CN1632106A - Hypothermal alkaline metal protease of marine microorganism and its enzyme producing strain - Google Patents

Abstract

The invention a unknown deformation of Pseudomonas QD 80 cultivated and separated from the bottom mud in the East Sea, which is preserved in CCTCC, and its CCTCC NO is M204100, and a low temperature alkaline metal proteinase of new sea microbe. They can be applied widely in fields like processions of washing agent, feed, wool.

Description

Novel marine microorganism low-temperature alkaline metalloprotease and enzyme-producing strain thereof

Technical Field

The invention relates to the field of microbial enzymes, in particular to a novel marine microorganism low-temperature alkaline metalloprotease and a pseudomonas unknown strain QD80 of the marine microorganism low-temperature alkaline metalloprotease with high yield.

Background

The ocean is wide, the microbial resources are rich, but due to the unique ocean environment including high salt, high pressure, low nutrition, low temperature and the like, the marine microbes have many specificities such as difficult culture, various forms, easy death in the preservation and transplanting process and the like which are different from those of terrestrial microbes, and are not beneficial to the deep research and development of the marine microbes, so that some simple, convenient and easy-to-operate microbial strains which can be more scientifically, accurately and quickly found to a certain extent and can be separated to obtain useful microbial strains are urgently needed to be established, and a foundation is laid for the development and utilization of the marine microbial resources. At present, macromolecular rRNA is a molecular index, widely used for the research of genetic and molecular differences of various microorganisms, and DNA of a large number of known microorganisms is measured and input into an international gene database to become a very useful reference system for microorganism identification and classification, so that the aim of rapidly and effectively identifying and classifying new strains can be achieved by measuring, comparing and analyzing DNA sequences of unknown microorganisms.

With the development of biotechnology in recent years, the search for enzymes with completely new properties has become a hot spot of international enzyme preparation research. Marine organisms can produce abundant extreme enzymes due to their growth in the unique environment of the ocean, particularly marine microorganisms and microalgae living in extreme environments. Compared with enzymes produced by terrestrial microorganisms, marine enzymes have more unique enzymological properties, thus causing great attention of researchers at home and abroad. Alkaline proteases are well known as an essential ingredient for improving the detergency of synthetic detergents. Over the past 10 years, however, world washing traditions have revolutionized. The global laundry is developing towards low temperature and water saving, and the traditional detergent enzyme (mainly alkaline protein)Enzymes) exhibit cleaning ability at relatively low temperatures that has been difficult to meet consumer demand, and there is a need for enzymes that have significant relative activity advantages in low temperature regions. Commercial low temperature alkaline proteases for detergents currently known include: the oxidative stable detergent alkaline protease is MaxapemCX (GENENCOR international, usa) under the brand names Properase CT (GENENCOR international, usa), savinase4.ot (NOVO, denmark). Wherein: the relative activity of the Savinase 4.0T enzyme is about 15 percent under the condition of pH 10.1, the optimal reaction temperature is 55 ℃ and the relative activity is 20 ℃. The relative activity of the Properase CT enzyme is about 42 percent under the condition of pH10, the optimal reaction temperature is 50-55 ℃ and the relative activity is 20 ℃. MaxapemCX enzyme at 20mM H under optimal conditions₂O₂Can be stabilized for 60 min. As can be seen, the relative activity of the proteases in the low temperature region is low under the respective recommended optimal conditions, so that the low temperature activity of these enzymesThe stability of the zones and towards oxidizing agents is still not satisfactory.

Disclosure of Invention

The invention aims to provide a pseudomonas unknown strain QD80 for high yield of novel marine microorganism low-temperature alkaline metalloprotease, which is obtained by culturing and separating in sea mud of the east China sea area.

Another object of the present invention is to provide a highly productive marine microorganism low temperature alkaline metalloprotease produced by the above Pseudomonas unknown species strainQD 80.

The novel pseudomonas unknown strain QD80 provided by the invention is a pseudomonas unknown strain QD80 which is obtained by culturing and separating sea mud in the east China sea area and has high yield of novel marine microorganism low-temperature alkaline metalloprotease (low-temperature alkaline metalloprotease for short). The pseudomonas unknown strain QD80 is preserved in China Wuhan type culture Collection (CCTCC for short) in 12.1.2004, with the preservation number of CCTCC NO: m204100.

