Strain for producing chitinase and application thereof

Abstract

The present invention relates to a strain for producing chitinase and application thereof. The class of the strain is named Streptomyces diastaticus CS1801 and the preservation number thereof is CCTCC NO: M2018263. The Streptomyces diastaticus CS1801 of the present invention is derived from naturally fermented prawn paste. By fermentation of prawns, the enzyme activity of the chitosan is as high as 57.3 U/L and the content of chitooligosaccharides is 0.58 mol/L. The present invention provides a new method for producing chitooligosaccharides and has a good application prospect.

Claims

1. A strain for producing chitinase, where the strain is Streptomyces diastaticus CS1801 and the preservation number thereof is CCTCC NO: M2018263.

2. A method for production of chitooligosaccharides, comprising: admixing Streptomyces diastaticus CS1801 with the preservation number CCTCC NO: M2018263 with a chitosan-containing substrate in a fermentation medium to produce a mixture; and fermenting the mixture for a time sufficient to produce chitooligosaccharides.

3. The method for production of chitooligosaccharides according to claim 2, wherein the fermenting the mixture is at a temperature of 30° C., and pH 6.5-7.

4. The method for production of chitooligosaccharides according to claim 2, wherein the fermentation medium is: liquid A: K.sub.2HPO.sub.4 1.4 g/L, KH.sub.2PO.sub.4 0.6 g/L, MgSO.sub.4.7H.sub.2O 1 g/L, NaCl 10 g/L, (NH.sub.4).sub.2SO.sub.4 20 g/L, deionized water 1000 mL, pH 6.5; and the chitosan-containing substrate is: liquid B: 10 g/L colloidal chitosan, pH 6.5; wherein, liquid A and liquid B are mixed in an equal volume.

5. The method for production of chitooligosaccharides according to claim 4, wherein the colloidal chitosan is prepared as follows: weigh 10 g of powdery chitosan, add 200 mL of concentrated hydrochloric acid, stir, filter the solution with glass wool after thorough dissolution to remove impurities, and add 1,000 mL of distilled water into the solution, centrifuge to obtain a precipitate and wash the precipitate with distilled water till neutral.

6. The method according to claim 2, wherein the fermentation medium is: liquid A: K.sub.2HPO.sub.4 1.4 g/L, KH.sub.2PO.sub.4 0.6 g/L, MgSO.sub.4.7H.sub.2O 1 g/L, NaCl 10 g/L, (NH.sub.4).sub.2SO.sub.4 20 g/L, deionized water 1000 mL, pH 6.5; and the chitosan-containing substrate is: liquid B: 100 g/L prawn powder, pH 6.5; wherein liquid A and liquid B are mixed in an equal volume.

7. The method according to claim 6, wherein the prawn powder is prepared as follows: defrost frozen prawns bake; pulverize the prawns to produce a powder, and sieve the powder through a 100 mesh sieve.

8. The method according to claim 2, wherein the chitosan-containing substrate is: 300 g/L wet prawns, pH 6.5.

9. The method according to claim 2, further comprising: (1) inoculating strain CS1801 into a PDA liquid medium, and culturing it under shaking at 30° C. for 2 to 3 d; (2) mixing the strain cultured at step (1) and the fermentation medium, and fermenting under shaking at 30 to 37° C. for 5 to 7 d to create a fermentation liquor; centrifuging the fermentation liquor, discarding the precipitate and collecting the supernate containing chitooligosaccharides.

10. The method according to claim 9, wherein the temperature of fermenting and culturing is 30° C.

11. The method according to claim 2, wherein the fermenting the mixture is at a temperature of 30° C., and pH 6.5-7.

12. The method of claim 8 wherein the wet prawns are prepared by defrosting frozen prawns.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is an image of Streptomyces diastaticus CS1801 observed under an electron microscope;

(2) FIG. 2 is microscopic morphology of Streptomyces diastaticus CS1801 after Gram staining;

(3) FIG. 3 is a phylogenetic tree of Streptomyces diastaticus CS1801;

(4) FIG. 4 is an ultra high performance liquid chromatogram of a fermentation liquor of Streptomyces diastaticus CS1801, where 4-A is a liquid chromatogram of a standard substance, and 4-B is a liquid chromatogram of the fermentation liquor;

(5) FIG. 5 is a mass spectrum of a fermentation liquor of Streptomyces diastaticus CS1801, where 5-A is a mass spectrum of a standard substance, and 5-B is a mass spectrum of the fermentation liquor;

(6) The biological material that the present invention relates to, with its class named Streptomyces diastaticus CS1801, preserved at China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, China, preservation number: CCTCC NO: M2018263, and date of preservation: May 10, 2018.

