CN109055260B

CN109055260B - Bacillus flexus alkaAU and method for producing urate oxidase, product and application

Info

Publication number: CN109055260B
Application number: CN201810897916.XA
Authority: CN
Inventors: 焦豫良; 王淑军; 吕明生; 房耀维; 刘姝
Original assignee: Huaihai Institute of Techology
Current assignee: Huaihai Institute of Techology
Priority date: 2018-08-08
Filing date: 2018-08-08
Publication date: 2021-11-09
Anticipated expiration: 2038-08-08
Also published as: CN109055260A

Abstract

The invention relates to a bacillus flexusBacillus flexus) alkaAU, CCTCC NO: m2018378. The strain has the following characteristics: gram-positive rod-shaped bacteria, wherein cells are in a long rod shape, are mesogenic or telogenic and can move; the strain has a growth temperature range of 15-70 ℃, a growth pH range of 4-12 and a growth NaCl concentration range of 0-15%; the invention also discloses an enzyme production method, a product and application thereof. The strain can produce various medical and industrial and agricultural enzymes including urate oxidase, arginase, pullulanase, isoamylase and atrazine hydrolase. The urate oxidase produced by the bacillus flexus reaches 0.52U/mL, the optimum temperature of the enzyme activity is 40 ℃, the optimum pH value is 10, the half-life period at the optimum temperature is about 12 h, and the urate oxidase is applied to the development of medicines and diagnostic reagents.

Description

Bacillus flexus alkaAU and method for producing urate oxidase, product and application

Technical Field

The present invention relates to a microorganism which is capable of producing,in particular to a bacillus flexus alkaAU (AlkaAU) separated from the Hongkong harbor sea area of Jiangsu province of ChinaBacillus flexusalkaAU), which has been preserved in China Center for Type Culture Collection (CCTCC) in 2018, 6 and 19 months, with the preservation number of CCTCC NO: m2018378; the invention also relates to a method for producing urate oxidase by using the strain and application of the urate oxidase.

Background

Urate oxidase (E.C. 1.7.3.3) can catalyze uric acid to be oxidized into allantoin and hydrogen peroxide, so that uric acid is not absorbed by renal tubules any more and is excreted, and the urate oxidase has good effect on hyperuricemia caused by nodal gout, urinary calculus and renal failure, and can also be used for preparing a blood uric acid detection kit. In addition, urate oxidase can be used for treating tumor lysis syndrome, relieving cardiovascular diseases caused by hyperuricemia, and is a medicinal enzyme preparation with wide application. The urate oxidase derived from microorganisms has the advantages of good activity, suitability for industrial production and the like, and has become a research hotspot in recent years. At present, microorganisms producing urate oxidase include fungi, yeasts and bacteria, but are not commercially effectively used due to low enzyme production level, low enzyme activity, unstable enzymology, high production cost and the like. In addition, the commercialized urate oxidase mainly comes from candida or aspergillus niger, the optimum enzyme activity temperature is lower than the human body temperature, the price is high, and the price of each 100 enzyme activity unit commercial enzyme (about 0.05 g) is about 1000 yuan RMB, so the commercial potential is huge. The active development of new sources of urate oxidase with new enzymatic properties remains an important breakthrough.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a novel bacillus flexus with high urate oxidase yield (aBacillus flexus）alkaAU。

Another object of the present invention is to provide a method for producing urate oxidase by using Bacillus flexus alkau.

It is a further object of the present invention to provide urate oxidase produced by the aforementioned method and its use.

The object of the present invention is achieved byThe technical scheme is realized. The invention relates to a bacillus flexusBacillus flexus) The alkaAU is characterized in that: the biological preservation number is CCTCC NO: m2018378.

Bacillus flexus of the invention (A)Bacillus flexus) The alkaAU is preserved in the China center for type microbiological culture Collection (CCTCC) in 2018, 6 months and 19 days, and the preservation number is CCTCC NO: m2018378, accession number: wuhan university Collection, Lodoku mountain, Wuchang, Wuhan, Hubei province.