The morphological characteristics of the pseudomonas unknown strain QD809 (referred to as strain QD80) provided by the invention are as follows:

morphological characteristics

Gram-negative bacteria were obtained by gram-staining the strain QD 80. The morphological observation of the microscope shows that the morphological characteristics are thick short rods; periphytic flagellum is aerobic; the bacterial colony is milky white, convex and smooth in edge; opaque, no pigment produced.

The QD 8016 SrDNA sequence of the strain is determined, and the total length is 1443 bp. The 16SrDNA sequence is submitted to GenBank for Blast homologous sequence search, and as a result, the first 400 sequences with similar relativity are all strains of pseudomonas, and QD80 has more than 98% homology with the strains.

Strain QD 8016 srRNA complete sequence

GCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGAGAGGTGCTTGCACCT

CTTGAGAGCGGCGGACGGGTGAGTAATACCTAGGAATCTGCCTGGTAGTGGGGG

ATAACGTTCGGAAACGGACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGG

GGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTG

AGGTAATGGCTCACCAAGGCTACGATCCGTAACTGGTCTGAGAGGATGATCAGTC

ACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATA

TTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTT

CGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCATTAACCTAATACGTTAGTG

TTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGT

AATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGG

TGGTTTGTTAAGTTGAATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCCAA

AACTGGCAAGCTAGAGTATGGTAGAGGGTAGTGGAATTTCCTGTGTAGCGGTGA

AATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACTACCTGGACTGAT

ACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTA

GTCCACGCCGTAAACGATGTCAACTAGCCGTTGGGAACCTTGAGTTCTTAGTGGC

GCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACT

CAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAA

GCAACGCGAAGAACCTTACCAGGCCTTGACATCCAATGAACTTTCCAGAGATGGA

TTGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGT

CGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCA

GCACGTAATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGT

GGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACA

ATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACC

GATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAG

TAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGC

CCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGAG

GACGGTTANCACGGTGTGATTCATG

The QD 8016 SrRNA gene sequence of the strain is mutated at certain sites with different probabilities, shows structural and functional conservation at the level of species, genus and the like, is called bacterial fossils, particularly has good clock property in the evolution, and is a timer for the biological evolution history. By using the 16SrRNA as a molecular index, the microorganisms can be classified and identified quickly, slightly, accurately and simply.

The marine microorganism low-temperature alkaline metalloprotease provided by the invention is a marine microorganism low-temperature alkaline metalloprotease with high yield by an unknown strain QD80 of pseudomonas, and has the characteristics that:

(1) genes for enzymes:

