CN116286512A - Bacillus for producing chitosanase and application thereof - Google Patents
Bacillus for producing chitosanase and application thereof Download PDFInfo
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- CN116286512A CN116286512A CN202310198854.4A CN202310198854A CN116286512A CN 116286512 A CN116286512 A CN 116286512A CN 202310198854 A CN202310198854 A CN 202310198854A CN 116286512 A CN116286512 A CN 116286512A
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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)
- C12Y302/01132—Chitosanase (3.2.1.132)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
Abstract
The invention belongs to the technical field of microorganisms, and relates to bacillus for producing chitosanase and application thereof. The strain is named as Bacillus HZ20-1, is preserved in China center for type culture collection, with a preservation number of CCTCC M2023172, a preservation date of 2023 and 22 days of 02 month, and a preservation address of the strain is in eight-path 299 universities of Wuchang district in Wuhan, hubei province, china. The strain provided by the invention can efficiently produce the chitosan enzyme under the optimal growth condition and the fermentation condition, the enzyme activity can reach 60U/mL, and the strain does not need chitosan induction in the enzyme production process and is not stressed by external environment. The enzyme belongs to constitutive enzyme and non-induced enzyme, and the continuous enzyme production effect can be achieved through the continuous amplification of thalli by optimizing the fermentation process of the thalli. The above shows that the strain has simple culture conditions and great application prospect, and provides a good method for industrially producing solid or liquid chitosan enzyme.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to bacillus for producing chitosanase and application thereof.
Background
The chitosan is nontoxic and odorless, is a macromolecular linear polymer, can be extracted from silkworm chrysalis, eggshells and shells of marine organisms, has good biocompatibility, and has wide sources, low cost and easy obtainment. The amino group on the molecular chain segment is easy to be protonated and has a large amount of positive charges, so that the cationic high molecular basic polymer is the only cationic high molecular basic polymer in nature. In recent years, the composition has important application in medical cosmetology, food and the like. Although chitosan has many advantages, chitosan is not easily soluble in water and alkali solutions, but only in dilute acids, limiting its wider application. The chitosan oligosaccharide obtained by degrading chitosan has the effects of efficient antibiosis, antioxidation, blood fat reduction, blood pressure reduction, infection prevention and anti-tumor, so the research of chitosan oligosaccharide obtained by degrading chitosan has become a development trend.
The degradation process of chitosan mainly comprises a chemical degradation method and an enzymatic hydrolysis method. Compared with the existing common chemical degradation method, the enzymatic degradation chitosan has the advantages of mild and controllable conditions, high yield of functional oligosaccharides, difficult environmental pollution and the like. Chitosanase is a key enzyme for catalyzing chitosan hydrolysis to produce chitosan oligosaccharide and glucosamine.
The sources of chitosanase are wide, and in recent years, researchers screen strains with potential application capability from bacteria, fungi and plants for producing the chitosanase by fermentation, but because the enzymes of most strains need inducers, the requirements on equipment of factories are higher, the enzyme yield and the enzyme activity are lower, the chitosanase is not suitable for wide industrial production, and the popularization of producing chitosan oligosaccharide by an enzyme method is limited.
Disclosure of Invention
Based on the problems of the prior art that an inducer is needed for enzyme production and the enzyme yield and the enzyme activity are low in the strain for producing the chitosanase, the invention provides bacillus for producing the chitosanase and application thereof.
The bacillus for producing chitosanase provided by the invention can exocytosis chitosanase. The bacillus provided by the invention can efficiently produce the chitosan enzyme without an inducer, and provides a basis for industrial application in the future.
The aim of the invention can be achieved by the following technical scheme:
the invention provides Bacillus for producing chitosanase, which is named as Bacillus HZ20-1 and is preserved in China center for type culture collection (CCTCC M2023172), the preservation date is 2023, 02 month and 22 days, and the preservation address is in eight-path 299-number Wuhan universities in Wuchang district of Wuhan, hubei province, china.
Bacillus HZ20-1 is in a rod shape, gram-positive, light yellow, opaque, no fold, slightly raised center, easy picking, matt surface and irregular edge.
