CN112458022B - Bacillus licheniformis Bl22 for high yield of chitin deacetylase and related products and application thereof - Google Patents
Bacillus licheniformis Bl22 for high yield of chitin deacetylase and related products and application thereof Download PDFInfo
- Publication number
- CN112458022B CN112458022B CN202011451720.1A CN202011451720A CN112458022B CN 112458022 B CN112458022 B CN 112458022B CN 202011451720 A CN202011451720 A CN 202011451720A CN 112458022 B CN112458022 B CN 112458022B
- Authority
- CN
- China
- Prior art keywords
- bacillus licheniformis
- chitin
- chitin deacetylase
- fermentation
- deacetylase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
-
- 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)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12P19/26—Preparation of nitrogen-containing carbohydrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01041—Chitin deacetylase (3.5.1.41)
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to the field of microorganisms, in particular to bacillus licheniformis Bl22 with high chitin deacetylase yield and related products and application thereof. The preservation number of the bacillus licheniformis is CGMCC No. 20490. The method has the advantages that the provided method for culturing the bacillus licheniformis Bl22 is simple, and the bacillus licheniformis Bl22 can be rapidly cultured and fermented to produce chitin deacetylase with high yield by using common carbon sources and nitrogen sources. The optimal catalysis temperature of the produced chitin deacetylase is relatively low, about 40 ℃, the chitin deacetylase shows high catalytic activity in a wide pH range (pH value of 6-10), and the chitin deacetylase can save energy, auxiliary materials and other costs in industrial application and has wide application prospect.
Description
Technical Field
The present invention relates to the field of microorganisms. More particularly, the invention relates to bacillus licheniformis Bl22 with high chitin deacetylase yield and related products and applications thereof.
Background
Chitin (chitin), also known as chitin and chitin, is a polysaccharide formed by connecting N-acetamido-D-glucose monomers through beta-1, 4-glycosidic bonds. It is the second largest natural macromolecular organic compound with a second content of cellulose in nature, and is widely found in exoskeletons of invertebrates (such as shrimps, crabs, insects, etc.) and cell walls of fungi and algae. Chitin is insoluble in water, dilute acid, dilute alkali, organic solvent and the like due to poor solubility, so that the utilization value of the chitin is greatly limited. For example, chitosan (chitosan) with greatly improved solubility can be obtained by deacetylating chitin (deacetylation degree is more than 55%). And the chitosan has multiple physiological functions of biodegradability, biocompatibility, nontoxicity, bacteriostasis, cancer resistance, lipid reduction, immunity enhancement and the like, can be widely applied to the fields of medicine, food, textile, agriculture, environmental protection, cosmetics and the like, and has high application value and development prospect.
At present, the industrial production method of chitosan is mainly a chemical method. Chitosan is typically prepared by high temperature treatment of chitin with 40% -60% concentrated sodium hydroxide. The method has the defects of high production cost, large environmental pollution, poor product stability and uniformity and the like. Chitin deacetylase (e.c. 3.5.1.41) can remove acetyl groups from chitin to produce chitosan products with stable deacetylation degree and narrow molecular mass distribution range. In addition, the enzymatic method for producing chitosan has mild reaction conditions, low energy consumption value and environmental protection, provides a new way for solving the problems existing in the chemical method for preparing chitosan, and is a future development direction of the chitosan production industry.
Since the first report in 1974 of Mucor rouxii (Mucor rouxii) chitin deacetylase, researchers have isolated a variety of chitin deacetylases from fungi, bacteria and insects. However, the chitin deacetylases reported at present generally have the problems of long fermentation time, low enzyme production activity, high catalytic temperature, poor deacetylation effect and the like, and most of them are enzymes derived from fungi and few of them are derived from bacteria. The bacteria have more advantages than fungi in the aspect of fermentation and enzyme production, the strain is easier to realize large-scale fermentation culture, and the produced enzyme is easier to separate and purify. Therefore, the development of microbial resources for producing the chitin deacetylase, particularly the screening of bacteria with high enzyme production, is expected to meet the industrial production requirement of preparing the chitosan by the enzyme method.