ATGATCGAATCCGTCGAGCACTTCCTTGCCCGCCTCAAAAAACGCGACCCAGACCAGCCA

GAATTCCACCAGGCGGTGGAAGAAGTCCTGCGCAGCCTGTGGCCTTTTCTCGAAGCCAAT

CCGCACTACCTGACTTCCGGGATTCTCGAACGTATTTGCGAACCTGAGCGGGCAATTGTG

TTTCGCGTGTCATGGGTGGATGACGAAGGCAAAGTGCGGGTTAACCGCGGCTTCCGCATT

CAGATGAACAGCGCCATTGGCCCTTACAAAGGCGGGTTGCGCTTCCACCCGTCGGTGAAT

TTGGGGGTGTTGAAGTTCTTGGCGTTTGAACAAACCTTCAAAAACTCCCTGACCTCGCTG

CCCATGGGCGGCGGTAAGGGTGGCTCGGATTTCAACCCCAAGGGCAAGAGCGACGCGGAA

GTCATGCGTTTCTGCCAGGCCTTCATGAGCGAGCTGTACCGTCATATCGGTTCGGACGTG

GACGTGCCCGCTGGCGATATCGGCGTCGGCGCCCGTGAGATTGGCTTCCTCTTTGGCCAA

TACAAACGCCTGAGCAACCAGTTCACCTCCGTGTTGACCGGCAAAGGCATGAGCTACGGC

GGCAGCCTGATTCGCCCGGAAGCCACCGGGTTTGGCTGCGTGTATTTTGCGCAGGAAATG

CTCAAGCGCAGCGGCCAGCGGATTGATGGCAAGCCGGTTGCGATTTCCGGCTCGGGTAAC

GTGGCGCAGTATGCCGCGCGCAAAGTCATGGACCTGGGCGGCAAAGTCATTTCGCTCTCG

GACTCCGAAGGCACCCTGTATTGCGAAGCCGGTCTGGACGACGCGCAGTGGGAAGCACTG

ATGGAGCTGAAAAACGTCAAGCGCGGACGTATCAGCGAACTGGCGGCGCAATTTGGTCTG

GAGTTTCTGGCGGGCCAGCATCCGTGGCATCTGCCCTGTGACATTGCGCTGCCTTGCGCA

ACACAGAACGAACTGGACGCCGAAGCCGCTCGCACATTGCTGAGCAATGGCTGTGGGTGC

GTGGCCGAAGGCGCCAACATGCCGACCACGCTGGAAGCGGTGGACCTGTTTATCGAGGCG

GGCATTTTGTTCGCACCGGGCAAAGCCTCCAATGCGGGCGGTGTGGCCGTGAGCGGTCTG

GAAATGTCGCAGAACGCCATGCGCTTGCTGTGGACGGCGGGTGAGGTGGACAGCAAGTTG

CACAACATCATGCAATCGATCCACCACGCCTGA。

(2) molecular weight, isoelectric point of the enzyme (referring to low temperature alkaline metalloprotease):

the apparent molecular weight of the enzyme was 49000. + -. 1000Dal as determined by SDS-PAGE gel electrophoresis. The enzyme molecular weight was measured to be 49320Dal by HPLC gel filtration. The isoelectric point of the enzyme is pH8.5 when the thin-layer polyacrylamide gel isoelectric focusing is measured.

(3) Substrate specificity of the enzyme:

the protease has low activation energy and high activity at low temperature below 35 deg.C, and can effectively degrade protein substances such as protein stain (milk, blood, grass juice) or peptide and protein such as casein, hemoglobin, fibrin, elastin, meat protein, fish protein, etc. at low temperature.

(4) Optimum reaction temperature and pH of enzyme:

the Folin-phenol method is adopted to measure the enzyme activity, and the influence of different pH values on the enzyme activity is measured through a pre-adjusted pH buffer system, so that the effective pH stability range of the enzyme is about 6-10 (the activity of the enzyme after heat preservation for 60min at 20 ℃ under different pH conditions); the optimum reaction pH is 9-10(20 ℃, reaction time is 10min).

The influence of different temperatures on the enzyme activity is measured by the same enzyme activity measuring method, and the effective temperature stability range of the enzyme is about below 20 ℃ (the activity of the enzyme after the temperature is preserved for 60min at pH9.5 under different temperature conditions); the optimal reaction temperature is 30 ℃ (pH9.5, reaction time is 10min).

(5) Compatibility of the enzyme with conventional reagents:

effect of metal ions on low temperature alkaline metalloproteases:

name (R)	Concentration Mm	Relative enzyme activity retention rate%
			Does not contain metal ions	0	100
Cu2+	5	19.42
			Ca2+	5	86.38
Mg2+	5	122
			Mn2+	5	141
Co2+	5	20.09
			Fe3+	5	2.74
Zn2+	5	36.58
			K+	5	97.86
Na+	5	94.5
			Ag+	5	1.22

Effect of compatible chemicals on enzyme activity:

name (R)	Concentration of	Relative enzyme activity retention rate%
			Does not contain metal ions	0	100
Ethanol	1％(V/V)	100
			Borax	1％(W/V)	120
Glutaraldehyde	1％(V/V)	30.9
			Urea	1％(W/V)	83
Polyoxyethylene (20) sorbitan Alcohol monooleate (Tween 80)	0.1％(W/V)	110
			Polyoxyethylene (20) sorbitan Alcohol monooleate (Tween 40)	0.1％(W/V)	77.32
KH2PO4	1％(W/V)	77.9
			Na2HPO4	1％(W/V)	32.9
Na2SO3	0.1％(W/V)	75.38
			Ethylene glycol phenyl ether	0.1％(W/V)	90.11
Tris hydroxymethyl aminomethane (Tris)	1mM	134
			Sodium Dodecyl Sulfate (SDS)	0.1mM	85
Sodium linear alkyl benzene sulfonate (LAS)	500ppm	83

