CN112553123B

CN112553123B - Microbial inoculum combination for high-yield chitin deacetylase and application thereof

Info

Publication number: CN112553123B
Application number: CN202011574278.1A
Authority: CN
Inventors: 蔡俊; 郭依依; 王常高; 杜馨; 赵泽鑫
Original assignee: Hubei University of Technology
Current assignee: Hubei University of Technology
Priority date: 2020-12-25
Filing date: 2020-12-25
Publication date: 2021-10-15
Anticipated expiration: 2040-12-25
Also published as: CN112553123A

Abstract

The invention discloses a microbial inoculum combination for high-yield chitin deacetylase, which comprises Rhodococcus HQcdag (HQcdag)Rhodococcus sp.) M2020336 and Bacillus cereus CJPE209 (with the preservation number of CCTCC NO) ((II))Bacillus cereus) The preservation number is CCTCC NO: M2015734. Compared with the single fermentation of rhodococcus, the two strains improve the enzyme activity of the chitin deacetylase by about 14 times through synergistic fermentation, and obviously shorten the fermentation time.

Description

Microbial inoculum combination for high-yield chitin deacetylase and application thereof

Technical Field

The invention belongs to the technical field of microbial fermentation, and particularly relates to a microbial inoculum combination for high-yield chitin deacetylase and application thereof.

Background

Chitin (C8H13O5N) N, also called chitin, is a poorly soluble N-acetyl-D-glucosamine polymer present in the shell and fungal cell walls of various crustaceans. Chitosan is obtained when the deacetylation rate of the chitin reaches 55% or more, and the chitosan is widely applied to the fields of industry (cloth, clothes, dye, paper, water treatment and the like), agriculture (pesticides and plant antiviral agents), fishery (fish-farming feed, cosmetic and cosmetic agents, hair protection, moisturizers and the like), medical industry (contact lenses, artificial skin, sutures, artificial dialysis membranes, artificial blood vessels and the like), and the like, and particularly has good application prospects in the aspects of tissue engineering, wound healing and biosensors due to good biocompatibility in the biomedical field.

The preparation method of chitosan mainly comprises a chemical method, a physical method, a fermentation method, an enzymatic method and the like. The most widely used method is a chemical method, and chitosan is prepared by soaking chitin in a strong alkali solution at high temperature to remove acetyl. The chemical method for preparing chitosan has large alkali consumption, pollutes environment and has unstable characteristics of the obtained product (the acetyl degree is not uniform, the molecular weight change is large, and the position of acetyl is not fixed); the fermentation method is to extract chitosan by using waste mycelia of a fermentation factory, and the method can comprehensively utilize industrial waste residues, but the obtained product has more impurities and more complicated separation and purification steps; the physical method mainly comprises a microwave method and an ultrasonic method, and has high energy consumption and poor deacetylation effect. The chitosan prepared by the enzyme method is expected to solve the problems of the method and has good application prospect.

In the process of preparing chitosan by chitin enzymatic deacetylation, commonly used enzymes include chitinase, chitosanase, chitin deacetylase, chitobiase and the like. From the action mechanism, the chitinase, the chitosanase and the chitobiase all have the function of cutting off sugar chain molecules, only the chitin deacetylase is the specific enzyme for deacetylating the chitin, and the chitin deacetylase cannot cut off the sugar chain of chitosan molecules in the deacetylation process. The synergistic effect of other enzymes belonging to the same chitinase system on chitin deacetylase has not been reported, but there is a possibility.

Chitin deacetylase (e.c. 3.5.41, hereinafter abbreviated as CDA) can act on chitin and related derivatives of chitin, such as chitin oligosaccharide, carboxymethyl chitin, and aliphatic diol of chitin, and can also act on chitin in different states, such as colloidal chitin, powdered chitin, amorphous chitin, and crystalline chitin. The research finds that the hydrolysis process of chitin deacetylase is approximately as follows: firstly, chitin deacetylase is randomly bonded to any sequence of a substrate molecular chain, then deacetylation is carried out along the chain by taking a non-reducing end of a bonding part as a starting point, after hydrolysis is finished, the chitin deacetylase is dissociated from a substrate, and then the chitin deacetylase is bonded to another substrate molecular chain to start a new round of hydrolysis. It was found that the substrate (molecular chain length) of action had a great influence on the effect of the deacetylation of CDA. Martinou et al have studied that when Mucor rouxii-derived chitin deacetylase acts on fully water-soluble partially N-acetylated chitosan, it was found that when the substrate is water-soluble chitosan (30-unit polymer), three or more deacetylation sites of the substrate can be hydrolyzed by the chitin deacetylase. When the substrate is chitosan oligosaccharide (2-7 unit polymer), the chitosan deacetylase has activity only on three or more chitosan oligosaccharide substrates.

