Bacillus tropicus for producing chitosanase and application thereof

Abstract

The application discloses a chitosanase-producing Bacillus tropicus strain and application thereof. The strain is named Bacillus tropicus LHLJW.A06, and the strain has been preserved in the Guangdong Microbial Culture Collection Center on December 21, 2021, with a preservation number of GDMCC NO.62150. The strain provided in the application can ferment to produce chitosanase. The produced chitosanase can act on chitosan under acid conditions and within a certain temperature range, and hydrolyze the chitosan into water-soluble chitooligosaccharide. The enzyme activity of the fermentation liquor is 7.23 U / mL, and the strain is a strain with great research and development value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a bacillus tropicus Bacillus tropicus LHLJW.A06, and particularly relates to a bacillus tropicus producing chitosanase and application. BACKGROUND

[0002] Chitosan is a natural cationic amino polysaccharide, which widely exists in fungi, crustaceans, insects and some algae. Due to its unique characteristics such as non-toxicity and wide biocompatibility, chitosan has been applied in many fields, such as food and cosmetic additives. However, the poor water solubility of chitosan limits its wider application. Chitooligosaccharide is water-soluble and has good anti-inflammatory, antioxidant, antitumor, antibacterial and other functional properties. Chitosan can be degraded into chitooligosaccharide by chemical or enzymatic method. Chemical method is mainly through acid hydrolysis of chitosan at high temperature to become chitooligosaccharide, and enzymatic method is through various enzymes to make chitosan become chitooligosaccharide, these enzymes mainly include specific enzyme chitosanase and non-specific enzymes such as carbohydrate enzymes, proteases, lipases, etc. Chitosanase can catalyze the hydrolysis of β-1, 4 glycosidic bond to make chitosan crack into chitooligosaccharide. The advantage of enzymatic method over chemical method is that the conditions are relatively mild, specific chitooligosaccharide with different degrees of polymerization can be generated, and no harmful waste liquid is produced.

[0003] Chitosanase is an enzyme that specifically hydrolyzes chitosan, mainly exists in bacterial and fungal cells, and various strains have been found to produce chitosanase. Some researchers have also genetically recombined different strains to increase the yield of chitosanase. The fermentation production of chitosanase can adopt liquid fermentation and solid fermentation. Wang Gang et al. used Bacillus subtilis (preserved number: CGMCC No: 14841) to ferment and produce chitosanase, and the chitosanase produced by the Bacillus subtilis had high yield, strong enzyme activity, different protein structure from ordinary chitosanase, and good activity in weak alkaline, neutral, weak acidic and relatively strong acidic conditions, and the reaction end product was dimeric glucosamine and trimeric glucosamine (patent application number: CN201810030676.3). Yu Feng used a strain of fungus Paecilomyces sp.JW1727 produces chitosanase in the liquid medium of chitosan-added potato, the culture method is simple, the enzyme production time is short, the enzyme production activity is high, and it is beneficial to the development and utilization of chitosanase (Patent Application No: CN201010250754.4). Wang Zhongyong et al. constructed a recombinant plasmid encoding the chitosanase gene sequence of Bacillus subtilis, and the recombinant plasmid was transformed into the Rhodopseudomonas palustris strain by contact transformation of Escherichia coli. The Rhodopseudomonas palustris recombinant strain was obtained, and the chitosanase was obtained by fermentation culture of the Rhodopseudomonas palustris recombinant strain. The Rhodopseudomonas palustris recombinant strain of the invention does not need chitosan induction and does not affect the growth of the recombinant strain. The expressed extracellular chitosanase can continuously hydrolyze chitosan to form chitooligosaccharide, improve the solubility of chitosan, and the fermentation agent containing chitooligosaccharide can be directly used (Patent Application No: CN202111268892.X). SUMMARY

[0004] The Bacillus tropicus provided by the present application Bacillus tropicus The LHLJW.A06 strain can produce chitosanase, which can hydrolyze chitosan to chitooligosaccharide. The reaction degree of chitosanase and chitosan substrate can be represented by reducing sugar determination method and viscosity method. The produced chitosanase can act on chitosan under acidic conditions within a certain temperature range, hydrolyzing chitosan to water-soluble chitooligosaccharide. The enzyme activity of the fermentation broth is 7.23 U / mL. The strain is a strain with great research and development value.

