CN113481186A

CN113481186A - GH18 chitinase ChiA and application thereof

Info

Publication number: CN113481186A
Application number: CN202110660700.3A
Authority: CN
Inventors: 王立梅; 李芹; 齐斌
Original assignee: Changshu Institute of Technology
Current assignee: Changshu Institute of Technology
Priority date: 2021-06-15
Filing date: 2021-06-15
Publication date: 2021-10-08
Anticipated expiration: 2041-06-15
Also published as: CN113481186B

Abstract

The invention relates to GH18 chitinase ChiA and application thereof. The present invention relates to the strain Amyloctase (A) and (B)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, 354 amino acids in total. The chitinase gene PROKKA01070 is cloned and connected with an expression vector pET32a (+), then escherichia coli BL21(DE3) is transformed, and after cultivation and induced expression, the recombinant chitinase ChiA is obtained. The optimum reaction temperature of the enzyme is 50 ℃, and the optimum reaction temperature is pH is 7.0, and the enzyme activity is most stable at 25 ℃ and in a buffer solution with pH of 7.0. Chitinase ChiA can degrade chitin to prepare chitosan oligosaccharide, has substrate specificity on colloidal chitin, provides a new way for producing the chitosan oligosaccharide by an enzyme method, and has important application prospect.

Description

GH18 chitinase ChiA and application thereof

Technical Field

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

Background

Chitinase belongs to glycosyl hydrolase family, and can specifically catalyze and hydrolyze beta-1, 4 glycosidic bond of chitin to generate chitosan oligosaccharide, chitosan and N-acetylglucosamine. The degradation products of chitin, such as chitooligosaccharide, chitooligosaccharide and N-acetylglucosamine, can be widely applied to the fields of biological medicine, food, cosmetics, textile, agriculture and the like, and have general biological activity and wide market development prospect. The chitinase method for producing the chitosan oligosaccharide, chitosan and the like has mild conditions and small environmental pollution, and is a development direction for preparing the chitosan oligosaccharide and the like.

At present, about 46 chitinase-producing microorganisms have been reported to belong to nearly 70 species, mainly bacteria, actinomycetes and fungi. However, the reported strains have low chitinase production amount, low production efficiency and poor tolerance, and have a certain distance from industrial production. Therefore, the discovery of a new chitinase gene and the breeding of the chitinase producing strain with strong tolerance and high chitinase activity have important guiding significance for the industrial work of chitinase. With the continuous progress of molecular biology technology, the engineering bacteria obtained by means of genetic engineering often have the characteristics of high expression level, strong product specificity, high production efficiency, short propagation period, easy separation and the like compared with wild strains, but the difficulty of constructing the genetic engineering bacteria lies in the construction of cloning expression vectors and the expression of heterologous proteins.

Disclosure of Invention

The invention aims to provide GH18 chitinase ChiA and application thereof, and the chitinase ChiA has high activity and can be used for catalytic production of chitosan oligosaccharide.

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

the GH18 chitinase ChiA has the nucleotide sequence of a gene PROKKA01070 for coding the GH18 chitinase shown as SEQ ID NO: 1 is shown.

The above GH18 chitinase ChiA is derived from the strain Streptomyces diastaticus CS1801, which has been disclosed in the applicant's prior application CN 109337843A.

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

The invention also provides a genetically engineered bacterium containing the recombinant vector.

Further, the engineering bacterium host cell is E.coli BL21(DE 3).

The invention also provides the application of the engineering bacteria in the enzymatic production of the chitosan oligosaccharide.

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

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

(1) inoculating the genetically engineered bacteria containing GH18 chitinase into an LB culture medium for seed culture;

(2) transferring the seed liquid into a fermentation culture medium for fermentation culture, and then adding an inducer to induce the expression of enzymes;

(3) centrifuging the fermentation liquor to obtain a precipitate, and carrying out ultrasonic crushing to obtain an enzyme solution;

(4) reacting the enzyme supernatant with neutral colloidal chitin to produce the chitooligosaccharide.

Further, in the above (4), Na is added to the enzyme solution⁺、Li⁺、Mg²⁺、Ca²⁺One or more of them.