The bacillus can produce a constitutive chitosanase, and chitosan induction is not needed in the enzyme production process, and the bacillus is not stressed by external environment. The effect of continuous enzyme production can be achieved through the continuous amplification of thalli.
The invention also provides a 16S rDNA gene of the bacillus for producing the chitosanase, and the sequence of the 16S rDNA gene is shown as SEQ ID NO. 1.
The length of the 16S rDNA gene sequence of Bacillus for producing chitosanase is 1442bp, and the Bacillus is determined to be Bacillus sp by comparing and analyzing the 16S rDNA gene sequence of the strain and establishing a evolutionary tree. The 16S rDNA gene is specifically as follows:
GGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAATGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTT
the invention also provides application of bacillus for producing chitosanase, which is fermented and cultured in a fermentation medium to obtain the chitosanase.
In one embodiment of the invention, the carbon source in the fermentation medium comprises sucrose, lactose, glucose, maltose and soluble starch.
In one embodiment of the invention, the nitrogen source in the fermentation medium comprises peanut meal, beef extract, peptone, yeast powder, soybean meal, bran, urea, ammonia water and ammonium sulfate.
In the fermentation culture process of bacillus for producing chitosanase, provided by the invention, the enzyme can be efficiently produced under the condition that no redundant trace elements are added in a fermentation culture medium.
In one embodiment of the invention, when the bacillus for producing the chitosanase is subjected to fermentation culture, a growth factor can be added into a fermentation medium, wherein the growth factor comprises V B1 、V B3 、V B5 、V B7 And V B12 Wherein V is B5 Is the optimal growth factor.
In one embodiment of the present invention, the conditions for fermentation culture are as follows: the pH range is 4.0-11.0, the temperature range is 28-42 ℃, the rotating speed is 50-250r/min, and the shake culture is carried out for 0-108 hours.
Preferably, the conditions for carrying out the fermentation culture are: the pH range is 4.0-10.0, the temperature range is 28-42 ℃, the rotating speed is 120-200r/min, and the shake culture is carried out for 0-108 hours.
In one embodiment of the invention, the step of fermentation culture of the bacillus for producing chitosanase comprises the following steps:
the bacillus for producing chitosanase is prepared by preserving the inclined plane of bacillus strain, inoculating 1-2 loop bacteria into a seed culture medium for activating culture to obtain activated bacterial liquid;
inoculating the activated bacterial liquid into a fermentation medium according to the inoculation amount of 2% -10% for fermentation culture.
In one embodiment of the invention, the bacillus for producing the chitosanase comprises lactose 2.5g/L, peptone 25g/L and V in a fermentation medium when the bacillus is subjected to fermentation culture B5 6mg/L, pH 6.2, and no trace elements.
The bacillus provided by the invention can efficiently produce the enzyme under the conditions of proper enzyme production and proper enzyme production culture medium, and the enzyme activity of the crude enzyme solution can reach 15-60U/mL. Under the optimal enzyme production condition and the optimal enzyme production culture medium condition, the enzyme activity of the crude enzyme liquid can reach more than 60U/mL.
The invention also provides an industrial production chitosan enzyme preparation, and bacillus containing the chitosan enzyme.
Compared with the prior art, the invention has the following advantages:
the invention uses bacillus bacteria to produce exocytosis chitosanase, optimizes the culture condition of bacteria, such as an enzyme production culture medium, optimizes a carbon source, a nitrogen source, trace elements and the like, screens out the optimal enzyme production culture condition, takes the enzyme yield and the enzyme activity as investigation indexes, optimizes the process condition of bacterial fermentation enzyme production, and thus obtains the optimal process condition of producing chitosanase by using bacillus bacteria, and carries out high-efficiency enzyme production, wherein the enzyme activity can reach 60U/mL.
The bacillus of the invention produces a structural chitosanase without substrate induction. The bacterium does not need chitosan induction in the enzyme production process, and is not stressed by external environment. The enzyme belongs to constitutive enzyme and non-induced enzyme through detection.