[0005] The technical scheme of the present application is as follows.

[0006] The Bacillus tropicus provided by the present application Bacillus tropicus LHLJW.A06 is separated from Guangdong shrimp shells. The strain has been preserved in the "Guangdong Microbial Culture Collection Center" on December 21, 2021, and the preservation number is GDMCC NO:62150.

[0007] The address of the Guangdong Microbial Culture Collection Center is: No. 59, 5th Floor, Building, 100, Martyrs Road, Guangzhou, Guangdong Province.

[0008] Further, the strain is isolated from Guangdong shrimp shells. The strain is isolated by the following method:

[0009] Fresh shrimp shells are taken into a triangular flask containing sterile water and glass beads, shaken, and the bacterial suspension is taken into a sterile centrifuge tube. Gradient dilution is performed and plated on chitosan screening medium. Invert constant temperature culture, pick the colonies with transparent circles for re-dilution and plating after single colonies grow, and then constant temperature culture. After culture, store in the refrigerator.

[0010] The specific method is:

[0011] Take 2 g of fresh shrimp shell into a 300 mL flask with 50 mL of sterile water and glass beads, shake for 12 h. Take 0.1 mL of the above bacterial suspension in a 10 mL sterile centrifuge tube, perform gradient dilution and spread on the chitosan screening medium, and incubate at 35 DEG C in an inverted constant temperature incubator. After single colonies grow, pick the colonies with transparent circles for re-dilution and spreading, and incubate at constant temperature. Then inoculate the single colonies into nutrient agar slant and incubate at 35 DEG C. After incubation, store in a refrigerator at 4 DEG C and send to a gene company for sequencing.

[0012] Further, the sequence of the strain is shown in the sequence table.

[0013] Further, the chitosan screening medium has the following composition:

[0014] Chitosan 5 g / L, agar 18 g / L, pH 5.5.

[0015] Further, the chitosanase is cultured by the following method:

[0016] Inoculate the slant strain into liquid seed culture medium, and incubate at 35 DEG C and 150 r / min for 12-24 h. Then inoculate into a shake flask fermentation medium, and shake flask ferment at 35-40 DEG C and 150 r / min for 4-5 d. Centrifuge the fermentation broth at 4000 r / min for 20 min, and filter through a 0.22 mu sterile filter to obtain a crude enzyme solution. First add 20% saturated ammonium sulfate to the crude enzyme solution, stand for 2 h, and centrifuge at 10000 rpm to remove impurities. Then add ammonium sulfate to the supernatant to 80% saturation, magnetically stir for 2 h, and stand for 8 h. Centrifuge at 10000 rpm to collect the precipitate, place the precipitate in a dialysis bag, dialyze in acetic acid buffer solution for 24 h, and finally freeze-dry the precipitate to obtain chitosanase.

[0017] The chitosanase produced by the application can act on chitosan under acidic conditions and within a certain temperature range to hydrolyze chitosan into water-soluble chitooligosaccharides. The enzyme activity of the fermentation broth is 7.23 U / mL. The strain is a strain with great research and development value.

[0018] Further, the medium has the following composition: glucose 10 g / L, peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, pH 7.4. The fermentation medium has the following composition: colloidal chitosan 1 g / L, glucose 30 g / L, yeast powder 5 g / L, (NH4)2SO4 10 g / L, ammonium chloride 5 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, MnCl2 0.1 g / L.

[0019] The application of the strain produces chitosanase, and a preparation method of chitooligosaccharide by hydrolyzing chitosan is as follows: mixing the fermentation crude enzyme liquid with chitosan solution at a ratio of 5%-20%(v / v), slowly stirring at a temperature of 30-70 DEG C, reducing the viscosity of the chitosan solution and increasing the content of reducing sugar in the solution, and obtaining a chitooligosaccharide solution.