Disclosure of Invention
The invention aims to provide Bacillus licheniformis (Bacillus licheniformis) capable of producing chitin deacetylase with high yield, and related products and applications thereof.
In order to achieve the aim, the invention provides a Bacillus licheniformis (Bacillus licheniformis) strain, wherein the preservation number of the Bacillus licheniformis strain is CGMCC No. 20490.
In a second aspect, the present invention provides a starter culture comprising the bacterial material of Bacillus licheniformis as described above.
In a third aspect, the present invention provides the use of a bacillus licheniformis enzyme as described above, or a starter culture as described above, in the production of chitin deacetylase and/or in the removal of acetyl groups from chitin.
In a fourth aspect, the present invention provides a chitin deacetylase derived from a bacillus licheniformis enzyme as described above, or a starter culture as described above.
In a fifth aspect, the present invention provides a method for preparing chitin deacetylase, comprising: inoculating the bacillus licheniformis seeds or the leaven into a fermentation culture medium for fermentation to obtain fermentation liquor, carrying out solid-liquid separation on the fermentation liquor to obtain thallus cell sediment, and then crushing the thallus cells to obtain a material containing chitin deacetylase.
In a sixth aspect, the present invention provides a product comprising a chitin deacetylase as described above, or prepared by a process as described above.
In a seventh aspect, the present invention provides the use of a chitin deacetylase as described above or a product as described above for deacetylation of chitin.
In an eighth aspect, the present invention provides a method for removing acetyl groups from chitin, the method comprising: bacillus licheniformis as described above, or a starter culture as described above, or a chitin deacetylase as described above, or a product as described above, is contacted with chitin to remove acetyl groups therefrom.
The invention has the advantages that: the provided Bacillus licheniformis Bl22 has simple culture method, can utilize common carbon source and nitrogen source to carry out rapid culture and ferment to produce chitin deacetylase with high yield. The optimal catalysis temperature of the produced chitin deacetylase is relatively low, about 40 ℃, the chitin deacetylase shows high catalytic activity in a wide pH range (pH value of 6-10), and the chitin deacetylase can save energy, auxiliary materials and other costs in industrial application and has wide application prospect.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Biological preservation
The Bacillus licheniformis (Bacillus licheniformis) is preserved in China general microbiological culture Collection center (address: No. 3 of West Luo No.1 of North Chen of the south Kogyo of Beijing, China institute of microbiology, postal code: 100101) (the abbreviation of the preservation unit is CGMCC) in 06.2020 and 08.s.2020, with the preservation number of CGMCC No.20490, abbreviated as Bl 22.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.
FIG. 1 is a graph showing the enzymatic activity of Bl22 chitin deacetylase under different temperature conditions.
FIG. 2 is a graph showing the enzymatic activity of Bl22 chitin deacetylase under different pH conditions.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In a first aspect, the invention provides a Bacillus licheniformis (Bacillus licheniformis), wherein the preservation number of the Bacillus licheniformis is CGMCC No.20490, abbreviated as Bl 22.
The bacillus licheniformis of the invention is separated from Jiehu city, Hunan province.
The sequence of the 16SrDNA of the bacillus licheniformis provided by the invention is shown in SEQ ID NO. 1.
SEQ ID NO.1：
The sequence was subjected to homologous alignment with sequences in GenBank databases, and found to have 99% similarity to 16SrDNA of the strain Bacillus licheniformis YEBFR6(Accession: MT 332717.1).
The invention relates to the amplification and analysis of the sequence of the strain Bacillus licheniformis Bl 2216 SrDNA as follows:
extracting to obtain a Bacillus licheniformis Bl22 genome by using a bacterial genome extraction kit produced by Tiangen Biochemical (Beijing) Co., Ltd, and reacting in a PCR mix system by using a universal primer for amplifying a prokaryotic microorganism 16SrDNA sequence.
The universal primers (synthesized by Shanghai Producer Co., Ltd.):
27F：5’-AGAGTTTGATCCTGGCTCAG-3’(SEQ ID NO.2)；
1492R:5’-GGTTACCTTGTTACGACTT-3’(SEQ ID NO.3)。
the reaction system (50. mu.l) was: premix (25. mu.l), ddH 2 O (22. mu.l), upstream and downstream primers (1. mu.l each), DNA template(1. mu.l). Reaction procedure: denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 30s, extension at 72 ℃ for 90s, and 35 cycles; final extension at 72 ℃ for 5 min.