The bacillus for producing chitosanase provides an dominant strain for industrial production of chitosanase preparations.
Drawings
FIG. 1 shows the cell morphology of a chitosanase-producing strain (HZ 20-1);
FIG. 2 shows the 16S rDNA sequencing result of the chitosanase-producing strain (HZ 20-1) and a phylogenetic tree constructed by homology.
FIG. 3 shows the optimization of the culture conditions of the thalli, and the influence of different carbon sources on the fermentation enzyme production is examined.
FIG. 4 shows the optimization of the culture conditions of the cells, and the influence of different nitrogen sources on the fermentation enzyme production is examined.
FIG. 5 shows the optimization of the culture conditions of the thalli, and the influence of different microelements on the fermentation enzyme production of the thalli is examined.
FIG. 6 shows the optimization of the culture conditions of the cells, and the influence of different growth factors on the fermentation and enzyme production of the cells is examined.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples.
Example 1: isolation and identification of chitosanase-producing Strain (HZ 20-1)
1.1 Experimental materials
1.1.1 Strain Source
The strain is separated from sludge at the bottom of a sea or river shrimp and crab shell sewage well.
1.1.2 Medium
Enrichment medium: yeast powder 1.0g/L, KH 2 PO 4 1.0g/L、MgSO 4 0.5g/L、NaCl 5.0g/L、(NH 4 ) 2 SO 4 5.0g/L，pH 6.5。
Primary screening plate: yeast powder 2.0g/L and glucose 1.0g/L, KH 2 PO 4 0.3g/L、MgSO 4 0.5g/L、NaCl 5.0g/L、(NH 4 ) 2 SO 4 10.0g/L，pH 6.5。
Slope preservation medium: 37g/L of brain heart infusion powder, 20.0g/L of agar powder and pH 7.2.
Liquid seed medium: 10.0g/L peptone, 5.0g/L, naCl 5.0.5.0 g/L yeast powder, pH 6.5.
1.2 Experimental methods
Enrichment: mixing the collected sample with sterile normal saline according to the proportion of 1:10, oscillating for 30min at room temperature, fully and uniformly mixing the sample with the normal saline, standing for 3h, taking the supernatant, inoculating the supernatant into an enrichment culture medium according to the inoculum size of 10% (v/v), and culturing for 48h at 37 ℃ and 200 r/min.
And (3) primary screening: diluting the enriched liquid to 10 0 、10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 And (3) coating 100uL of enrichment solution with different dilution gradients on a preliminary screening plate, inversely culturing at 37 ℃, observing the growth condition of colonies on the plates with different culture times, dipping the plates with an inoculating loop to obtain the colonies with relatively large transparent rings after the culture is finished, marking the colonies on a blank plate for separation and purification until the same strain has single colonies twice, carrying out inclined plane preservation until the same strain appears, and naming the strain as Bacillus HZ20-1, preserving the strain in China center for type culture collection, wherein the preservation number is CCTCC M2023172, the preservation date is 2023 and 22 months, and the preservation address is in eight-path 299 of Wuchang area Wuchang university school in Hubei province, china.
Culturing the strain HZ20-1 obtained by separation and re-screening at 37 ℃ for 16-24 hours at 200r/min, streaking a flat plate, carrying out morphological observation after single bacterial colonies grow out, carrying out gram staining on the strain, and observing through a microscope.
Genomic DNA of the strain was extracted and submitted to Shanghai bioengineering Co.Ltd. The sequencing results of the 16S rDNA were subjected to similarity alignment using BLAST program, and a phylogenetic tree of the strain was constructed using MEGA7.0 software (Neighbor-Joining method).
1.3 experimental results
Referring to FIG. 1, strain HZ20-1 was in the form of a rod, gram-positive, pale yellow opaque colonies, no wrinkles on the colonies, slightly raised center, easy picking, matt surface, and irregular edges.
Referring to FIG. 2, the length of the gene sequence was 1442bp, and the 16S rDNA gene sequence of the strain was subjected to correlation alignment, and the strain was determined to be Bacillus.