The invention also discloses the bacillus flexus (in the technical scheme)Bacillus flexus) A method for producing cell wall-bound urate oxidase by alkaAU, comprising the steps of:

(1) inoculating Bacillus flexus alkau into a seed culture medium, rotating at 150-180 rpm, filling liquid at 20-30%, and culturing at 35-45 ℃ for 12-24 h to obtain a seed liquid; the seed culture medium comprises the following components: 0.5% of yeast powder, 1% of tryptone, 1% of arginine and distilled water;

(2) inoculating the seed solution into an enzyme production culture medium according to the inoculation amount of 1% of the volume percentage content, culturing at 35-45 ℃ for 36-72 hours at 180 rpm and 10-20% of the liquid loading amount to obtain urate oxidase fermentation liquor; the enzyme production culture medium comprises the following components: 0.5 percent of yeast powder, 1 percent of fish peptone, 0.5 percent of uric acid and 0.5 mol/L phosphate buffer solution, and the pH value is 6.

The cell content of the seed liquid is preferably 10⁷cells/mL.

The invention also discloses urate oxidase, which is characterized in that: the urate oxidase adopts the bacillus flexus (B) of the technical schemeBacillus flexus) and (3) fermenting the alkaAU to produce the enzyme. Or the urate oxidase is prepared by the enzyme production method of the technical scheme. The urate oxidase can be prepared into enzyme preparations for medical use. The medical enzyme preparation is a medicine for treating hyperuricemia caused by nodular gout, urinary calculus and kidney disease failure. Or a reagent for detecting blood uric acid, or a medicine for preparing a medicine for treating tumor lysis syndrome or a medicine for relieving cardiovascular diseases caused by high uric acid.

The bacillus flexus is separated from sea mud in a nuclear power station of a bay of continuous cloud harbor of Jiangsu province in China. And screening the bacillus curvatus by using a screening culture medium. The screening medium consists of: 0.3% of yeast powder, 0.5% of oyster powder, 0.5% of fish meal, 1% of arginine, 2% of agar and distilled water. And carrying out shake flask culture on the strain, and carrying out uricoxidase activity identification on the fermentation liquor of the strain.

The present invention relates to Bacillus flexusBacillus flexus) and (3) identifying the strain as the campylobacter by 16S rDNA sequence analysis and combining colony morphology, cell microscopic morphology and physiological and biochemical reaction results. The physiological and biochemical tests show that said strain possesses the capability of producing several medical and industrial and agricultural enzymes, including: urate oxidase, arginase, pullulanase, isoamylase and atrazine hydrolase.

The invention can prepare the urate oxidase by taking the curvibacillus alkaAU as a production strain through seed culture and liquid shallow fermentation.

The invention also discloses a method for preparing the bacillus flexusBacillus flexus) The method for hydrolyzing the pullulan by the alkaAU is characterized by comprising the following steps: adding pullulan with the final concentration of 1% into a beef extract peptone culture medium, and inoculating campylobacter alkaAU; at 180 rpm, filling 20% of liquid, culturing at 37 ℃ for 72 h, centrifuging and precipitating cells, collecting supernatant, dialyzing with 0.01 mol/L pH7 phosphate buffer at 4 ℃ to remove reducing sugar, and obtaining pullulanase extracting solution.

The invention also discloses a method for preparing the bacillus flexusBacillus flexus) The method for hydrolyzing arginine by alkaAU is characterized by comprising the following steps: adding arginine with the final concentration of 1% into a beef extract peptone culture medium, and inoculating campylobacter alkaAU; culturing at 33 ℃ for 12 h at 180 rpm, centrifuging and precipitating cells, adding 10 times of sterile water for resuspension, carrying out cell ultrasonic crushing in an ice bath, carrying out ultrasonic power of 250-300W for 30 min, crushing for 2s at intervals of 3 s, centrifuging at 10000 rpm for 10 min, and collecting supernatant to be used as arginase extracting solution.

The invention also discloses a method for preparing the bacillus flexusBacillus flexus) The method for hydrolyzing amylopectin by alkaAU is characterized by comprising the following steps: adding amylopectin with final concentration of 1% into beef extract peptone culture medium, and inoculating Bacillus flexus alkau; culturing at 35 deg.C for 72 h at 180 rpm and liquid loading of 20%, centrifuging to precipitate thallus, collecting supernatant, dialyzing with 0.05 mol/L sodium acetate buffer solution (pH 7) at 4 deg.C to remove reducing sugar to obtain isoamylase extract.