Influence of inhibiting substances on enzyme Activity

Name (R)	Concentration of	Relative enzyme activity retention rate%
			Contains no inhibitor	0	100
PMSF (tosyl fluoride)	0.016％(W/V)	100
			EDTA	0.04％(W/V)	55.82
Na2O6S4.2H2O	0.04％(W/V)	77.58
			TLCK (Trypsin inhibitor)	0.04％(W/V)	54
TPCK (chymotrypsin inhibitor)	0.04％(W/V)	100

Effect of stabilizers (thickeners) on enzyme Activity

Name (R)	Concentration (%)	Relative enzyme activity retention (%)
			Contains no thickener	0	100
Glycerol	1(w/v)	102.2

Ethylene glycol	1(w/v)	98.7
			Polyethylene glycol	1(w/v)	126.5
Polyvinyl alcohol	1(w/v)	97.7
			Mannitol	1(w/v)	107.5
Sodium alginate	1(w/v)	94.0
			Dextrin	1(w/v)	107.2
Emulsifier OP	1(w/v)	100.0
			Gelatin	1(w/v)	115.4
Olive oil	1(w/v)	101.1
			Agar-agar	1(w/v)	86.8

(6) Stability of the enzyme

The low-temperature alkaline metalloprotease can satisfy one of the following conditions:

① Folin-phenol reagent method for determining the activity of the alkaline metalloprotease at low temperature, at 25 deg.C in the presence of hydrogen peroxide (H)₂O₂) Buffer solution (50Mm borax salt buffer, pH10, H)₂O₂20mM、Ca²⁺5mM) for 60min, the residual enzyme activity is not less than 95%.

② the low temperature alkaline gold is measured by Folin-phenol reagent methodActivity of a protease, containing 1% (W/V) sodium perborate (NaBO) at 25 ℃₃) Buffer solution (50mM borax salt buffer, pH10, Ca)²⁺5mM) for 60min, the residual enzyme activity is not less than 90%.

③ the activity of the low temperature alkaline metalloprotease was measured by Folin-phenol reagent method at 25 deg.C in a buffer solution containing sodium linear alkyl benzene sulfonate (LAS) (50mM borax salt buffer, pH10, LAS500ppm, Ca)²⁺5mM) for 60min, the residual enzyme activity is not less than 65%.

④ the activity of the low temperature alkaline metalloprotease was measured by Folin-phenol reagent method at 25 deg.C in the presence of sodium sulfite (Na)₂SO₃) Buffer solution (50mM borax salt buffer, pH10, Na)₂SO₃8mM、Ca²⁺5mM) for 60min, the residual enzyme activity is not less than 90%.

⑤ the activity of the low temperature alkaline metalloprotease was measured by Folin-phenol reagent method at 10 deg.C in a buffer solution containing 5% (W/V) sodium chloride (NaCl) (50mM borax salt buffer, pH10, Ca)²⁺5mM) for 12 hours, the residual enzyme activity is not less than 70 percent.

⑥ the activity of the low temperature alkaline metalloprotease was measured by Folin-phenol reagent method at 25 deg.C in a buffer solution containing 2% (V/V) ethanol (50mM borax salt buffer, pH10, Ca)²⁺5mM)The residual enzyme activity after standing for 121 hours is not less than 70 percent.

(7) Enzyme kinetics study

① basic kinetic parameters of the enzyme:

casein with different concentrations as substrate is added into 0.05M glycine-sodium hydroxide pH9.5 buffer solution, the temperature of the reaction system is 18 ℃, and the initial velocity v of the reaction is measured₀To [ S]]Plotting, the results are as follows

② Effect of temperature on enzyme reaction kinetic parameters

Casein as substrate, in 0.05M glycine-sodium hydroxide pH9.5 buffer solution, according to Folin method, the steady kinetic parameters of the reaction V, K at different temperatures_mIn (1).