Further, the preparation method of the colloidal chitin comprises the following steps: pulverizing flaky chitin, sieving, weighing powdery chitin, slowly adding concentrated hydrochloric acid, stirring rapidly, dissolving completely, filtering with glass wool to remove impurities, adding distilled water into the solution, centrifuging to obtain precipitate, and washing with distilled water to neutrality.

Specifically, the method comprises the following steps: the engineering bacteria cultured in LB culture medium for 8-12 h at 37 ℃ and 200r are inoculated into the fermentation culture medium in an inoculum size of 2%. Fermenting OD600 to 0.6-0.8, adding IPTG to a final concentration of 0.4-1 mmol/L, culturing for 24h at 16 ℃ under 200r, 10000r, centrifuging for 5min, adding 10mL PBS, 200uL lysozyme solution, 400uL PMSF according to 1g wet weight of thallus, carrying out ice bath for 30min, 40%, super 6s, spacing for 4s, carrying out ultrasound for 25min, centrifuging to obtain enzyme solution, centrifuging for 20min at 10000r/min, taking 200uL of cell-broken enzyme supernatant after induction by ChiA/pET-32a (+)/BL21(DE3), reacting with 800uL 1% colloidal chitin at 50 ℃ under a shaker for 1 h, adding 2mL DNS, boiling for 5min, cooling to room temperature in ice water, adding 2mL deionized water, centrifuging for 2min at 10000r/min, and measuring absorbance at 540 nm.

Further, the fermentation medium comprises the following components:

10g/L of tryptone, 5g/L of yeast powder, 10g/L of NaCl and 7.2-7.4 of pHs.

The invention uses the bioinformatics analysis method to obtain the chitinase gene PROKKA01070 from the amylase streptomycete CS1801(Streptomyces diastaticus CS1801) of genome sequencing, the total length is 1065bp, and the chitinase ChiA coded by the chitinase gene comprises 354 amino acids; the gene is a novel chitinase gene and has low similarity with other chitinase genes. The chitinase gene PROKKA01070 is cloned and connected with an expression vector pET32a (+), then escherichia coli BL21(DE3) is transformed, and after cultivation and induced expression, the recombinant chitinase ChiA is obtained. The chitinase activity is proved to be 132U/L by measuring the enzyme activity, analyzing the enzymology property and analyzing the substrate specificity. Chitinase has optimal reaction temperature and pH of 50 deg.C and 7.0, highest stability at 25 deg.C and pH of 5.0, specificity to colloidal chitin, Zn²⁺SDS has strong inhibitory action on the enzyme, while Na⁺And Li⁺The enzyme is promoted at any concentration. The enzyme has the advantages that the enzyme can be subjected to reaction for a long time under the condition of keeping higher activity without being limited by temperature. Chitinase ChiA can degrade chitin to prepare the chitooligosaccharide, the content of the chitooligosaccharide obtained by conversion is 201.4 mu g/mL, and the chitinase ChiA provides a new way for producing the chitooligosaccharide by an enzyme method and has important application prospect.

Drawings

FIG. 1 shows the band of interest in the PCR amplification with chitinase.

FIG. 2 shows the target band of TA clone identification PCR amplification.

FIG. 3 shows PCR identification of pET32a (+) -ChiA expression vector bacterial liquid.

FIG. 4 shows the double restriction enzyme identification of pET32a (+) -ChiA expression vector.

FIG. 5 is an SDS-PAGE electrophoresis of E.coli.

FIG. 6 is a standard curve for chitinase activity assays.

FIG. 7 shows the pH optimum and pH stability of chitinase; a influence of pH on the activity of recombinant chitinase; b pH stability of recombinant chitinase.

FIG. 8 shows the optimal reaction temperature and temperature stability of chitinase; a effect of temperature on recombinant chitinase activity; b temperature stability of recombinant chitinase.

Detailed Description

Example 1

This example illustrates a PCR amplification method for the ChiA gene of GH18 chitinase derived from Amylomycete.

Taking the amylase streptomycete CS1801 stored on the inclined surface of the test tube for plate activation, inoculating a single colony into an LB liquid culture medium, and culturing for 2-3 days at 30 ℃. Taking the culture solution, centrifuging at 8000r for 2min, collecting thallus, extracting total genome with bacterial genome extraction kit, and extracting according to the instruction of bacterial genome extraction kit of Shanghai worker.