Take 4. mu.l PCR amplification product, detect by 1% agarose gel electrophoresis. Sequencing of the PCR amplification product was entrusted to Shanghai Biotech.
The morphological characteristics of the strain Bacillus licheniformis Bl22 related by the invention are as follows:
the strain is gram-positive bacteria, has a straight rod shape (the length is 2.0-6.0 mu m, the width is 0.3-1.0 mu m), has spores and does not expand; the single colony growing on the LB culture medium is yellowish white, opaque, wrinkled, slightly raised in the middle, irregular in edge, 3-5mm in size and easy to pick.
The bacillus licheniformis provided by the invention can produce a large amount of live bacteria of the bacillus licheniformis after being cultured, the culturing method has no special requirement as long as the bacillus licheniformis can be proliferated, for example, 10 can be added 7 The order of magnitude of viable bacteria is inoculated in a bacillus licheniformis culture medium, and cultured for 12-72 hours at the temperature of 35-37 ℃ to obtain a bacteria culture solution. The Bacillus licheniformis culture medium can be various known culture media suitable for culturing Bacillus licheniformis, and can be LB liquid culture medium with or without inorganic salt.
According to a preferred embodiment of the present invention, the seed of Bacillus licheniformis Bl22 can be inoculated into a fermentation medium for fermentation culture, thereby obtaining a fermentation liquid containing live thallus of Bacillus licheniformis Bl 22.
Wherein the fermentation medium may be a normal liquid LB medium, but according to a preferred embodiment of the present invention, the fermentation medium contains glucose, yeast powder, tryptone and K 2 HPO 4 The pH value is 6.5-7.2; wherein glucose can be 35-45g/l, yeast powder can be 20-30g/l, tryptone can be 10-20g/l, K 2 HPO 4 Can be 2-4g/L, and the balance is water.
Wherein, the fermentation culture conditions can be as follows: the temperature is 30-37 ℃ and the time is 20-24 h. Further preferably, the culture is carried out under stirring (100-. After the culture, the content of the chitin deacetylase in the fermentation liquor reaches 1800 plus 2100U/ml through detection.
Therefore, the bacillus licheniformis Bl22 provided by the invention can utilize common carbon sources (such as glucose) and nitrogen sources (such as yeast powder) to carry out rapid culture and ferment to produce chitin deacetylase with high yield.
The chitin deacetylase content of the fermentation broth can be determined by conventional methods, for example, by the p-nitroaniline color development method.
The present invention can further separate viable bacteria of bacillus licheniformis in the culture solution, the method for separating is not particularly limited as long as the bacteria can be enriched from the culture solution, and for example, the separation can be realized by a centrifugation and/or filtration method, and the conditions of the centrifugation and the filtration can be known conditions, and the present invention is not repeated herein.
According to the present invention, it is also preferred that the Bacillus licheniformis Bl22 is activated, e.g., by stage 1-3 activation, prior to inoculation into the fermentation medium, which can be done in conventional Bacillus licheniformis medium.
Preferably, the strain preserved at ultralow temperature is streaked on an LB solid culture medium, and is placed in a constant-temperature incubator for culture and activation at the temperature of 30-37 ℃ for 18-24 hours. Wherein the LB solid culture medium can contain yeast extract 4-6g/L, tryptone 8-12g/L, NaCl 8-12g/L, agar powder 15-25g/L, and water in balance, and the pH value is 6.5-7.2.
After the plate activation, it is further preferable that a single colony is picked from the activation plate and placed in a seed medium for constant temperature shaking culture activation at a stirring speed of 100 and 200rpm at a temperature of 30-37 ℃ for 6-12 h. Wherein the seed culture medium may contain: yeast extract 4-6g/L, tryptone 8-12g/L, NaCl 8-12g/L, and water in balance, and the pH value is 6.5-7.2.
In a second aspect, the present invention provides a starter culture comprising the bacterial material of Bacillus licheniformis as described above.