[0020] Compared with the prior art, the application has the advantages that:

[0021] The chitosanase produced by the strain can act on chitosan under acidic conditions in a wide temperature range, hydrolyze chitosan into water-soluble chitooligosaccharide, has high enzyme activity of fermentation liquid, and does not produce additional harmful substances in the hydrolysis process, and has environmental friendliness. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 A viscosity change curve diagram of hydrolyzing chitosan with different crude enzyme liquid dosages;

[0023] Figure 2 A viscosity change curve diagram of hydrolyzing chitosan under different temperature conditions;

[0024] Figure 3 A DNS reducing sugar determination method standard curve diagram;

[0025] Figure 4 A fermentation liquid enzyme activity curve diagram under different pH conditions;

[0026] Figure 5 A fermentation liquid enzyme activity curve diagram under different temperature conditions;

[0027] Figure 6 A Bacillus tropicus A phylogenetic tree diagram of LHLJW.A06. DETAILED DESCRIPTION

[0028] The application will be further specifically and specifically described below in combination with specific embodiments, but the embodiments of the application are not limited thereto, and for the process parameters not specially marked, the conventional technology can be referred to.

[0029] Example 1: Screening of bacillus tropicus strain

[0030] Take 2 g of fresh shrimp shell into a 300 mL flask with 50 mL of sterile water and glass beads, shake for 12 h. Take 0.1 mL of the above bacterial suspension into a 10 mL sterile centrifuge tube, perform gradient dilution and spread on chitosan screening medium, 35°C inverted constant temperature culture, after single colony growth, pick transparent circle colonies for re-dilution and spread, constant temperature culture, then inoculate single colonies into nutrient agar slant at 35°C constant temperature culture, after culture, store in 4°C refrigerator and send to gene company for sequencing. Finally, a strain of Bacillus tropicus was obtained, and the strain was named Bacillus tropicus Bacillus tropicus LHLJW.A06, which has been deposited at the "Guangdong Microbial Culture Collection Center" on December 21, 2021, with the accession number GDMCC NO:62150.

[0031] Example 2 Liquid fermentation of GDMCC NO:62150 strain to produce chitosanase

[0032] Inoculate the slant strain into liquid seed culture medium, 35°C, 150 r / min culture for 12-24 h, then inoculate into shake flask fermentation medium, 35-40°C, 150 r / min shake flask fermentation for 4-5 d. Centrifuge the fermentation broth at 4000 r / min for 20 min, then filter through a 0.22 μm sterile filter to obtain crude enzyme liquid. First add 20% saturated ammonium sulfate to the crude enzyme liquid, stand for 2 h, then centrifuge at 10000 rpm to remove impurities, add ammonium sulfate to 80% saturation to the supernatant, magnetically stir for 2 h, then stand for 8 h, centrifuge at 10000 rpm to collect the precipitate, place the precipitate in a dialysis bag and dialyze in acetic acid buffer solution for 24 h, finally freeze-dry the precipitate to obtain chitosanase.

[0033] The seed culture medium consists of: glucose 10 g / L, peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, pH 7.4. The fermentation medium consists of: colloidal chitosan 1 g / L, glucose 30 g / L, yeast powder 5 g / L, (NH4)2SO4 10 g / L, ammonium chloride 5 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, MnCl2 0.1 g / L.

[0034] Example 3 Viscosity changes of chitosan hydrolysis with different amounts of crude enzyme liquid

[0035] Prepare a 2% chitosan solution, add crude enzyme liquid at a ratio of 5%-20%(v / v) to the chitosan solution, slowly stir in a 45°C water bath for 1.5 h, and record the viscosity changes, the results are shown in Figure 1 .

[0036] Example 4 Viscosity change of hydrolysis of chitosan at different temperatures

[0037] A 2% chitosan solution was prepared, and the crude enzyme solution was added in the chitosan solution at a proportion of 10% (v / v), and the reaction was stirred slowly in a water bath at different temperatures for 1.5 h, and the viscosity change was recorded, and the results are shown in Table 1. Figure 2 .

