CN113481186B - GH18 chitinase ChiA and application thereof - Google Patents

Abstract

The invention relates to a GH18 chitinase ChiA and application thereof. The present invention relates to the strain Streptomyces diastaticus (Streptomyces diastaticus) CS1801 found a novel chitinase gene PROKKA01070, the gene sequence of which is shown in SEQ ID NO:1, the total length is 1065bp, and the coded chitinase ChiA amino acid sequence is shown as SEQ ID NO:2, comprises 354 amino acids in total. The chitinase gene PROKKA01070 is cloned and connected with an expression vector pET32a (+) to transform escherichia coli BL21 (DE 3), and after culture and induced expression, the recombinant expression chitinase ChiA is obtained. The optimal reaction temperature of the enzyme is 50 ℃, the optimal pH value is 7.0, and the enzyme activity is most stable in a buffer solution with the pH value of 7.0 and the temperature of 25 ℃. Chitinase ChiA can degrade chitin to prepare chitooligosaccharide, has substrate specificity on colloidal chitin, provides a new way for producing chitooligosaccharide by an enzyme method, and has important application prospect.

Description

A kind of GH18 chitinase ChiA and its application

technical field

The invention relates to the technical field of microorganisms and genetic engineering, in particular to a GH18 chitinase ChiA and an application thereof.

Background technique

Chitinase belongs to the glycosyl hydrolase family, which can specifically catalyze the hydrolysis of β-1,4 glycosidic bonds of chitin to generate chitooligosaccharides, chitosan and N-acetylglucosamine. The degradation products of chitin, chitooligosaccharides, chitooligosaccharides and N-acetylglucosamine, can be widely used in the fields of biomedicine, food, cosmetics, textiles, agriculture, etc., and have universal biological activity and broad market development prospects. The use of chitin enzymatically to produce chitooligosaccharides and chitosan has mild conditions and little environmental pollution, and is a development direction for the preparation of chitooligosaccharides and the like.

At present, about 46 genera and nearly 70 species of microorganisms that produce chitinase have been reported, mainly including bacteria, actinomycetes and fungi. However, the reported strains have low chitinase production, low production efficiency, and poor tolerance, and there is still a certain distance from industrial production. Therefore, the discovery of new chitinase genes and the selection of chitinase-producing strains with strong tolerance and high chitinase activity have important guiding significance for the industrialization of chitinase. With the continuous advancement of molecular biology technology, compared with wild strains, engineering bacteria obtained by means of genetic engineering often have the characteristics of high expression level, strong product specificity, high production efficiency, short reproduction cycle, and easy separation. However, the difficulty in constructing genetically engineered bacteria lies in the construction of cloning expression vectors and the expression of heterologous proteins.

Contents of the invention

The purpose of the present invention is to provide a GH18 chitinase ChiA and its application. The chitinase has high activity and can be used for catalytic production of chitooligosaccharides.

In order to realize the above-mentioned technical purpose, the present invention adopts following technical scheme:

A GH18 chitinase ChiA, the nucleotide sequence of the gene PROKKA01070 encoding the GH18 chitinase is shown in SEQ ID NO:1.

The aforementioned GH18 chitinase ChiA is derived from Streptomyces diastaticus CS1801, which has been disclosed in the applicant's previous application CN109337843A.

The present invention also provides a recombinant vector, which comprises the gene PROKKA01070 encoding GH18 chitinase and an expression vector. The expression vector is pET-32a(+). The above genes were inserted between the multiple cloning sites of the expression vector pET-32a(+).

The present invention also provides a genetically engineered bacterium comprising the above-mentioned recombinant vector.

Further, the engineering bacteria host cell is E.coli BL21(DE3).

The present invention also provides the application of the above engineering bacteria in the enzymatic production of chitosan oligosaccharides.

Preferably, the GH18 chitinase ChiA uses colloidal chitin as a substrate to produce chitooligosaccharides.

Further, the present invention provides a specific application mode, including:

(1) inserting genetically engineered bacteria comprising GH18 chitinase into LB medium for seed culture;

(2) The seed liquid is transferred to the fermentation medium for fermentation and cultivation, and then an inducer is added to induce the expression of enzymes;

(3) centrifuging the fermented liquid to obtain a precipitate, and ultrasonically crushing to obtain an enzyme liquid;

(4) Reacting the enzyme supernatant with neutral colloidal chitin to produce chitooligosaccharides.