The leaven contains Bacillus licheniformis CGMCC No.20490The content of the viable bacteria is preferably 10 per gram of the leavening agent 5 -10 10 CFU, more preferably 10 7 -10 9 CFU。
Here, CFU (Colony-Forming Units) means the number of viable bacteria. In viable bacteria culture counting, a colony formed by growing and propagating a single bacterial cell or a plurality of bacterial cells aggregated into a mass on a solid medium is called a colony forming unit, and the number of viable bacteria is expressed by the colony forming unit. Can be obtained by diluting the culture solution after the culture to an appropriate level and plating the culture solution.
According to the present invention, the fermentation agent can be prepared according to a conventional method, for example, the fermentation culture can be carried out according to the method of the first aspect as described above to obtain the somatic cells of Bacillus licheniformis CGMCC No.20490, and then the fermentation agent can be prepared according to a conventional method.
The fermentation agent may be in a liquid state or a solid state, and is usually in a solid state. Specifically, after the fermentation culture is finished, if the fermentation liquid can be used within a short period of time, for example, within 1 month, the obtained fermentation liquid can be directly used or appropriately concentrated to be used as a fermentation agent, and in this case, the fermentation agent may be in a liquid state; however, if the fermentation broth is stored for a long time and then used, the fermentation broth can be prepared into a solid fermentation agent, for example, by subjecting the fermentation broth to solid-liquid separation to obtain a bacterial cell precipitate, mixing the bacterial cell precipitate with a lyoprotectant, and lyophilizing the mixture to obtain a solid fermentation agent.
Wherein the freeze-drying protective agent can be at least one of skimmed milk powder, maltodextrin, trehalose, dextran, glycerol and the like.
In addition, the leavening agent can be in a form preserved in glycerin tube, for example, by inoculating the strain in logarithmic growth phase into glycerin tube, wherein the glycerin tube usually contains 25-35 vol%, for example, 30 vol% of glycerin.
As described above, the Bacillus licheniformis Bl22 provided by the invention can utilize common carbon sources and nitrogen sources to carry out rapid culture and ferment to produce chitin deacetylase with high yield. Based on the method, the invention also provides the following application.
In a third aspect, the present invention provides the use of a bacillus licheniformis enzyme as described above, or a starter culture as described above, in the production of chitin deacetylase and/or in the removal of acetyl groups from chitin.
In a fourth aspect, the present invention provides a chitin deacetylase derived from a bacillus licheniformis enzyme as described above, or a starter culture as described above.
In a fifth aspect, the present invention provides a method for preparing chitin deacetylase, comprising: inoculating the bacillus licheniformis seeds or the leaven into a fermentation culture medium for fermentation to obtain a fermentation liquid, carrying out solid-liquid separation on the fermentation liquid to obtain a thallus cell precipitate, and then crushing the thallus cell to obtain a material containing chitin deacetylase.
The method for preparing the fermentation liquid according to the present invention has been described in detail in the above first aspect, and will not be described herein again in order to avoid unnecessary repetition.
According to the present invention, the method of subjecting the fermentation broth to solid-liquid separation may be a conventional method, for example, centrifugation, filtration, or the like. According to a preferred embodiment of the present invention, the fermentation broth is centrifuged for 2-10min at 8000-.
According to the present invention, the method for disrupting the bacterial cells is not particularly limited, and conventional procedures in the art may be employed, for example, ultrasonication, enzymatic disruption, surfactant disruption, high-temperature disruption, and the like. According to a preferred embodiment of the present invention, the bacterial cell pellet is disrupted by ultrasonication. For example, the amplitude bar of the ultrasonic crusher is set to be 2, the power is 20%, the ultrasonic frequency is 2s/2s (namely crushing for 2s, pausing for 2s), and the total crushing time is 5 min.
According to the invention, the supernatant of the feed liquid obtained after the thalli are crushed and subjected to solid-liquid separation is the chitin deacetylase crude enzyme liquid of the bacillus licheniformis.
Among them, the method of subjecting the liquid obtained by disrupting the cells to solid-liquid separation may be a conventional method, for example, centrifugation, filtration, or the like. According to a preferred embodiment of the present invention, the liquid obtained after cell disruption is centrifuged at 8000-.