The invention also discloses a method for preparing the bacillus flexusBacillus flexus) The method for hydrolyzing atrazine by alkaAU is characterized by comprising the following steps: adding atrazine with the final concentration of 0.03% into a beef extract peptone culture medium, and inoculating campylobacter alkaAU; 180 rpm, liquid loading capacity of 20%, culturing at 37 ℃ for 36 h, centrifuging to collect cells, washing with sterile distilled water for 3 times, then adding 10 times of sterile water for heavy suspension, carrying out cell ultrasonic crushing in an ice bath, carrying out ultrasonic power of 250-300W for 30 min, crushing for 2s at intervals of 3 s, centrifuging at 10000 rpm for 10 min, and collecting supernatant to be used as an atrazine hydrolase extracting solution.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a Bacillus flexus alkAU for high yield of urate oxidase, and the activity of urate oxidase reaches 0.52U/mL. The optimum pH value of the urate oxidase produced by the strain is 10, the optimum temperature of the enzyme activity is 40 ℃, and the half-life period is about 12 hours. The urate oxidase produced by the invention can be used in the industries of medicines and diagnostic reagents. In addition, the strain Bacillus flexus alkau also produces various medical and industrial and agricultural enzymes including arginase, pullulanase, isoamylase and atrazine hydrolase, and has wide application potential.

Drawings

FIG. 1 is a cell morphology map of the strain alkaAU (1000-fold; spore staining);

FIG. 2 is a morphological diagram of a single colony of the strain alkaAU;

FIG. 3 is a phylogenetic relationship diagram of a strain alkaAU constructed based on 16S rDNA;

FIG. 4 is a graph showing the effect of different temperatures on the growth of the species alkAU;

FIG. 5 is a graph of the effect of different pH on the growth of the species alkAU;

FIG. 6 is a graph showing the effect of different NaCl concentrations on the growth of the strain alkAU;

FIG. 7 is a graph showing the effect of different reaction temperatures on enzyme activity;

FIG. 8 is a graph showing the effect of different temperatures on the thermostability of an enzyme;

FIG. 9 is a graph showing the effect of different reaction pH on enzyme activity.

Detailed Description

The invention is further illustrated by the following examples to enable a person skilled in the art to better understand the invention without constituting a limitation to the rights of the invention.

Example 1A Bacillus flexus bacterium (Bacillus flexus) The biological preservation number of the alkaAU is CCTCC NO: m2018378. Isolation of Bacillus curvatus alkaAU by the following steps:

selecting a part of sea mud 10 g dug 5 cm below the ground in a low-tide region of a nuclear power station of a Hongkou Bay of continuous cloud, adding the sea mud into a triangular flask filled with 90 mL of sterile water under aseptic operation, and oscillating for 30 min to fully break up a sample. And (3) sucking 1.0 mL for gradient dilution, selecting a proper dilution, coating the dilution on a screening medium plate, culturing at 37 ℃ for 36 h, and selecting a colony with a transparent ring. And selecting colonies, inoculating the colonies into an enzyme production culture medium, culturing at 37 ℃ for 36 h, centrifuging the culture at 5000 rpm for 10 min to obtain a supernatant, and detecting the activity of the uricoxidase in the supernatant. 0.3 mmol/L uric acid solution was prepared using 0.2 mol/L boric acid buffer solution (pH 8) as a substrate solution. Setting a reaction group and a control group, wherein the reaction group comprises adding 2 mL of substrate solution into a 4 mL sterile centrifuge tube, adding 1 mL of fermentation liquor, carrying out water bath at 37 ℃ for 30 min, transferring the substrate solution into a quartz cuvette after the water bath, and measuring the absorbance value of 293 nm on an ultraviolet spectrophotometer. In the control group, 1 mL of fermentation broth was directly mixed with 2 mL of substrate solution, and the absorbance value was immediately measured without water bath. The absorbance value of the reaction group is obviously lower than that of the control tube, which indicates that the urate oxidase is detected to be positive, namely, a strain for producing urate oxidase is obtained by screening.

The screening medium consists of: 0.3% of yeast powder, 0.5% of oyster powder, 0.5% of fish meal, 1% of uric acid, 2% of agar and distilled water.