Most of chitin deacetylase producing strains are fungi, which are detected in more than ten fungi such as mucor, rhizopus, mortierella, Absidia, yeast and the like, and are first discovered in Mucor rouxii by Araki and Ito in 1974; as the bacteria producing this enzyme, there are known a few, including Brevibacterium, Rhodococcus erythropolis, Bacillus anthracis, Bacillus subtilis and the like. For example, the Colletotrichum lindemunianum strain discovered by Kauss and the like can secrete chitin deacetylase to the outside of cells, and the chitin deacetylase can be directly extracted from a culture solution, and the specific activity of pure protein can be as high as 72U/ml; the actinomycete as chitin deacetylase producing strain produces antibiotic to reduce contamination probability and inhibit enzyme activity. The actinomycete, micromonospora citriodora, screened by Wang Yao and the like can produce various antibacterial antibiotics such as gentamicin, roxamicin and the like, and has good deacetylation effect.

The breeding of the bacterial strain for producing the chitin deacetylase mainly has three approaches: 1. manually screening, and separating SUN and the like from the Hongkong soil sample to obtain a rhodococcus with the enzyme activity of 238.89U/ml; the two fungi with the strongest enzyme-producing ability, i.e., Aspergillus nidulans and Absidia coerulea, were identified from dozens of fungi deposited in the laboratory by Zea Jun et al. 2. Mutation breeding, Qin Wang Yan and the like, a high-yield CDA strain UV-210S is obtained by taking a marine filamentous fungus (Penicillium janthinellum)1-5-2 as an initial strain through ultraviolet mutation, and the enzyme activity can reach 16.76U/ml at most. 3. Construction of genetically engineered bacteria, Carole Gauthier et al, introduced three chitinase genes (RC, D2, and 13/2) derived from Rhizopus circinans into Pichia pastoris for expression, found that only one Recombinase (RC) had activity, presumably due to different secretion signal cleavage sites of the three enzyme proteins (RC and I3/2 are located upstream), and mis-selection of this site resulted in misfolding of the latter two recombinases.