Example 2: enzyme activity of bacillus HZ20-1 enzyme under different carbon sources
The carbon source is a nutrient substance which constitutes a carbon source in microbial cells and metabolites, and is an essential part for the growth of the strain.
Step 1: strain activation
The strain is taken from a glycerol tube for preserving bacillus HZ20-1 and cultured on a blank slant culture medium for 16 hours at 30 ℃, and the blank slant culture medium is: yeast extract 5.0g/L, peptone 10.0g/L and sodium chloride 5.0g/L, pH6; inoculating into shake flask containing seed culture medium, and activating and culturing at 30deg.C and shaking rate of 100r/min for 16 hr, wherein the formula of seed culture medium is: 5.0g/L of yeast extract, 10.0g/L of peptone and 5.0g/L of sodium chloride, and pH6 to obtain a seed solution.
Step 2: fermentation culture
Inoculating the seed solution into a fermentation culture medium according to the percentage mentioned by 4%, fermenting and culturing at 30 ℃ and 180r/min for 36 hours, wherein the fermentation culture medium respectively comprises the following components according to different carbon sources:
fermentation medium (base): 25g/L of ammonium sulfate, 8.0g/L of dipotassium hydrogen phosphate, 0.5g/L of anhydrous magnesium sulfate and pH value of 6.2.
Fermentation medium (sucrose): sucrose 2.5g/L, ammonium sulfate 25g/L, dipotassium hydrogen phosphate 8.0g/L, anhydrous magnesium sulfate 0.5g/L and pH 6.2.
Fermentation medium (lactose): lactose 2.5g/L, ammonium sulfate 25g/L, dipotassium hydrogen phosphate 8.0g/L, anhydrous magnesium sulfate 0.5g/L, and pH 6.2.
Fermentation medium (maltose): maltose 2.5g/L, ammonium sulfate 25g/L, dipotassium hydrogen phosphate 8.0g/L, anhydrous magnesium sulfate 0.5g/L and pH 6.2.
Fermentation medium (glucose): glucose 2.5g/L, ammonium sulfate 25g/L, dipotassium hydrogen phosphate 8.0g/L, anhydrous magnesium sulfate 0.5g/L and pH 6.2.
Fermentation medium (water-soluble starch): 2.5g/L of water-soluble starch, 25g/L of ammonium sulfate, 8.0g/L of dipotassium hydrogen phosphate, 0.5g/L of anhydrous magnesium sulfate and pH value of 6.2.
Step 3: after fermentation culture is finished, centrifuging and collecting supernatant to obtain crude enzyme liquid of the chitosan enzyme, and detecting the enzyme activity of the crude enzyme liquid.
The enzyme activity of the crude enzyme solution of the chitosanase prepared by the method is measured by a DNS method, and the result is shown in figure 3, wherein the enzyme activity of lactose is the highest, and the enzyme activity is 53U/mL.
Example 3: enzyme activity of bacillus HZ20-1 enzyme under different nitrogen sources
Step 1: strain activation
The strain is taken from a glycerol tube for preserving bacillus HZ20-1 and cultured on a blank slant culture medium for 16 hours at 30 ℃, and the blank slant culture medium is: yeast extract 5.0g/L, peptone 10.0g/L and sodium chloride 5.0g/L, pH6; inoculating into shake flask containing seed culture medium, and activating and culturing at 30deg.C and shaking rate of 100r/min for 16 hr, wherein the formula of seed culture medium is: 5.0g/L of yeast extract, 10.0g/L of peptone and 5.0g/L of sodium chloride, and pH6 to obtain a seed solution.
Step 2: fermentation culture
Inoculating the seed solution into a fermentation culture medium according to the percentage mentioned by 4%, fermenting and culturing at 30 ℃ and 180r/min for 36 hours, wherein the fermentation culture medium respectively comprises the following components according to different nitrogen sources:
fermentation medium (base): lactose 2.5g/L, ammonium sulfate 25g/L, dipotassium hydrogen phosphate 8.0g/L, anhydrous magnesium sulfate 0.5g/L, and pH 6.2.