The identification of Bacillus curvatus alkaAU comprises the following steps:

(1) microscopic observation of Bacillus flexus alkaAU showed that: the strain is gram-positive rod-shaped bacteria; observed under an oil lens: the cells are in two forms of long rod shape and bent rod shape, and spores are mesogenic or terminal, and can move; the size (width x length) of the cells is about 0.45 to 1.09 μm x 1.61 to 4.30 μm; singly or in a chain arrangement, see fig. 1; the size (width. times. length) of the spore is 0.61 to 1.16 μm.times.0.80 to 1.74. mu.m.

(2) The colony morphology characteristic observation is carried out on the campylobacter alkaAU, and after the campylobacter alkaAU is cultured for 48 hours at 37 ℃, the colony characteristic of the strain alkaAU on a beef extract peptone culture medium is as follows: the diameter of a single colony is 3 mm-10 mm, the colony is white and opaque, and the edge of the colony is irregular, which is shown in figure 2; the culture medium around the colony can see a transparent ring.

Beef extract peptone medium: beef extract 0.3%, peptone 1%, NaCl 0.5%, distilled water, pH 7.0.

(3) The physiological and biochemical performance of the campylobacter alkaAU is measured, and the result is that:

the bacterial strain alkaAU has negative reactions such as arginine double hydrolase reaction, methyl red reaction, VP reaction, lysine decarboxylase reaction, ornithine decarboxylase reaction, H2S reaction, nitrate reduction reaction, citric acid growth, oxidase and the like, and has positive reactions such as urease, casein hydrolysis reaction, beta-galactosidase reaction, gelatin hydrolysis, starch hydrolysis, catalase, atrazine hydrolysis, glutathione peroxidase, metallo beta-lactamase, isoamylase and the like. Can be grown by using pullulan, D-fructose, alpha-D-glucose, D-raffinose, D-lactose, D-trehalose, etc. See table 1 for results.

TABLE 1 physiological and biochemical Properties of Bacillus curvatus alkaAU

Experimental project	Results	Experimental project	Results
				Cell morphology	Rod and bent rod shapes	Gram stain	Positive for
Form spores	+	Contact enzyme	+
				Arginine double hydrolase	-	Thiol peroxidase	+
Arginase	+	Oxidase enzyme	-
				Methyl Red reaction	-	VP reaction	-
Lysine decarboxylase	-	Starch hydrolysis	+
				Hydrolysis of casein	+	Hydrolysis of gelatin	+
Beta-galactosidase enzyme	+	Atrazine hydrolysis	+
				Urate oxidase	+	Urease	+
Citric acid growth	-	Arsenate reduction	+
				Ornithine decarboxylase	-	Nitrate reduction	-
Glutathione peroxidase	+	H₂S	-
				Metallo-beta-lactamase	+	Isoamylase	+
Pullulan sugar	+	D-raffinose	-
				D-fructose	+	Malonic acid salts	-
D-glucose	+	Maltose	+
				Glycerol	+	Sucrose	+
Tween 40	+	D-lactose	-
				Growth pH range	4-12	D-mannitol	+
Growth NaCl Range	0-15%	D-xylose	-
				Temperature range of growth	20-70℃	D-trehalose	+

Note: +: positive; -: and (4) negativity.

(4) Bacterial 16S rDNA sequence universal primers 27F and 1492R are used for amplification by taking a strain genome as a template. The primer sequence is as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3' are provided. The PCR conditions were all: 25 mu L of reaction system, and pre-denaturation at 94 ℃ for 2 min; 94 ℃, 20 s; 45 s at 55 ℃; 72 ℃ for 45 s; for a total of 35 cycles, a final extension of 7 min at 72 ℃. Obtaining 16S rDNA amplification product, sending to Shanghai biological sequencing with length of 1462 bp. Germ line relationship analysis was performed on the species alkau based on the 16S rDNA sequence using Mega 5 software, see fig. 3.

Comprehensively analyzing colony morphology, cell morphology and 16S rDNA sequence to identify the strain alkaAU as campylobacterBacillus flexus。The sequence thereof is shown in a sequence table.

The growth characteristics of Bacillus curvatus alkaAU are determined by the following steps:

(1) preparing a seed solution:

inoculating Bacillus flexus alkaAU into seed culture medium, rotating at 180 rpm, filling 20%, and culturing at 37 deg.C for 18 h to obtain seed solution.

Seed culture medium: 0.5 percent of yeast powder, 1 percent of tryptone, 1 percent of arginine and distilled water.