[0038] Example 5 Determination of enzyme activity in fermentation broth by reducing sugar determination method

[0039] (1) Determination of standard curve:

[0040] 0.516 g of glucosamine hydrochloride was accurately weighed, dissolved in distilled water, and finally diluted to 100 mL to obtain a standard solution with a concentration of 24 μmol / mL. According to the parameters in the following table, the solution was added to test tubes with the same specifications numbered 1-11, and then boiled in a water bath for 10 min. After cooling, distilled water was added to make up to 25 mL. The absorbance was measured at a wavelength of 540 nm using a UV spectrophotometer, and a standard curve was plotted. The standard curve is shown in Figure 1. Figure 3 .

[0041]

[0042] (2) Enzyme activity determination: 1 mL of fermentation broth was centrifuged at 5000 r / min for 10 min, and 0.2 mL of the supernatant was taken into test tubes, which were blank and experimental groups. The blank group was inactivated by strong alkali, and 1.0 mL of acetic acid-sodium acetate buffer solution and 1.0 mL of 1% colloidal chitosan were added. The experimental group was directly added with 1.0 mL of acetic acid-sodium acetate buffer solution and 1.0 mL of 1% colloidal chitosan in the crude enzyme solution, and both were placed in a water bath at 50°C. During the water bath, the test tubes were constantly shaken to ensure that the enzyme solution and chitosan colloids were in full contact. After 15 min, the experimental group was added with strong alkali to inactivate the enzyme. After the test tubes were cooled, 1.5 mL of DNS reagent was added to the blank and experimental groups, respectively, and shaken well. Then, the mixture was boiled in a water bath for 10 min to allow the DNS to fully react with the reducing sugar in the test tube. After boiling, the mixture was centrifuged at 3500 r / min for 10 min, and the supernatant was diluted to 25 mL. The absorbance was measured at a wavelength of 540 nm, and the enzyme activity of chitosanase was calculated to be 7.23 U / mL.

[0043] Enzyme activity unit (U): the amount of enzyme required to catalyze the production of 1 μmol of glucosamine per minute.

[0044] Enzyme activity formula: Enzyme activity (U / mL) =

[0045] Wherein, n - amount of glucosamine hydrochloride (pmol); N - dilution fold; t - reaction time (min); V - fermentation liquid volume (mL)

[0046] Example 6 Enzyme activity of fermentation liquid under different pH conditions

[0047] The chitosanase fermentation liquid obtained above was used as the enzyme liquid to be tested, and the enzyme activity was measured at pH 3.0-6.5. The highest value of enzyme activity was taken as 100%, and the relative enzyme activity under different pH conditions was calculated respectively. The results are shown in Table 2. Figure 4 The optimum pH was 5.0.

[0048] Example 7 Enzyme activity of fermentation liquid under different temperature conditions

[0049] The chitosanase fermentation liquid obtained above was used as the enzyme liquid to be tested, and the enzyme activity was measured at 30-70℃. The highest value of enzyme activity was taken as 100%, and the relative enzyme activity under different temperature conditions was calculated respectively. The results are shown in Table 3. Figure 5 The optimum temperature was 50℃.

[0050] Example 8 Establishment of strain phylogenetic tree

[0051] The gene sequence of the strain was compared with the standard database through fast local sequence alignment (BLAST) comparison on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), and the gene sequence of the related strain with the highest similarity, coverage and minimum E value was searched. BLAST analysis showed that the isolated strain had the highest homology with Bacillus tropicus (GenBank accession number NR 157736.1). Using molecular evolution genetics analysis software MEGA11, a phylogenetic tree was constructed for the highly homologous DNA sequences using the neighbor-joining method, as shown in Figure 2. Figure 6 .

[0052] It should be understood that the above detailed description of the technical solutions of the present application by means of the optimization examples is illustrative rather than limiting, and the specific embodiments of the present application should not be construed as being limited thereto. For ordinary skilled persons in the technical field to which the present application belongs, modifications to the technical solutions described in the embodiments, or equivalent replacement of some technical features, without departing from the concept of the present application, should be considered as belonging to the patent protection scope determined by the claims submitted by the present application.