In the reported literature at present, the literature related to the production of chitin deacetylase by synergistic fermentation is not found. The existing patent literature on chitin deacetylase mainly focuses on three aspects: 1. an enzyme method or microbial fermentation method is used for extracting chitosan from shrimp and crab shells or insect skin shells and other raw materials, for example, CN107653294A discloses a method for simultaneously preparing shrimp and crab melanin and chitosan from shrimp and crab wastes, which utilizes the synergistic action of bacillus subtilis and gluconobacter oxydans and glucose to ferment the shrimp and crab shells and heads of the shrimp and crab to desalt and deproteinize, then utilizes a streptococcus thermophilus, lactobacillus acidophilus and lactobacillus bulgaricus symbiotic system to ferment for a week to decalcify and decolor, utilizes chitin deacetylase to treat chitin, and utilizes gram-negative bacteria plesiomonas shigelloides to treat glucosamine. The method does not need to add a large amount of acid and alkali in the fermentation process, reduces the environmental pollution, realizes the reutilization of the waste raw materials, and is economical and feasible. CN101078023B discloses a method for preparing chitin/chitosan from the shells of silkworm chrysalis and fly maggots, which comprises the following steps: crushing and pulverizing silkworm pupa and fly maggot hulls by a dry method or a wet method; carrying out enzymolysis reaction on the obtained fine powder by using a lipase crude enzyme liquid and a protease crude enzyme liquid from microorganisms to fully remove fat and protein in the pupa skin/maggot skin raw material; decolorizing by a two-step method; collecting decolorized materials, washing and drying to prepare a chitin product; performing a circulating deacetylation reaction by a rhizopus oryzae whole-cell immobilized bioreactor to obtain chitosan; performing liquid submerged fermentation on chitosan by acid-producing bacteria to obtain highly water-soluble chitosan oligosaccharide. The method utilizes the immobilized cells to perform cyclic deacetylation, thereby greatly improving the product quality and the production efficiency. 2. CN101608177A uses shrimp shell culture soil as a screening raw material, uses antifungal drugs to inhibit the growth of fungi, sequentially adopts methods of plate coating, plate scribing and the like to purify, simultaneously uses N-p-nitroacetanilide (PN) -casein plate to carry out CDA activity verification, separates to obtain bacillus cereus capable of secreting CDA, and prepares CDA through liquid fermentation, the CDA activity of fermentation liquor supernatant is 0.598-0.912U/ml, the strain is suitable for large-scale industrial fermentation, and can be applied without processing and transformation. In addition, patent documents of other fungi and bacteria producing chitin deacetylase have also been reported. 3. CN102676485A discloses a method for preparing chitin deacetylase, which uses Scopulariopsis brevicaulis as a chitin deacetylase producing strain, after activation, the strain is inoculated into a fermentation container containing a fermentation culture medium, the strain is fermented for 90-100 hours on a shaking table with the pH value of 6.5-7.0, the temperature of 27-29 ℃ and the rotating speed of 200 plus materials of 240rpm, and the fermentation liquor is separated, salted out and purified to obtain the product of the chitin deacetylase, wherein the activity unit of each ml of the fermentation liquor of the chitin deacetylase prepared by the method can reach 36U at most. CN101659960A discloses a biological preparation method of chitin deacetylase derived from mucor racemosus, which comprises the following steps: (a) preparing a recombinant expression vector containing the chitin deacetylase with a gene sequence shown in SEQ ID NO. 1; (b) preparing a transformant containing the recombinant expression vector of step (a); (c) expressing and purifying chitin deacetylase. The invention designs gene specificity degenerate primers, adopts reverse transcription-polymerase chain reaction and rapid amplification cDNA fragment end technology, combines a recombinant DNA method, utilizes the prior prokaryotic expression vector to obtain an escherichia coli strain capable of producing the chitin deacetylase, provides a reliable source for preparing the chitin deacetylase by a biological method, greatly shortens the production period, reduces the extraction cost, and obtains the product enzyme with high yield and high quality.

Disclosure of Invention

Because the naturally bred bacterial strain for producing the chitin deacetylase has low enzyme yield, mostly is intracellular enzyme, is not easy to separate and purify, and is not easy to act on natural chitin, on one hand, a Rhodococcus Hqcdag (Rhodococcus sp) for producing the chitin deacetylase is screened out, on the other hand, the bacterial strain and Bacillus cereus CJPE209(Bacillus cereus) are found to be subjected to synergistic fermentation to obviously improve the enzyme activity of the chitin deacetylase, the chitin deacetylase can be produced at high yield by a synergistic fermentation method, and the fermentation time is greatly shortened.

In order to achieve the purpose, the invention adopts the following technical scheme:

the microbial inoculum composition for high yield of chitin deacetylase comprises Rhodococcus HQcdag (Rhodococcus sp.), with the preservation number of M2020336, and Bacillus cereus CJPE209(Bacillus cereus), with the preservation number of M2015734.

Further, in order to improve the enzyme activity of the chitin deacetylase, the ratio of viable bacteria of the bacillus cereus CJPE209 to the viable bacteria of the rhodococcus HQcdag is 1-2: 1-4; optimally, the ratio of viable bacteria of the bacillus cereus CJPE209 to the viable bacteria of the rhodococcus erythropolis HQcdag is 1: 4.