ResultsDetermination of kinetic parameters V, K by Hanes mapping method_m。

T(℃) 5.0 15.0 20.0 25.0 30.0 35.0 40.0

V(10^-4g·l^-1·s^-1) 1.53 6.21 13.41 16.98 23.37 33.05 27.65

K_m(g·l^-1) 0.19 2.11 5.37 6.29 10.65 14.51 10.48

According to the Arrhenius formula:

k＝A·e^-Ea/RT

the above formula can be rewritten as: logk ═logA-E_a/2.3RT

Plotting logK against 1/T

Determining E from the slope of the straight line in FIG. 1_a33242J/mol. The results are as follows:

activation energy of reaction catalyst (J/mol)

H⁺66944

Hydrolysis of OH by butyl acetate^-41840

Pancreatic lipase 18828

H⁺83680

Casein hydrolysis

Trypsin 50208

H⁺104600

Hydrolysis of sucrose

Yeast sucrase 33472

With K_eAnd K_HRepresents an enzyme and H⁺The catalyzed reaction rate constant, then:

$\log \mathrm{ke}=\log A-\frac{E{a}_e}{2.3\mathrm{RT}}$

$\log k_H=\log A-\frac{E{a}_H}{2.3\mathrm{RT}}$

$\log \frac{\mathrm{ke}}{k_H}=\frac{{\mathrm{Ea}}_H-{\mathrm{Ea}}_e}{2.3\mathrm{RT}}=\frac{83680-33242}{2.3\×8.28\×303}=8.7$

$\frac{\mathrm{ke}}{k_H}=5.0\×{10}^8$

temperature coefficient of reaction Q₁₀(Q₁₀Representing the temperature increase of 10 ℃, the fold increase in reaction rate).

logQ₁₀＝logk_T2/k_T2

＝E_a(1/T₁-1/T₂)/(2.3R)

＝[1/(T₁··T₂)]10×33242/(2.3×8.28)

＝17455[1/(T₁··T₂)]

(3) Effect of pH on enzyme kinetic parameters

Casein as substrate, and in 0.05M buffer solution (disodium hydrogen phosphate and sodium dihydrogen phosphate pH 6-7; Tris-hydrochloric acid pH 8; glycine pH9-11) with different pH values, the Folin method was used to determine the steady kinetic parameters V, K of the reaction at different pH values_mThe reaction system was controlled at 30 ℃.

Kinetic parameters V, K at different pH values determined by Hanes mapping method_m。

pH 6.0 7.0 8.0 8.5 9.0 9.5 10.0 10.5 11

V_ap(10^-4g·l^-1·s^-1) 3.45 5.83 10.57 12.07 13.16 23.10 23.32 25.85 15.17

K_ap(g·l^-1) 1.31 2.50 3.80 4.48 5.06 10.46 10.53 11.64 7.12

The log of the measured V was plotted against pH to obtain a pH curve of the reaction as shown in FIG. 2.

From the data measured in FIG. 2, pK was determined_a＝9.3、pK_b10.7, pH optimum for low temperature alkaline metalloprotease catalyzed casein reaction₀＝10.0。

(4) Effect of inhibitors on enzymatic reactions

The V, K degree of reaction was determined by adding various concentrations of EDTA to casein as substrate in 0.05M glycine-sodium hydroxide pH9.5 buffer_mAnd the temperature of the reaction system is 18 ℃. The results were analyzed using the Kinetics software,

EDTA enzyme inhibition data analysis

Competitive 8.390.511.67

Non-competitive 0.800.050.06

Mixing 0.100.060.09

According to the analysis result, the EDTA inhibits the enzyme competitively in reversible inhibition, and the reaction of the enzyme reaction system in the presence of the reversible inhibitor can be expressed as follows:

in this inhibition, the enzyme cannot bind to the substrate, inhibitor simultaneously, i.e.it cannot form EIS, or K'_iOr K'_s∞, its kinetic equation is:

$v_i=\frac{V\·\left[S\right]}{\left[S\right]+K_m(1+[I]/K_i)}$

after EDTA has bound the enzyme, K_mIncrease (1+ [ I]]/K_i) Double, and the affinity of the enzyme and the substrate is reduced (1+ [ I]]/K_i) Doubling; [ I]of]High disease, K_iThe smaller the size, the higher the inhibitor concentration, the stronger the binding force between the enzyme and the inhibitor, K_mThe increase, the further decrease in the affinity of the enzyme to the substrate, and the corresponding decrease in the speed of the enzyme reaction. The degree of inhibition depends on [ I]]And [ S]]、K_sAnd K_iRelative size of (1/v)_i-1/v) when the degree of inhibition is expressed, there are:

$\frac{1}{v_i}-\frac{1}{v_i}=\frac{1}{V}\·\frac{K_s}{\left[S\right]}\·\frac{\left[I\right]}{K_i}$

shows that the substrate and the inhibitor have competition relation in concentration and affinity with enzyme, and the substrate has protection effect. EDTA is combined with the active site of the marine low-temperature alkaline protease, prevents the further combination of the enzyme and the substrate, thereby inhibiting the activity of the enzyme, and because EDTA is a characteristic reagent with strong chelation on metal, metal ions may exist in the active center of the enzyme, and the protease is the low-temperature alkaline metalloprotease. The protease can be widely applied to the fields of detergents, feeds, wool fabric treatment and the like.