(2) Effect of temperature on growth of strain alkau:

mixing the seed liquid (10)⁷cells/mL) were inoculated in a beef extract peptone medium at 1% (v/v) at 180 rpm in a 20% liquid loading, incubated at different temperatures, respectively, and after 48 h the OD of the cell suspension was determined at 600 nm. The result shows that the growth temperature range of the strain is 15-70 ℃, the optimal growth temperature is 40 ℃, and the optimal growth temperature is shown in figure 4.

(3) Effect of pH on growth of strain alkau:

preparing a beef extract peptone culture medium with the pH value of 3-8 by using a phosphate buffer solution, and preparing a culture medium with the pH value of 9-12.5 by using a boric acid buffer solution. The seed solution was inoculated in culture media of different pH at an inoculum size of 1%, cultured at 37 ℃ for 48 hours, and the cell concentration was determined. The result shows that the growth pH range of the strain is 4-12, the optimum growth pH is 6, and the figure is 5.

(4) Effect of NaCl on growth of the strain alkau:

beef extract peptone culture media with different NaCl concentrations are prepared, so that the concentration of the beef extract peptone culture media is 0% -20%. The seed solution was inoculated in culture media of different NaCl concentrations at an inoculum size of 1%, cultured at 37 ℃ for 48 hours, and the cell concentration was determined. The result shows that the concentration of NaCl which can grow by the strain is 0-15%, and the concentration of NaCl which is most suitable for growth is 0.5%, which is shown in figure 6.

Example 2, the method of producing urate oxidase by Bacillus flexus alkau described in example 1, the steps are as follows:

seed solutions were prepared as in example 1. Mixing the seed liquid (10)⁷cells/mL) was inoculated in 1% (v/v) inoculum size in an enzyme-producing medium. 180 rpm, liquid loading capacity of 20%, culturing at 37 ℃ for 72 h, centrifuging or filtering cells, and collecting supernatant to obtain urate oxidase fermentation liquor. The enzyme activity of the fermentation broth was measured to be 0.52U/mL according to the method in example 1. The enzyme amount required for converting 1 mu mol of uric acid per minute is 1 enzyme activity unit.

Enzyme production culture medium: 0.5 percent of yeast powder, 1 percent of fish peptone, 0.5 percent of uric acid and 0.5 mol/L phosphate buffer solution (pH 6).

Properties of urate oxidase produced by Bacillus flexus alkau:

(1) effect of reaction temperature on enzyme activity:

the reaction system for Bacillus flexus alkAU urate oxidase was as described in example 1. When the influence of the reaction temperature on the enzyme activity is measured, the reaction system is placed at different temperatures for reaction for 30 min, the enzyme activity is measured, the result is shown in figure 7, the optimal action temperature of the enzyme is 40 ℃, and the catalytic activity is more than 70% in the temperature range of 35-50 ℃.

(2) Thermostability of the enzyme:

taking 2 mL of urate oxidase fermentation broth, respectively preserving heat at different temperatures, taking a group of samples every 8 h, rapidly freezing and storing in a refrigerator at-40 ℃, uniformly measuring residual enzyme activity after heat preservation is finished, and setting the enzyme activity of the fermentation broth which is not subjected to temperature treatment as 100%, wherein the result is shown in figure 8. The urate oxidase produced by the strain alkAU has better thermal stability, the residual enzyme activity can still keep more than 50 percent after heat preservation is carried out for 12 hours at 40 ℃, and the residual enzyme activity is 0 after 48 hours.

(3) Effect of reaction pH on enzyme activity:

buffer solutions with different pH values are used for preparing 1% pullulan solution, and after enzyme solution is added, the reaction system is as follows: 25 mu L of urate oxidase fermentation liquor and 75 mu L of buffer solution, and enzyme activity determination is carried out at 45 ℃, wherein the buffer solutions with different pH values are as follows: 20 mmol/L phosphate buffer (pH7.0-8.0); 50 mmol/L glycine-sodium hydroxide buffer solution (pH9.0-10.0); 0.1 mol/L boric acid buffer (pH11.0-12.0). The results are shown in FIG. 9, pH10 where the enzyme activity is highest. The enzyme activity is 70% at the pH of 9-11.

