CN116656548A - Bacillus marinus strain for producing chitinase and fermentation process and application thereof - Google Patents

Bacillus marinus strain for producing chitinase and fermentation process and application thereof Download PDF

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CN116656548A
CN116656548A CN202310589956.9A CN202310589956A CN116656548A CN 116656548 A CN116656548 A CN 116656548A CN 202310589956 A CN202310589956 A CN 202310589956A CN 116656548 A CN116656548 A CN 116656548A
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fermentation
chitinase
chitin
culture
gxmu
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申乃坤
谢俊杰
张红岩
姜明国
殷豆豆
刘睿
杨可欣
许霞
张重庆
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Guangxi University for Nationalities
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Guangxi University for Nationalities
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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    • A01N63/22—Bacillus
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2442—Chitinase (3.2.1.14)
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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    • C12P19/12—Disaccharides
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
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    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01014—Chitinase (3.2.1.14)
    • C—CHEMISTRY; METALLURGY
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    • C12R2001/07—Bacillus
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture

Abstract

The invention relates to the technical field of microbial fermentation engineering, and provides a bacillus marinus strain for producing chitinase, a fermentation process and application thereof. Bacillus marinus (Bacillus haynesii) GXMU-J23.1 was deposited at 10.14 of 2022 with the Guangdong province microbiological culture Collection center (GDMCC) under the accession number GDMCCNo.62891. The invention also relates to application of bacillus marinus in producing chitinase and degrading chitin. The strain fermentation process is simple to operate, improves the production efficiency, reduces the cost, provides possibility for high-density fermentation culture of fermentation engineering bacteria for producing chitinase, and has huge application potential.

Description

Bacillus marinus strain for producing chitinase and fermentation process and application thereof
Technical Field
The invention relates to the technical field of microbial fermentation engineering, in particular to a bacillus marinus strain for producing chitinase, and a fermentation process and application thereof.
Background
With the progress of the social science and technology in China, the industry is increasingly promoted to emphasize the exploration of new green, circulating and sustainable modes. In the nature and processing industry, however, there are waste of resources and lack of deep processing systems. In particular, in fishery production, shrimp and crab shells are usually directly buried or dumped into the ocean as waste, which not only costs to dispose of, but also can cause serious pollution to the ecological environment. The shrimp and crab shell contains chitin, protein, inorganic calcium and partial astaxanthin, and the components can provide raw materials for the production of foods, cosmetics, medical treatment, chemical industry and other industries. Therefore, development and utilization of beneficial resources in shrimp and crab shells, especially full play of the resource value of chitin as the second largest natural polysaccharide, has very important significance.
Chitin (chitin) is also called chitin, and is formed by connecting N-acetylglucosamine together through beta- (1, 4) glycosidic bond, and is the second large natural high molecular polysaccharide with the content inferior to that of cellulose in nature. Initially the presence of native chitin was found in fungal cell walls, and later further studies found that chitin was also widely distributed in the cell walls of lower plant algae, with the presence of large amounts of chitin as an important component in the shells of crustacean shrimp, crab and arthropods, such as the exoskeleton of most insects. However, further research has found that natural chitin exists in a stable crystalline form, is insoluble in common solvents, and the property of difficult dissolution and processing limits the commercial application value of the chitin. However, a large amount of chitin resources generated in the natural world and in the human production and cultivation process need to be further developed and utilized, so that more and more researchers focus on finding sustainable methods for transforming and utilizing natural chitin.
Traditional chitin treatment methods include chemical and physical: acid-base treatment, heat treatment and the like, and is characterized by high energy consumption, high pollution, low accuracy and difficult control. Due to the limitation of the traditional method, the development and utilization of chitin resources are limited. Compared with the traditional method, the microbial fermentation enzyme production technology can catalyze indissolvable chitin to degrade into micromolecule monosaccharide or oligosaccharide through enzymatic reaction, thereby providing convenience for further converting chitin resources into other biochemical components, greatly reducing the dependence on strong acid and strong alkali, enabling the reaction process to be milder, enabling the catalytic degradation process to be more accurate and controllable, and improving the catalytic conversion efficiency of chitin resources on the basis of environmental protection.