the application of the microbial inoculum combination in the production of chitin deacetylase or the degradation of chitin. In the specific embodiment of the invention, the optimal conditions (shake flask culture) for the synergistic fermentation of rhodococcus HQcdag and Bacillus cereus CJPE209 are optimized, and specifically: the fermentation time is 24h, the temperature is 35 ℃, the total inoculum size is 3%, the rotating speed is 160r/min, the liquid loading capacity is 50ml (the shake flask is 250ml), and the components of the culture medium are as follows: 2.5g/L yeast extract powder, 2.5g/L peptone and 5g/L, NaCl5g/L, K colloidal chitin₂HPO₄ 1g/L、KH₂PO₄ 1g/L、MgSO₄0.5g/L, and the enzyme activity of the chitin deacetylase under the condition is 421.4U/ml.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the naturally bred bacterial strain for producing the chitin deacetylase produces most of enzymes which are intracellular enzymes, the enzyme activity is not high, the natural chitin with a compact crystal structure is slowly degraded, and the fermentation period is long. The method for producing the chitin deacetylase by the synergistic fermentation of the two strains is adopted, so that the enzyme activity is increased to 226.27U/mL from 16.37U/mL of the single fermentation of the rhodococcus HQcdag, and the fermentation time is shortened to 24h from 72h reported in most documents.

Drawings

FIG. 1 is a growth curve of Rhodococcus HQcdag (Rhodococcus sp.).

FIG. 2 is a growth curve of Bacillus cereus CJPE209(Bacillus cereus).

FIG. 3 shows the result of the optimized ratio of Bacillus cereus CJPE209 to Rhodococcus HQcdag seed liquid.

FIG. 4 shows the effect of fermentation period on the enzymatic activity of chitin deacetylase.

FIG. 5 is a graph showing the effect of total inoculum size on chitin deacetylase enzymatic activity.

FIG. 6 shows the effect of fermentation speed on the enzymatic activity of chitin deacetylase.

FIG. 7 shows the effect of liquid loading on the enzymatic activity of chitin deacetylase.

FIG. 8 is a graph showing the effect of fermentation temperature on the enzymatic activity of chitin deacetylase.

FIG. 9 is an HPLC plot of oligosaccharide standards.

FIG. 10 is an HPLC chart of the degradation product of crude chitin enzyme solution.

Detailed Description

The foregoing aspects of the present invention are described in further detail below by way of examples, but it should not be construed that the scope of the subject matter of the present invention is limited to the following examples, and that all the technologies realized based on the above aspects of the present invention are within the scope of the present invention.

The first embodiment is as follows: breeding of strains

(1) Bacterial strains

Rhodococcus HQcdag (Rhodococcus sp.)

(2) Culture medium

Every 1L of the enrichment medium contains: NaCl 0.5g, K₂HPO₄ 1g、KH₂PO₄ 1g、MgSO₄0.1g of colloidal chitin and 5g of liquid culture medium with natural pH;

each 1L of the screening medium contained: NaCl 0.5g, K₂HPO₄ 1g、KH₂PO₄ 1g、MgSO₄0.1g of colloidal chitin, 5g of p-nitroacetanilide and 20g of agar, wherein the pH of the flat solid culture medium is natural;

every 1L of the seed medium on the shake flask contained: 10g of peptone, 3g of beef extract and 5g of sodium chloride, wherein the pH of the liquid culture medium is natural;

each 1L of the solid slant medium contains: 10g of peptone, 3g of beef extract, 5g of sodium chloride and 20g of agar, and sterilizing for 20min under 0.1MPa, wherein the pH of the slant solid culture medium is natural;

every 1L of the shake flask is re-screened with a fermentation medium containing: 2.5g yeast extract powder, 2.5g peptone, 5g colloidal chitin, 5g NaCl, K₂HPO₄ 1g、KH₂PO₄ 1g、MgSO₄0.5g, and the pH of the fermentation medium is 7.0.

(3) Solutions used in the experiments

Sterile physiological saline: dissolving 8.5g sodium chloride in 1L distilled water, and sterilizing at 0.1MPa for 20 min.

(4) Screening method

15 parts of soil sample are collected from a crayfish breeding factory. Firstly, taking a certain sample from each sample, and scattering the sample by using sterile normal saline; then culturing for 1d at 30 ℃ by using an enrichment culture medium; then diluting the suspension into different soil suspensions by using physiological saline in a gradient manner, coating 0.1ml of the suspension on a screening plate, culturing the suspension for 2-3d at the temperature of 37 ℃, and selecting a strain with a large and obvious color circle as a primary screening strain; picking out single colony on the plate, and inoculating the single colony into a slant solid culture medium for preservation and standby.