Chitinase gene amplification primers are designed as follows:

F:5’-GTGTACGACCGGAACTACCACG-3’

R:5’-TCAGTCGATCGCGTGGATCAG-3’

the extracted genome of the amylase streptomycete is used as a template, the primer and a PCR amplification kit with high GC content of Shanghai workers are adopted for amplification, but Taq enzyme is not added.

The specific amplification procedure was as follows, pre-denaturation at 95 ℃ for 3min, and addition of Taq enzyme. Denaturation at 95 ℃ for 30s, annealing at 55 ℃, annealing time for 30s, extension temperature for 72 ℃, extension time for 1min, 29 cycles of this process, and final extension at 72 ℃ for 30 min. And (4) taking the product to perform agarose gel electrophoresis, and cutting and recovering the gel of the target band and storing the recovered gel. Gel recovery kits were purchased from manufacturers.

Example 2

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

The chitinase gene amplification primer with the enzyme cutting site is designed as follows:

F:5’-CCGGAATTCGTGTACGACCGGAACTACCACG-3’

R:5’-CCGCTCGAGTCAGTCGATCGCGTGGATCAG-3’

the gel recovered product in example 1 was used as a template, and the above primers and the PCR amplification kit with high GC content of Shanghai worker were used for amplification.

The specific amplification procedure was as follows, pre-denaturation 95 ℃ for 3 min. Denaturation at 95 ℃ for 30s, annealing at 55 ℃, annealing time for 30s, extension temperature for 72 ℃, extension time for 1min, 30 cycles in total, and final extension at 72 ℃ for 30 min. And (3) taking the product to carry out agarose gel electrophoresis, cutting and recovering gel of a target band as shown in figure 1, and then carrying out sequencing to obtain a sequence SEQ ID NO: 1, the coded chitinase ChiA amino acid sequence is shown as SEQ ID NO: 2, 354 amino acids in total.

Example 3

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

The recovered product of the gel in the example 2 is connected with a T4 vector, after the recovered product is transformed into Top10, a positive clone is selected for verification, and after a plasmid is extracted, the verification is carried out by sequencing.

Example 4

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

The plasmid in example 3 is subjected to double enzyme digestion by XhoI and EcoRI, a target band is recovered, meanwhile, the pET32a (+) vector is subjected to double enzyme digestion by XhoI and EcoRI, a larger fragment in the vector is recovered, the recovered target gene fragment and the vector fragment are connected, the target gene fragment and the vector fragment are introduced into a host cell Escherichia coli Top10, and after resistance screening, a positive clone is selected for sequencing verification.

Example 5

This example illustrates a method for constructing chitinase ChiA genetically engineered bacteria.

The plasmid of the positive clone with correct sequencing in the embodiment 4 is extracted and directly transformed and introduced into host cell escherichia coli BL21(DE3), the chitinase gene engineering bacteria are successfully constructed, and inducers such as IPTG and the like are added in the fermentation process to induce the chitinase protein to be efficiently expressed. The success of the expression of the fusion protein was verified by SDS-PAGE. The SDS-PAGE electrophoresis picture is shown in figure 5, lane 1 is non-induced E.coli pET32a-ChiA, lane 2 is recombinant Escherichia coli E.coli pET32a-CHiA whole thallus which is induced for 12h by IPTG,

lanes

3 and 4 are recombinant Escherichia coli E.coli pET32a-ChiA kit extracted protein which is induced for 24h by IPTG, and

lanes

5 and 6 are recombinant Escherichia coli E.coli pET32a-ChiA ultrasonication supernatant which is induced for 24h by IPTG. Compared with the lane 1,

lanes

2, 3, 4, 5 and 6 have obvious bands at the molecular weight of 38k Da, and the size of the fusion expressed protein on the removed plasmid is consistent with the size of the predicted target protein, which indicates that the chitinase is successfully expressed in the recombinant bacteria.

Example 6

Characterization of recombinant chitinase

1. Optimum reaction pH and pH stability of recombinant enzyme

Determination of the optimum reaction pH for the recombinant enzymes: the recombinase is tested for enzyme activity in substrates with different pH values (pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0) at 50 ℃ to determine the optimum pH value.

Determination of recombinase pH stability: and (3) preserving the recombinase in buffer solutions with different pH values (pH 3.0, 5.0, 7.0, 9.0 and 11.0) for 0.5h, 1.0h, 1.5h, 2.0h, 2.5h and 3.0h respectively, and then taking the enzyme solution to measure the enzyme activity of the recombinase respectively at the optimal temperature and pH.

