameters of the Tam-chit1 gene-encoded protein obtained in this invention. The average value of three replicates for each reaction is taken, and the percentage ratio of sample activity to the untreated group represents the relative activity. Detailed Implementation

[0048] The present invention relates to a chitinase with broad-spectrum acid and alkali tolerance. This chitinase has good thermal stability, and although its optimal pH is 6.0, it still retains more than 80% of its catalytic activity after treatment with 50 mM buffer solution at pH 3.0 to 11.0 for 24 h. Compared with other existing chitinases, this tolerance and stability in a wider pH range enables it to adapt to more complex catalytic environments and has special value in the study of catalytic mechanisms.

[0049] Example 1: Induction and determination of chitinase activity

[0050] Pick colonies and place them in 20 mL of BMGY culture medium (shake flask volume ≥ 100 mL), or inoculate the seed culture into 20 mL of BMGY culture medium (shake flask volume ≥ 100 mL) at an inoculation rate of 1%–2%. Incubate for 16–20 h (shake flask conditions: 30℃, 200–250 rpm), centrifuge (5000 rpm, 5 min), discard the supernatant, and transfer the bacterial pellet to 40 mL of BMMY culture medium (shake flask volume ≥ 200 mL). Incubate for several days (shake flask conditions: 30℃, 200–250 rpm). Add 0.5% methanol every 24 h until the optimal induction period ends, centrifuge (5000 rpm, 5 min), and the supernatant is the fermentation broth / crude enzyme solution.

[0051] Mix 0.2 mL of colloidal chitin (w:v 0.2%), 0.3 mL of buffer (50 mM pH 6.0 citrate-sodium citrate buffer), and 0.1 mL of crude enzyme solution in a 2 mL EP tube to obtain the reaction solution. Place the reaction solution in a 60 °C water bath for 2 h, then boil at 100 °C for 10 min to inactivate the enzyme. Centrifuge (10000 rpm, 5 min, ACC=1), collect the supernatant, add 0.4 mL of DNS, mix well, and boil for 10 min. Use a pipette to transfer 0.2 mL of the boiled liquid into a 96-well plate, and measure the OD value at 540 nm using a microplate reader.

[0052] Under the above reaction conditions, the amount of enzyme protein required to produce 1 μg of N-acetylglucosamine reducing sugar per minute is defined as one enzyme activity unit.

[0053] Example 2: Determination of optimal pH

[0054] The prepared pH buffer solutions (50mM) are as follows: citric acid-sodium citrate pH 3.0~6.0, Tris-HCl pH 7.0~8.0, and glycine-sodium hydroxide pH 9.0~11.0.

[0055] After adding the expressed enzyme to the reaction system, absorbance values ​​were obtained using the method in Example 1 at 60°C and different pH values ​​(pH 3.0~11.0), and the relative enzyme activity was measured. The optimal pH for Tam-chit1 was 6.0 (citric acid-sodium citrate buffer, 50 mM). Figure 2 (as shown)

[0056] Example 3: Determination of the optimal temperature

[0057] After adding the expressed enzyme to the reaction system, absorbance values ​​were obtained using the method in Example 1 under different temperatures (30~80℃) in 50mM pH 6.0 citrate-sodium citrate buffer. The relative enzyme activity was then measured. The optimal temperature for Tam-chit1 was 60℃ (e.g., ...). Figure 3 (as shown)

[0058] Example 4: Thermal stability determination

[0059] Take 1 mL of enzyme solution and incubate it in a 60℃ water bath for 20 min. Add 0.1 mL of the treated enzyme solution to the reaction system. After obtaining the absorbance value using the method in Example 1, and taking the untreated group as 100%, measure the remaining enzyme activity. The thermal stability of Tam-chit1 is 62.04% (e.g., ...). Figure 4 (As shown).

[0060] Example 5: pH stability determination

[0061] 1 mL of pH buffer (50 mM) was mixed with 1 mL of crude enzyme solution and incubated at 30 °C for 24 h. 0.1 mL of the treated enzyme solution was then added to the reaction system. The absorbance was obtained using the method in Example 1, and the remaining enzyme activity was measured with the untreated group as 100%. Tam-chit1 exhibited good pH stability (e.g., residual enzyme activity) from pH 3.0 to 11.0. Figure 5 (As shown).

