A chitinase-producing Penicillium oxalicum H13 and its application

Abstract

The present invention discloses a chitinase-producing Penicillium oxalicum H13 and its application, belonging to the field of enzyme engineering technology. The strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on April 19, 2024, with a preservation address of 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, and a preservation number of GDMCC NO: 64525. The present invention provides a chitinase-producing Penicillium oxalicum H13, which can produce chitinase. The obtained enzyme has high activity and can directly degrade suspended chitin powder without pre-swelling the chitosan with dilute acid. The chitosan in the environment can be degraded into chitosan oligosaccharides of various chain lengths, so that the production process of substances such as amino sugars and oligosaccharides avoids the use of acid solution. The Penicillium oxalicum H13 of the present invention also has a good inhibitory effect on Escherichia coli, Staphylococcus aureus, and Vibrio parahaemolyticus.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, in particular to a chitinase-producing Penicillium oxalicum H13 and application thereof. Background Art

[0002] Shrimp and crab shells contain a large amount of chitosan (about 35%, w / w). my country is the world's largest chitosan processing country, and its products include chitosan, glucosamine, chitosan oligosaccharides, etc. In theory, there are acid hydrolysis, enzymatic hydrolysis, microwave method, ultrasonic method, etc. for chitosan degradation, but acid hydrolysis is the most widely used. Concentrated hydrochloric acid is generally used for hydrolysis, and a large amount of concentrated hydrochloric acid is consumed in the process. Taking the preparation of glucosamine as an example, for every ton of glucosamine, about 8.5 tons of hydrochloric acid with a concentration of 30% is consumed. The processing wastewater is super acidic (pH <0.5), like soy sauce, and has a very high chloride ion concentration (about 5%), which brings great environmental pressure and governance costs to enterprises, and restricts the development of the industry.

[0003] Compared with the acid hydrolysis method, the enzymatic hydrolysis method has the advantages of mild reaction conditions, easy control of the degradation process and the relative molecular weight distribution of the degradation products, no need for desalination of the products, easy control of the process, and no pollution to the environment. It is an ideal degradation method. Researchers have been screening various enzyme preparations and trying to use enzymatic hydrolysis instead of acid hydrolysis. The enzymes currently available include ChiA, PbChi67, BpNagZ, ChiC, Chi1, SaHEX and MrChi3B, but these enzymes have a common defect that they can only degrade chitin swollen by dilute acid and cannot completely avoid the use of acid. In order to solve these problems, researchers have been screening efficient chitin-degrading enzymes in various microorganisms.

[0004] Mangroves grow on saline soil flooded by coastal tides, forming a unique ecological feature. They are rich in fungal resources, accounting for about one-sixth of the total marine fungi, and their exoenzymes show unique biochemical characteristics. Fungal exoenzymes are protein compounds secreted by fungi into the surrounding environment during growth. These compounds decompose substances in the environment into small molecules for absorption by fungal cells. Depending on the different substances available in the environment, fungi can secrete different types of enzymes, including proteases, pectinases, cellulases, hemicellulases, lipases, amylases, chitinases, etc. For example, if there are available protein resources in the environment, proteases will be secreted. However, there are no reports on the use of fungi in mangrove environments for the production of chitin. Summary of the invention

[0005] The purpose of the present invention is to provide a chitinase-producing Penicillium oxalicum H13 and its application, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a chitinase-producing Penicillium oxalicum H13, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 19, 2024, with a preservation address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and a preservation number of GDMCC NO: 64525.

[0008] The invention separates a chitinase high-yield strain from mangrove wetlands, and its exoenzyme can directly degrade wet shrimp and crab shells. Compared with the existing enzymes, it has very important cutting-edge and unique features.

[0009] The present invention also provides a microbial agent, comprising the Penicillium oxalicum H13 and / or its metabolites.

[0010] The invention also provides a chitinase produced by metabolism of the Penicillium oxalicum H13.

[0011] Preferably, the amino acid sequence is as shown in SEQ ID NO.1.