According to the invention, the obtained crude enzyme solution of the deacetylase of the bacillus licheniformis can be further purified according to actual conditions to obtain the chitin deacetylase with higher purity, the purification method is well known by the technical personnel in the field, and repeated details are not repeated here.
In a sixth aspect, the present invention provides a product comprising a chitin deacetylase as described above, or prepared by a process as described above.
According to the present invention, the product may be, for example, the crude enzyme solution of chitin deacetylase described above, a purified enzyme solution of chitin deacetylase, or an enzyme product prepared by artificially adding chitin deacetylase to a predetermined medium. The concentration of chitin deacetylase in the product may be from 0.01 to 99.99% by weight.
In a seventh aspect, the present invention provides the use of a chitin deacetylase as described above or a product as described above for deacetylation of chitin.
In an eighth aspect, the present invention provides a method for removing acetyl groups from chitin, the method comprising: bacillus licheniformis as described above, or a starter culture as described above, or a chitin deacetylase as described above, or a product as described above, is contacted with chitin to remove acetyl groups therefrom.
Preferably, the method for removing acetyl groups on chitin comprises the following steps: chitin deacetylase, as described above, or a product as described above, is contacted with chitin to remove acetyl groups therefrom.
The conditions of the contacting can be selected within a wide range, with a preferred pH of 4-13 (e.g., can be 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13), preferably 7-10; the temperature is 25-55 deg.C (for example, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C and 55 deg.C), preferably 30-45 deg.C.
According to the present invention, the enzyme activity under different temperature conditions is shown in FIG. 1, and it can be seen that Bacillus licheniformis Bl22 chitin deacetylase activity is highest at 40 ℃.
According to the present invention, the enzyme activity curves under different pH conditions are shown in FIG. 2, which shows that Bacillus licheniformis Bl22 chitin deacetylase has high activity at pH 6-10.
The enzyme activity as described above refers to the enzyme activity measured using a crude enzyme solution of Bacillus licheniformis deacetylase.
Wherein, the crude enzyme solution can be prepared by the following method: collecting fermentation liquor, performing solid-liquid separation, pouring out culture medium supernatant, and fully suspending thallus in ultrasonication buffer (45-55mM Tris-cl, 15-25mM NaOH, pH 7.5-8.5); and (3) placing the heavy suspension bacteria in an ice water mixed bath, carrying out ultrasonic crushing, carrying out solid-liquid separation on the crushed liquid, and obtaining the supernatant, namely the crude enzyme liquid of the bacillus licheniformis deacetylase.
The optimal catalysis temperature of the chitin deacetylase produced by the bacillus licheniformis Bl22 provided by the invention is relatively low, about 40 ℃, the chitin deacetylase shows very high catalytic activity in a wide pH range (pH value of 6-10), the cost of energy and auxiliary materials can be saved in industrial application, and the chitin deacetylase has a wide application prospect.
Examples
The following examples further illustrate the invention but are not intended to limit the invention thereto.
Example 1
This example illustrates the production of chitin deacetylase using Bacillus licheniformis Bl22
(1) Strain activation
The culture method comprises the following steps: taking a proper amount of ultra-low temperature preservation strains to an LB activation flat plate, marking, and placing in a constant temperature incubator for culturing at the temperature of 35 +/-2 ℃ for 20 hours.
Activating a culture medium: 5g/L of yeast extract, 10g/L of tryptone, 10g/L of L, NaCl 10g/L of agar powder and the balance of water, and the pH value is 6.5-7.2.
(2) Seed culture
The culture method comprises the following steps: picking single colony from the activation plate, placing the single colony in a seed culture medium for constant temperature shaking culture at the stirring speed of 150rpm and the temperature of 35 +/-2 ℃ for 9 hours.
Seed culture medium: 5g/L of yeast extract, 10g/L, NaCl 10g/L of tryptone and the balance of water, and the pH value is 6.5-7.2.