Further, in (4), one or more of Na ⁺ , Li ⁺ , Mg ²⁺ , and Ca ²⁺ is added to the enzyme solution.

Further, the preparation method of the colloidal chitin is as follows: crush and sieve the flake chitin, weigh the powdered chitin, slowly add concentrated hydrochloric acid, stir rapidly, and filter out impurities with glass wool after it is completely dissolved. , the solution was added to distilled water, centrifuged to obtain a precipitate, washed with distilled water until neutral.

Specifically, the method includes: inserting engineering bacteria cultivated in LB medium for 8-12 hours at 37° C. and 200 r for 8 to 12 hours into the fermentation medium with an inoculum size of 2%. After fermentation OD600 to 0.6-0.8, add IPTG to a final concentration of 0.4-1mmol/L, culture at 16°C, 200r for 24h, 10000r, centrifuge for 5min, add 10ml of PBS according to the wet weight of 1g of bacteria, 200ul of lysozyme solution, 400ul of PMSF, ice bath 30min, 40%, super 6s, interval 4s, ultrasonic 25min, centrifuge to obtain enzyme solution, centrifuge at 10000r/min for 20min, take ChiA/pET-32a(+)/BL21(DE3) induced wall-broken enzyme supernatant 200uL and 800uL1% Colloidal chitin was reacted at 50°C under a 200r shaker for 1 hour, added 2mL DNS and boiled for 5min, cooled to room temperature in ice water, added 2mL deionized water, centrifuged at 10000r/min for 2min, and measured the absorbance at 540nm.

Further, the components of the fermentation medium are:

Tryptone 10g/L, yeast powder 5g/L, NaCl 10g/L, pH7.2-7.4.

The present invention uses the bioinformatics analysis method to obtain the chitinase gene PROKKA01070 from the genome sequenced Streptomyces diastaticus CS1801 (Streptomyces diastaticus CS1801), the full length is 1065bp, and the chitinase ChiA encoded by it contains 354 amino acid; this gene is a novel chitinase gene with little similarity to other chitinase genes. By cloning the chitinase gene PROKKA01070 and connecting it to the expression vector pET32a(+), transforming Escherichia coli BL21(DE3), culturing and inducing expression, the recombinantly expressed chitinase ChiA was obtained. By measuring enzyme activity, enzymatic property analysis, and substrate specificity analysis, it was proved that the chitinase activity was 132U/L. The optimal reaction temperature and pH of chitinase are 50°C and 7.0, and the stability is the highest at 25°C and pH 5.0. It is specific to colloidal chitin, and Zn ²⁺ and SDS can strongly inhibit the enzyme effect, while Na ⁺ and Li ⁺ can promote the enzyme at any concentration. The advantage of the enzyme is that it is not limited by temperature, and can maintain high activity for a long time under lower temperature conditions. Chitinase ChiA can degrade chitin to prepare chitooligosaccharides, and the content of chitooligosaccharides obtained by the conversion is 201.4 μg/mL. This chitinase ChiA provides a new way for the enzymatic production of chitooligosaccharides, with Important application prospects.

Description of drawings

Figure 1 is the target band amplified by chitinase PCR.

Fig. 2 is the target band of PCR amplification identified by TA clone.

Figure 3 is the PCR identification of pET32a(+)-ChiA expression vector bacterial liquid.

Figure 4 is the double enzyme digestion identification of pET32a(+)-ChiA expression vector.

Figure 5 is the SDS-PAGE electrophoresis diagram of Escherichia coli.

Figure 6 is the standard curve for the determination of chitinase activity.

Fig. 7 shows the optimal reaction pH and pH stability of chitinase; a. the effect of pH on the activity of recombinant chitinase; b the pH stability of recombinant chitinase.

Figure 8 shows the optimum reaction temperature and temperature stability of chitinase; a. the influence of temperature on the activity of recombinant chitinase; b the temperature stability of recombinant chitinase.

detailed description

Example 1

This example illustrates the PCR amplification method of the GH18 chitinase ChiA gene derived from Streptomyces amylase.