Example 3 hydrolysis of pullulan with Bacillus curvatus alkau the procedure was as follows:

adding pullulan with the final concentration of 1% into a beef extract peptone culture medium, and inoculating the campylobacter alkaAU. At 180 rpm, filling 20% of liquid, culturing at 37 ℃ for 72 h, centrifuging and precipitating cells, collecting supernatant, and dialyzing with 0.01 mol/L phosphate buffer (pH 7) at 4 ℃ to remove reducing sugar to obtain pullulanase extract. Taking 100 mu L of enzyme extract, adding 100 mu L of phosphate buffer solution with pH7, then adding 100 mu L of 1% pullulan solution (prepared by phosphate buffer solution with pH 7), mixing uniformly, and reacting for 30 min at 37 ℃; meanwhile, a negative control group is prepared, 100 mu L of enzyme extract is taken, 100 mu L of phosphate buffer solution with pH7 is added, then boiling water bath is carried out for 10 min, and then substrate is added, wherein the reaction conditions are the same as above; after the reaction, 100. mu.L of DNS was added to both groups, and boiling water bath was carried out for 10 min. The absorbance at 540 nm of the reaction mixture was measured. The enzyme activity of the pullulanase extracting solution is 39.18U/mL according to the calculation of a maltose standard curve. Definition of enzyme activity: the amount of enzyme required to decompose pullulan to 1. mu. mol reducing sugar per unit time (h) at 45 ℃ was one activity unit (U). Enzyme activity (U) = Wx1000/MxT xV; in the formula, W: maltose (mg), standard curve; m: maltose molecular weight, 360.32; t: reaction time (h); v: enzyme dosage (mL).

Example 4 hydrolysis of arginine using Bacillus curvatus aikaAU, the procedure was as follows:

preparing ornithine solutions with different concentrations, taking 200 mu L, sequentially adding 200 mu L of glacial acetic acid, 200 mu L of mixed acid solution and 200 mu L of ninhydrin color developing agent, adjusting the pH to about 1, reacting in a boiling water bath for 15 min, measuring the absorbance value at 515 nm, and drawing a concentration-absorbance value standard curve. Adding arginine with the final concentration of 1% into a beef extract peptone culture medium, and inoculating the campylobacter alkaAU. Culturing at 33 ℃ for 12 h at 180 rpm, centrifuging and precipitating cells, adding 10 times of sterile water for resuspension, carrying out cell ultrasonic crushing in an ice bath, carrying out ultrasonic power of 250-300W for 30 min, crushing for 2s at intervals of 3 s, centrifuging at 10000 rpm for 10 min, and collecting supernatant to be used as arginase extracting solution. mu.L of the enzyme solution was added to 180. mu.L of arginine solution (0.1 mol/L L-arginine, 50 mmol/L glycine-NaOH buffer, pH 9.5), reacted at 60 ℃ for 10 min with precise timing, and 0.1 m L1 mol/L of TCA solution was added to terminate the reaction. Sequentially adding 200 μ L of glacial acetic acid, 200 μ L of mixed acid and 200 μ L of ninhydrin, adjusting pH to about 1, reacting in boiling water bath for 15 min, calculating absorbance value of the reaction solution at 515 nm, calculating L-ornithine generation amount according to standard curve, and measuring arginase activity of the extractive solution to be 0.57U/mL. The reaction system of the enzyme which is added into a boiling water bath and heated for 10 min to be inactivated is used as a blank. The enzyme activity unit is defined as: the enzyme activity is the unit of the enzyme amount needed for catalyzing and generating 1 mmol/L-ornithine within 1 min.

6 mol/L phosphoric acid solution: 41 mL of 85% phosphoric acid was slowly added to distilled water, and the volume was adjusted to 100 mL using a 100 mL volumetric flask to obtain a 100 mL phosphoric acid solution.

Mixed acid solution: mixing the freshly prepared 6 mol/L phosphoric acid solution with glacial acetic acid according to a volume ratio of 1: and 3, preparing the required mixed acid solution.

25 mg/mL ninhydrin color reagent: weighing 1.25 g ninhydrin, adding into 50 mL mixed acid, dissolving in 70 deg.C water bath, transferring into brown reagent bottle, and storing in 4 deg.C refrigerator in dark place.

