Preparation method and application of novel halophilic archaea chitinase

Abstract

The invention belongs to the field of gene engineering, and relates to a preparation method and application of novel halophilic archaea chitinase. The method comprises the following steps: firstly, performing database comparison based on a genome of halophilic archaea HN-29 to obtain a chitinase gene chiDHap; amplifying by utilizing a PCR (Polymerase Chain Reaction) technology to obtain a target gene segment; then, a target gene segment is connected with a pTA04 vector through a molecular cloning technology, and a recombinant plasmid pTA04-chiDHap is obtained; the method comprises the following steps of: preparing a Hv-YPC liquid culture medium, converting the Hv-YPC liquid culture medium into a halophilic archaea expression host, inoculating the Hv-YPC liquid culture medium with the halophilic archaea expression host, culturing, centrifuging, collecting thalli, re-suspending by using a cell lysis solution, ultrasonically crushing, centrifuging, collecting supernate, and finally purifying to obtain chitinase. The obtained chitinase has good enzymatic characteristics and stability, and can tolerate a variety of metal ions, organic solvents and surfactants; the method can be applied to degradation of chitin, and degradation products can be applied to preparation of functional foods.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and specifically relates to a preparation method of a novel halophilic archaea chitinase and application thereof. Background Art

[0002] Chitin is the second largest renewable natural resource in nature after cellulose. It is mainly found in the shells of crustaceans, insect exoskeletons and fungal cell walls. Chitin is a linear polysaccharide formed by N-acetyl-D-glucosamine (GlcNAc) connected by β-1,4 glycosidic bonds. It contains a large number of hydrogen bonds and has a very stable structure. Therefore, chitin is difficult to dissolve in water and is not easy to degrade, which limits the application of chitin. Therefore, the value of chitin can be better realized by degrading chitin into chito-oligosaccharides with lower molecular weight and easy to dissolve in water. It is reported that chito-oligosaccharides have many biological activities, including antibacterial effects, anti-inflammatory properties, anti-tumor ability, antioxidant and immunomodulatory. Chito-oligosaccharides have certain applications in medicine, health care, agricultural production, food industry and other fields. The seafood processing industry produces a large amount of chitin waste, which is easy to cause environmental pollution and waste of resources. For this type of salt-containing chitin resources, salt-tolerant chitinase is needed to degrade them.

[0003] Halophilic archaea mainly grow in high-salt environments, such as saline-alkali land, salt lakes, and salt pans. The enzymes they produce usually have good tolerance, such as resistance to salt and alkali, high temperature, and organic solvents. Therefore, halophilic archaea provide a good source for the excavation of broad-spectrum chitinases. At present, the research on chitinases from halophilic archaea is very limited, limited to Halobacterium salinarum and Haloferax mediterranei, and the characterization is insufficient. At present, the partial purification methods of chitinase still have limitations, such as ammonium sulfate precipitation method, low purity; ion exchange chromatography method, high operating conditions; gel filtration chromatography method, long time and high cost. Summary of the invention

[0004] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a novel halophilic archaea chitinase gene and a method for preparing halophilic archaea chitinase. The present invention fills the gap in the research of halophilic archaea chitinase; compared with the reported preparation methods of halophilic archaea chitinase, the present invention is simpler and more efficient to operate and can obtain good purity. In addition, the prepared chitinase has good enzymatic properties and stability, can tolerate a variety of metal ions, organic solvents and surfactants; and can be used for the degradation of chitin, especially salt-containing chitin; its degradation products can be used to make functional foods, and have good market application prospects.

[0005] The present invention obtains a chitinase gene of Halophilic Archaea (Haladaptatus sp.) HN-29 based on the genome, named chiD Hap The nucleotide sequence is shown in SEQ ID No. 1, and the encoded chitinase is named ChiD Hap , and its amino acid sequence is shown in SEQ ID No. 2. The Halophilic Archaea (Haladaptatus sp.) HN-29 used is a conventional public strain purchased from the General Microbiological Center of China Microbiological Culture Collection Administration, with the number CGMCC 1.63101.