The method for measuring the enzymatic activity of the chitin deacetylase comprises the following steps: a test tube was taken and 1ml of 200mg/l p-nitroacetanilide solution, 1ml of cell disruption supernatant of appropriate concentration and 3ml of 0.05mol/l phosphate buffer solution pre-incubated at 50 ℃ were added in this order to make the reaction volume 5 ml. Reacting in 50 deg.C water bath for 30min, terminating the enzymatic reaction in boiling water bath, centrifuging, and measuring the light absorption value (A) of the supernatant₄₀₀). The blank control system was added with 1ml of the same concentration of the inactivated enzyme solution, and the absorbance of the supernatant (A) was measured₀) One blank for each sample.

One enzyme activity unit (U) is determined as: the amount of the enzyme required to produce 1. mu.g of p-nitroaniline per hour at 45 ℃ using p-nitroacetanilide as a substrate was defined as one unit of the enzyme activity.

Enzyme activity (U/ml) ═ A₄₀₀-A₀) Xenzyme dilution/KXT (K is determined from a standard curve, T means the enzymatic reaction time, h)

A Rhodococcus erythropolis HQcdag (Rhodococcus sp.) with strong capability of producing chitin deacetylase is obtained through a shake flask re-screening fermentation test, is preserved in China center for type culture Collection (preservation Address: Wuhan university, China) in 7-21 months in 2020, and has the preservation number of CCTCC NO: M2020336.

Example two: seed liquid proportioning optimization of double-strain synergistic fermentation

(1) The strain is as follows: example I selected strains of Rhodococcus erythropolis HQcdag (Rhodococcus sp.) and Bacillus cereus CJPE209(Bacillus cereus) with a preservation number of CCTCC NO: M2015734.

(2) The method comprises the following steps:

the culture medium is seed culture medium (peptone 10g/L, beef extract 3g/L, sodium chloride 5 g/L). The culture conditions were: the natural pH value, the inoculation amount of 1 percent and the liquid loading amount of 100ml/300ml, 37 ℃ and 180 r/min.

a. Optimization test of mixing time of two strains

The growth curves of the two strains were measured separately, and their logarithmic growth phases were found as shown in FIGS. 1 and 2.

b. Optimization experiment of double-strain inoculation amount ratio

The number of viable bacteria is measured at the 20 th hour of the logarithmic growth phase of the two strains in the growth period, and then the viable bacteria are converted into a volume ratio according to a certain ratio (9: 1, 8: 2, 7: 3, 6: 4, 5: 5, 4: 6, 3: 7, 2: 8, 1: 9 and 0: 10) and inoculated into a fermentation medium for fermentation. As shown in FIG. 3, the ratio of viable bacteria of Bacillus cereus to Rhodococcus erythropolis was 2: when 8, the enzyme activity of the chitin deacetylase is the highest, and is 226.27U/ml.

Example three: culture condition optimization of double-strain synergistic fermentation

(1) The strain is as follows: example one of the screened strains Rhodococcus erythropolis HQcdag (Rhodococcus sp.) and Bacillus cereus CJPE209(Bacillus cereus) have a viable count ratio of Bacillus cereus to Rhodococcus 2: 8.

(2) the method comprises the following steps:

the fermentation medium is initial medium (yeast extract powder 2.5g/L, peptone 2.5g/L, colloidal chitin 5g/L, NaCl5g/L, K)₂HPO₄ 1g/L、KH₂PO₄ 1g/L、MgSO₄0.5 g/L). The initial fermentation conditions were: natural pH, the inoculum size of 1 percent, the liquid loading amount of 50ml/250ml, 37 ℃ and 180r/min shake flask fermentation for 24 h.

The influence of the fermentation period, the total inoculation amount, the fermentation speed, the liquid loading amount and the fermentation temperature of the double-strain synergistic fermentation on the yield of the chitin deacetylase is inspected by adopting a single-factor test.

a. Preferred testing of fermentation cycles

And other fermentation conditions are initial fermentation conditions, sampling every 12h to measure the activity of the chitin deacetylase, and determining the optimal fermentation period of the double-strain synergistic fermentation.