(3) Fermentation culture
The culture method comprises the following steps: inoculating the seed solution to a fermentation culture medium according to the inoculation amount of 1 volume percent, and performing constant-temperature shaking culture at the stirring speed of 150rpm and the temperature of 35 +/-2 ℃ for 20-24 h;
fermentation medium: 40g/L of glucose, 25g/L of yeast powder, 15g/L of tryptone, K2HPO 43 g/L and the balance of water, and the pH value is 6.5-7.2.
After 20-24h of fermentation, the chitin deacetylase content in the fermentation broth reaches 1800-. The test conditions were 40 ℃ temperature and 8.0 pH.
Example 2
This example illustrates the preparation of a crude chitin deacetylase solution from Bacillus licheniformis Bl22
Collecting 10mL fermentation liquor fermented for 23 hours, centrifuging at 12000rpm for 5min, pouring out culture medium supernatant, taking 10mL ultrasonication buffer (50mM Tris-cl, 20mM NaOH, pH8.0) to suck and beat up and down, and fully resuspending thalli; averagely dividing 10mL of the resuspended bacterial liquid into 10 parts, transferring each part of the resuspended bacterial liquid into 10 centrifugal tubes with the volume of 1mL, placing each centrifugal tube into a fixed ice-water mixed bath, and independently carrying out ultrasonic crushing, wherein an amplitude rod of an ultrasonic crusher is set to be 2, the power is set to be 20%, the ultrasonic frequency is set to be 2s/2s (namely 2s of crushing, 2s of pause), and the total time of crushing is 5 min; centrifuging all the crushed solutions at 12000rpm for 5min, and collecting the supernatant to a new 15ml centrifuge tube to obtain the crude enzyme solution of the bacillus licheniformis deacetylase.
Example 3
This example illustrates the enzymatic activity of chitin deacetylase under different conditions
The enzyme activity detection method comprises the following steps:
taking a 15ml centrifuge tube, adding 1ml of 200mg/l paranitroacetanilide aqueous solution, 1ml of crude enzyme solution diluted by proper times and 3ml of pre-insulated phosphate buffer solution with the concentration of 0.05M to ensure that the final volume of the reaction solution is 5ml, carrying out water bath reaction at 50 ℃ for 0.5h, carrying out boiling water bath for 5min, stopping enzymatic reaction, adding water to fix the volume to 10ml, mixing uniformly, centrifuging at 12000rpm for 5min, and measuring the light absorption value A400 of the supernatant at 400 nm. The blank control system was supplemented with 1ml of the same concentration of inactivated enzyme solution, and the absorbance A0 of the supernatant was measured as above for each sample.
Definition of enzyme activity unit: the amount of enzyme required to produce 1. mu.g of p-nitroaniline per hour under the above reaction conditions was defined as one unit of enzyme activity. The enzyme activity calculation formula is as follows:
enzyme activity (U/ml) ((A400-A0) × 10 × n)/KT
A400-absorbance of the sample at 400 nm; a0 — absorbance of blank; 10-volume of solution 10 ml; n is the dilution multiple of enzyme solution; k-linear coefficient (0.0648); t-reaction time, h
1) Optimum catalytic temperature of enzyme
Preparing a plurality of 1.5ml centrifuge tubes, adding 200 mul of 200mg/l p-nitroacetanilide aqueous solution, 200 mul of the crude enzyme solution (diluted by a proper amount) and 600 mul of phosphate buffer solution (pH8.0) with the concentration of 0.05M into the centrifuge tubes respectively, wherein the total volume of the reaction solution is 1ml, placing the centrifuge tubes into water baths with the temperature of 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃ and 55 ℃ respectively, placing 3 parallel centrifuge tubes under each temperature condition, reacting for 0.5h, boiling the water bath for 5min, terminating the enzymatic reaction, centrifuging at 12000rpm for 5min, and measuring the light absorption value A400 of the supernatant at 400 nm. The blank control system was added with 200. mu.l of the inactivated enzyme solution, and the absorbance A0 of the supernatant was measured as above for the rest, and a blank was set for each temperature condition.
The enzyme activity curves under different temperature conditions are shown in figure 1, the chitinase activity of the Bacillus licheniformis Bl22 is highest at 40 ℃, the enzyme activity of the chitinase at the temperature is defined as 100% (2100U/ml crude enzyme liquid), and the ratio of the enzyme activity data measured under the other temperature conditions to the enzyme activity at 40 ℃ is the corresponding relative enzyme activity.