[0006] The present invention relates to a method for preparing halophilic archaea chitinase, which specifically comprises the following steps:

[0007] (1) Construction of pTA04-chiD Hap Recombinant expression plasmid;

[0008] Firstly, a chitinase gene chiD of Halophilic Archaea (Haladaptatus sp.) HN-29 was obtained after genome comparison with database. Hap , whose nucleotide sequence is shown in SEQ ID No.1; then the target gene fragment is amplified by PCR technology; then the amplified target gene fragment is connected to the pTA04 vector by molecular cloning technology to construct a recombinant plasmid pTA04-chiD Hap ;

[0009] (2) Transformation of Haloferax volcanii with the recombinant plasmid;

[0010] The recombinant plasmid pTA04-chiD obtained in step (1) was Hap Transform the halophilic archaea expression host, inoculate the transformed recombinant host cells into Hv-YPC liquid medium for cultivation, centrifuge the cell fluid cultured to the stable phase, collect the bacterial cells, and store them for future use;

[0011] (3) Purification of chitinase from halophilic archaea;

[0012] The cells collected in step (2) were resuspended in a cell lysis buffer, the cells were ultrasonically disrupted, and the supernatant was collected by centrifugation and purified by nickel column affinity chromatography to obtain high-purity halophilic archaeal chitinase, which was recorded as ChiD Hap , and its amino acid sequence is SEQ ID NO.2.

[0013] Preferably, the halophilic archaea expression host in step (2) is Haloferax volcanii, purchased from Japan Culture Collection of Microorganisms (JCM), with the culture collection number of JCM 8879.

[0014] Preferably, the Hv-YPC liquid culture medium in step (2) comprises, per liter, 5.0 g yeast extract, 1.0 g soy peptone, 1.0 g acid hydrolyzed casein, 4.2 g KCl, 33.0 g MgSO4·7H2O, 30.0 g MgCl2·6H2O, 144.0 g NaCl, 12.0 mL 1M, pH 8.0 Tris-HCl, and 0.33 g CaCl2. The above components are diluted to 1 L with distilled water and the pH is adjusted to 7.5.

[0015] Preferably, the culture conditions in step (2) are 37° C. and the rotation speed is 160 rpm.

[0016] Preferably, the storage temperature in step (2) is -20°C.

[0017] Preferably, the cell lysis solution in step (3) comprises: 2M NaCl, 50mM Tris-HCl, pH 8.0.

[0018] Preferably, the conditions for ultrasonic cell disruption in step (3) are: ultrasonic time 3 s, interval 5 s, power 200 W, and total time 30 min.

[0019] Preferably, the steps of purifying by nickel column affinity chromatography in step (3) are: washing the column with 20 times the column volume of ddH2O, and then balancing the nickel column with 20 times the column volume of cell lysate; centrifuging the cell lysate and taking the supernatant onto the column; eluting impurities with 20 ml of buffer I; and eluting the target protein with 10 ml of buffer II.

[0020] Preferably, the components of the buffer I are: 40 mM imidazole, 2 M NaCl, 50 mM Tris-HCl, pH 8.0.

[0021] Preferably, the components of the buffer II are: 100 mM imidazole, 2 M NaCl, 50 mM Tris-HCl, pH 8.0.

[0022] The novel halophilic archaeal chitinase ChiD prepared by the present invention Hap The invention can be applied to the degradation of chitin resources, especially the degradation of salt-containing chitin; the chitin resources include shrimp shells and crab shells; and the degradation products can be applied to the preparation of functional foods.

[0023] Preferably, the degradation reaction system applied to chitin resources is: chitin resources, chitinase ChiD Hap (enzyme solution, concentration is 1mg / ml) and enzyme activity assay buffer; chitin resources, chitinase ChiD HapThe dosage relationship of the enzyme activity assay buffer is 1 mg: 4 μL: 96 μL. The enzyme activity assay buffer composition is: 1 M NaCl, 0.1 M phosphate buffer at pH 7.5;

[0024] Specific operation: the reaction system was reacted in a 45°C water bath for 30 minutes; after the reaction was completed, the reaction was terminated under the conditions of 100°C, 3 minutes in a metal bath, thereby achieving the degradation of chitin resources.

[0025] The invention is used for degrading chitin resources and can effectively improve the utilization rate of chitin resources.