The results show that the optimal reaction pH of the recombinant chitinase is 7.0, the recombinant chitinase is most stable at pH5.0, and the experimental results are shown in FIG. 7.

Determination of optimum reaction temperature and thermal stability of recombinase

Determination of the optimum reaction temperature of the recombinase: the purified recombinase reacts in a substrate with the optimum pH value at different temperatures (20 ℃, 25 ℃, 30 ℃, 37 ℃, 42 ℃, 50 ℃, 60 ℃ and 70 ℃) to measure the enzyme activity so as to determine the optimum reaction temperature.

Determination of recombinase thermal stability: the recombinase is subjected to heat preservation for 0.5h, 1.0h, 1.5h, 2.0h, 2.5h and 3.0h at different temperatures (25 ℃, 37 ℃, 42 ℃, 50 ℃, 60 ℃ and 70 ℃), and then the enzyme activity of the recombinase is respectively measured under the conditions of the optimal pH and temperature.

The result shows that the optimal reaction temperature of the recombinant chitinase is 50 ℃, and more than 80 percent of enzyme activity can be still maintained at 25 ℃. The results of the experiment are shown in FIG. 8.

Preparing 1.0% (w/v) colloidal chitin, fine powder chitin, shrimp shell powder, crab shell powder, chitosan and sodium carboxymethylcellulose (CMC-Na), and determining the activity of the recombinant enzyme under the optimal reaction condition. The enzyme activity of the substrate which is colloidal chitin is set as 100 percent, and the relative enzyme activities of other substrates are calculated. The results show that chitinase has substrate specificity for colloidal chitin.

The results of the experiment are shown in table 1.

TABLE 1 substrate specificity assay of ChiA

2. 1m M, 5m M and 10m M of a salt solution containing the following metal ions and a metal ion chelating agent: MgCl₂，FeCl₃，ZnCl₂，CaCl₂，KCl，NaCl、C₂H₃O₂Li, EDTA, mercaptoethanol and SDS are added into the recombinant enzyme respectively, and the activity of the recombinant enzyme is determined under the optimal reaction condition by taking neutral colloidal chitin with the final concentration of 1 percent as a substrate.

The results show that Zn²⁺SDS has strong inhibitory effect on the enzyme under three concentrations, while Na⁺And Li⁺Promoting the enzyme at any concentration, Mg²⁺And Ca²⁺The recombinant enzyme is promoted at a concentration of 1 mmoL/L. The results of the experiment are shown in table 2.

TABLE 2 Effect of different ions and chemical reagents on the enzyme activity of the recombinase ChiA

Example 7

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

(1) Inoculating the genetic engineering bacteria cultured in LB culture medium at 37 ℃ and 200r for 8-12 h into a fermentation culture medium in an inoculum size of 2%.

(2) After fermenting for 4h, IPTG is added to the final concentration of 0.4-1 mmol/L, and fermentation culture is carried out for 24h under the conditions of 16 ℃ and 200 r.

(3)10000r, 5min, centrifuging to collect thalli, adding 10mL PBS, 200uL lysozyme solution, 400uL PMSF according to the wet weight of 1g thalli, carrying out ice bath for 30min, carrying out 40%, exceeding 6s, carrying out interval s, carrying out ultrasonic treatment for 25min, centrifuging to obtain enzyme solution, and centrifuging for 20min at 10000 r/min.

(4) 200uL of the wall-broken supernatant after ChiA/pET-32a (+)/BL21(DE3) induction and 800uL of 1% neutral chitin colloid are taken to react for 30min at 200r in a shaking table at 50 ℃. After the reaction was complete, 2mL of DNS solution was added, boiled for 5min and cooled to room temperature in an ice-water mixture. Then 2m deionized water is added, centrifugation is carried out for 2min at 1000r/min, and the absorbance is measured at 540 nm. The blank control is ChiA/pET-32a (+)/BL21(DE3) supernatant after 30min boiling for inducing wall breaking. The standard substance is N-acetylglucosamine with content of 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18mg, and the standard curve is Y-3.3106X-0.0774, R2-0.9959 (Y is absorbance, and X is N-acetylglucosamine content). Chitinase enzyme activity unit definition: under the condition of 50 ℃ water bath heat preservation, the enzyme quantity required by releasing 1 mu g of acetylglucosamine per minute per liter of solution is determined as one enzyme activity unit. The enzyme activity can reach 132U/L through determination, the activity is greatly improved compared with the original enzyme activity (50.3U/L), and the content of the produced chitosan oligosaccharide is 201.4 mu g/mL.