Take Streptomyces amylase CS1801 stored on the slant of the test tube for plate activation, inoculate a single colony into LB liquid medium, and culture at 30°C for 2 to 3 days. Take the culture solution, centrifuge at 8000r for 2min, collect the bacteria, and use the bacterial genome extraction kit to extract the total genome. The extraction steps refer to the instructions of the Shanghai Sangong bacterial genome extraction kit.

Chitinase gene amplification primers were designed as:

F:5'-GTGTACGACCGGAACTACCACG-3'

R: 5'-TCAGTCGATCGCGTGGATCAG-3'

Using the extracted Streptomyces amylase genome as a template, use the above primers and Shanghai Sangong's high GC content PCR amplification kit to amplify without adding Taq enzyme.

The specific amplification procedure is as follows, pre-denaturation at 95°C for 3 minutes, and adding Taq enzyme. Denaturation at 95°C for 30s, annealing temperature at 55°C, annealing time for 30s, extension temperature at 72°C, extension time for 1min, this process was performed for 29 cycles, and finally extended at 72°C for 30min. The product was taken for agarose gel electrophoresis, and the target band was cut and recovered for preservation. Gel recovery kit was purchased from Sangong.

Example 2

This example illustrates the PCR amplification method of chitinase ChiA gene with restriction sites.

Chitinase gene amplification primers with restriction sites are designed as follows:

F:5'-CCGGAATTCGTGTACGACCGGAACTACCACG-3'

R: 5'-CCGCTCGAGTCAGTCGATCGCGTGGATCAG-3'

Using the gel recovery product in Example 1 as a template, the above primers and Shanghai Sangon's high GC content PCR amplification kit were used for amplification.

The specific amplification procedure is as follows, pre-denaturation at 95°C for 3min. Denaturation at 95°C for 30s, annealing temperature at 55°C, annealing time for 30s, extension temperature at 72°C, extension time for 1min, this process was carried out for 30 cycles, and finally extended at 72°C for 30min. The product was taken for agarose gel electrophoresis. The target band was shown in Figure 1. The target band was cut and recovered by gel cutting and sent to Sangong for sequencing to obtain the sequence SEQ ID NO: 1. The amino acid sequence of chitinase ChiA encoded by it is shown in SEQ ID NO: 2, containing a total of 354 amino acids.

Example 3

This example illustrates the construction method of the recombinant cloning vector of chitinase ChiA.

The gel recovery product in Example 2 was connected to the T4 vector, and after being transformed into Top10, positive clones were picked for verification, and the plasmid was extracted and sent to Sangon for sequencing verification.

Example 4

This example illustrates the construction method of the recombinant expression vector of chitinase ChiA.

The plasmid in Example 3 was double-digested with XhoI and EcoRI and the target band was recovered. At the same time, the pET32a(+) vector was double-digested with XhoI and EcoRI, and the larger fragment in the carrier was recovered. The recovered target gene fragment Ligated with the vector fragment, introduced into the host cell Escherichia coli Top10, after resistance screening, picked positive clones for sequencing verification.

Example 5

This example illustrates the construction method of chitinase ChiA genetically engineered bacteria.

Extract the plasmid of the positive clone with correct sequencing in Example 4, and directly transform it into the host cell Escherichia coli BL21 (DE3). Efficient expression of chitinase protein. The successful expression of the fusion protein was verified by SDS-PAGE. SDS-PAGE electrophoresis is shown in Figure 5, lane 1 is uninduced E.coli pET32a-ChiA, lane 2 is the whole recombinant E. coli pET32a-CHiA induced by IPTG for 12 hours, lanes 3 and 4 Protein was extracted from the recombinant E. coli pET32a-ChiA kit induced by IPTG for 24 hours, and lanes 5 and 6 were the sonicated supernatants of recombinant E. coli pET32a-ChiA induced by IPTG for 24 hours. Compared with lane 1, lanes 2, 3, 4, 5, and 6 have obvious bands at the molecular weight of 38k Da, excluding the fusion expressed protein on the plasmid, which is consistent with the predicted target protein size, indicating that chitinase is recombined successfully expressed in bacteria.