[0026] Beneficial effects of the present invention:

[0027] (1) The present invention discovered a novel halophilic archaeal chitinase gene and expressed a halophilic archaeal chitinase. Currently, there is little research on halophilic archaeal chitinase, and this invention can fill the gap in the research on halophilic archaeal chitinase. Compared with the reported chitinase preparation methods, the present invention is simpler and more efficient and can obtain good purity.

[0028] (2) Halophilic archaeal chitinase ChiD prepared by the present invention Hap It has good enzymatic properties; it has high enzyme activity in the range of 40-60℃, and has good stability at 40℃ and 60℃; it maintains high activity in the pH range of 4.0-8.0, and has good stability under acidic, neutral and alkaline conditions; it can tolerate a variety of metal ions, organic solvents and surfactants. Halophilic archaeal chitinase ChiD with excellent properties Hap It can be applied to the degradation of salt-containing chitin, such as the degradation of shrimp shell powder. Its degradation products can be used to make functional foods and have broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The figure is the restriction enzyme cutting verification diagram of the successful construction of plasmid.

[0030] Figure 2 pTA04-chiD Hap Validation diagram of transformants of Haloferax volcanii.

[0031] Figure 3 ChiD purified by nickel column affinity chromatography Hap SDS-PAGE electrophoresis diagram.

[0032] Figure 4 ChiD Hap Effect diagram of catalytic activity.

[0033] Figure 5 is the effect of NaCl concentration on ChiDHap Effect diagram of catalytic activity.

[0034] Figure 6 pH to ChiD Hap Effect diagram of catalytic activity.

[0035] Figure 7 ChiD Hap Figure 2. Stability analysis results at different temperatures.

[0036] Figure 8 ChiD Hap Figure 2. Stability analysis results at different NaCl concentrations.

[0037] Fig. 9 ChiD Hap Figure 2. Stability analysis results under different pH conditions.

[0038] Fig.10 Different metal ions are ChiD Hap Effect diagram of catalytic activity.

[0039] Fig.11 Effects of different organic solvents and surfactants on ChiD Hap Effect diagram of catalytic activity.

[0040] Fig.12 ChiD Hap Enzyme kinetics curve using shrimp shell powder as substrate. DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to the accompanying drawings and specific implementation examples.

[0042] Halophilic archaeon Haladaptatus sp.HN-29 is a common public strain purchased from the General Microbiological Center of China Microbiological Culture Collection Administration, with the number CGMCC 1.63101.

[0043] Performance testing:

[0044] (1) Halophilic Archaea Chitinase ChiD Hap Characterization of enzymatic properties;

[0045] Determination of chitinase ChiD using shrimp shell powder as substrate Hap The enzyme activity is used to characterize the enzymatic properties, including the optimum temperature of the enzyme catalytic reaction; the optimum NaCl concentration; the optimum pH; the effect of different metal ions on ChiD Hap Effects of different organic solvents and surfactants on the activity of ChiD HapThe influence of the activity and the determination of the enzyme kinetic parameters; wherein the determined ranges of the temperature, NaCl concentration and pH are 30-65°C, 0-4M, and pH 3.0-10.5 respectively; the different metal ions are: Ca 2+ , Sr 2 + 、Zn 2+ , K + 、Ni 2+ Mg 2+ , Ba 2+ , Mn 2+ , Cu 2+ , Fe 3+ 、Co 2+ The different organic solvents and surfactants are: methanol, ethanol, acetonitrile, glycerol, isopropanol, acetone, DMF (dimethylformamide), DMSO, Tween 20, Tween 80, and Triton X-100.

[0046] (2) Halophilic Archaea Chitinase ChiD Hap Stability characterization;

[0047] The chitinase ChiD Hap After incubating for a certain period of time at different temperatures, NaCl concentrations and pH values, the residual activity is determined using shrimp shell powder as a substrate; wherein the different temperatures are: 40°C, 60°C, 80°C, the NaCl concentrations are: 0.5M, 2.0M, 3.5M, the pH values ​​are: 5.0, 7.0, 9.0, and the incubation time is: 30min, 60min, 90min.