2) Optimum catalytic pH of the enzyme
A96-well deep-well plate is taken, 200. mu.l of 200mg/l p-nitroacetanilide aqueous solution, 200. mu.l of the above crude enzyme solution (diluted by an appropriate factor) and 600. mu.l of phosphate buffer solutions with different pH values (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0 and 13.0) are respectively added into each well, the total volume of the reaction solution is 1ml, the reaction solution is placed in a shaking table at 40 ℃ for oscillation reaction for 0.5h, a boiling water bath is carried out for 5min, the enzymatic reaction is stopped, centrifugation is carried out at 12000rpm for 5min, and the absorbance A400 of the supernatant at 400nm is measured. The blank control system was added with 200. mu.l of the inactivated enzyme solution, and the absorbance A0 of the supernatant was measured as above, and a blank was set for each pH condition.
The graph of the enzyme activity curves under different pH values is shown in FIG. 2, when the pH value is 8, the activity of the chitin deacetylase of Bacillus licheniformis Bl22 is the highest (2100U/ml crude enzyme solution), the enzyme activity of the chitin deacetylase under the condition is defined as 100%, and the ratio of the enzyme activity data measured under the other pH values to the enzyme activity when the pH value is 8 is the corresponding relative enzyme activity. The bacillus licheniformis Bl22 chitin deacetylase has high activity at the pH value of 6-10, and the relative enzyme activity is 91.6-100%.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Claims (9)
1. Bacillus licheniformisBacillus licheniformis) Characterized in that the Bacillus licheniformisThe preservation number is CGMCC number 20490.
2. A fermentation product comprising the viable cell of Bacillus licheniformis according to claim 1.
3. The starter culture according to claim 2, wherein the content of the viable bacteria per gram of the starter culture is 10 5 -10 10 CFU。
4. The starter culture according to claim 2, wherein the content of the viable bacteria per gram of the starter culture is 10 7 -10 9 CFU。
5. Use of a bacillus licheniformis enzyme according to claim 1 or a leavening agent according to any of the claims 2-4 for producing chitin deacetylase and/or deacetylation of chitin.
6. A method for preparing chitin deacetylase, comprising: inoculating the bacillus licheniformis of claim 1 or the leavening agent of any one of claims 2-4 into a fermentation medium for fermentation to obtain a fermentation liquid, performing solid-liquid separation on the fermentation liquid to obtain a bacterial cell precipitate, and then crushing the bacterial cell to obtain a material containing chitin deacetylase.
7. A method for removing acetyl groups from chitin, the method comprising: contacting the Bacillus licheniformis of claim 1 or the leavening agent of any of claims 2-4 with chitin to deacetylate the same.
8. The method of claim 7, wherein the conditions of the contacting comprise: the pH value is 4-13; the temperature is 25-55 ℃.
9. The method of claim 7 or 8, wherein the conditions of contacting comprise: the pH value is 7-10; the temperature is 30-45 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011451720.1A CN112458022B (en) | 2020-12-09 | 2020-12-09 | Bacillus licheniformis Bl22 for high yield of chitin deacetylase and related products and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011451720.1A CN112458022B (en) | 2020-12-09 | 2020-12-09 | Bacillus licheniformis Bl22 for high yield of chitin deacetylase and related products and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112458022A CN112458022A (en) | 2021-03-09 |
| CN112458022B true CN112458022B (en) | 2022-08-16 |
Family
ID=74800671
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011451720.1A Active CN112458022B (en) | 2020-12-09 | 2020-12-09 | Bacillus licheniformis Bl22 for high yield of chitin deacetylase and related products and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112458022B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112175974A (en) * | 2020-09-23 | 2021-01-05 | 深圳润康生态环境股份有限公司 | Chitin deacetylase gene, chitin deacetylase and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630193A (en) * | 2013-11-06 | 2015-05-20 | 中国科学院海洋研究所 | Chitin deacetylase, and construction method and application thereof |