The fermentation medium comprises the following components:

10g/L of tryptone, 5g/L of yeast powder and 10g/L of NaCl, adjusting the pH value to 7.2-7.4, and fixing the volume of distilled water to 1L.

The preparation method of the colloidal chitin comprises the following steps: crushing and sieving flaky chitin, weighing 10g of powdery chitin, slowly adding 200mL of concentrated hydrochloric acid, rapidly stirring, dissolving completely, filtering with glass wool to remove impurities, adding 1000mL of distilled water into the solution, centrifuging to obtain precipitate, and washing with distilled water to neutrality.

Sequence listing

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Val Tyr Asp Arg Asn Tyr His Val Lys Asn Ile Glu Thr Ser Gly Ser

1 5 10 15

Ala Asp Lys Leu Thr His Ile Asn Tyr Ser Phe Gly Asn Val Gln Gly

20 25 30

Gly Lys Cys Ala Leu Gly Asp Gly Phe Ala Ala Thr Glu Lys Ala Tyr

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Thr Ala Glu Glu Ser Val Asp Gly Glu Ala Asp Ala Trp Asp Gln Pro

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Leu Arg Gly Asn Phe Asn Gln Leu Leu Lys Leu Lys Glu Leu His Pro

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Asp Leu Lys Val Leu Trp Ser Phe Gly Gly Trp Thr Trp Ser Gly Gly

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Phe Thr Glu Ala Ala Lys Asp Pro Ala Ala Phe Ala Glu Ser Cys His

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Asp Leu Val Glu Asp Pro Arg Trp Ala Asp Val Phe Asp Gly Ile Asp

115 120 125

Val Asp Trp Glu Tyr Pro Asn Ala Cys Gly Leu Thr Cys Asp Glu Ser

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Ala Gly Val Pro Ala Ser Lys Leu Leu Leu Gly Leu Gly Phe Tyr Gly

245 250 255

Arg Gly Trp Thr Gly Val Ser Glu Ala Ala Pro Gly Gly Ser Ala Ser

260 265 270

Gly Pro Ala Ala Gly Val His Glu Gln Gly Ile Glu Asp Tyr Lys Val

275 280 285

Leu Lys Glu Arg Cys Pro Ala Thr Gly Glu Ala Gly Gly Thr Ala Tyr

290 295 300

Ala Leu Cys Asp Gly Glu Trp Trp Ser Tyr Asp Thr Pro Glu Thr Ile

305 310 315 320

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

325 330 335

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

340 345 350

Ile Asp

Claims

1. The GH18 chitinase ChiA, wherein the nucleotide sequence of the gene coding the GH18 chitinase ChiA is shown as SEQ ID NO: 1 is shown.

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 of claim 3.

5. The genetically engineered bacterium of claim 4, wherein the host cell is Escherichia coliE.coli BL21(DE3)。

6. The use of GH18 chitinase ChiA of claim 1 for the production of chitooligosaccharides.

7. The use of claim 6, wherein the GH18 chitinase ChiA produces chitooligosaccharides using colloidal chitin as a substrate.

8. The use according to claim 6, comprising:

(3) centrifuging the fermentation liquor to obtain a precipitate, and obtaining an enzyme solution by ultrasonic crushing;

(4) reacting the enzyme solution with neutral colloidal chitin to produce the chitooligosaccharide.

9. The use according to claim 6, further comprising, in the step (4), adding Na to the enzyme solution⁺、Li⁺、Mg²⁺、Ca²⁺One or more of them.

10. The use of claim 6, wherein the colloidal chitin is formulated by a method comprising: pulverizing flaky chitin, sieving, weighing powdery chitin, slowly adding concentrated hydrochloric acid, stirring rapidly, dissolving completely, filtering with glass wool to remove impurities, adding distilled water into the solution, centrifuging to obtain precipitate, and washing with distilled water to neutrality.