[0048] Embodiment 1:

[0049] Construction of recombinant plasmids based on restriction endonucleases

[0050] (1) Amplification of target gene based on PCR technology

[0051] The inoculation loop was used to pick up the bacteria and put them into 50 μL ddH2O. The bacteria were boiled at 100℃ for 10 minutes to lyse the cells, which were used as templates for DNA amplification. The PCR products were detected by agarose gel electrophoresis under UV light, and the target fragments were cut out and purified using a DNA gel recovery kit.

[0052] Primer chiD Hap -F-EcoRI: 5'-CGCGAATTCGCGGACTGCACGGTTAC-3';

[0053] d Hap -R-BamHI:5'-ATAGGATCCGAGATGGTCGAGCATCGC-3'.

[0054] PCR amplification system (25 μL):

[0055]

[0056] PCR amplification procedure:

[0057]

[0058]

[0059] (2) Enzyme digestion of target gene and vector

[0060] The purified target gene and pTA04 vector were double-digested in a metal bath using BamHI and EcoRI restriction endonucleases at 37°C for 30 min. The digested product was purified again using a DNA gel recovery kit to obtain the purified target gene.

[0061] (3) Ligation of the target fragment with the pTA vector

[0062] The purified target gene and pTA04 vector were ligated with T4 DNA ligase at 16°C for 2 h.

[0063] Ligation system (10 μL):

[0064]

[0065] (4) Transformation of Escherichia coli DH5α

[0066] Add 10 μL of the ligation system to 100 μL of E. coli DH5α competent cells, place on ice for 30 min, heat shock at 42°C in a metal bath for 90 s, take out and place on ice again for 2 min, then add 500 μL of LB liquid culture medium to the competent cells added with the ligation system in an ultra-clean workbench, and culture on a shaker for 50 min (37°C, 160 rpm), take 200 μL of the bacterial solution, evenly spread it on an LB agar plate containing ampicillin, and culture it at 37°C overnight (12-15 h) to obtain the positive transformants verified by PCR.

[0067] (5) Plasmid extraction and verification

[0068] Add the positive transformants verified by PCR to the LB liquid culture medium supplemented with ampicillin, culture in an incubator overnight (37°C, 12-15h), extract the plasmid using a plasmid DNA small-scale extraction kit for enzyme digestion verification, and observe the enzyme digestion products under ultraviolet light by agarose gel electrophoresis to determine whether the plasmid is successfully constructed.

[0069] Enzyme digestion system (10 μL):

[0070]

[0071]

[0072] The digestion products were verified by agarose gel electrophoresis. Figure 1 As shown, it is the restriction enzyme digestion verification diagram of the successful construction of the plasmid; Lane M represents the DNA marker, and Lane 1 represents the plasmid double restriction enzyme digestion result; Figure 1 It can be seen that the target gene chiD can be obtained after enzyme digestion of the plasmid Hap The plasmid was successfully constructed by using pTA04 vector and sequenced and stored at -20℃.

[0073] Embodiment 2:

[0074] Halophilic Archaea Chitinase ChiD Hap Expression

[0075] The correct recombinant plasmid was transferred into Haloferax vocanii by PEG-mediated transformation, and the red transformants on the Hv-YPC plate were verified by PCR. The verification conditions were the same as those in Example 1 (1), and the correct positive transformants were finally obtained. The correct positive transformants were inoculated into Hv-YPC liquid culture medium. The liquid culture medium of the inoculated positive transformants was cultured at 37°C and 160rpm for 3 days, and then inoculated into 200mL Hv-YPC liquid culture medium with a 1% (v / v) inoculation amount. After culturing to the stable period, the cells were centrifuged and the centrifuged bacteria were stored at -20°C. Figure 2 As shown, pTA04-chiD Hap Haloferax volcanii transformant validation diagram, lane M in the figure represents DNA marker, lane 1 represents the transformant validation result; Figure 2 It can be seen that the recombinant plasmid was successfully transformed into Haloferax vocanii.

[0076] Embodiment 3:

[0077] Halophilic Archaea Chitinase ChiD Hap Purification

[0078] (1) The cells collected in Example 2 were resuspended in 40 mL of cell lysis buffer (2 M NaCl, 50 mM Tris-HCl, pH 8.0), placed on ice for ultrasonic disruption, and the supernatant was collected after centrifugation at 4°C for subsequent purification.