Drawings

FIG. 1 is a graph of logK-1/T

FIG. 2 is a graph lgV plotting pH (30 ℃ C.)

Detailed Description

The invention is illustrated by the following examples, but the invention is not limited by the scope of protection

Example 1: culture of enzyme-producing strain and production of marine microorganism low-temperature alkaline metalloprotease

The Pseudomonas unknown species strain QD80 of the present invention was added to 500ml of a mixture containing 3.5% soybean flour, 2% wheat bran, 0.4% NaH at 20 deg.C₂PO₄、0.03％ K₂HPO₄、0.2％ Ca(Cl)₂The medium of (4) is shake-cultured for 48 hours (the pH of the medium is adjusted to 7.5 to 7.7 with 1M sodium hydroxide), and then the low-temperature alkaline metalloprotease can be produced and secreted in the medium. The culture was centrifuged at 1000G at 4 ℃ to obtain a supernatant containing the enzyme of the present invention. The activity of the enzyme in the supernatant was approximately 700u/ml (determined using the Folin-phenol reagent method described above).

Example 2: purification of marine microbial low temperature alkaline metalloproteases

The supernatant of the culture obtained in example 1 was again brought to 30-65% saturation (NH)₄)₂SO₄And (4) carrying out fractional precipitation, and centrifuging to collect the precipitate. The precipitate was dissolved in distilled water and dialyzed for 24 hours. Passing the dialysate through diethylaminoethyl-agarose (DEAE-Sepharose) FF anion exchange chromatography column, gradient-eluting with 0-1M NaCl-phosphate buffer (20mM, pH7.0), collecting active components, passing through CM-Sepharose FF gel chromatography column, gradient-eluting with 0-1M NaCl-phosphate buffer (10mM, pH5.0), and collecting active components. And then carrying out sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE identification, wherein the sample is dyed into a single band by Coomassie brilliant blue, and the purity of the sample is more than 95% as a result of laser gray scanning.

Claims (5)

1. An unknown strain QD80 of Pseudomonas is obtained by culturing and separating from sea mud in the east China sea area, the unknown strain QD80 is a high-yield marine microorganism low-temperature alkaline metalloprotease, the strain is preserved in the Wuhan type culture Collection in China, and the preservation number is CCTCC NO: m204100.

2. The Pseudomonas unknown strain QD80, according to claim 1, characterized in that the strain has morphological and biochemical characteristics:

gram staining is carried out on the strain, and the strain is gram-negative bacteria; the morphological observation of the microscope shows that the morphological characteristics are thick short rods; periphytic flagellum is aerobic; the bacterial colony is milky white, convex and smooth in edge; opaque, no pigment produced.

3. The Pseudomonas unknown strain QD80 according to claim 1, wherein the strain QD 8016 srRNA has the following full sequence, and the full length is 1443bp

GCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGAGAGGTGCTTGCACCTCTTGAGAGCG

GCGGACGGGTGAGTAATACCTAGGAATCTGCCTGGTAGTGGGGGATAACGTTCGGAAACGGACG

CTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAG

CCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCTACGATCCGTAACTGGTCT

GAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGG

GGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGG

ATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCATTAACCTAATACGTTAGTGTTTTGACGTTAC

CGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTT

AATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGAATGTGAAATCCCCGG

GCTCAACCTGGGAACTGCATCCAAAACTGGCAAGCTAGAGTATGGTAGAGGGTAGTGGAATTTC

CTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACTACCTGGAC

TGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCAC

GCCGTAAACGATGTCAACTAGCCGTTGGGAACCTTGAGTTCTTAGTGGCGCAGCTAACGCATTA

AGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCAC

AAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATCCA

ATGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTGTCG

TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTAC

CAGCACGTAATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGA

CGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAAAGGG

TTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAA

CTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCC

CGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAA

CCTTCGGGAGGACGGTTANCACGGTGTGATTCATG

The 16SrRNA gene sequence can be mutated at certain positions with different probabilities, and the structural and functional conservation is shown at the level of species, genus and the like.