As can be seen from FIG. 4, the enzyme activity of chitin deacetylase was the highest at 222.4U/ml when the fermentation time reached 24 hours, so the fermentation period was selected to be 24 hours.

b. Preferred test of total inoculum size

And (3) taking the optimal result determined by the test in the fermentation time, and inspecting the influence of the total inoculation amount on the synergistic fermentation of the double strains to produce the chitin deacetylase when the other fermentation conditions are initial fermentation conditions and the total inoculation amounts are 1%, 3%, 5%, 7% and 9% respectively.

As can be seen from FIG. 5, when the total inoculum size was 3%, the enzyme activity of the chitin deacetylase was the highest at 234.7U/ml, and the total inoculum size was selected to be 3%.

c. Optimization test of fermentation rotation speed

The fermentation time and the inoculation amount are measured to obtain the optimal result determined by the test, other fermentation conditions are initial fermentation conditions, the fermentation rotating speeds are adjusted to be 140r/min, 160r/min, 180r/min, 200r/min and 220/min respectively, the enzyme activity of the chitin deacetylase is measured after shake flask fermentation culture, and the influence of the fermentation rotating speeds on the production of the chitin deacetylase by the synergistic fermentation of the double strains is inspected.

As can be seen from FIG. 6, when the fermentation speed is 160r/min, the enzyme activity of the chitin deacetylase reaches 292.9U/ml, so the fermentation speed is selected to be 160 r/min.

d. Optimization test of liquid loading of fermentation flasks

Taking the optimal results determined by the tests on the fermentation time, the inoculation amount and the rotating speed, taking other fermentation conditions as initial fermentation conditions, adopting 250ml shake flask liquid loading, and observing the influence of the liquid loading on the production of the chitin deacetylase by the synergistic fermentation of the double strains when the liquid loading of the shake flask is respectively 25ml, 50ml, 75ml, 100ml and 125 ml.

As is clear from FIG. 7, the liquid filling amount was selected to be 50ml (250 ml in the shake flask volume) because the enzyme activity of the chitin deacetylase was 308.6U/ml when the liquid filling amount was 50ml (250 ml in the shake flask volume).

e. Preferred test of fermentation temperature

Measuring the optimal result determined by the test on the fermentation time, the inoculation amount, the rotating speed and the liquid loading amount, taking other fermentation conditions as initial fermentation conditions, performing fermentation culture in a shaking table at 30 ℃, 35 ℃, 37 ℃, 40 ℃ and 45 ℃ respectively, determining the enzymatic activity of the chitin deacetylase, and inspecting the influence of the temperature on the production of the chitin deacetylase by the synergistic fermentation of the double strains.

As can be seen from FIG. 8, when the fermentation temperature was 35 ℃, the enzyme activity of the chitin deacetylase was 401.28U/ml, and thus 35 ℃ was selected as the fermentation temperature.

f. Optimum culture conditions

The culture conditions comprise 24h of fermentation time, 35 ℃ of temperature, 3 percent of total inoculum size, 160r/min of fermentation speed and 50ml of liquid loading capacity (250 ml of shake flask); the medium components (per 1L contained): 2.5g yeast extract powder, 2.5g peptone, 5g colloidal chitin, 5g NaCl, K₂HPO₄ 1g、KH₂PO₄ 1g、MgSO₄0.5g, fermentation verification test is carried out. The enzyme activity of the chitin deacetylase in the fermentation liquor obtained after fermentation is 421.4U/ml.

Example four: verification experiment for promoting rhodococcus to produce chitin deacetylase by using chitinase and HQcdag

a. Verification experiment for chitin enzyme produced by fermentation of bacillus cereus

Bacillus cereus CJPE209(Bacillus cereus) is used as a fermentation strain; the culture conditions are the same as the optimal culture conditions in the third embodiment, and the culture solution is centrifuged at 6000r/min for 10min to remove the precipitate, so as to obtain the fermentation supernatant. 0.5mL of appropriately diluted enzyme solution and 1.5mL of 1.0% colloidal chitin (the colloidal chitin is dissolved in 0.05mol/LpH7 phosphate buffer solution) are mixed uniformly and reacted for 30min at 50 ℃. After the reaction, the reaction was terminated by boiling water bath for 10 min. 3mL of DNS solution is added, the mixture is cooled immediately after boiling water bath for 10min, and deionized water is used for constant volume until the volume is 25 mL. And taking the inactivated crude enzyme solution as a blank control, measuring light absorption values at 490nm, and taking an average value for each group of three parallels. The amount of reducing sugars for the enzyme and substrate reactions was calculated using D-glucosamine hydrochloride as a standard curve. Definition of enzyme activity: under certain conditions, enzymatic degradation of the substrate yields 1. mu. mol of reducing sugars per minute as 1U. The activity of the chitinase is measured to be 1.91U/ml.