DETAILED DESCRIPTION

Embodiment 1

(7) This embodiment describes methods for screening, purifying and identifying Streptomyces diastaticus CS1801.

(8) The screening sample is prawn paste of Lianyungang Haiwa Food Co., Ltd. 25 g of the prawn paste and 225 mL of normal saline are taken to prepare a bacterial suspension, and the suspension is diluted by 10.sup.−1, 10.sup.−2, 10.sup.−3 and 10.sup.−4 times respectively. The original bacterial suspension, 10.sup.−1 diluted bacterial suspension, 10.sup.−2 diluted bacterial suspension, 10.sup.−3 diluted bacterial suspension and 10.sup.−4 diluted bacterial suspension are spread on the primary screening medium respectively. After growth at 37° C. for 1 to 7 d, single colony, which grows well, is picked, and lines are drawn on the primary screening medium for isolation. A single colony produced on the primary screening medium and having an obvious transparent circle around it is picked, inoculated to a liquid medium and cultured in a 37° C., 200 r/min shaker for 1 to 7 d. Primary screening medium: Liquid A: K.sub.2HPO.sub.4 1.4 g/L, KH.sub.2PO.sub.4 0.6 g/L, MgSO.sub.4.7H.sub.2O 1 g/L, NaCl 10 g/L, (NH.sub.4).sub.2SO.sub.4 20 g/L, agar 40 g/L, deionized water 1000 mL, pH 6.5. Liquid B: 10 g/L colloidal chitosan, pH 6.5. Before use, liquid A and liquid B are mixed in an equal volume.

(9) Secondary screening medium: Liquid A: K.sub.2HPO.sub.4 1.4 g/L, KH.sub.2PO.sub.4 0.6 g/L, MgSO.sub.4.7H.sub.2O 1 g/L, NaCl 10 g/L, (NH.sub.4).sub.2SO.sub.4 20 g/L, deionized water 1000 mL, pH 6.5. Liquid B: 10 g/L colloidal chitosan, pH 6.5. Before use, liquid A and liquid B are mixed in an equal volume.

(10) A slide is inserted on the PDA solid medium and cultured for 7 days. Then the slide is cut to a suitable size, directly sprayed with gold and scanned, and observed under an electron microscope. The spore chain runs straight or flexible and has branches, and the spores are oval, with a smooth surface (FIG. 1).

(11) A ring of bacteria are picked from the primary screening medium plate, mixed with the water beads on the slide and quenched. After primary staining by crystal violet, enzyme staining by an iodine solution, decoloring by ethanol and counterstaining by safflower, the bacteria are observed under a microscope. The bacteria are Gram negative bacteria (FIG. 2).

(12) After determination of the sequence of 16SrDNA part of the foregoing strain and BLAST comparison, MEGA 5.1 is used to construct N-J phylogenetic tree for analysis, its 16SrDNA sequence is as shown in SEQ ID NO.1, and the phylogenetic tree is as shown in FIG. 3. Thus it is identified as Streptomyces, and after the identification and preservation of the microorganism preservation program, its class is named Streptomyces diastaticus CS1801, and its preservation number is CCTCC M2018263.

Embodiment 2

(13) This embodiment specifically describes the application of strain CS1801 for producing chitooligosaccharides through fermentation by colloidal chitosan.

(14) (1) Inoculate a strain CS1801 to a PDA liquid medium, and culture it under shaking at 30° C. for 2 to 3 d; and

(15) (2) Lead the strain cultured at step (1) to a fermentation medium, and culture it under shaking at 30° C. for 7 d; centrifuge the fermentation liquor to discard the precipitate and take the supernate, and determine the enzyme activity of chitinase is 117.4 U/L, and the content of chitooligosaccharides is 1.18 mol/L.