[0012] The production and separation and purification of fungal exoenzymes mainly involve three steps: fermentation, extraction and purification. Fermentation methods are mainly divided into two types: liquid fermentation and solid fermentation. Liquid fermentation is carried out in a fermentation tank, and the temperature, pH and oxygen supply can generally be precisely controlled. It is widely used in large-scale industrial production; solid fermentation can be carried out in a bed or vertical column fermenter. The fungus is inoculated into a solid substrate containing a certain amount of water, and the fermentation of the substrate is completed in a relatively open environment. Both methods are widely used in fungal production, and the specific choice depends on the specific requirements of production and the nature of the fungus.

[0013] After the fermentation is completed, the fermentation liquid (or eluent) is centrifuged or filtered to separate the bacteria and the culture liquid. Exocrine enzymes are mainly present in the culture liquid, and their crude extracts can be prepared by salting out, organic solvent precipitation, affinity adsorption and other processes, and then refined by ultrafiltration, column chromatography and other technologies to obtain high-purity and high-activity enzyme products.

[0014] The invention uses Penicillium oxalicum H13 as a raw material, induces it to produce chitinase through a solid static fermentation method, and prepares a high-purity chitinase product through salting out and ultrafiltration technology.

[0015] The present invention also provides the use of the Penicillium oxalicum H13, the microbial agent or the chitinase in preparing a product for decomposing chitin.

[0016] The present invention also provides a product for decomposing chitin, comprising the Penicillium oxalicum H13, the microbial agent or the chitinase.

[0017] The present invention also provides a method for decomposing chitin, which uses the Penicillium oxalicum H13, the microbial agent, the chitinase or the product to treat chitin or a substance containing chitin.

[0018] The invention aims at the problem of using a large amount of concentrated hydrochloric acid in the process of industrial acid hydrolysis of chitosan, produces chitinase through fungal solid fermentation, and lays a foundation for the establishment of an enzymatic chitosan hydrolysis process.

[0019] The present invention also provides the use of the Penicillium oxalicum H13, the microbial agent or the chitinase in preparing a product for inhibiting bacterial growth.

[0020] Preferably, the bacteria include Escherichia coli, Staphylococcus aureus and Vibrio parahaemolyticus.

[0021] The present invention also provides an antibacterial agent, comprising the Penicillium oxalicum H13, the microbial agent or the chitinase.

[0022] Based on the above technical solution, the present invention has the following technical effects:

[0023] The invention provides a chitinase-producing Penicillium oxalicum H13, which can produce chitinase, and the obtained enzyme has high activity, and can directly degrade suspended chitin powder without pre-swelling the chitin with dilute acid. The chitin in the environment can be degraded into chitosan oligosaccharides of various chain lengths, so that the production process of substances such as amino sugars and oligosaccharides avoids the use of acid solution, which can not only reduce the processing cost, but also has mild reaction conditions and is environmentally friendly, and has great application value and practical needs. Solid static fermentation requires less raw materials and energy consumption, and is suitable for industrial production. The Penicillium oxalicum H13 of the invention also has a good inhibitory effect on Escherichia coli, Staphylococcus aureus, and Vibrio parahaemolyticus. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 Electrophoresis analysis of proteins at each stage of chitinase purification process;

[0026] Figure 2 To verify the activity of purified chitinase by hydrolysis circle method;

[0027] Figure 3The product of chitin hydrolysis by purified chitinase was analyzed by liquid chromatography-mass spectrometry;

[0028] Figure 4 The inhibitory effect of purified exogenous chitinase from Penicillium oxalicum on Staphylococcus aureus (left), Escherichia coli (middle) and Vibrio parahaemolyticus (right). DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0035] Example 1

[0036] Isolation and Purification of Penicillium oxalicum H13

[0037] Soil samples were collected from three sites (nearshore, intermediate, and nearland) in the mangroves of Leizhou Peninsula (21°68'N, 110°33'E) before the tide rose. The sampling depth was 0-10 cm below the surface sediment. The sampling was performed by hand to remove large debris using a sterilized hand shovel (approximately 4 cm × 6 cm with an 18 cm long handle). Samples from all sites were mixed well and then transferred to sterilized 500 ml sealed glass jars and transported to the laboratory in ice boxes.