[0079] (2) Wash the column with 20 times the column volume of ddH2O, and then equilibrate the nickel column with 20 times the column volume of cell lysate.

[0080] (3) Load the supernatant onto the column and repeat twice.

[0081] (4) Add 20 mL of buffer I to wash away impurities and retain 1 mL of the sample.

[0082] (5) Add 10 mL of buffer II to elute the target protein, collect 1 mL of the eluate in each tube, and obtain high-purity halophilic archaeal chitinase, which is recorded as ChiD Hap , stored at 4℃.

[0083] The obtained protein was analyzed by SDS-PAGE. Figure 3 ChiD purified by nickel affinity chromatography Hap SDS-PAGE electrophoresis diagram, where: Lane M: Marker; Lane 1: supernatant after cell lysis and centrifugation; Lane 2: sample flow-through; Lane 3: impurity protein eluted by buffer I; Lane 4: target protein eluted by buffer II; Figure 3 It can be seen that buffer II can be used to Hap The eluted bands are relatively single.

[0084] Embodiment 4:

[0085] Enzyme activity assay

[0086] Determination of chitinase ChiD using shrimp shell powder as substrate Hap The reaction system includes 1 mg shrimp shell powder, 4 μL chitinase ChiD Hap (enzyme solution, concentration is 1 mg / ml) and 96 μL enzyme activity assay buffer; wherein the enzyme activity assay buffer composition is: 2M NaCl, 50mM Tris-HCl, pH 8.0.

[0087] Specific operation: The reaction system was reacted in a 37℃ water bath for 30min; no chitinase ChiD was added to the control group. Hap (Replaced by an equal amount of buffer II); after the reaction was completed, the reaction was terminated under the conditions of 100°C and 3 min in a metal bath.

[0088] After the reaction was terminated, 40 μL of supernatant was collected by centrifugation. 80 μL of 0.05% (M / V) potassium ferrocyanide solution (prepared with 0.5M Na2CO3) was added to the supernatant of the experimental group and the control group, mixed, kept at 100°C for 10 minutes, centrifuged, and the supernatant was collected to measure the absorbance at 420 nm. ddH2O was used as a blank control.

[0089] A standard curve was prepared using N-acetylglucosamine (GlcNAc), and the reducing sugar concentration in the reaction solution was calculated.

[0090] Definition of enzyme activity (U): The amount of reducing sugar produced per minute under the above conditions is one activity unit.

[0091] Embodiment 5:

[0092] Chi Hap Analysis of optimal reaction conditions;

[0093] (1) Chitinase ChiD Hap The optimum reaction temperature was determined in the range of 30 to 65°C;

[0094] The reaction temperature was set at 30, 35, 40, 45, 50, 55, 60, and 65° C. The method for determining the enzyme activity was the same as in Example 4 except for the reaction temperature. The relative enzyme activity at other temperatures was calculated with the highest enzyme activity as 100%, and each group was tested three times in parallel. Figure 4 ChiD Hap The results show that ChiD Hap The optimum reaction temperature is 45°C.

[0095] (2) Chitinase ChiD Hap The optimal reaction NaCl concentration was determined in the range of 0 to 4 M;

[0096] The final concentration of NaCl in the enzyme activity assay buffer was prepared to be 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 M, and the chitinase activity was measured at the optimum temperature (45° C.). The determination method was the same as in Example 4, except that the final concentration of NaCl was changed. The relative enzyme activity of other salt concentrations was calculated with the highest enzyme activity as 100%, and each group was tested three times in parallel. Figure 5 is the effect of NaCl concentration on ChiD Hap The effect diagram of catalytic activity showed that the optimal NaCl concentration for the enzyme reaction was 1.0 M.