(16) The composition of the fermentation medium is the same as that of the foregoing secondary screening medium.

(17) The method for determining enzyme activity of chitinase is as follows: The fermentation liquor is centrifuged at 3000 r for 10 min, and the supernate is taken as a test sample. The test sample, horse radish peroxidase (HRP)-labeled detection antibody are added in turn into the micropores that are coated with chitinase antibody in advance, cultured at 37° C. for 1 h and then thoroughly washed. Color development is performed using substrate 3,3′,5,5′-tetramethyl benzidine (TMB) and the color turns blue under the catalysis of HRP and turns yellow in the end under the action of acid. The OD value at 450 nm is measured with a microplate reader, and the sample activity is calculated through a standard curve. The standard substances are 0, 1.5, 3, 6, 12, 24 U/L enzyme solutions prepared from pure chitinase. The enzyme activity is defined as: at 37° C., the amount of enzyme that decomposes chitosan per mg of protein per hour to produce 1 mg of N-acetylglucosamine is an enzyme activity unit U. The chitinase ELISA detection kit was purchased from Wuhan Chundu Biotechnology Co., Ltd.

Embodiment 3

(18) This embodiment specifically describes the types of products of chitooligosaccharides produced from strain CS1801 through fermentation by colloidal chitosan.

(19) (1) Inoculate a strain CS1801 to a PDA liquid medium, and culture it under shaking at 30° C. for 2 to 3 d; and

(20) (2) Lead the strain cultured at step (1) to a fermentation medium, and culture it under shaking at 30° C. for 7 d; centrifuge the fermentation liquor to discard the precipitate and take the supernate.

(21) The composition of the fermentation medium is the same as that of the foregoing secondary screening medium.

(22) The composition of the fermentation liquor is analyzed by ultra-high performance liquid chromatography-mass spectrum (UPLC-Q-TOF-MS) technology to determine that chitosan is decomposed into chitobiose, chitotriose, chitotetraose, chitopentaose and chitohexaose under the action of Streptomyces CS1801. The ultra-high performance liquid chromatogram is shown in FIG. 4, and the mass spectrum is shown in FIG. 5.

Embodiment 4

(23) This embodiment specifically describes the application of strain CS1801 for producing chitooligosaccharides through fermentation by prawn powder.

(24) (1) Inoculate a strain CS1801 to a PDA liquid medium, and culture it under shaking at 30° C. for 2 to 3 d; and

(25) (2) Lead the strain cultured at step (1) to a fermentation medium, and ferment it under shaking at 35° C. for 5 d; centrifuge the fermentation liquor to discard the precipitate and take the supernate, and determine the enzyme activity of chitinase is 57.3 U/L, and the content of chitooligosaccharides is 0.58 mol/L. The enzyme activity of chitinase is determined as described in Embodiment 2.

(26) The fermentation medium is as follows:

(27) Liquid A: K.sub.2HPO.sub.4 1.4 g/L, KH.sub.2PO.sub.4 0.6 g/L, MgSO.sub.4.7H.sub.2O 1 g/L, NaCl 10 g/L, (NH.sub.4).sub.2SO.sub.4 20 g/L, deionized water 1000 mL, pH 6.5. Liquid B: 100 g/L prawn powder, pH 6.5. Liquid A and liquid B are mixed in an equal volume.

Embodiment 5

(28) This embodiment specifically describes the application of strain CS1801 for producing chitooligosaccharides through fermentation by wet prawns.

(29) (1) Inoculate a strain CS1801 to a PDA liquid medium, and culture it under shaking at 30° C. for 2 to 3 d;

(30) (2) Lead the strain cultured at step (1) to a fermentation medium, and ferment it under shaking at 37° C. for 7 d; centrifuge the fermentation liquor to discard the precipitate and take the supernate, and determine the enzyme activity of chitinase is 50.3 U/L, and the content of chitooligosaccharides is 0.43 mol/L. The enzyme activity of chitinase is determined as described in Embodiment 2.

(31) The fermentation medium is as follows: 300 g/L wet prawns, pH 6.5.