[0038] Within 30 minutes after collection, suspend the collected soil in 4-9 liters of sterile seawater at 100-300 grams, homogenize and let stand. Take the supernatant and centrifuge at 200-500g, and dilute the centrifuged supernatant in a gradient (10-fold interval dilution). Transfer 50-100 μl of the bacterial solution to yeast extract peptone dextrose agar (YPD) prepared with sterile seawater, to which ampicillin, kanamycin and streptomycin were added (final concentrations were 10-50 μg / mL, 10-50 μg / mL and 5-25 μg / mL, respectively). Use a sterile spreading stick to apply a small amount of the bacterial solution to the culture medium, and then incubate at 25-35°C.

[0039] The morphological characteristics of a single colony were observed and combined with ITS characteristic sequence detection, it was determined to be Penicilliumoxalicum. The bacterial lawn was cut from the culture medium (each piece was about 4 mm×4 mm), immersed in 35% glycerol solution (v / v), and stored at -80°C for a long time.

[0040] The oxalic acid Penicillium H13 provided by the present invention was preserved in the Guangdong Provincial Microbiological Culture Collection Center on April 19, 2024, with the preservation address being 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC NO: 64525.

[0041] Example 2

[0042] (1) Fungal pre-culture

[0043] One month before use, Penicillium oxalicum H13 was inoculated in Sabouraud agar medium (SDA) containing 4% sodium chloride, cultured at 35-45°C for 5-9 days, and transferred to a new SDA medium. Each time the medium was transferred, the weight of glucose in the medium was reduced by 30%, and the same weight of chitin was added. The transfer was repeated three times. Finally, a sterilized 0.05% Tween solution was used to collect spores, and the concentration of the spore solution was calculated using a hemocytometer.

[0044] (2) Chitin pretreatment

[0045] The chitosan is crushed into particles with a diameter of 200-350 μm, placed in a high-temperature oven, and sterilized at a temperature of 160° C.-180° C. for 20-40 minutes.

[0046] (3) Preparation of culture medium

[0047] Dissolve protein powder, glucose and sodium chloride in deionized water and sterilize at 121°C for 15-20 minutes. Then spray the solution on chitin under sterile conditions, preferably without visible water flow, until the moisture content in chitin is between 18% and 30%. The amount of protein powder added is about 1 / 7-1 / 4 of the dry weight of chitin, glucose is about 1 / 16-1 / 10 of the dry weight of chitin, and sodium chloride is about 1 / 9-1 / 5 of the dry weight of chitin.

[0048] (4) Sterilization and filling of vertical column fermenters

[0049] The column fermenter was sterilized by steam, and the moist chitosan was evenly and slowly filled into the fermenter through a 35-mesh vibrating screen. Fungal spores were sprayed into the filler every 8-20 minutes. The dosage was about 1.0×10 9 -1.2×10 10 / cubic meter, fill to 2 / 3 of the column height.

[0050] (5) Fermentation

[0051] Start the fermentation program, control the temperature within the range of 40±5℃, and let the air pass through a filter valve with a pore size of 0.22μm before entering the fermenter. The ventilation flow rate is 230-450L / min. In the first 9 days, the moisture content in the fermenter is controlled between 18% and 30%. After that, sterile water is injected into the tank at a rate of 2%-6% every day. The longest fermentation time is 21 days.

[0052] (6) Collection of fermentation products

[0053] The fermenter was opened and the outflowing liquid was collected through the collecting pipe. Then 1.5-3 times the volume of phosphate buffer (0.1-0.2M, pH 6.8-7.4) was injected into the three-time tank, the contents of the tube were fully stirred, and the outflowing liquid was collected through the collecting pipes respectively, all the outflowing liquids were combined, centrifuged at room temperature (9000-14000g) by a disc centrifuge, and the supernatant was collected.