Example 6

Characterization of recombinant chitinases

1. The optimal reaction pH and pH stability of the recombinant enzyme

Determination of the optimal reaction pH of the recombinant enzyme: measure the enzyme activity of the recombinant enzyme in substrates with different pH (pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0) at 50°C to Determine its optimum pH.

Determination of the pH stability of the recombinant enzyme: after incubation of the recombinant enzyme in buffers with different pH values (pH 3.0, 5.0, 7.0, 9.0, 11.0) for 0.5h, 1.0h, 1.5h, 2.0h, 2.5h, 3.0h , Take the enzyme solution and measure its enzyme activity at the optimum temperature and pH respectively.

The results showed that the optimum reaction pH of the recombinant chitinase was 7.0, and it was most stable at pH 5.0. The experimental results are shown in FIG. 7 .

Determination of Optimum Reaction Temperature and Thermal Stability of Recombinase

Determination of the optimal reaction temperature of the recombinant enzyme: put the purified recombinant enzyme in the optimal pH substrate at different temperatures (20°C, 25°C, 30°C, 37°C, 42°C, 50°C, 60°C, 70°C ) to determine the optimum reaction temperature.

Determination of the thermal stability of the recombinant enzyme: the recombinant enzyme was incubated at different temperatures (25°C, 37°C, 42°C, 50°C, 60°C, 70°C) for 0.5h, 1.0h, 1.5h, 2.0h, 2.5h, 3.0 After h, the enzyme activities were measured under the optimal pH and temperature conditions.

The results showed that the optimal reaction temperature of the recombinant chitinase was 50°C, and more than 80% of the enzyme activity could be maintained at 25°C. The experimental results are shown in Figure 8.

The preparation concentration is 1.0% (w/v) colloidal chitin, fine powder chitin, shrimp shell powder, crab shell powder, chitosan, carboxymethylcellulose sodium (CMC-Na), under the optimum reaction conditions The recombinase activity was measured. The enzyme activity of the substrate as colloidal chitin was set as 100%, and the relative enzyme activities of other substrates were calculated. The results showed that chitinase has substrate specificity for colloidal chitin.

The experimental results are shown in Table 1.

Table 1 Determination of substrate specificity of ChiA

2. Mix 1mM, 5mM and 10mM salt solutions containing the following metal ions and metal ion chelating agents: MgCl ₂ , FeCl ₃ , ZnCl ₂ , CaCl ₂ , KCl, NaCl, C ₂ H ₃ O ₂ Li, EDTA , mercaptoethanol, and SDS were added to the recombinase respectively, and the neutral colloidal chitin with a final concentration of 1% was used as the substrate, and the activity of the recombinase was determined under the optimum reaction conditions.

The results showed that Zn ²⁺ and SDS could strongly inhibit the enzyme at three concentrations, while Na ⁺ and Li ⁺ could promote the enzyme at any concentration, and Mg ²⁺ and Ca ²⁺ could At a concentration of 1mmoL/L, the recombinant enzyme can be promoted. The experimental results are shown in Table 2.

Table 2 Effects of different ions and chemical reagents on the activity of recombinase ChiA

Example 7

This example illustrates the application of chitinase genetically engineered bacteria in the production of chitooligosaccharides.

(1) Insert the genetically engineered bacteria cultivated in LB medium at 37° C. for 8 to 12 hours at 200 r with an inoculum size of 2% into the fermentation medium.

(2) After 4 hours of fermentation, IPTG was added to a final concentration of 0.4-1 mmol/L, and fermented at 16°C and 200r for 24 hours.

(3) 10000r, 5min, collect the bacteria by centrifugation, add 10mL PBS, 200uL lysozyme solution, 400uL PMSF according to the wet weight of 1g of the bacteria, ice bath for 30min, 40%, super 6s, interval s, ultrasonic 25min, centrifuge to obtain the enzyme solution , 10000r/min centrifugal 20min.