[0062] Example 6: Effects of metal ions on chitinase

[0063] Add 0.6 μL and 6 μL of 1M metal ion solution to the reaction system, respectively, to make final concentrations of 1 mM and 10 mM. After obtaining absorbance values ​​using the method in Example 1, with the untreated group (no metal ions added) as 100%, measure the remaining enzyme activity (e.g., ...). Figure 6 (As shown). Under low concentrations of metal ions (1 mM), Co 2+ Mn 2+ Fe 3+ It has a certain promoting effect on this enzyme, while the promoting or inhibiting effects of other metal ions are not obvious. At high concentrations of metal ions (10 mM), Co... 2+ Mn 2+ Fe 2+ Fe 3+ It has a promoting effect on Tam-chit1, while the promoting or inhibiting effects of other metal ions are not obvious.

[0064] Example 7: Effects of organic matter on chitinase

[0065] The following organic compounds were added to the reaction system:

[0066] Low-concentration organic matter group: 1% methanol, 1% ethanol, 1% acetone, 0.1% SDS, 1mM urea, 1mM β-mercaptoethanol, 1mM EDTA, 1mM vitamin C; High-concentration organic matter group: 10% methanol, 10% ethanol, 10% acetone, 1% SDS, 10mM urea, 10mM β-mercaptoethanol, 10mM EDTA, 10mM vitamin C. After obtaining absorbance values ​​using the method in Example 1, the untreated group (without added organic matter) was used as 100%, and the remaining enzyme activity was measured (results are shown in Figure 1). Figure 7 (As shown).

[0067] In low concentrations of organic matter, acetone and β-mercaptoethanol have a certain promoting effect on Tam-chit1, while SDS and vitamin C have an inhibitory effect on Tam-chit1. For high concentrations of organic matter, most organic matter has a significant inhibitory effect on Tam-chit1, while β-mercaptoethanol has no significant effect on Tam-chit1.

[0068] Example 8: HPLC Product Analysis

[0069] The product generated from colloidal chitin catalyzed by Tam-chit1 was subjected to ultrafiltration and filtration, and then analyzed by HPLC for component composition (results are shown below). Figure 8 (As shown). HPLC conditions were: Agilent 1260, 4.6 mm x 250 mm amino liquid chromatography column, mobile phase V(acetonitrile):V(water) = 7:3, ELSD detection (drift tube temperature 50℃, nebulizer gas flow rate 1 mL / min), flow rate 0.5 mL / min, column temperature 30℃, injection volume 10 μL.

[0070] Example 9: Dynamic Parameter Analysis:

[0071] Protein purification was performed using Ni TED Agarose Beads 6FF from Jiangsu Qianzhusong Biotechnology Co., Ltd., and the kinetic parameters of the purified enzyme were calculated.

[0072] The reaction system consisted of 50 μL purified enzyme + 200 μL colloidal chitin of different concentrations + 350 μL buffer (50 mM pH 6.0 citrate-sodium citrate buffer). The reaction time was 1 h, and the reaction conditions were 60℃ and 50 mM pH 6.0 citrate-sodium citrate buffer. The results are shown in Table 1. Tam-chit1 4.669 26.347 130.145

Claims

1. A gene encoding a chitinase with broad-spectrum acid-base tolerance, characterized in that: It can be efficiently expressed in Pichia pastoris, and the expressed protein can catalyze the production of N-acetylglucosamine and a small amount of chitobiose from chitin.

2. The gene encoding a broad-spectrum acid-base tolerant chitinase as described in claim 1, characterized in that: The optimal pH is 6.0 and the optimal temperature is 60℃. After treatment at 60℃ for 20 min, the remaining enzyme activity is 62.04%. After treatment in 50 mM pH 3.0-11.0 buffer for 24 h, the remaining enzyme activity is still higher than 80%.

3. The gene encoding the chitinase with broad-spectrum acid-base tolerance as described in claim 1, characterized in that: It has the DNA sequence shown in the sequence listing in this article.

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