[0054] (7) Ultrafiltration concentration

[0055] The supernatant obtained in step (6) is first separated by a polyamide membrane or a polyetherketone membrane with a molecular cutoff of 300-400 kDa, and the filtrate is collected, and then separated by a polyethersulfone membrane with a molecular cutoff of 3-10 kDa, and the retentate is collected.

[0056] (8) Salting out

[0057] Add dried and crushed ammonium sulfate to the retentate obtained in step (7), stir rapidly in the same direction (150-200 rpm) while adding, stop adding when the ammonium sulfate saturation reaches 85%, continue stirring for 1-2 min. Let stand for 10-15 min, take the upper turbid liquid, centrifuge and dehydrate (>14000 g), collect the paste, and obtain the crude chitinase extract. It can be subsequently refined by conventional column chromatography.

[0058] (9) Isolation and purification of chitinase

[0059] The chitinase crude extract was redissolved with phosphate buffer (pH 7.2, 0.05 mol / L), and the redissolved enzyme solution was concentrated and desalted using a microporous filter membrane (molecular cutoff 300-900 Da). The concentrate was purified by Sephadex G-150 dextran gel chromatography, Sephadex G-75 dextran gel chromatography, and DEAE-32 anion exchange chromatography, and gradient elution (NaCl 0-1.0 mol / L) was performed using PBS buffer containing sodium chloride (NaCl) to collect components with chitinase activity. After ultrafiltration concentration (polyethersulfone ultrafiltration membrane, molecular cutoff 30000-90000 Da), the concentrate was vacuum freeze-dried to obtain chitinase dry powder.

[0060] Edman sequencing analysis showed that the amino acid sequence of the chitinase is shown in SEQ ID NO.1:

[0061] SEQ ID NO.1: Mvftktiaaiaalsslvvpsialnvdaktnvavywgqgpdqkrlrdfcaetdldiinlafvngfpa gvnaypgsnfgnqcdgtkypnsdllsgchqivndipvckamgktlmlsiggayttgqvtndaqarwfadflwysfgpvntavanqyprpfgdnsvdgfdfdiehggpvgwktmidrlrfrfqehadkkfyisaapqcttpdaqlgsaiansaldfiwvqfyntpgcsardfadgtghfnfdewaaiikaspnpnaklfiglpaapaaanqgyyleasevytlvrwymawypdifggvmlweatyaennknaagltyyeqvkeilkscapppvqtttiivtptpvhtsaaststpadttstvssavvssstpvvhtsstpvisststpvlshtstpagsspvgsssstpvvshtstpvgsgsstpvvsptstpvgsgsstpvvshtstpvgsgsstpvvshtstpvgsgsstpvvsptstpvgsgssspvvshtstpvgsgsstpvvshtstpagsipvgsgsstpagstpvvshtstpvgsgsstplvshtstpagsspagptstpvgsgsvtsssvpvvsgsstplipsqstttssvpagpsssdcddsstsvtgthtetslglsttssavsvsatsgthgtgsstssdvgaigtssattvpgatgsgpsgitstvnispsvttaptsttpaavttvivtsytdicptgfttitttitttvcpeatasvtgpagsvatttpsipegwtttvtvcthcaatpttvtltkpmstpavqttsvplapegtetvteawtttvsfckecgptgstvtvtvpytllatptgasagtaap。

[0062] Based on the amino acid sequence, bioinformatics analysis showed that the theoretical molecular weight of this enzyme was 84.4 kDa, which was close to the result of electrophoretic analysis ( Figure 1 ).

[0063] The purified chitinase was resuspended in sterile water and its activity was verified by the hydrolysis circle method. The results showed that the purified enzyme could degrade chitin in the plate and produce obvious hydrolysis circles ( Figure 2 ).

[0064] Chitinase purified product (0.1%, w / w) was added to the chitin suspension, incubated at 45°C for 2 hours, and the product was analyzed by liquid chromatography-mass spectrometry. The results showed that the product of chitin hydrolysis by the purified product was mainly glucosamine ( Figure 3 ).