Example 5 hydrolysis of amylopectin with Bacillus curvatus aikaAU, the procedure was as follows:

amylopectin with the final concentration of 1% is added into beef extract peptone medium, and Bacillus flexus alkaAU is inoculated. Culturing at 35 deg.C for 72 h at 180 rpm and liquid loading of 20%, centrifuging to precipitate thallus, collecting supernatant, dialyzing with 0.05 mol/L sodium acetate buffer solution (pH 7) at 4 deg.C to remove reducing sugar to obtain isoamylase extract. mu.L of 1% pullulan solution was added with 50. mu.L of sodium acetate buffer (0.05 mol/L, pH 7), and then 50. mu.L of enzyme extract was added and reacted at 40 ℃ for 30 min. mu.L of hydrochloric acid solution (3 mmol/L) and 2.5 mL of iodine solution (2%) were mixed together, 100. mu.L of the reaction solution was then added, and the absorbance value at 620 nm was measured to calculate the enzyme activity. The mixture of the inactivated enzyme solution and substrate was heated in a boiling water bath for 10 min as a blank. The enzyme activity of the enzyme extract was measured to be 27.14U/mL. The enzyme activity unit is defined as: the absorbance value at 620 nm is increased by 0.01 caused by amylose generated in enzyme minutes, namely 1 enzyme activity unit.

Example 6 hydrolysis of atrazine with bacillus curvatus alkaAU, the procedure was as follows:

preparing a gradient solution of the triazine herbicide atrazine by using methanol, measuring an absorption peak area value under 228 nm by using a high pressure liquid chromatography, and drawing a concentration-peak area standard curve. Adding atrazine with the final concentration of 0.03% into a beef extract peptone culture medium, and inoculating the curvulus alkaAU. 180 rpm, liquid loading capacity of 20%, culturing at 37 ℃ for 36 h, centrifuging to collect cells, washing with sterile distilled water for 3 times, then adding 10 times of sterile water for heavy suspension, carrying out cell ultrasonic crushing in an ice bath, carrying out ultrasonic power of 250-300W for 30 min, crushing for 2s at intervals of 3 s, centrifuging at 10000 rpm for 10 min, and collecting supernatant to be used as an atrazine hydrolase extracting solution. Adding 100 μ L of the enzyme extract into 300 μ L of Tri-HCl solution (1 mol/L, pH 7.5) with concentration of atrazine 40 mmol/L, and reacting at 30 deg.C for 10 min. And (3) preparing a negative control group, taking 100 mu L of enzyme extract, preserving the temperature at 100 ℃ for 15 min, and then adding a substrate for reaction. And extracting the reaction solution by using dichloromethane, detecting residual atrazine by using a high pressure liquid chromatography, and calculating the enzyme activity of the enzyme extracting solution to be 2.31U/mL. The enzyme activity was defined as the amount of enzyme required to convert 1. mu. mol atrazine per minute at 30 ℃.

Sequence listing

<110> Huaihai institute of Industrial and Marine science

<120> Bacillus flexus alkau, method for producing urate oxidase, product and application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1462

