CN115125227B - Chitin complex enzyme and application thereof - Google Patents

Abstract

The invention relates to a chitinase complex enzyme and application thereof, wherein the chitinase complex enzyme is a mixed enzyme of Chi-h, chtI and ChtII, solid chitin is taken as a substrate, the chitinase complex enzyme is adopted for hydrolysis, the total amount is 1 mu M, the amount of produced glucosamine is measured by a potassium ferricyanide method, and the conversion of more than 75% of the substrate can be realized in 4 hours through calculation. The invention utilizes the synergistic effect of various enzymes in the insect chitin degradation system to realize the efficient degradation of chitin in a short time and produce glucosamine.

Description

Chitin complex enzyme and application thereof

Technical Field

The invention relates to an enzymolysis reaction, in particular to a chitin complex enzyme and application thereof.

Background

Chitin is a natural polysaccharide polymerized from N-acetylglucosamine through beta-1, 4 glycosidic linkages, also known as chitin or chitin. Chitin is the nitrogenous polysaccharide with the highest annual yield in nature, about 100 hundred million tons, and is mainly present in the cell walls of fungi, the exoskeletons of arthropods such as shrimps and crabs, and the epidermis of insects, and is an important biomass resource. The degradation product amino sugar can be widely applied to the fields of food, agriculture, medicine and the like, and has important economic value.

At present, chitin can be degraded by a chemical method or a biological enzyme method, but the common chemical degradation method consumes a large amount of strong acid and alkali, has huge environmental pollution, complex components and poor quality of products, is environment-friendly by adopting the biological enzyme method to degrade chitin, has little pollution, and meets the requirement of green sustainable development. One major problem in degrading chitin by bioenzyme method is that chitin existing under natural conditions exists in a compact solid form, and a large number of hydrogen bonds exist between internal molecules, so that the efficiency of degrading chitin by adopting single enzyme is very low, and the application of chitin as biomass resource is limited. Chitin degrading systems are found in microorganisms that use chitin as a carbon source and in organisms that contain chitin, such as insects, and include a variety of different types of enzymes, such as, for example, the lytic chitinase monooxygenase (LPMO), chitinase (Cht), and beta-N-acetylhexosaminidase (Hex). The Chinese patent 'a method for degrading N-acetylglucosamine of chitin living beings by using insect chitinase' (Zl 2018100442700) discloses that chitinase H and chitinase I are adopted as mixed enzymes for enzymolysis of chitin microfilaments, and the chitinase H and the chitinase I have synergistic effect and can realize high-efficiency degradation of crystalline chitin. However, the solid chitin must be broken into chitin microfilaments to be degraded, and chitinase H and chitinase I as mixed enzymes hardly degrade the solid chitin.

Disclosure of Invention

Aiming at the problems existing in the chitin degradation process, the invention provides a chitin complex enzyme, which utilizes the synergistic effect of a plurality of enzymes in an insect chitin degradation system to realize the efficient degradation of chitin in a short time and a method for producing glucosamine. The reaction process has mild condition and is easy to separate products and realize the reaction process.

The invention adopts the specific technical scheme that:

a chitin complex enzyme consists of chitinase Chi-h, chu I and Chu II-B4C1, and the molar ratio is 1: (0.5-3): (0.5-3).

The molar ratio of the chitinase Chi-h, the chitI and the Chul-B4C 1 is 1:1 to 3:1 to 3.

The molar ratio of the chitinase Chi-h, the chitI and the Chul-B4C 1 is 1:1:3.

a method for producing glucosamine by degrading solid chitin with insect chitin degradation system uses solid chitin as substrate, and adopts the chitin complex enzyme to degrade and separate to obtain glucosamine.

The substrate is suspended in a buffer solution with the pH value of 6.0-7.0 and the total adding concentration of the chitin complex enzyme is 1 mu M, the substrate concentration is 0.5-2mg/mL, and the degradation time is 4-24 hours.

The solid chitin is defatted shrimp shell waste powder, the degradation is carried out in two steps, a substrate is pretreated by protease to hydrolyze protein, the precipitate is collected after centrifugation, and then the chitin complex enzyme is adopted for enzymolysis precipitation.

The pretreatment is to suspend a substrate in a buffer solution with pH of 7.0-8.0, and adding protease with final concentration of 100000U/g for pretreatment hydrolysis, wherein the enzymolysis is to suspend a precipitate obtained by separation after pretreatment in a buffer solution with pH of 6.0-7.0 and adding chitin complex enzyme for enzymolysis, and the addition concentration of the chitin complex enzyme is 1 mu M.

The pretreatment hydrolysis condition is that the temperature is 20-40 ℃, the hydrolysis time is 2-6 hours, the enzymolysis condition is that the temperature is 20-40 ℃, and the enzymolysis time is 4-24 hours.