The modern biotechnology is mainly applied to biomass conversion methods including a direct fermentation method and a microbial enzyme method, and a microbial enzyme method is studied, chitin is converted and split by using enzymes generated by microorganisms to obtain enzymatic reaction products such as chitosan oligosaccharide, N-acetylglucosamine (amine) and the like, and the method has the outstanding advantages of environmental pollution, low toxicity and the like, but the method has the application limitation due to the need of adding procedures and cost for preparing an enzyme preparation. Compared with the enzyme method, the direct fermentation method directly carries out catalytic conversion on the fermentation substrate by the enzyme generated in the microbial fermentation process to generate the corresponding enzymolysis product, the process is simple and convenient to process, the process flow is less, the enzyme production and the catalysis are carried out simultaneously, the time and the cost are saved, and the fermentation enzyme liquid is convenient for further processing, refining and extracting the needed small molecular components or is used as an agricultural microbial inoculum in crop production.
Chitinase of different sources has different stereoselectivity, can stereospecifically split chitin to generate chitooligosaccharide or N-acetylglucosamine, and the microorganism strain for producing the chitinase mainly comprises the following components: chitin degrading enzyme systems have been found in the genus vibrio, alteromonas, bacillus, serratia marcescens, etc., and also in the genus proteus, firmicutes, actinomycetes, archaebacteria, fungi, viruses, animals and plants. These microorganisms provide a resource basis for the wide selection of high-yield chitinase strains, and provide a convenient operating tool for chitin bioconversion. The existence of the related enzyme system enables the bioconversion of chitin to be deeper and more comprehensive, and the realization of the high yield of the target enzyme is particularly critical through research and control of the fermentation process and conditions.
However, the existing microorganism strain for producing chitinase has the problems of low fermentation density and low chitinase expression amount, so that the chitinase catalytic efficiency is low.
Disclosure of Invention
The invention aims to provide a bacillus marinus strain for producing chitinase, a fermentation process and application thereof, which effectively improve the biological expression quantity of the strain for producing the chitinase and the enzyme activity of thalli.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a bacillus marinus (Bacillus haynesii) GXMU-J23.1 which is deposited with the Guangdong province microbiological bacterial collection center (GDMCC) on the 10 th month 14 of 2022, wherein the deposit number is GDMCC No.62891. Wherein the 16S rRNA sequence of the bacillus marinus (Bacillus haynesii) GXMU-J23.1 is shown in SEQ ID No: 1.
The invention provides application of bacillus marinus GXMU-J23.1 in preparing chitinase and degrading chitin.
Preferably, the chitinase is a chitobiose exonuclease.
The invention provides a fermentation process for preparing chitinase by utilizing bacillus marinus GXMU-J23.1, which comprises the following steps:
(1) Placing bacillus marinus in a seed culture medium for fermentation to obtain seed liquid;
(2) Inoculating the seed solution obtained in the step (1) into a fermentation medium for culture to obtain fermentation bacteria solution.
Preferably, the seed culture medium in step (1) uses water as a solvent, and comprises the following components in concentration: KH of 0.2-0.4 g/L 2 PO 4 K of 0.6-0.8 g/L 2 HPO 4 FeSO of 0.01-0.02 g/L 4 ·7H 2 O, 0.4-0.6 g/L MgSO 4 ·7H 2 2, 22-28 g/L of colloidal chitin with mass fraction of 1 percent and 1-3 g/L of yeast powder; the pH of the seed culture medium is 6-8.
Preferably, the Bacillus marinus of step (1) is inoculated at a concentration of 0.5X10 8 cfu/ml～1.5×10 8 cfu/ml, and the inoculation amount of the cfu/ml inoculated into the seed culture medium is 0.5-5.0% (v/v); the temperature of the culture after inoculation is 32-38 ℃; the culture time is 24-96 hours; the rotation speed of the shaking table for culture is 150-220 rpm.