4. A marine microorganism low-temperature alkaline metalloprotease, which is produced by a Pseudomonas unknown strain QD 80.

5. A marine microorganism low-temperature alkaline metalloprotease has the characteristics of

(1) Genes for enzymes:

ATGATCGAATCCGTCGAGCACTTCCTTGCCCGCCTCAAAAAACGCGACCCAGACCAGCCA

GAATTCCACCAGGCGGTGGAAGAAGTCCTGCGCAGCCTGTGGCCTTTTCTCGAAGCCAAT

CCGCACTACCTGACTTCCGGGATTCTCGAACGTATTTGCGAACCTGAGCGGGCAATTGTG

TTTCGCGTGTCATGGGTGGATGACGAAGGCAAAGTGCGGGTTAACCGCGGCTTCCGCATT

CAGATGAACAGCGCCATTGGCCCTTACAAAGGCGGGTTGCGCTTCCACCCGTCGGTGAAT

TTGGGGGTGTTGAAGTTCTTGGCGTTTGAACAAACCTTCAAAAACTCCCTGACCTCGCTG

CCCATGGGCGGCGGTAAGGGTGGCTCGGATTTCAACCCCAAGGGCAAGAGCGACGCGGAA

GTCATGCGTTTCTGCCAGGCCTTCATGAGCGAGCTGTACCGTCATATCGGTTCGGACGTG

GACGTGCCCGCTGGCGATATCGGCGTCGGCGCCCGTGAGATTGGCTTCCTCTTTGGCCAA

TACAAACGCCTGAGCAACCAGTTCACCTCCGTGTTGACCGGCAAAGGCATGAGCTACGGC

GGCAGCCTGATTCGCCCGGAAGCCACCGGGTTTGGCTGCGTGTATTTTGCGCAGGAAATG

CTCAAGCGCAGCGGCCAGCGGATTGATGGCAAGCCGGTTGCGATTTCCGGCTCGGGTAAC

GTGGCGCAGTATGCCGCGCGCAAAGTCATGGACCTGGGCGGCAAAGTCATTTCGCTCTCG

GACTCCGAAGGCACCCTGTATTGCGAAGCCGGTCTGGACGACGCGCAGTGGGAAGCACTG

ATGGAGCTGAAAAACGTCAAGCGCGGACGTATCAGCGAACTGGCGGCGCAATTTGGTCTG

GAGTTTCTGGCGGGCCAGCATCCGTGGCATCTGCCCTGTGACATTGCGCTGCCTTGCGCA

ACACAGAACGAACTGGACGCCGAAGCCGCTCGCACATTGCTGAGCAATGGCTGTGGGTGC

GTGGCCGAAGGCGCCAACATGCCGACCACGCTGGAAGCGGTGGACCTGTTTATCGAGGCG

GGCATTTTGTTCGCACCGGGCAAAGCCTCCAATGCGGGCGGTGTGGCCGTGAGCGGTCTG

GAAATGTCGCAGAACGCCATGCGCTTGCTGTGGACGGCGGGTGAGGTGGACAGCAAGTTG

CACAACATCATGCAATCGATCCACCACGCCTGA；

(2) molecular weight, isoelectric point of the enzyme:

the apparent molecular weight of the enzyme is 49000 +/-1000 Dal, the molecular weight of the enzyme is 49320Dal, and the isoelectric point of the enzyme is pH8.5;

(3) substrate specificity of the enzyme:

the protease has low activation energy and high activity at the low temperature of below 35 ℃, and can effectively degrade protein substances at the low temperature;

(4) optimum reaction temperature and pH of enzyme:

the optimum reaction pH is 9-10, the effective pH stability range of the enzyme is 6-10, and the optimum reaction temperature is 30 ℃;

(5) compatibility of the enzyme with conventional reagents:

effect of metal ions on low temperature alkaline metalloproteases: name (R) Concentration Mm Relative enzyme activity retention rate% Does not contain metal ions 0 100 Cu²⁺ 5 19.42 Ca²⁺ 5 86.38 Mg²⁺ 5 122 Mn²⁺ 5 141 Co²⁺ 5 20.09 Fe³⁺ 5 2.74 Zn²⁺ 5 36.58 K⁺ 5 97.86 Na⁺ 5 94.5 Ag⁺ 5 1.22