[0065] Example 3

[0066] Process for preparing glucosamine and other chitosan oligosaccharides by degrading chitin suspension with chitinase from Penicillium oxalicum H13

[0067] The supernatant of the culture of Penicillium oxalicum H13, or its purified exotoxinase, is mixed with chitin powder in a ratio of 1000:15-1000:200 and 1000:160-1000:6000 (volume-to-mass ratio), and reacted at 30-80°C and pH 6-11 for 30-300 minutes, and at the same time, 0.2%-0.9% copper sulfate and 0.45%-1.1% ferric chloride (mass-to-volume ratio) are added to the reaction solution. After the reaction time is reached, centrifugation is performed at room temperature (9000-14000g), and the supernatant is collected.

[0068] The supernatant is first separated by a polyamide membrane or polyetherketone membrane with a molecular cutoff of 300-400 kDa, and the filtrate is collected. Then, it is separated by a polyethersulfone with a molecular cutoff of 3-10 kDa, and the retentate is collected. The operating temperature is controlled between 4-20°C. Trichloroacetic acid is added to the retentate, stirred evenly, allowed to stand for no more than 10 minutes, centrifuged (9000-14000g), and the supernatant is collected.

[0069] Add 2.5-4.5 times the volume of 95% ethanol solution to the supernatant, let stand at 4-10°C for 24-48 hours, centrifuge (6000-12000g), and collect the precipitate to obtain a mixture of aminoglucosamine and a small amount of other chitosan oligosaccharides.

[0070] Example 4

[0071] The Penicillium oxalicum chitinase was prepared into a solution with an activity of 2-21U / mL and added to a liquid containing 1.0×10 4 -1.0×10 9 Incubated in a suspension of 100 bacterial cells at 25-40°C, it has a good inhibitory effect on Escherichia coli, Staphylococcus aureus, and Vibrio parahaemolyticus ( Figure 4 ).

[0072] In summary, the present invention provides a chitinase-producing Penicillium oxalicum H13, which can produce chitinase, and the obtained enzyme has high activity, and can directly degrade suspended chitin powder without pre-swelling the chitin with dilute acid. The chitin in the environment can be degraded into chitosan oligosaccharides of various chain lengths, so that the production process of substances such as amino sugars and oligosaccharides avoids the use of acid solution, which can not only reduce processing costs, but also has mild reaction conditions and is environmentally friendly, and has great application value and practical needs. Solid static fermentation, required raw materials and energy consumption are small, and it is suitable for industrial production. The Penicillium oxalicum H13 of the present invention also has a good inhibitory effect on Escherichia coli, Staphylococcus aureus, and Vibrio parahaemolyticus.

[0073] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A chitinase-producing Penicillium oxalicum ( Penicillium oxalicum ) H13, characterized in that, The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 19, 2024. The collection address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the collection number is GDMCC NO: 64525.

2. A microbial agent, characterized in that: Including the Penicillium oxalicum described in claim 1.

3. A chitinase, characterized in that It is produced by the metabolism of Penicillium oxalicum described in claim 1, and its amino acid sequence is shown in SEQ ID NO.

1.

4. Use of the Penicillium oxalicum according to claim 1, the microbial agent according to claim 2 or the chitinase according to claim 3 in preparing a product that decomposes chitin.

5. A product for decomposing chitin, characterized in that: It comprises the Penicillium oxalicum described in claim 1, the microbial agent described in claim 2 or the chitinase described in claim 3.

6. A method for decomposing chitin, characterized in that: Chitosan or a substance containing chitosan is treated by using the Penicillium oxalicum described in claim 1, the microbial agent described in claim 2, the chitinase described in claim 3 or the product described in claim 5.

7. Use of the Penicillium oxalicum according to claim 1, the microbial agent according to claim 2 or the chitinase according to claim 3 in the preparation of a product for inhibiting bacterial growth, characterized in that: The bacteria include Escherichia coli, Staphylococcus aureus and Vibrio parahaemolyticus.

8. An antibacterial agent, characterized in that It comprises the Penicillium oxalicum described in claim 1, the microbial agent described in claim 2 or the chitinase described in claim 3.

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