Preferably, the seed solution in step (2) is inoculated into the fermentation medium in an inoculum size of 0.5 to 5% (v/v).
Preferably, the fermentation medium in the step (2) uses water as a solvent, and comprises the following components in concentration: KH of 0.1-0.5 g/L 2 PO 4 0.3 to 1.0g of K 2 HPO 4 FeSO of 0.01-0.05 g/L 4 ·7H 2 O, 0.2-0.8 g/L MgSO 4 ·7H 2 O, 1-5 g/L yeast powder, 5-50 g/L powder chitin; the pH of the fermentation medium is 6.5-7.5.
Preferably, the temperature of the culture in the step (2) is 28-45 ℃; the culture time is 1-5 d; the rotation speed of the shaking table for culture is 100-300 rpm.
The invention also provides application of the bacillus marinus GXMU-J23.1 in promoting plant growth and improving crop stress resistance.
The bacillus marinus (Bacillus haynesii) GXMU-J23.1 is separated from a high-salt environment of the crab paste, has a wider adaptive NaCl range, can produce extracellular chitinase, and has the characteristic of tolerating alkaline environment. Besides being convenient for separation and purification, the strain is an ideal material for chitinase gene cloning, and has potential theoretical and practical significance for constructing chitinase decomposition engineering strains and exploring chitinase action mechanisms.
According to the invention, the bacillus marinus GXMU-J23.1 with high chitinase yield is selected as an enzyme-producing strain, the components and the proportion of a fermentation culture medium are optimized, and the culture conditions are regulated and controlled, so that the nutrition conditions and the external environment required by the rapid growth and metabolism of the strain are ensured, the method has the advantages of larger thallus yield and high expression level of chitinase protein, simple process, improved production efficiency and reduced cost, and the method provides possibility for high-density fermentation culture of fermentation engineering bacteria with chitinase yield, and has great application potential.
Chitinase obtained by fermenting bacillus marinus GXMU-J23.1 has high enzyme activity, and can degrade chitin in fermentation broth into chitosan. The fermentation product is rich in chitinase, amino acid, chitosan oligosaccharide and the like, can be prepared into a microbial inoculum for agricultural application, can be used for extracting bioactive components and preparing chitinase, and has wide application prospect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is the size of the plate transparent circle of strain GXMU-J23.1 in example 1;
FIG. 2 is a plate purification morphology of strain GXMU-J23.1 of example 1;
FIG. 3 is a gram staining result of the strain GXMU-J23.1 in example 1;
FIG. 4 is a scanning electron micrograph of the strain GXMU-J23.1 of example 1 after staining;
FIG. 5 is a phylogenetic tree constructed based on the 16S rRNA sequence of the strain GXMU-J23.1 in example 1;
FIG. 6 shows the results of thin layer chromatography of chitin enzymatic hydrolysate produced by strain GXMU-J23.1 in Experimental example 1, wherein 1: a standard sample; 2:1% colloidal chitin sample; 3: adding enzyme into 1% of colloidal chitin sample to react for 0min;4: adding enzyme into 1% colloidal chitin sample for reaction for 10min. G1: n-acetylglucosamine; and G2: a chitosan; and G3: chitotriose; and G4: chittetrose; and G5: chitosan; g6: chitosan is used as a chitosan.
Preservation description
Bacillus marinus (Bacillus haynesii) GXMU-J23.1, which is deposited in the microorganism strain collection center of Guangdong province, and has an address of Guangzhou Md. 100 institute of first and second, building 5; the preservation time is 2022.10.14; deposit No. GDMCC NO:62891.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Screening and identifying bacillus marinus GXMU-J23.1 strain producing chitinase
In the embodiment, 4 strains of chitinolytic bacteria are obtained by carrying out enrichment culture and chitin screening flat plate separation and purification on a crab paste sample produced in North Guangxi sea, and a strain of bacteria GXMU-J23.1 with high chitinase secretion is obtained after liquid fermentation and is subjected to primary strain identification.