b. Verification experiment for promoting rhodococcus to produce deacetylase by using chitin enzyme enzymolysis product

Taking Bacillus cereus CJPE209(Bacillus cereus) as a fermentation strain, fermenting for 96h, centrifuging and taking supernatant to obtain the crude chitinase solution. Respectively numbering 3 test tubes as 1, 2 and 3, and adding 10ml of 0.5% colloidal chitin. Adding 3ml of crude chitinase enzyme solution into a test tube 1, adding 3ml of inactivated crude chitinase enzyme solution into a test tube 2, adding 3ml of water into a test tube 3, and incubating at 30 ℃ for 72 hours to respectively obtain a raw material 1, a raw material 2 and a raw material 3. Respectively preparing fermentation culture medium 1 (culture medium components comprise yeast extract powder 2.5g/L, peptone 2.5g/L, NaCl5g/L, K)₂HPO₄ 1g/L、KH₂PO₄ 1g/L、MgSO₄0.5g/L + raw material 1), 2 (the components of the culture medium are yeast extract powder 2.5g/L, peptone 2.5g/L, NaCl5g/L, K₂HPO₄ 1g/L、KH₂PO₄ 1g/L、MgSO₄0.5g/L + raw material 2), 3 (the components of the culture medium are yeast extract powder 2.5g/L, peptone 2.5g/L, NaCl5g/L, K₂HPO₄1g/L、KH₂PO₄ 1g/L、MgSO₄0.5g/L + raw material 3) was used for Rhodococcus HQcdag (Rhodococcus sp.) fermentation under the same optimum culture conditions as in example three. After 24 hours of fermentation, the enzyme activities of the chitin deacetylase are measured to be 120.35U/ml, 18.29U/ml and 14.53U/ml respectively. The colloidal chitin treated by the crude enzyme solution of the chitinase can promote the rhodococcus to produce the chitin deacetylase.

c. Analysis of degradation products of chitinase

Taking the raw material 1 prepared in the step b to pass through a liquid phase special organic film with the diameter of 0.22 mu m for standby. Configuring GlcNAc (GlcNAc) with concentration of 1mg/ml₂、(GlcNAc)₃、(GlcNAc)₄、(GlcNAc)₅、(GlcNAc)₆And (4) standard sample liquid. The enzymatic hydrolysate was assayed using a prominine LC-20AD (Shimadzu, Japan) and a 4.6mm X250 mm amino liquid chromatography column (Elite, China) with a mobile phase composition of V (acetonitrile): v (water) ═ 7: 3, selecting an ultraviolet detector as the detector, wherein the wavelength is 195nm, the flow rate is 0.5mL/min, the column temperature is 30 ℃, and the sample injection amount is 10 μ L. As can be seen from FIG. 9, the crude chitin degradation product showed a peak at 11.36min and in the standard (GlcNAc)₂The peak time of the chitin is close, which indicates that the chitin enzymolysis product is (GlcNAc)₂。

Claims

1. The microbial inoculum combination for high yield of chitin deacetylase is characterized by comprising Rhodococcus HQcdag (HQcdag)Rhodococcussp.) M2020336 and Bacillus cereus CJPE209 (with the preservation number of CCTCC NO) ((II))Bacillus cereus) The preservation number is CCTCC NO: M2015734.

2. The microbial inoculant combination of claim 1, wherein the ratio of viable bacteria of bacillus cereus CJPE209 to that of rhodococcus HQcdag is 1-2: 1-4.

3. The microbial inoculant combination of claim 1 or 2, wherein the ratio of viable bacteria of bacillus cereus CJPE209 and rhodococcus HQcdag is 1: 4.

4. use of a combination of agents according to any one of claims 1 to 3 for the production of chitin deacetylase.

5. Use of a combination of agents according to any one of claims 1 to 3 for degrading chitin.