The pH7.0-8.0 and the pH6.0-7.0 buffer solution is phosphate buffer solution or ammonium acetate buffer solution.

The solid chitin is solid granular chitin in a crystalline state, and the granularity is smaller than 50 meshes.

The chitinase complex enzyme is a mixed enzyme of Chi-h, chu I and Chu II, and the total addition amount is 1 mu M. The amount of glucosamine produced was determined by the potassium ferricyanide method and calculated to achieve a substrate conversion of more than 75% at 4 hours.

The solid chitin referred to in the present invention, also called alpha-chitin, refers to solid particulate chitin that is mainly in a crystalline state, preferably with a particle size that can be sieved through a 50 mesh screen. The solid chitin is not treated by acid, and has compact structure. There is a great difference from colloidal chitin which is loose in structure and does not contain a dense structure.

The chitinase complex enzyme can be used for degrading shrimp and crab shells, and is prepared by suspending a substrate in a buffer solution with pH of 7.0-8.0, adding protease with the final concentration of 100000U/g of the substrate for pretreatment, and carrying out solid-liquid separation to obtain a first supernatant and a first precipitate, wherein the first supernatant is shrimp shell powder protein hydrolysate, and the first precipitate is chitin.

The protease is preferably a complex animal protease, which is available directly (Guangxi Pang Bo organism). The temperature is preferably 40 ℃. The hydrolysis time is preferably 2 to 6 hours;

the particle size of the defatted shrimp shell waste powder is preferably 50 mesh.

The first precipitate chitin is suspended in 20mM buffer, pH 6.0-7.0. The addition concentration of the main effect part is 1 mu M, so that the conversion of the substrate exceeding 75% can be realized in 4 hours;

in the practical application process, a person skilled in the art can select a proper gene expression vector according to self experimental conditions, and express and prepare the three enzymes. In the invention, the insect chitin complex enzyme is obtained by utilizing the existing pichia pastoris engineering strain to respectively express insect chitinase ofChi-h, chitinase ofChut I and chitinase ofChut II, and then separating the recombinant protein from the supernatant of yeast fermentation liquor. More specifically, the separation is to separate the corresponding recombinant protein from the supernatant of the yeast fermentation broth by metal chelate chromatography.

Proteases are commercially available products.

The chitinase complex enzyme is a mixed enzyme of Chi-h, chu I and Chu II, and the three enzymes are synergistic with each other to improve the enzymolysis rate of chitin and the enzymolysis efficiency of chitin; the conversion of more than 75% of the substrate to glucosamine was calculated to be able to be achieved in 4 hours, degrading solid chitin to glucosamine. The reaction process has mild condition and is easy to separate products and realize the reaction process.

Advantageous effects

1. There is currently no study of the combined use of a number of different types of insect chitin degrading enzymes for chitin degradation. The invention combines and applies a plurality of insect chitin degrading enzymes for the first time.

2. And the multienzyme system is adopted, the synergistic effect of the multienzyme system is utilized for degradation, and the reaction speed is high.

2. The method adopts various enzymes to degrade the shrimp shells, avoids the use of strong acid and strong alkali reagents in the traditional process, and is environment-friendly.

Drawings

FIG. 1 is a graph showing the effect of hydrolysis of different chitinase ratios.

FIG. 2 is a graph showing the effect of chitinase mixture on the synergistic hydrolysis of chitin after optimizing the ratio.

FIG. 3 is a graph showing the effect of protease on degrading proteins in shrimp and crab shells.

FIG. 4 is a graph showing the effect of chitinase mixture in optimized ratio on degrading chitin.

FIG. 5 is a graph showing the effect of protease and chitinase on degrading shrimp shell.

Detailed Description

The present invention will be described in further detail with reference to examples.

Example 1 optimization of chitinase

Table 1{3,5} simplex method examined chitin degrading activities of different chitinase ratios

The {3,5} simplex method is used to realize the optimization of the proportion of chitinase. Different enzyme ratios are set, solid alpha-chitin is used as hydrolysis substrate, and the change of hydrolysis effect of three chitinase under different ratios is explored. Phosphate buffer, pH 6.5, was added to the alpha-chitin to a chitin concentration of 2mg/mL and total chitinase addition of 1. Mu.M. The reaction was carried out at 30℃for 6 hours with shaking at 200 rpm. After the reaction, the amount of chitinase hydrolysate was measured by the potassium ferricyanide method, and 60. Mu.L of the sample was taken, 180. Mu.L of potassium ferricyanide solution (2 mg/mL) was added thereto, and the mixture was boiled for 15 minutes. Centrifugation was performed at 17000 Xg for 5 minutes, 200. Mu.L of the supernatant was placed in a 96-well plate, and absorbance at 420nm was measured. Substituting the absorbance into a standard curve to convert the yield of the reducing sugar. As a result, the optimal ratio of chitinase was determined to be a ratio of Chi-h, chu I and Chu II-B4C1 of 1:1:3 (FIG. 1). When the ratio of the three enzymes is in the area A in FIG. 1, the yield can reach more than 90% of the maximum value, and a more obvious effect can be obtained.