[0097] (3) Chitinase ChiD Hap The optimum reaction pH was determined in the range of 3.0 to 10.5;

[0098] 0.1M citric acid-trisodium citrate buffer (Citrate buffer) with a pH range of 3.0 to 6.0; 0.1M phosphate buffer (Phosphate buffer) with a pH range of 6.0 to 7.5; 0.1M Tris-HCl buffer with a pH range of 7.5 to 9.0; and 0.1M CHES-NaOH buffer with a pH range of 9.0 to 10.5 were prepared respectively. The chitinase ChiD was determined at the optimal reaction temperature of 45°C and the optimal NaCl concentration of 1.0M in the buffer systems with different pH values. HapThe enzyme activity was determined by the same method as in Example 4, except that pH 8.0, 50 mM Tris-HCl was used instead. The highest enzyme activity was taken as 100% to calculate the relative enzyme activity at other pH values, and each group was tested three times in parallel. Figure 6 pH to ChiD Hap The results show that ChiD Hap The optimal reaction pH is 7.5 (phosphate buffer).

[0099] Halophilic Archaea Chitinase ChiD Hap The optimal reaction conditions were 45°C, 1.0 M NaCl, and 0.1 M phosphate buffer, pH 7.5.

[0100] Embodiment 6:

[0101] Chi Hap Stability analysis;

[0102] (1) Chitinase ChiD Hap Thermal stability analysis;

[0103] Chitinase ChiD Hap Incubate at 40, 60, and 80°C for 0, 30, 60, and 90 min, and determine the residual chitinase activity under the optimal reaction conditions. The enzyme activity at 0 min was taken as 100% to calculate the relative enzyme activity at other times under each temperature gradient, and each group was tested in parallel three times. Figure 7 ChiD Hap The results of stability analysis at different temperatures show that the enzyme can still maintain more than 83% activity after incubation at 40 and 60°C for 90 minutes, and has good thermal stability.

[0104] (2) Chitinase ChiD Hap NaCl stability analysis;

[0105] Chitinase ChiD Hap The chitinase residual activity was determined under the optimal reaction conditions in the optimal pH buffer containing 0.5, 2.0, and 3.5 M NaCl at room temperature for 0, 30, 60, and 90 min. The relative enzyme activity at other times was calculated with the enzyme activity at 0 min as 100% under each NaCl gradient, and each group was tested in triplicate. Figure 8 ChiD Hap The results of stability analysis at different NaCl concentrations show that the enzyme can still maintain more than 95% activity after incubation for 90 minutes at 0.5, 2.0 and 3.5 M NaCl concentrations, and has good NaCl stability.

[0106] (3) Chitinase ChiD Hap pH stability analysis;

[0107] The enzyme was placed in a buffer containing 1M NaCl at pH 5.0 (citric acid-trisodium citrate buffer), pH 7.0 (phosphate buffer) and pH 9.0 (CHES-NaOH buffer) at room temperature for 0, 30, 60, and 90 minutes, and the residual chitinase activity was determined under the optimal reaction conditions. The relative enzyme activity at other times was calculated with the enzyme activity at 0 min as 100% under each pH gradient, and each group was tested in triplicate. Fig. 9 ChiD Hap The results of stability analysis under different pH conditions show that after incubation for 90 minutes at pH 5.0, pH 7.0 and pH 9.0, the relative enzyme activity still remains above 83%. The enzyme has good stability under acidic, neutral and alkaline conditions, indicating that the enzyme has good pH stability.

[0108] Embodiment 7:

[0109] Chi Hap Tolerance analysis;

[0110] (1) Chitinase ChiD Hap Analysis of tolerance to metal ions;

[0111] 5 mM of different metal ions Ca were added to the reaction system. 2+ , Sr 2+ 、Zn 2+ , K + 、Ni 2+ Mg 2+ , Ba 2+ , Mn 2+ , Cu 2+ , Fe 3+ 、Co 2+ The enzyme activity was determined under the optimal reaction conditions. In the positive control system, ddH2O was used instead of metal ions. The positive control result was taken as 100% enzyme activity. The relative enzyme activity of the reaction system containing metal ions was compared. Each group was tested three times in parallel. Fig.10 Different metal ions are ChiD Hap The catalytic activity effect diagram shows that K + , Fe 3+ 、Co 2+ It has a promoting effect on the activity of the enzyme, among which K + Can increase enzyme activity by about 30%; Ba 2+ There is almost no effect on enzyme activity; Ca 2+ , Sr 2+ 、Zn 2+ 、Ni 2+ Mg 2+ , Mn2+ , Cu 2+ The enzyme has different degrees of inhibition, among which Ni 2+ and Mn 2+ The inhibitory effect was the strongest, inhibiting ChiD Hap Almost all enzyme activities, Mg 2+ and Cu 2+ The inhibition rates of ChiD Hap The relative enzyme activity can still maintain more than 60% activity. These results show that the enzyme has good tolerance to metal ions under certain conditions.