[0053] The above embodiments of the present application are merely exemplary but not intended to limit the embodiments of the present application. Based on the above description, those skilled in the art can make other variations and modifications of the present application in different forms. There is no need and it is also not intended to describe all the embodiments of the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall fall within the scope of the claims of the present application. SEQUENCE LISTING <110> South China University of Technology <120> A Bacillus tropicus producing chitosanase and application <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1408 <212> DNA <213> Bacillus tropicus <400> 1 tgcagtcgag cgaatggatt aagagcttgc tcttatgaag ttagcggcgg acgggtgagt 60 aacacgtggg taacctgccc ataagactgg gataactccg ggaaaccggg gctaataccg 120 gataacattt tgaaccgcat ggttcgaaat tgaaaggcgg cttcggctgt cacttatgga 180 tggacccgcg tcgcattagc tagttggtga ggtaacggct caccaaggca acgatgcgta 240 gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg 300 aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag 360 tgatgaaggc tttcgggtcg taaaactctg ttgttaggga agaacaagtg ctagttgaat 420 aagctggcac cttgacggta cctaaccaga aagccacggc taactacgtg ccagcagccg 480 cggtaatacg taggtggcaa gcgttatccg gaattattgg gcgtaaagcg cgcgcaggtg 540 gtttcttaag tctgatgtga aagcccacgg ctcaaccgtg gagggtcatt ggaaactggg 600 agacttgagt gcagaagagg aaagtggaat tccatgtgta gcggtgaaat gcgtagagat 660 atggaggaac accagtggcg aaggcgactt tctggtctgt aactgacact gaggcgcgaa 720 agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct 780 aagtgttaga gggtttccgc cctttagtgc tgaagttaac gcattaagca ctccgcctgg 840 ggagtacggc cgcaaggctg aaactcaaag gaattgacgg gggcccgcac aagcggtgga 900 gcatgtggtt taattcgaag caacgcgaag aaccttacca ggtcttgaca tcctctgaca 960 accctagaga tagggcttct ccttcgggag cagagtgaca ggtggtgcat ggttgtcgtc 1020 agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gatcttagtt 1080 gccatcatta agttgggcac tctaaggtga ctgccggtga caaaccggag gaaggtgggg 1140 atgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggacggt 1200 acaaagagct gcaagaccgc gaggtggagc taatctcata aaaccgttct cagttcggat 1260 tgtaggctgc aactcgccta catgaagctg gaatcgctag taatcgcgga tcagcatgcc 1320 gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccacgag agtttgtaac 1380 acccgaagtc ggtggggtaa ccttttgg 1408

Claims

1. A chitosanase-producing Bacillus strain, characterized in that, The strain is named Bacillus tropicus Bacillus tropicus LHLJW.A06, with the preservation number of GDMCC NO: 62150.

2. The chitosanase-producing Bacillus of claim 1 is applied to ferment chitosanase.

3. Use according to claim 2, characterized in that, The chitosanase is prepared by the following method: The slant strain is inoculated into liquid seed culture medium, cultured at 35℃ and 150 r / min for 12-24 h, then inoculated into flask fermentation medium, shaken at 35-40℃ and 150 r / min for 4-5 d, the fermentation liquor is centrifuged at 4000 r / min for 20 min, then filtered through a 0.22 μm sterile filter to obtain crude enzyme liquor; 20% saturated ammonium sulfate is added to the crude enzyme liquor, after standing for 2 h, the impurities are removed by centrifugation at 10000 rpm, then 80% saturated ammonium sulfate is added to the supernatant, after magnetic stirring for 2 h and standing for 8 h, the precipitate is collected by centrifugation at 10000 rpm, the precipitate is placed in a dialysis bag and dialyzed in acetic acid buffer solution for 24 h, finally the precipitate is freeze-dried to obtain chitosanase.

4. Use according to claim 3, characterized in that, The seed culture medium consists of 10 g / L glucose, 10 g / L proteose peptone, 5 g / L yeast powder, 10 g / L sodium chloride, pH 7.4; the fermentation medium consists of 1 g / L colloidal chitosan, 30 g / L glucose, 5 g / L yeast powder, 10 g / L (NH4)2SO4, 5 g / L ammonium chloride, 2 g / L KH2PO4, 0.5 g / L MgSO4, 0.1 g / L MnCl2.

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