Fermentation medium (urea): lactose 2.5g/L, urea 25g/L, dipotassium hydrogen phosphate 8.0g/L, anhydrous magnesium sulfate 0.5g/L, and pH 6.2.
Fermentation medium (ammonia): lactose 2.5g/L, ammonia water 25g/L, dipotassium hydrogen phosphate 8.0g/L, anhydrous magnesium sulfate 0.5g/L and pH 6.2.
Fermentation medium (peptone): lactose 2.5g/L, peptone 25g/L, dipotassium hydrogen phosphate 8.0g/L, anhydrous magnesium sulfate 0.5g/L, and pH 6.2.
Fermentation medium (yeast powder): lactose 2.5g/L, yeast powder 25g/L, dipotassium hydrogen phosphate 8.0g/L, anhydrous magnesium sulfate 0.5g/L and pH 6.2.
Fermentation medium (peanut cake meal): lactose 2.5g/L, peanut cake powder 25g/L, dipotassium hydrogen phosphate 8.0g/L, anhydrous magnesium sulfate 0.5g/L and pH 6.2.
Fermentation medium (soybean cake powder): lactose 2.5g/L, soybean cake powder 25g/L, dipotassium hydrogen phosphate 8.0g/L, anhydrous magnesium sulfate 0.5g/L and pH 6.2.
Fermentation medium (bran): lactose 2.5g/L, bran 25g/L, dipotassium hydrogen phosphate 8.0g/L, anhydrous magnesium sulfate 0.5g/L, and pH 6.2.
Fermentation medium (beef extract): lactose 2.5g/L, beef extract 25g/L, dipotassium hydrogen phosphate 8.0g/L, anhydrous magnesium sulfate 0.5g/L, and pH 6.2.
The enzyme activity of the crude enzyme solution of the chitosanase prepared by the method is measured by a DNS method, and the result is shown in figure 4, wherein the enzyme activity of peptone is the highest, and the enzyme activity is 59U/mL.
Example 4: enzyme activity of bacillus HZ20-1 enzyme under different trace elements
Step 1: strain activation
The strain is taken from a glycerol tube for preserving bacillus HZ20-1 and cultured on a blank slant culture medium for 16 hours at 30 ℃, and the blank slant culture medium is: yeast extract 5.0g/L, peptone 10.0g/L and sodium chloride 5.0g/L, pH6; inoculating into shake flask containing seed culture medium, and activating and culturing at 30deg.C and shaking rate of 100r/min for 16 hr, wherein the formula of seed culture medium is: 5.0g/L of yeast extract, 10.0g/L of peptone and 5.0g/L of sodium chloride, and pH6 to obtain a seed solution.
Step 2: fermentation culture
Inoculating the seed solution into a fermentation culture medium according to the percentage mentioned by 4%, fermenting and culturing at 30 ℃ and 180r/min for 36 hours, wherein the fermentation culture medium respectively comprises the following components according to the difference of microelements:
fermentation medium (base): lactose 2.5g/L, peptone 25g/L, dipotassium hydrogen phosphate 8.0g/L, anhydrous magnesium sulfate 0.5g/L, and pH 6.2.
Fermentation medium (blank): lactose 2.5g/L, peptone 25g/L, pH 6.2.
Fermentation medium (calcium ion): lactose 2.5g/L, peptone 25g/L, dipotassium hydrogen phosphate 8.0g/L, anhydrous magnesium sulfate 0.5g/L, calcium chloride 5mM/L, pH 6.2.
Fermentation medium (manganese ion): lactose 2.5g/L, peptone 25g/L, dipotassium hydrogen phosphate 8.0g/L, anhydrous magnesium sulfate 0.5g/L, manganese sulfate 5mM/L, pH 6.2.
The enzyme activity of the crude enzyme solution of the chitosanase prepared by the method is measured by a DNS method, and the result is shown in figure 5, wherein the enzyme activity without adding any trace elements is the highest, and the enzyme activity is 59U/mL.