[0112] (2) Chitinase ChiD Hap Analysis of resistance to organic solvents and surfactants;

[0113] 15% (v / v) methanol, ethanol, acetonitrile, glycerol, isopropanol, acetone, DMF, DMSO, Tween 20, Tween 80, Triton X-100 were added to the reaction system. The enzyme activity was determined under the optimal reaction conditions, and ddH2O replaced the organic solvent and surfactant in the positive control system. The positive control result was taken as 100% enzyme activity, and the relative enzyme activity containing organic solvents and surfactants was compared, and each group was tested three times in parallel. Fig.11 Effects of different organic solvents and surfactants on ChiD Hap The results show that ethanol and glycerol promote the enzyme activity. DMSO has almost no effect on the enzyme activity. Other organic solvents and surfactants show different degrees of inhibitory effects on the enzyme. Acetonitrile, isopropanol and Tween 80 can maintain more than 60% activity; methanol can maintain about 50% activity; DMF can maintain more than 50% activity; ChiD Hap It is relatively sensitive to acetone, Tween 20 and Triton X-100, among which Tween 20 can only maintain about 10% of its activity. The results show that the enzyme has good tolerance to organic solvents and surfactants.

[0114] Embodiment 8:

[0115] Analysis of enzyme kinetic parameters

[0116] Determination of chitinase ChiD using shrimp shell powder as substrate Hap The specific operation of the enzymatic reaction kinetic curve is as follows: add shrimp shell powder with a mass concentration of 4, 6, 8, 10, 12, 15, 20, 25, and 30 mg / mL to the reaction system, and measure the enzyme activity under the optimal reaction conditions. The chitinase ChiD was calculated according to the Michael-Menten equation. Hap The maximum reaction rate V max and the kinetic constant Km . Fig.12 Chitinase ChiD Hap Enzyme kinetics curve of chitinase ChiD with shrimp shell powder as substrate Hap Taking shrimp shell powder as reaction substrate, the kinetic constant K m is 22.379 mg / mL, and the maximum reaction rate V max It is 0.2709μg / min / μg.

[0117] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, although this specification has described the present invention in detail with reference to the above embodiments, a person skilled in the art should understand that the present invention can still be modified or replaced by equivalents; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

[0118] d Hap Nucleotide sequence: SEQ ID No.1

[0119]

[0120] ChiD Hap Amino acid sequence: SEQ ID No. 2

[0121] ADCSGYSDWQSDVAYTDGDRVVYDGALWEAQWWTQANEPETSDSVWVKVGPCDGSGGDGGDGGDGGDGGDGGGNAAPTASFTANPISPAPGETATFDASGSSDSDGSVASYDWTFGDGSSATGQSVTHSYGSAGSYTVTLTVTDDSGATGSATKTITVADGGTSLDQRVTGYYMQWSQWDRDYYPGDIPVDKVSHVNYAFLTVEQDGTVDYIQESAAMNVLQPKSWQDWTGFDSLVDDPNTKFLFSIGGWSDSTHFSDAANTQANRERFASTAIDIMQENDFDGIDIDWEYPGGGGKSGNVVRDGDKARFTKLLQEVRSQLDDAEAADGKEYLLTAAMSADPDKAEGLDHAANAEALDYASIMAYDYHGAFDDYTNHDAPLYAKSNDPSPRASDFNVNASMNYWANTAFDASQLSMGMPFYGRSFANVTSSTNDGLYQSFDGAASGTWGQDNGVKEFWDINQNLEPSSAYDYYWDDTAKVPWLYSPSKDIFITYDNERSIGAKTDYAVDNDFGGVMFWAFANDKNEVLLDAMLDHL