TABLE 2 computer simulation of chitin hydrolase ratios

Example 2 insect chitinase hydrolysis of chitin

The substrate used in the examples was 2mg/ml chitin, suspended in 20mM phosphate buffer pH 6.5, and the total amount of chitinase added was 1. Mu.M, and the ratio of Chi-h, chu I and Chu II-B4C1 was 1:1:3, and reacted by shaking at 200rpm at 30 ℃. Samples were taken at 6, 12 and 24 hours, respectively, and the content of reducing sugar produced in the system was measured by the potassium ferricyanide method. 24 hours can produce 2.33mM of reducing sugar (FIG. 2).

Example 3 Combined enzymatic degradation of shrimp Shell powder

The substrate used in the examples was defatted shrimp shell waste powder. The peptide bond in the protein is broken down into amino acids by proteases (Nanning Pang Bo bioengineering Co., ltd., neutral protease, complex protease, bromelain). Adding phosphate buffer solution with pH of 7.5 into shrimp shell waste powder to make the concentration of the shrimp shell powder be 2mg/L, adding substrate with enzyme quantity of 100000U/g, oscillating at 200rpm for 4 hours at 40 ℃, boiling in boiling water for 5 minutes after the reaction, centrifuging at 12000rpm for 5 minutes, wherein the obtained supernatant is shrimp shell powder protein hydrolysate, and precipitating to be chitin which can be used as a substrate for the next reaction. The amount of protein in the supernatant was determined by the forskolin method. Residual = total protein-proteolytic amount. Under the optimal reaction condition, the effect of the compound protease in hydrolyzing the shrimp shell meal is better than that of other proteases (figure 3), the protein residue after 4 hours is only 9.76%, and the compact structure of protein-chitin in the shrimp shell is fully destroyed.

The shrimp shell is hydrolyzed by composite protease, the sediment is resuspended by adding deionized water, repeatedly washed for 3 times, phosphate buffer solution with pH of 6.5 is added to the original volume, at the moment, the chitin concentration is about 0.5mg/ml, the total addition amount of the chitinase is 1 mu M, the ratio of Chi-h, chu I and Chu II-B4C1 is 1:1:3, and the shaking reaction is carried out at 200rpm under the condition of 30 ℃. Samples were taken at 2, 4, 6, 12 and 24 hours, respectively. After the reaction, the amount of chitinase hydrolysate was measured by potassium ferricyanide method. The 6-hour yield of reducing sugar was calculated to reach 1.05mM. Chitin degradation rate was over 75% as measured by weight loss method (fig. 4). The crab shell can be effectively degraded by the combination of protease and compound chitinase, and the remainder is ash (figure 5).

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (3)

1. A chitin complex enzyme consists of chitinase Chi-h, chu I and Chu II-B4C1, and the molar ratio is 1:1 to 3:1 to 3.

2. The chitin complex enzyme of claim 1, wherein the molar ratio of chitinase Chi-h, chtI, and ChtI-B4C 1 is 1:1:3.

3. a method for producing glucosamine by degrading solid chitin by utilizing an insect chitin degradation system, which takes the solid chitin as a substrate, adopts the chitin complex enzyme of any one of claims 1-2 to degrade, and separates to obtain the glucosamine;

the substrate is suspended in a buffer solution with the concentration of 20mM and the pH value of 6.0-7.0, the total addition concentration of the chitin complex enzyme is 1 mu M, the concentration of the substrate is 0.5-2mg/mL, and the degradation time is 4-24 hours;

the solid chitin is defatted shrimp shell waste powder, the degradation is carried out in two steps, a substrate is pretreated by protease to hydrolyze protein, the precipitate is collected after centrifugation, and then the chitin complex enzyme is adopted for enzymolysis precipitation;

the pretreatment is to suspend a substrate in a buffer solution with pH of 7.0-8.0, and adding protease with final concentration of 100000U/g for pretreatment hydrolysis, wherein the enzymolysis is to suspend a precipitate obtained by separation after pretreatment in a buffer solution with pH of 20mM and pH of 6.0-7.0, and adding chitin complex enzyme for enzymolysis, wherein the addition concentration of the chitin complex enzyme is 1 mu M;

the pretreatment hydrolysis condition is that the temperature is 20-40 ℃, the hydrolysis time is 2-6 hours, the enzymolysis condition is that the temperature is 20-40 ℃, and the enzymolysis time is 4-24 hours;

the pH7.0-8.0 and the pH6.0-7.0 buffer solution is phosphate buffer solution or ammonium acetate buffer solution;

the solid chitin is solid granular chitin in a crystalline state, and the granularity is smaller than 50 meshes.