Effect of compatible chemicals on enzyme activity: . Name (R) Concentration of Relative enzyme activity retention rate% Does not contain metal ions 0 100 Ethanol 1％(V/V) 100 Borax 1％(W/V) 120 Glutaraldehyde 1％(V/V) 30.9 Urea 1％(W/V) 83 Polyoxyethylene (20) sorbitan Monooleate ester 0.1％(W/V) 110 Polyoxyethylene (20) sorbitan Monooleate ester 0.1％(W/V) 77.32 KH₂PO₄ 1％(W/V) 77.9 Na₂HPO₄ 1％(W/V) 32.9 Na₂SO₃ 0.1％(W/V) 75.38 Ethylene glycol phenyl ether 0.1％(W/V) 90.11 Tris (hydroxymethyl) aminomethane 1mM 134 Sodium dodecyl sulfate 0.1mM 85 Sodium Linear alkyl benzene sulfonate 500ppm 83

Influence of inhibiting substances on enzyme Activity Name (R) Concentration of Relative enzyme activity retention rate% Contains no inhibitor 0 100 Tosyl fluoride 0.016％(W/V) 100 EDTA 0.04％(W/V) 55.82 Na₂O₆S₄.2H₂O 0.04％(W/V) 77.58 Trypsin inhibitor 0.04％(W/V) 54 Chymotrypsin inhibitors 0.04％(W/V) 100

Effect of stabilizers on enzyme activity: name (R) Concentration (%) Relative enzyme activity retention (%) Contains no thickener 0 100 Glycerol 1(w/v) 102.2 Ethylene glycol 1(w/v) 98.7 Polyethylene glycol 1(w/v) 126.5 Polyvinyl alcohol 1(w/v) 97.7 Mannitol 1(w/v) 107.5 Sodium alginate 1(w/v) 94.0 Dextrin 1(w/v) 107.2 Emulsifier OP 1(w/v) 100.0 Gelatin 1(w/v) 115.4 Olive oil 1(w/v) 101.1 Agar-agar 1(w/v) 86.8

(6) Stability of the enzyme

The low-temperature alkaline metalloprotease can satisfy one of the following conditions:

① the activity of the low temperature metallo-alkaline protease was measured by Folin-phenol reagent method at 25 deg.C in buffered solution containing hydrogen peroxide (50Mm borax salt buffer, pH10, H)₂O₂20mM、Ca²⁺5mM) for 60min, the residual enzyme activity is not less than 95 percent;

② the activity of the low temperature alkaline metalloprotease was measured by Folin-phenol reagent method at 25 deg.C in a buffer solution containing 1% (W/V) sodium perborate (50mM borax salt buffer, pH10, Ca)²⁺5mM) for 60min, the residual enzyme activity is not less than 90 percent;

③ the activity of the low temperature alkaline metalloprotease is measured by Folin-phenol reagent method at 25 deg.CSodium alkyl benzene sulfonate buffer solution (50mM borax salt buffer solution, pH10, sodium linear alkyl benzene sulfonate 500ppm, Ca)²⁺5mM) for 60min, the residual enzyme activity is not less than 65%;

④ the activity of the low temperature alkaline metalloprotease was measured by Folin-phenol reagent method at 25 deg.C in sodium sulfite-containing buffer solution (50mM borax salt buffer, pH10, Na)₂SO₃8mM、Ca²⁺5mM) for 60min, the residual enzyme activity is not less than 90 percent;

⑤ the activity of the low temperature alkaline metalloprotease was measured by Folin-phenol reagent method at 10 deg.C in a buffer solution containing 5% (W/V) sodium chloride (50mM borax salt buffer, pH10, Ca)²⁺5mM) for 12 hours, the residual enzyme activity is not less than 70 percent;

⑥ the activity of the low temperature alkaline metalloprotease was measured by Folin-phenol reagent method at 25 deg.C in a buffer solution containing 2% (V/V) ethanol (50mM borax salt buffer, pH10, Ca)²⁺5mM) for 121 hours, the residual enzyme activity is not less than 70%.

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