Claims

1. A method for preparing chitinase from halophilic archaea, characterized in that: The specific steps are as follows: (1) Construction of pTA04-chiD Hap Recombinant expression plasmid; Firstly, a chitinase gene chiD of Halophilic Archaea (Haladaptatus sp.) HN-29 was obtained after genome comparison with database. Hap , whose nucleotide sequence is shown in SEQ ID No.1; then the target gene fragment is amplified by PCR technology; then the amplified target gene fragment is connected to the pTA04 vector by molecular cloning technology to construct a recombinant plasmid pTA04-chiD Hap ; (2) Transformation of Haloferax volcanii with the recombinant plasmid; The recombinant plasmid pTA04-chiD obtained in step (1) was Hap Transform the halophilic archaea expression host, inoculate the transformed recombinant host cells into Hv-YPC liquid medium for cultivation, centrifuge the cell fluid cultured to the stable phase, collect the bacterial cells, and store them for future use; (3) Purification of chitinase from halophilic archaea; The cells collected in step (2) were resuspended in a cell lysis buffer, the cells were ultrasonically disrupted, and the supernatant was collected by centrifugation and purified by nickel column affinity chromatography to obtain high-purity halophilic archaeal chitinase, which was recorded as ChiD Hap , and its amino acid sequence is SEQ ID NO.

2.

2. The method for preparing chitinase from halophilic archaea according to claim 1, characterized in that: The halophilic archaea expression host in step (2) is Haloferax volcanii, which was purchased from Japan Culture Collection of Microorganisms with a culture collection number of JCM 8879.

3. The method for preparing a halophilic archaea chitinase according to claim 1, characterized in that: The Hv-YPC liquid culture medium in step (2) comprises, per liter, 5.0 g yeast extract, 1.0 g soy peptone, 1.0 g acid hydrolyzed casein, 4.2 g KCl, 33.0 g MgSO4·7H2O, 30.0 g MgCl2·6H2O, 144.0 g NaCl, 12.0 mL 1M, pH 8.0 Tris-HCl, and 0.33 g CaCl2. The above components are diluted to 1 L with distilled water and the pH is adjusted to 7.

5.

4. The method for preparing chitinase from halophilic archaea according to claim 1, characterized in that: The culture conditions in step (2) are 37° C. and a rotation speed of 160 rpm.

5. The method for preparing chitinase from halophilic archaea according to claim 1, characterized in that: The storage temperature in step (2) is -20°C.

6. The method for preparing a halophilic archaea chitinase according to claim 1, characterized in that: The cell lysis solution in step (3) comprises: 2M NaCl, 50mM Tris-HCl, pH 8.

0.

7. The method for preparing chitinase from halophilic archaea according to claim 1, characterized in that: The conditions for ultrasonic cell disruption in step (3) are: ultrasonic time 3 s, interval 5 s, power 200 W, and total time 30 min.

8. The method for preparing chitinase from halophilic archaea according to claim 1, characterized in that: The steps of purifying by nickel column affinity chromatography in step (3) are as follows: washing the column with 20 times the column volume of ddH2O, and then balancing the nickel column with 20 times the column volume of cell lysate; centrifuging the cell lysate and applying the supernatant to the column; eluting the impurities with 20 ml of buffer I; and eluting the target protein with 10 ml of buffer II; The components of the buffer I are: 40 mM imidazole, 2 M NaCl, 50 mM Tris-HCl, pH 8.0; The components of the buffer II are: 100 mM imidazole, 2 M NaCl, 50 mM Tris-HCl, pH 8.

0.

9. Use of the halophilic archaea chitinase prepared according to the method of any one of claims 1 to 8 for the degradation of chitin resources, characterized in that: The chitin resource is salt-containing chitin, including shrimp shells and crab shells; the reaction system used for the degradation of chitin resources is: chitin resources, ChiD Hap and enzyme activity assay buffer.

10. The use according to claim 9, characterized in that Chitin resources and ChiD in the reaction system Hap The dosage of enzyme activity assay buffer is 1 mg: 4 μL: 96 μL, where ChiD Hap The concentration was 1 mg / ml; the enzyme activity assay buffer composition was: 1 M NaCl, 0.1 M phosphate buffer at pH 7.5; The steps are: reacting the reaction system in a 45°C water bath for 30 minutes; and terminating the reaction under the conditions of a 100°C, 3-minute metal bath after the reaction is completed, thereby achieving the degradation of chitin resources.

Images