(4) Take ChiA/pET-32a(+)/BL21(DE3) induced broken supernatant 200uL and 800uL 1% neutral chitin colloid to react in a shaker at 50°C and 200r for 30min. After the reaction was completed, 2 mL of DNS solution was added, boiled for 5 min, and then cooled to room temperature in an ice-water mixture. Then add 2m deionized water, centrifuge at 1000r/min for 2min, and measure the absorbance at 540nm. The blank control was the supernatant of ChiA/pET-32a(+)/BL21(DE3) after wall-breaking induced by boiling for 30 min. The standard product is N-acetylglucosamine with content of 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18 mg, and the standard curve is: y=3.3106x-0.0774, R2=0.9959 (Y is absorbance, X is N-acetylglucosamine glucose content). Definition of chitinase enzyme activity unit: under the condition of 50°C water bath, the amount of enzyme required to release 1 μg of acetylglucosamine per minute per liter of solution is defined as an enzyme activity unit. The measured enzyme activity can reach 132U/L, which is greatly improved compared with the original enzyme activity (50.3U/L), and the content of chitooligosaccharide produced is 201.4μg/mL.

Described fermentation medium composition is:

Tryptone 10g/L, yeast powder 5g/L, NaCl 10g/L, adjust the pH to 7.2-7.4, distilled water to 1L.

The preparation method of colloidal chitin: crush and sieve flake chitin, weigh 10g of powdered chitin, slowly add 200mL of concentrated hydrochloric acid, stir rapidly, and filter out impurities with glass wool after it is completely dissolved, add 1000mL of the solution Distilled water, centrifuged to obtain a precipitate, washed with distilled water until neutral.

sequence listing

<110> Changshu Institute of Technology

<120> A kind of GH18 chitinase ChiA and its application

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Gly Pro Glu Ala Phe Gly Glu Val Val Ser Ala Leu Arg Ala Glu Phe

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Gly Asp Asp Tyr Leu Val Thr Ala Ala Ile Pro Ala Asp Ala Ser Glu

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Gly Gly Lys Leu Asp Ala Thr Asp Tyr Ala Gly Ala Ala Gln Tyr Val

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Asp Trp Tyr Asn Pro Met Thr Tyr Asp Tyr Tyr Gly Ala Trp Asp Ala

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Gln Gly Pro Thr Ala Pro His Ser Pro Leu Thr Ala Tyr Glu Ala Ile

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Pro Asp Ala Ser Phe Thr Thr Glu Ala Thr Ile Ala Lys Leu Lys Glu

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Ala Gly Lys Met Ala Phe Lys Glu Arg Glu Gly Leu Ala Gly Thr Phe

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Phe Trp Glu Leu Ser Gly Asp Thr Asp Asp Gly Glu Leu Ile His Ala

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Ile Asp

Claims (10)

1. A GH18 chitinase ChiA, characterized in that the nucleotide sequence of the gene encoding the GH18 chitinase ChiA is shown in SEQ ID NO:1. 2. A recombinant vector comprising the gene of claim 1. 3. The recombinant vector according to claim 2, wherein the expression vector is pET32a(+), and the gene is inserted between the multiple cloning sites of the expression vector pET-32a(+). 4. A genetically engineered bacterium comprising the recombinant vector according to claim 3. 5. The genetically engineered bacterium according to claim 4, wherein the host cell is Escherichia coli E.coli BL21(DE3). 6. The application of the GH18 chitinase ChiA described in claim 1 in the production of chitooligosaccharides. 7. The application according to claim 6, wherein the GH18 chitinase ChiA uses colloidal chitin as a substrate to produce chitooligosaccharides. 8. The application according to claim 6, characterized in that it comprises: (1) Insert the engineered bacteria containing the coding gene of GH18 chitinase into LB medium for seed culture; (2) The seed liquid is transferred to the fermentation medium for fermentation and culture, and then an inducer is added to induce the expression of enzymes; (3) Centrifuge the fermentation broth to obtain the precipitate, and obtain the enzyme liquid by ultrasonic crushing; (4) React the enzyme solution with neutral colloidal chitin to produce chitooligosaccharides. 9. The application according to claim 6, further comprising, in (4), adding one or more of Na ⁺ , Li ⁺ , Mg ²⁺ , Ca ²⁺ to the enzyme solution . 10. The application according to claim 6, wherein the preparation method of the colloidal chitin is as follows: crush and sieve the flake chitin, weigh the powdered chitin, slowly add concentrated hydrochloric acid, and stir rapidly , after it is completely dissolved, filter with glass wool to remove impurities, add distilled water to the solution, centrifuge to obtain a precipitate, and wash with distilled water until neutral.

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