Example 5: bacillus HZ20-1 enzyme activity under different growth factors
Step 1: strain activation
The strain is taken from a glycerol tube for preserving bacillus HZ20-1 and cultured on a blank slant culture medium for 16 hours at 30 ℃, and the blank slant culture medium is: yeast extract 5.0g/L, peptone 10.0g/L and sodium chloride 5.0g/L, pH6; inoculating into shake flask containing seed culture medium, and activating and culturing at 30deg.C and shaking rate of 100r/min for 16 hr, wherein the formula of seed culture medium is: 5.0g/L of yeast extract, 10.0g/L of peptone and 5.0g/L of sodium chloride, and pH6 to obtain a seed solution.
Step 2: fermentation culture
Inoculating the seed solution into a fermentation culture medium according to the percentage mentioned by 4%, fermenting and culturing at 30 ℃ and 180r/min for 36 hours, wherein the fermentation culture medium respectively comprises the following components according to different growth factors:
fermentation medium (base): lactose 2.5g/L, peptone 25g/L, pH 6.2.
Fermentation media (V) B1 ): lactose 2.5g/L, peptone 25g/L, V B1 6mg/L，pH 6.2。
Fermentation media (V) B3 ): lactose 2.5g/L, peptone 25g/L, V B3 6mg/L，pH 6.2。
Fermentation media (V) B5 ): lactose 2.5g/L, peptone 25g/L, V B5 6mg/L，pH 6.2。
Fermentation media (V) B7 ): lactose 2.5g/L, peptone 25g/L, V B7 6mg/L，pH 6.2。
Fermentation media (V) B12 ): lactose 2.5g/L,Peptone 25g/L, V B12 6mg/L，pH 6.2。
The enzyme activity of the crude enzyme solution of chitosanase obtained by the preparation is measured by adopting a DNS method, the result is shown in figure 6, wherein V is added B5 The highest enzyme activity was found to be 60U/mL.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (10)
1. The Bacillus strain for producing chitosanase is named as Bacillus HZ20-1, and is preserved in China center for type culture collection (CCTCC M2023172), the preservation date is 2023, 02 month and 22 days, and the preservation address is in eight-path 299-No. Wuhan university schools in Wuchang district of Wuhan, hubei province, china.
2. The 16S rDNA gene of bacillus for producing chitosanase according to claim 1, wherein the sequence is shown in SEQ ID NO. 1.
3. The use of a bacillus for producing chitosanase according to claim 1, characterized in that the bacillus for producing chitosanase is fermented in a fermentation medium to obtain chitosanase.
4. The use according to claim 3, wherein the carbon source in the fermentation medium is selected from one or a combination of several of sucrose, lactose, glucose, maltose or soluble starch.
5. The use according to claim 3, wherein the nitrogen source in the fermentation medium is selected from one or more of peanut meal, beef extract, peptone, yeast powder, soybean meal, bran, urea, ammonia water or ammonium sulphate.
6. The use according to claim 3, wherein the enzyme is produced with high efficiency without adding surplus trace elements to the fermentation medium.
7. The use according to claim 3, wherein the bacillus for producing chitosanase is fermented and cultured by adding a growth factor to the fermentation medium, the growth factor comprising V B1 、V B3 、V B5 、V B7 And V B12 。
8. The use according to claim 3, wherein the fermentation culture is carried out under the following conditions: the pH range is 4.0-11.0, the temperature range is 28-42 ℃, the rotating speed is 50-250r/min, and the shake culture is carried out for 0-108 hours.
9. The use according to claim 3, wherein the step of fermentation culture of the bacillus for chitosanase production comprises:
the bacillus for producing chitosanase is prepared by preserving the inclined plane of bacillus strain, inoculating the strain into a seed culture medium for activating culture to obtain activated bacterial liquid;
inoculating the activated bacterial liquid into a fermentation medium according to the inoculation amount of 0.1% -10% for fermentation culture.
10. The use according to claim 3, wherein the bacillus for producing chitosanase comprises lactose 2.5g/L, peptone 25g/L, V when fermentation culture is carried out in a fermentation medium B5 6mg/L, pH 6.2, and no trace elements.
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