Inoculating strain GXMU-J23.1 onto chitin plate (formula of chitin plate is KH) 2 PO 4 0.3g/L，K 2 HPO 4 0.7g/L，FeSO 4 ·7H 2 O 0.01g/L，MgSO 4 ·7H 2 O0.5 g/L,1% colloidal chitin 25g/L, yeast powder 2g/LAgar powder 20g/L, pH 7.0), colony and transparent circle size was measured after culturing at 35℃for 72 hours, and the transparent circle size of strain GXMU-J23.1 was D/d=4.37, which was larger than that of the other 3 strains, as shown in FIG. 1.
Further screening to obtain seed solution with 4 strains of bacteria reaching 1.0X10 8 At cfu/ml, inoculating 2% (v/v) of the inoculum size into a fermentation medium, and fermenting at 35 ℃ for 3d at a shaking table rotation speed of 200rpm, wherein the shaking liquid filling amount is 30ml/100ml, so as to obtain a fermentation broth. Wherein the fermentation medium takes water as a solvent and comprises the following components in concentration: KH 0.3g/L 2 PO 4 0.7g of K 2 HPO 4 FeSO 0.01g/L 4 ·7H 2 O,0.5g/L MgSO 4 ·7H 2 O,2g/L yeast powder, 10g/L powdery chitin; the pH of the fermentation medium was 7.2. Centrifuging the fermentation broth at 12000rpm for 5min, and collecting the supernatant to obtain crude enzyme, wherein the enzyme activity of the strain GXMU-J23.1 crude enzyme is as follows: 139.87U/mL, higher than the enzyme activity of other 3 strains. GXMU-J23.1 was thus used as the subsequent experimental strain.
After the strain GXMU-Q5.3 flat plate is streaked on LB and cultured for 48 hours at 35 ℃, as shown in figure 2, the colony form is observed to be circular, the surface is waxy, the color is milky and opaque, the edge of the colony is irregular, the strain is dyed, as shown in figure 3, the obtained colony is small, gram-positive in dyeing and spore production; the scanning electron microscope results showed that the strain was rod-shaped (as shown in FIG. 4).
Further physiological and biochemical identification shows that the strain GXMU-J23.1 is positive in gelatin liquefaction, catalase, cellulase and amylase test, and negative in MR-test, indole test and oxidase test, and shows that the strain is bacillus.
TABLE 1 physiological and biochemical test results of Bacillus marinus GXMU-J23.1
The strain GXMU-J23.116S rRNA has the highest similarity with the Bacillus marinus (Bacillus haynesii) NRRLB4132716S rRNA, reaching 99% (as shown in FIG. 5).
Comprehensive morphological identification, physiological biochemical identification and 16S rRNA sequence analysis, the strain is identified as Bacillus marinus (Bacillus haynesii) GXMU-J23.1. The strain is preserved in Guangdong province microorganism strain collection (GDMCC), and the preservation number is as follows: GDMCC NO:62891, classified and named: bacillus haynesii.
Example 2
A fermentation process for preparing chitinase by utilizing bacillus marinus GXMU-J23.1 comprises the following steps:
the Bacillus marinus GXMU-J23.1 obtained in example 1 was concentrated at a concentration of 1.0X10 8 Inoculating cfu/ml and 1% (v/v) of inoculum size to a seed culture medium, and activating at a shaking table rotation speed of 200rpm for 1d at 35 ℃ to obtain seed liquid for later use;
the seed culture medium takes water as a solvent and comprises the following components in concentration: KH 0.3g/L 2 PO 4 K at 0.7g/L 2 HPO 4 FeSO 0.01g/L 4 ·7H 2 O,0.5g/L MgSO 4 ·7H 2 O,25g/L of colloidal chitin with mass fraction of 1%, and 2g/L of yeast powder; the pH of the seed medium was 7.0.
Inoculating the activated seed liquid into a fermentation culture medium with an inoculum size of 2% (v/v), and fermenting at 35 ℃ for 3d at a shaking table rotation speed of 200rpm, wherein the shaking liquid filling amount is 30ml/150ml, so as to obtain a fermentation bacteria liquid.
Wherein the fermentation medium takes water as a solvent and comprises the following components in concentration: KH 0.3g/L 2 PO 4 0.7g of K 2 HPO 4 FeSO 0.01g/L 4 ·7H 2 O,0.5g/L MgSO 4 ·7H 2 O,2g/L yeast powder, 50g/L powdery chitin; the pH of the fermentation medium was 7.2.
Centrifuging fermentation bacteria liquid at 12000rpm for 5min, taking 0.4mL of supernatant crude enzyme liquid, adding the supernatant crude enzyme liquid into a 2mL EP tube, adding 0.4mL of 2% colloidal chitin into the EP tube, reacting for 20min under the water bath condition of 50 ℃, taking 0.2mL of reaction liquid and 0.3mL of DNS, simultaneously adding into the 2mL EP tube, uniformly mixing, boiling water bath for 10min, cooling to 28 ℃, centrifuging at 12000rpm for 5min, taking supernatant to remove redundant chitin, and measuring the OD value of the solution by adopting an enzyme marker. The boiling inactivating enzyme liquid is used as a control, and the enzyme activity is calculated to be 317U/mL.
The enzyme activity units are defined as: under the above conditions, the amount of enzyme required to release 1. Mu. M N-acetylglucosamine per minute was defined as 1 enzyme activity unit (U).
Comparative example 1
As compared with example 2, the difference is only that the nitrogen source is different, the component of the fermentation medium is KH of 0.3g/L 2 PO 4 K at 0.7g/L 2 HPO 4 FeSO 0.01g/L 4 ·7H 2 O,0.5g/L MgSO 4 ·7H 2 O,10g/L of powdery chitin, 10g/L of soybean powder, pH 7.0;
the crude enzyme liquid obtained by measurement has the enzyme activity of 126U/mL, compared with the example 2, the comparative example 1 replaces the nitrogen source by the yeast powder with the concentration of 2g/L and the concentration of 10g/L, which shows that the nitrogen source has a great influence on the chitinase activity, and the chitinase activity is the highest when the yeast powder is the nitrogen source for enzyme-producing fermentation of the strain.
Comparative example 2
The difference compared with example 2 is that the colloidal chitin concentration is different, the component of the fermentation medium is KH of 0.3g/L 2 PO 4 K at 0.7g/L 2 HPO 4 0.2g/L NaCl,10g/L powdery chitin, 2g/L yeast powder, and pH 7.0;
as compared with example 2, the crude enzyme liquid obtained by measurement has the enzyme activity of 66U/mL, and compared with example 2, the comparative example 2 has the powder chitin concentration of 50g/L replaced by 10g/L, which shows that the powder chitin concentration has a great influence on the chitinase activity, and the chitinase activity is the highest when the powder chitin concentration is 50 g/L.
Comparative example 3
The only difference compared with example 2 is the fermentation time, the composition of the fermentation medium is KH of 0.3g/L 2 PO 4 K at 0.7g/L 2 HPO 4 FeSO 0.01g/L 4 ·7H 2 O,0.5g/L MgSO 4 ·7H 2 O,50g/L of powdery chitin, 2g/L of yeast powder and pH 7.0; will be activatedThe fermentation time of inoculating the seed liquid to the fermentation medium is 4d;
the crude enzyme liquid enzyme activity obtained by measurement is 267U/mL, and compared with the example 2, the fermentation time is replaced by 4d from 3d in the comparative example 2, which shows that the fermentation time has a great influence on the chitinase activity, and the chitinase activity is highest when the fermentation time is 3 d.
Comparative example 4
The only difference compared with example 2 is that the initial pH of the fermentation is different, the composition of the fermentation medium is KH of 0.3g/L 2 PO 4 K at 0.7g/L 2 HPO 4 FeSO 0.01g/L 4 ·7H 2 O,0.5g/L MgSO 4 ·7H 2 O,50g/L of powdery chitin, 2g/L of yeast powder and pH 6.0;
the crude enzyme liquid obtained by measurement had an enzyme activity of 74U/mL, and compared with example 2, comparative example 2 was changed from 7.0 to 6.0, which indicates that the fermentation initiation pH had a large influence on chitinase activity, and the chitinase activity was highest when the fermentation initiation pH was 7.0.
Comparative example 5
The difference from example 2 was that the seed liquid was inoculated at 1% (v/v) and the fermentation medium had a KH content of 0.3g/L 2 PO 4 K at 0.7g/L 2 HPO 4 FeSO 0.01g/L 4 ·7H 2 O,0.5g/L MgSO 4 ·7H 2 O,50g/L of powdery chitin, 2g/L of yeast powder and pH 7.0;
as compared with example 2, the inoculation amount of comparative example 2 was replaced by 1% (v/v) with 156U/mL of crude enzyme liquid, which shows that the inoculation amount has a large influence on chitinase activity, and the chitinase activity is highest when the inoculation amount is 2% (v/v).
Comparative example 6
As compared with example 2, the difference is only that the shaking bottle liquid amount is different, the component of the fermentation medium is KH of 0.3g/L 2 PO 4 K at 0.7g/L 2 HPO 4 FeSO 0.01g/L 4 ·7H 2 O,0.5g/L MgSO 4 ·7H 2 O,50g/L of powdery chitin, 2g/L of yeast powder and pH 7.0; the liquid filling amount of the shake flask is 80ml/150ml。
As compared with example 2, the crude enzyme liquid obtained by measurement has the enzyme activity of 121U/mL, and the shake flask liquid filling amount is replaced by 80mL/150mL from 30mL/150mL in comparative example 2, which shows that the liquid filling amount has a great influence on chitinase activity, and the enzyme activity is highest when the liquid filling amount is 30mL/150mL.
As is clear from comparison of example 2 with comparative examples 1 to 6, the fermentation broth obtained from the fermentation medium used in example 2 was centrifuged, and the crude enzyme broth was subjected to enzyme activity measurement, the enzyme activity being the highest, indicating that the fermentation medium and the culture conditions in example 2 were used optimally. Comparative example 1 is an optimization of nitrogen source, demonstrating that yeast powder is the most suitable nitrogen source for the strain; comparative example 2 is an optimization of the chitin concentration of the powder, showing that 50g/L is the optimal addition, and comparative example 3 is an optimization of the fermentation time, showing that the optimal fermentation time of the strain is 3d; comparative example 4 is an optimization of fermentation initiation pH, which indicates an optimum fermentation initiation pH of 7.0, and comparative example 5 is an optimization of seed amount, which indicates an optimum seed amount of 2% (v/v); comparative example 6 shows the optimum liquid loading of 30ml/150ml by optimizing the liquid loading. As can be seen from the comparative examples, example 2 is the optimal condition for strain fermentation.
Experimental example 1
The fermentation broth obtained in example 2 was selected for analysis of chitinase-producing products, as follows:
after 50mL of fermentation broth was centrifuged at 12000rpm for 10min at 4 ℃, 40g of ammonium sulfate powder was added to 50mL of the supernatant to make the final concentration of ammonium sulfate 70% (m/v), the mixture was precipitated overnight at 4℃and centrifuged at 12000rpm for 10min, and the obtained precipitate was stirred and mixed with 10mL of PBS buffer with pH of 7.0 to obtain a crude enzyme solution.
2% (m/v) of colloidal chitin is taken as a substrate and the volume ratio of the colloidal chitin to the crude enzyme solution is 1:1, respectively reacting for 0, 5 and 10min, then carrying out boiling water bath for 5min, and centrifuging for 10min at 8000r/min, and collecting supernatant.
And (3) spotting (a small number of spotting is performed for a plurality of times, the next spotting can be performed after the last spotting is performed, and after the spotting is finished, the silica gel plate is placed in a chromatographic bar, and a spreading agent (isopropanol: ammonia water=2:1, V/V) is added for slow spreading. And (3) when the chromatographic liquid is 1cm away from the upper edge of the silica gel plate, taking the silica gel plate out of the chromatographic bar, drying, uniformly spraying 1% ninhydrin solution, and drying at 110 ℃ for 5min for color development. As shown in FIG. 6, the chitinase crude enzyme obtained from the fermentation broth obtained by the fermentation process in example 2 is used as a catalyst, and the chitinase produced by the strain is mainly chitobiose, so that the chitinase produced by the strain is chitobiose exoenzyme.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. Bacillus marinus (Bacillus haynesii) GXMU-J23.1 was deposited at the Guangdong province microbiological bacterial collection center (GDMCC) at 10.14 of 2022 under accession number GDMCC No.62891.
2. Use of bacillus marinus GXMU-J23.1 as claimed in claim 1 for the preparation of chitinase and degradation of chitin.
3. The use according to claim 2, wherein the chitinase is a chitobiose exonuclease.
4. A fermentation process for preparing chitinase using bacillus marinus GXMU-J23.1 as claimed in claim 1, comprising the steps of:
(1) Placing bacillus marinus in a seed culture medium for fermentation to obtain seed liquid;
(2) Inoculating the seed solution obtained in the step (1) into a fermentation medium for culture to obtain fermentation bacteria solution.
5. The fermentation process of claim 4, wherein the species of step (1)The sub-culture medium takes water as a solvent and comprises the following components in concentration: KH of 0.2-0.4 g/L 2 PO 4 K of 0.6-0.8 g/L 2 HPO 4 FeSO of 0.01-0.02 g/L 4 ·7H 2 O, 0.4-0.6 g/L MgSO 4 ·7H 2 2, 22-28 g/L of colloidal chitin with mass fraction of 1 percent and 1-3 g/L of yeast powder; the pH of the seed culture medium is 6-8.
6. The fermentation process of claim 5, wherein the Bacillus marinus of step (1) is inoculated at a concentration of 0.5X10 8 cfu/ml～1.5×10 8 cfu/ml, and the inoculation amount of the cfu/ml inoculated into the seed culture medium is 0.5-5.0% (v/v); the temperature of the culture after inoculation is 32-38 ℃; the culture time is 24-96 hours; the rotation speed of the shaking table for culture is 150-220 rpm.
7. The fermentation process of claim 6, wherein the seed solution of step (2) is inoculated into the fermentation medium in an amount of 0.5 to 5% (v/v).
8. The fermentation process of claim 7, wherein the fermentation medium of step (2) is water-soluble and comprises the following concentrations of components: KH of 0.1-0.5 g/L 2 PO 4 0.3 to 1.0g of K 2 HPO 4 FeSO of 0.01-0.05 g/L 4 ·7H 2 O, 0.2-0.8 g/L MgSO 4 ·7H 2 O, 1-5 g/L yeast powder, 5-50 g/L powder chitin; the pH of the fermentation medium is 6.5-7.5.
9. The fermentation process of claim 8, wherein the temperature of the culturing in step (2) is 28-45 ℃; the culture time is 1-5 d; the rotation speed of the shaking table for culture is 100-300 rpm.
10. Use of bacillus marinus GXMU-J23.1 as claimed in claim 1 for promoting plant growth and improving stress resistance of crops.
CN202310589956.9A 2023-05-24 2023-05-24 Bacillus marinus strain for producing chitinase and fermentation process and application thereof Pending CN116656548A (en)

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