<212> DNA

<213> Bacillus flexus alkaAU

<400> 1

taaggccaag cggcctgcta taatgcaagt cgagcgaact gattagaagc ttgcttctat 60

gacgttagcg gcggacgggt gagtaacacg tgggcaacct gcctgtaaga ctgggataac 120

tccgggaaac cggagctaat accggataac attttctctt gcataagaga aaattgaaag 180

atggtttcgg ctatcactta cagatgggcc cgcggtgcat tagctagttg gtgaggtaac 240

ggctcaccaa ggcaacgatg catagccgac ctgagagggt gatcggccac actgggactg 300

agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa tggacgaaag 360

tctgacggag caacgccgcg tgagtgatga aggctttcgg gtcgtaaaac tctgttgtta 420

gggaagaaca agtacaagag taactgcttg taccttgacg gtacctaacc agaaagccac 480

ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttat ccggaattat 540

tgggcgtaaa gcgcgcgcag gcggtttctt aagtctgatg tgaaagccca cggctcaacc 600

gtggagggtc attggaaact ggggaacttg agtgcagaag agaaaagcgg aattccacgt 660

gtagcggtga aatgcgtaga gatgtggagg aacaccagtg gcgaaggcgg ctttttggtc 720

tgtaactgac gctgaggcgc gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 780

ccacgccgta aacgatgagt gctaagtgtt agagggtttc cgccctttag tgctgcagct 840

aacgcattaa gcactccgcc tggggagtac ggtcgcaaga ctgaaactca aaggaattga 900

cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta 960

ccaggtcttg acatcctctg acaactctag agatagagcg ttccccttcg ggggacagag 1020

tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca 1080

acgagcgcaa cccttgatct tagttgccag catttagttg ggcactctaa ggtgactgcc 1140

ggtgacaaac cggaggaagg tggggatgac gtcaaatcat catgcccctt atgacctggg 1200

ctacacacgt gctacaatgg atggtacaaa gggctgcaag accgcgaggt caagccaatc 1260

ccataaaacc attctcagtt cggattgtag gctgcaactc gcctacatga agctggaatc 1320

gctagtaatc gcggatcagc atgccgcggt gaatacgttc ccgggccttg tacacaccgc 1380

ccgtcacacc acgagagttt gtaacacccg aagtcggtgg ggtaaccttt atggagccag 1440

ccgcctaatg gtgacccggg tg 1462

Claims

1. A Bacillus flexus (B) ((B))Bacillus flexus) an alkau characterized by: the biological preservation number is CCTCC NO: m2018378.

2. A Bacillus curvatus according to claim 1 (B)Bacillus flexus) A method for producing cell wall-bound urate oxidase by alkaAU, which is characterized by comprising the following steps:

(2) inoculating the seed solution into an enzyme production culture medium according to the inoculation amount of 1% of the volume percentage content, culturing at 35-45 ℃ for 36-72 hours at 180 rpm and 10-20% of the liquid loading amount to obtain urate oxidase fermentation liquor; the enzyme production culture medium comprises the following components: 0.5 percent of yeast powder, 1 percent of fish peptone, 0.5 percent of uric acid and 0.5 mol/L phosphate buffer solution, wherein the pH value is 6; the cell content of the seed liquid is preferably 10⁷cells/mL.

3. A method of using the Bacillus flexus of claim 1Bacillus flexus) The method for hydrolyzing the pullulan by the alkaAU is characterized by comprising the following steps: adding pullulan with the final concentration of 1% into a beef extract peptone culture medium, and inoculating campylobacter alkaAU; at 180 rpm, filling 20% of liquid, culturing at 37 ℃ for 72 h, centrifuging and precipitating cells, collecting supernatant, dialyzing with 0.01 mol/L pH7 phosphate buffer at 4 ℃ to remove reducing sugar, and obtaining pullulanase extracting solution.

4. A method of using the Bacillus flexus of claim 1Bacillus flexus) The method for hydrolyzing arginine by alkaAU is characterized by comprising the following steps: adding arginine with the final concentration of 1% into a beef extract peptone culture medium, and inoculating campylobacter alkaAU; culturing at 33 ℃ for 12 h at 180 rpm, centrifuging and precipitating cells, adding 10 times of sterile water for resuspension, carrying out cell ultrasonic crushing in an ice bath, carrying out ultrasonic power of 250-300W for 30 min, crushing for 2s at intervals of 3 s, centrifuging at 10000 rpm for 10 min, and collecting supernatant to be used as arginase extracting solution.

5. A method of using the Bacillus flexus of claim 1Bacillus flexus) The method for hydrolyzing amylopectin by alkaAU is characterized by comprising the following steps of: adding amylopectin with final concentration of 1% into beef extract peptone culture medium, and inoculating Bacillus flexus alkau; culturing at 35 deg.C for 72 hr at 180 rpm and liquid loading of 20%, centrifuging to precipitate thallus, collecting supernatant, dialyzing at 4 deg.C with 0.05 mol/L pH7 sodium acetate buffer solution to remove reducing sugar to obtain isoamylase extractAnd (4) liquid.

6. A method of using the Bacillus flexus of claim 1Bacillus flexus) The method for hydrolyzing atrazine by alkaAU is characterized by comprising the following steps: adding atrazine with the final concentration of 0.03% into a beef extract peptone culture medium, and inoculating campylobacter alkaAU; 180 rpm, liquid loading capacity of 20%, culturing at 37 ℃ for 36 h, centrifuging to collect cells, washing with sterile distilled water for 3 times, then adding 10 times of sterile water for resuspending, carrying out cell ultrasonic crushing in ice bath, carrying out ultrasonic power of 250-300W for 30 min, crushing for 2s at intervals of 3 s, centrifuging at 10000 rpm for 10 min, and collecting supernatant as an atrazine hydrolase extracting solution.