| CN106929456A (en) * | 2017-05-05 | 2017-07-07 | 淮海工学院 | A kind of temmoku acinetobacter calcoaceticus MCDA01 and its method for preparing chitin deacetylase |
| CN110093298A (en) * | 2019-05-17 | 2019-08-06 | 淮海工学院 | Ester perfume (or spice) microbacterium MCDA02 and its method for producing chitin deacetylase |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039193B (en) * | 2015-01-27 | 2016-08-17 | 安徽正方生物科技有限公司 | A kind of fermentable produces bacterial strain and the method for glucosamine |
-
2020
- 2020-12-09 CN CN202011451720.1A patent/CN112458022B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630193A (en) * | 2013-11-06 | 2015-05-20 | 中国科学院海洋研究所 | Chitin deacetylase, and construction method and application thereof |
| CN106929456A (en) * | 2017-05-05 | 2017-07-07 | 淮海工学院 | A kind of temmoku acinetobacter calcoaceticus MCDA01 and its method for preparing chitin deacetylase |
| CN110093298A (en) * | 2019-05-17 | 2019-08-06 | 淮海工学院 | Ester perfume (or spice) microbacterium MCDA02 and its method for producing chitin deacetylase |
Non-Patent Citations (2)
| Title |
|---|
| 几丁质脱乙酰酶产生菌的筛选;刘丽等;《中国酿造》;20110615(第06期);摘要 * |
| 培养条件对嗜热芽孢杆菌HU1合成几丁质降解酶及脱乙酰基酶的影响;戴德慧等;《安徽农业科学》;20110210(第05期);摘要 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112458022A (en) | 2021-03-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110257410B (en) | Gene for encoding algin lyase | |
| CN103468624B (en) | Genetic engineering bacteria used for high efficient production of mycose | |
| CN108018234B (en) | Bacterial strain for producing alginate lyase and application thereof | |
| CN110484471B (en) | Acid-resistant strain for high yield of bacterial cellulose and method for producing bacterial cellulose by using acid-resistant strain | |
| CN113684157B (en) | Bacillus subtilis for producing levansucrase and application thereof | |
| CN111961619B (en) | Vibrio maritima capable of producing alginate lyase with good thermal stability and application | |
| CN108546660B (en) | Chitin deacetylase high-yield strain and application thereof | |
| CN106635920B (en) | Marine alternans for high yield of fucosidase and application thereof | |
| CN112458022B (en) | Bacillus licheniformis Bl22 for high yield of chitin deacetylase and related products and application thereof | |
| CN107118980B (en) | Microbacterium keratanolyticum MCDA02 from sea, and its enzyme production method and product | |
| CN112111473A (en) | Chitosanase with high activity, preparation and application thereof | |
| JP2023534879A (en) | N-Acetylglucosamine-producing strain and method for construction and use thereof | |
| CN108913629B (en) | Bacterium for producing cellulase, preparation method and application thereof | |
| CN110093297B (en) | Nitrate reducing bacteria MCDA3-3 and method for producing chitin deacetylase by using same | |
| CN103468606A (en) | Klebsiella oxytoca and application thereof in allitol production | |
| CN112662572A (en) | Bacterial strain for high production of chitosanase and application thereof | |
| CN116731934B (en) | Escherichia coli and application thereof in production of glucosamine | |
| CN117229979B (en) | Extended microbubble strain for producing algin lyase and application thereof | |
| CN113151060B (en) | Bacillus lysine prolonging ZCGT05 and application thereof | |
| CN110656140A (en) | Method for improving chitin degradation rate by pretreating chitin with alkali freeze-thaw system | |
| CN116555094B (en) | Polysaccharide degrading bacteria of vibrio alginolyticus and culture method and application thereof | |
| CN115491321B (en) | Pseudomonas for efficiently producing extracellular polysaccharide, and preparation method and application thereof | |
| Ma et al. | Isolation and characterization of a thermostable alkaline chitinase-producing Aeromonas strain and its potential in biodegradation of shrimp shell wastes | |
| CN114134076B (en) | Bacillus licheniformis for producing extracellular polysaccharide, flocculant and application of flocculant in sewage treatment | |
| Ravikumar et al. | Production, optimization and characterization of chitin deacetylase from marine bacteria Bacillus cereus TK19 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |