CN110452835B

CN110452835B - Pseudomonas fish killing and application thereof

Info

Publication number: CN110452835B
Application number: CN201910610964.0A
Authority: CN
Inventors: 冉晓勇; 许敏; 刘东晓
Original assignee: Weihai Yinhe Biological Technology Co ltd
Current assignee: Weihai Galaxy Biotechnology Co ltd
Priority date: 2019-07-08
Filing date: 2019-07-08
Publication date: 2023-06-20
Anticipated expiration: 2039-07-08
Also published as: CN110452835A

Abstract

The invention provides a fish killing pseudoalteromonas and application thereof, in particular discloses a fish killing pseudoalteromonas (Pseudoalteromonas piscicida) X-21 from a barnacle epiphyte, which has higher protease production capacity, and the protease activity of fermentation liquor can reach 441.18U/mL under the initially optimized fermentation condition; and the protease produced by the X-21 strain can effectively tolerate Fe ²⁺ 、Ca ² 、Mg ²⁺ The divalent ions greatly reduce the requirement of enzyme on an acting object, and the optimal pH reaches 10, so that the method is very suitable for being applied to the washing industry. Meanwhile, the alkali-resistant characteristic of the protease is also suitable for the gelatin preparation and deep processing industries.

Description

Pseudomonas fish killing and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to 3. Pseudoalteromonas fish killing and application thereof.

Background

Protease is an enzyme for hydrolyzing protein, and is widely used in the fields of ecological environment treatment and the like. Proteases include plant, animal and microbial proteases, and among them, microbial proteases are becoming a major source of production due to their high efficiency of microbial growth and ease of large-scale fermentation production.

Proteases account for over 65% of the market share of enzyme preparations, where microbial protease production accounts for about 40% of the total industrial enzymes in the world. The protease is mainly used for enzyme-added detergents, and can be applied to industries such as food, medical treatment, leather making, silk and the like. Because of the wide application of protease, low enzyme production efficiency and the like, the protease in China mainly depends on import.

The method is a large ocean country, has wide ocean area and rich resources. Marine microorganisms are important components of biological resources in the ocean, and the types of the marine microorganisms can reach 2-10 hundred million, but the development and the utilization of the marine microorganisms are less than 1% of the marine microorganisms. The exploration, research and utilization of marine microbial resources in China is gradually pursuing developed countries. Thus, the search for and utilization of marine-derived protease-producing microorganisms is of great research, application and commercial value.

Those skilled in the art are working on the development of novel protease producing strains and proteases, which on the one hand wish to increase the production efficiency of the protease and on the other hand wish to obtain proteases with different properties to meet the demands of different fields of application.

Disclosure of Invention

The invention aims to provide a protease-producing pseudoalteromonas fish (Pseudoalteromonas piscicida) and application thereof.

In a first aspect of the present invention, there is provided a protease producing microbial strain, the microbial strain being:

strain 1: pseudoalteromonas fish (Pseudoalteromonas piscicida) X-21 is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.17513; or (b)

Strain 2: the strain 2 is a derivative strain of the strain 1, for example, the strain 1 is obtained by genetic mutation or genetic engineering.

In a second aspect of the invention, there is provided a protease produced by fermentation of pseudoalteromonas fish (Pseudoalteromonas piscicida).

In another preferred embodiment, the protease is produced by fermentation of a strain of microorganism according to the first aspect of the invention.

In a third aspect of the present invention, there is provided a method of preparing a protease, the method comprising the steps of:

(1) Fermenting the microbial strain of the first aspect of the invention;

(2) Separating protease from the fermentation broth.

In another preferred embodiment, in the step (1), the carbon source used for fermentation is soluble starch, sucrose, or maltose.

In another preferred embodiment, in step (1), the fermentation temperature is 15-30 ℃, preferably about 25 ℃.

In another preferred embodiment, in the step (1), the fermentation initiation pH is 7.0 to 8.0; preferably about 7.5.

In another preferred embodiment, in the step (1), the fermentation salinity is 5-20 per mill (w/w); preferably about 15 per mill (w/w).

In a fourth aspect of the invention, there is provided a biological enzyme preparation comprising a protease according to the second aspect of the invention.

In another preferred embodiment, the bio-enzyme preparation further comprises one or more bio-enzymes selected from the group consisting of:

xylanase, pectinase, lipase, cellulase, and esterase.

In another preferred embodiment, the biological enzyme preparation is a solid particle.

In another preferred embodiment, the biological enzyme preparation is a liquid preparation.

In a fifth aspect of the invention there is provided the use of pseudoalteromonas fish (Pseudoalteromonas piscicida) for the preparation of a protease.

In another preferred embodiment, the pseudoalteromonas fish (Pseudoalteromonas piscicida) is a microbial strain according to the first aspect of the invention.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

Fig. 1: transparent circles were screened on the X-21 strain caseinase medium.

Fig. 2: x-21 strain electron microscopy.

Fig. 3: growth curve of the X-21 strain.

Fig. 4: influence of carbon source on the growth of the X-21 strain.

Fig. 5: effect of initial pH of the medium on growth of X-21 strain.

Fig. 6: effect of the salinity of the culture medium on the growth of the X-21 strain.

Fig. 7: effect of culture temperature on growth of X-21 strain.

Fig. 8: tyrosine standard curve.

Detailed Description

The inventor obtains a pseudoalteromonas fish killing (Pseudoalteromonas piscicida) X-21 from the gamboge epiphyte through extensive and intensive research, has higher protease production capacity, and the protease activity of fermentation liquor can reach 441.18U/mL under the initially optimized fermentation condition; moreover, the protease produced by the X-21 strain can effectively tolerate Fe ²⁺ 、Ca ² 、Mg ²⁺ The divalent ions greatly reduce the requirement of enzyme on washing water, and the optimal pH reaches 10, so that the method is very suitable for being applied to the washing industry. Meanwhile, the alkali-resistant characteristic of the protease is also suitable for the gelatin preparation and deep processing industries.

Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

As used herein, the term "strain of the present invention" refers to a strain deposited in the China general microbiological culture Collection center (CGMCC), having a accession number of CGMCC No.17513 or a derivative strain derived from the strain, including a strain obtained by subculturing.

Barnacles (Balanus) are filter-feeding arthropods with clustered grey, hard shells attached to seaside rocks. The larvae of copepods and tendrils in zooplankton are mainly predated. The food source contains a large amount of protein substances, so the inventor guesses that protease-producing strains exist in the gamboge epiphyte so as to help the gamboge epiphyte digest and utilize food.

The strain of the invention is obtained as follows:

(a) Isolating epiphyte from the barnacle in coastal polluted sea areas of Shandong;

(b) Screening the separated epiphyte strain to successfully screen a strain with excellent protease activity, namely the pseudoalteromonas fish killing (Pseudoalteromonas piscicida) X-21.

Preservation of bacterial species

The strain is preserved in China general microbiological culture Collection center (China General Microbiological Culture Collection Center, CGMCC) in 4 months and 2 days of 2019, and has the preservation number of CGMCC No.17513, and the name: pseudoalteromonas fish (Pseudoalteromonas piscicida) X-21.

The invention has the main advantages that:

(1) The pseudoalteromonas fish killing (Pseudoalteromonas piscicida) X-21 has higher protease production capacity, and under the initially optimized fermentation condition, the protease activity of the X-21 strain can reach 441.18U/mL;

(2) The protease produced by the X-21 strain has higher pH tolerance, and the optimal pH reaches 10.0, so that the protease is suitable for alkaline conditions.

(3) The protease produced by the X-21 strain of the invention can effectively tolerate Fe ²⁺ 、Ca ² 、Mg ²⁺ The divalent ions greatly reduce the requirement of enzyme on washing water, and the optimal pH reaches 10, so that the method is very suitable for being applied to the washing industry.

The present invention will be described in further detail with reference to the following examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are not to be construed as limiting the details of the experimental procedure, and are generally carried out under conventional conditions such as those described in the guidelines for molecular cloning laboratory, sambrook.J.et al, (Huang Peitang et al, beijing: scientific Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.

EXAMPLE 1 screening of protease-producing Strain and optimization of culture conditions

1.1 materials and reagents

1.1.1 materials

Cane kettle: coastal polluted sea areas (north latitude 37°31 '59', east longitude 122°3 '43') of yellow sea, wihai, shandong; pseudoalteromonas tetrodotoxin (Pseudoalteromonas tetraodonis); georgia (Marinobacterium georgiense); pseudoalteromonas fish killing (Pseudoalteromonas piscicida); kangshi bacteria (Kangiella japonica).

1.1.2 reagents

Fulin reagent: sigma Co., USA; the other chemical reagents are analytically pure and the biological reagents are biologically pure.

1.1.3 Medium

Enrichment medium, seed medium, fermentation medium: 2216E liquid medium; plate separation solid medium: 2216E solid medium; a caseinase (Casease) medium.

1.3 method

1.3.1 collection of samples

The sample is collected on the coastal reef of the yellow sea pollution coast area of the Weihai, and the sample is collected in a sterile mode.

1.3.2 treatment of samples

Peeling the striated barnacle body, homogenizing for 30s, and inoculating in the enriched culture solution. Shake culturing at 28deg.C at 110r/min, and spreading at 0, 7, 12, 24, and 36 d.

1.3.3 isolation and purification of the epiphyte of the Trichinella gracilis

Enrichment broth was run for 10 ^-1 ～10 ^-n And (3) carrying out gradient dilution, namely respectively coating bacterial solutions on a plate of a separation solid culture medium by using a coating rod, culturing at the constant temperature of 28 ℃ for 48 hours, and screening target bacteria according to colony morphology.

1.3.4 screening of high protease-producing Strain

Preparing a bacterial suspension of the purified strain, adding 20ul of bacterial liquid to a plate of a spotted culture medium containing 1% of casein enzyme,

protease was used as positive control and buffer at pH7.2 was used as negative control. The diameter of the protease hydrolysis circle was measured.

1.3.5 high Propulsion enzyme Strain electron microscope ultrathin section observation and identification

Electron microscopic observation of the strain: the resolution of the optical microscope is only 0.2 mu m, so that the optical microscope cannot clearly observe the substance particles with the internal diameter of the strain being less than 0.2 mu m, and the resolution of the electron microscope reaches 1-2 nm. Therefore, high magnification electron microscopy was used to accurately observe the cells. Shooting by an electron microscope room of a medical college of Qingdao university.

The strain morphology, physiological and biochemical tests and molecular biological assays were carried out by reference (Buchanan R E, gibbens N E. Berjie bacteria identification handbook [ M ].8 edition. Beijing: science Press, 1984:274-325; huang Wenfang. Serratia marcescens (Serratia marcens) study I-Serratia marcescens9-2 strain isolation, classification identification and morphological characteristics [ J ]. University of North China university: nature science edition, 2003 (3): 108-111).

And amplifying 16S rDNA by PCR, and sequencing by using Huada genes after product electrophoresis verification. Results were aligned in the GenBank database.

1.3.6 determination of growth curves of high Propulsion protease Strain

Resuscitating and fermenting culture of strains: purification was performed from individual colonies isolated and stored in 1.3.3.

Growth curve measurement: 1mL of seed solution is inoculated into a fermentation liquid culture medium, and fermentation culture is carried out at 28 ℃ and 110 r/min. OD was measured at 0, 3, 6, 9, 12, 15, 18, 21h of culture, respectively ₆₀₀ Values were measured in duplicate 3 times per time.

The culture time of four bacterial strains is taken as the abscissa, and the average OD of the bacterial strains is measured ₆₀₀ Values are on the ordinate and growth curves are plotted.

1.3.7 optimization of fermentation conditions of high protease producing Strain

Under the condition that the fermentation quantity of each strain reaches the maximum culture time, a single-factor test design is adopted to explore the influence of a carbon source, an initial pH value, salinity and temperature on the growth of the strain.

Based on the initial fermentation medium, the effect of different carbon sources (glucose, maltose, sucrose, lactose and soluble starch) on the strain growth was investigated. The initial pH of the medium was adjusted and the effect of the initial pH of the medium (4, 5, 6, 7, 8, 9, 10) on the growth of the strain was investigated. The influence of different culture medium salinity (5%o, 10%o, 15%o, 20%o, 25%o, 30%o, 35%o) on the growth of the strain is studied. The effect of different culture temperatures (15 ℃,20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃) on strain growth was examined. And finally, culturing the strain under the optimal conditions to determine the protease activity.

1.3.8 determination of protease Activity

Tyrosine standard curve: the method of GB/T23527-2009 protease preparation is adopted. And drawing a standard curve by taking the tyrosine concentration c as an abscissa and the absorbance A as an ordinate. Reference is made to the preparation of crude enzyme solutions [20].

Measurement of protease Activity: reference is made to GB/T23527-2009 Pronase preparation method.

Protease activity is represented by X, calculated as follows:

(1)X＝c×4×N/1×10。

wherein: c is the activity of the final dilution of the sample, μg/mL, obtained from the standard curve; 4 is the total volume of the reaction reagent, mL;

n is the dilution of the sample; 1 is the enzyme quantity participating in the reaction, mL;10 is the reaction time, min.

2 results and analysis

2.1 isolation, screening and identification of Strain

2.1.1 isolation and purification of the epiphyte of the Trichinella gracilis

After separation and purification, 54 strains, numbered X1-X54, were obtained from the barnacles.

2.1.2 screening results of high protease-producing Strain

Four high-yield protease strains, namely X-8, X-18, X-21 and X-48, are obtained by comparing the ratio of the diameter of a casein hydrolysis ring of the epiphyte strain of the ampelopsis grossedentata to the diameter of a bacterial colony, and the hydrolysis rings of the strains on a casein enzyme culture medium are shown in figure 1. Fig. 1: transparent circles were screened on the X-21 strain caseinase medium.

2.1.3 electron microscope observations

The electron microscope photographs of the four strains are shown in FIG. 2.

As can be seen from FIG. 2, X-21 is a rod-shaped bacterium having flagellum.

2.1.4 morphological observations and physiological Biochemical identification results

Preliminary attribution of strains is carried out according to the morphology and physiological and biochemical detection results of the strains (reference document Zheng Chaocheng, etc. A strain producing high Wen Danbai enzyme in sludge is separated, identified and its enzymatic properties [ J ]. Environmental science, report 2012, 32 (3): 577-583). The X-21 bacteria produce orange colonies, the surfaces are smooth, and the edges are irregular. The physiological and biochemical characteristics of the strains are shown in Table 1.

TABLE 1 physiological Biochemical results of strains

Note that: "+" indicates positive results; "-" indicates negative results.

2.1.5 molecular biological identification

The sequence of the X18 strain has up to 99% homology with the 16S rDNA sequence of pseudoalteromonas fish killing after sequencing and alignment. And combining an electron microscope with morphological observation and physiological and biochemical characteristic test results, identifying the strain X-21 as pseudoalteromonas fish killing (Pseudoalteromonas piscicida).

X-21 Strain 16S rDNA:

AGGCCCGGGAACGTATTCACCGCAACATTCTGATTTGCGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACAGGCTTTAAGGGATTCGCTCACTATCGCTAGCTCGCTGCTCTCTGTACCTGCCATTGTAGCACGTGTGTAGCCCTACACGTAAGGGCCATGATGACTTGACGTCGTCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCCTTAGAGTTCCCGACCGAATCGCTGGCAACTAAGGATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTATCAGAGCTCCCGAAGGCACCAAACCATCTCTGGTAAGTTCTCTGTATGTCAAGTGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCTACTTAATGCGTTAGCTTTGGAAAAGTTGTCCGAAGACCCCAGCTCCTAGTAGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTACATGAGCGTCAGTGTTGACCCAGGTGGCTGCCTTCGCCATCGGTATTCCTTCAGATCTCTACGCATTTCACCGCTACACCTGAAATTCTACCACCCTCTATCACACTCTAGTTTGCCAGTTCGAAATGCAGTTCCCAGGTTAAGCCCGGGGCTTTCACATCTCGCTTAACAAACCGCCTGCGTACGCTTTACGCCCAGTAATTCCGATTAACGCTCGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGTCAGTAACGTCACAGCTAGCAGGTATTAACTACTAACCTTTCCTCCTGACTGAAAGTGCTTTACAACCCGAAGGCCTTCTTCACACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGATCATCCTCTCAAACCAGCTAGGGATCGTCGCCTTGGTGAGCCTTTACCTCACCAACTAGCTAATCCCACTTGGGCCAATCTAAAGGCGAGAGCCGAAGCCCCCTTTGGTCCGTAGACATTATGCGGTATTAGCCATCGTTTCCAATGGTTGTCCCCCACCTCAAGGCATGTTCCCAAGCA(SEQ ID NO.:1)

2.2 growth curves of high protease-producing strains

OD of X-21 bacteria cultured for 0, 3, 6, 9, 12, 15, 18, 21h under shaking culture conditions was measured ₆₀₀ Values were measured 3 times in duplicate at each time point and averaged. The growth curve of the strain is shown in FIG. 3.

Bacterial liquidIs proportional to the turbidity, the greater the turbidity, the measured OD ₆₀₀ The larger the value, the larger the concentration of the bacterial liquid and the larger the bacterial number. As can be seen from FIG. 3, the OD of the strain was increased with the prolonged culture time ₆₀₀ The values underwent a process of increasing and then gradually decreasing, indicating that the bacterial numbers of the three strains underwent a process of increasing and then decreasing with increasing culture time.

As shown in FIG. 3, pseudomonas fish-killing bacteria X-21 has logarithmic phase in 0-9 hr, plateau phase in 9-21 hr and decay phase after 21 hr. Therefore, when this bacterium was studied, 21 hours was selected as the optimal culture time.

2.3 optimization of fermentation conditions of high-yield protease Strain

2.3.1 Effect of carbon sources on Strain growth

The results of the effect of different carbon sources (content 2%o) on strain growth on the basis of the optimal fermentation time are shown in figure 4.

As can be seen from FIG. 4, when sucrose is used as a carbon source, pseudomonas fish-killing bacteria X-21 can obtain the maximum culture density, and the secondary concentration of soluble starch and glucose.

2.3.2 Effect of the initial pH of the Medium on the growth of the Strain

The results of the effect of different initial pH values of the liquid fermentation medium on the growth of the strain on the basis of the optimal fermentation time are shown in FIG. 5.

As can be seen from FIG. 5, the concentration of the Pseudomonas fish-killing bacteria X-21 increases gradually with increasing initial pH value; the concentration drop is evident at an initial pH > 8.

2.3.3 Effect of Medium salinity on Strain growth

The results of the effect of different salinity of the liquid fermentation medium on the strain growth on the basis of the optimal fermentation time are shown in FIG. 6.

As can be seen from FIG. 6, the concentration of the Pseudomonas fish-killing bacteria X-21 is gradually increased along with the increase of the salinity; when the salinity is more than 15 per mill, the bacterial concentration decreases.

2.3.4 Effect of culture temperature on Strain growth

The results of the effect of different culture temperatures of the liquid fermentation medium on the growth of the strain on the basis of the optimal fermentation time are shown in FIG. 7.

As shown in FIG. 7, the density of the Pseudomonas fish killing X-21 bacteria liquid gradually increases and the bacterial number increases with the increase of the culture temperature; when the temperature is more than 25 ℃, the turbidity of the bacterial liquid starts to decrease, and the bacterial quantity starts to decrease. Therefore, 25℃is the optimum temperature for fermentation.

2.4 determination of protease Activity

2.4.1 tyrosine standard curve

According to the operation method in the measurement of the protease activity, a regression equation of a tyrosine standard curve is obtained, which is shown in fig. 8.

2.4.2 Fulin assay for protease Activity

According to the method for measuring the protease activity of 1.3.8, under the optimized post-fermentation condition, the calculated protease activity of the X-21 strain reaches 441.18U/mL.

The present study screened high protease producing strain from the barnacle in coastal contaminated areas of Shandong, number X-21, identified as Pseudomonas fish (Pseudoalteromonas piscicida).

The highest fermentation amount of the strain X-21 is determined to be 21h by measuring the growth curve of the strain.

Based on the optimal culture time, the optimal fermentation conditions for the X-21 strain were obtained by a single-factor multi-level test: sucrose 5.0%o, initial pH 7.0-8.0, culture medium salinity 15%o, culture temperature 25 ℃. Under the preliminary optimized fermentation condition, the protease activity of the X-21 strain reaches 441.18U/mL.

Example 2 purification of enzyme and Property Studies

Strain X-21 was cultured under the culture conditions optimized in example 1. And (3) freezing and centrifuging the fermentation liquor for 10min at 3900r/min to obtain crude enzyme liquor. The crude enzyme solutions obtained were salted out stepwise with 30%, 80% saturation ammonium sulfate, respectively, and then dialyzed overnight at 4 ℃. Concentrating the dialyzed sample, and then sequentially passing through a Sephadex G-25 chromatographic column and a Sephadex G-100 chromatographic column for gel filtration to obtain the purified protease. The protease obtained by purification was single band as detected by SDS-PAGE.

(1) Optimum pH and pH stability test

The purified protease is subjected to enzyme activity measurement at different pH values, and the result shows that the protease X-21 can retain more than 90% of enzyme activity within the pH range of 7.5-11.0, and the pH value of 10.0 is the optimal pH value.

The enzyme solutions were diluted 10-fold in different pH buffers and tested for pH stability. Residual enzyme activity was determined after standing overnight at 4 ℃. The results show that protease X-21 is capable of maintaining more than 75% of the enzyme activity at pH 7.0 to pH 7.5; can maintain more than 85% of enzyme activity under the condition of pH8.0 to pH 11; the loss of enzyme activity is large at pH11.5 or below and at pH 6.5.

(2) Optimum temperature and temperature stability test

The protease activity of the purified protease was measured at various temperatures (30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃ and 70 ℃), and the result shows that the protease X-21 of the invention has an optimal reaction temperature of 55 ℃ and can maintain more than 80% of the enzyme activity under the condition of 40 ℃ to 60 ℃.

The enzyme solution was incubated at different temperatures (4 ℃, 10 ℃,20 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃) for the temperature stability test of the protease, and the residual enzyme activity was measured after incubation for 1 hour. The result shows that the protease of the invention has better stability under the condition of below 35 ℃, can still maintain more than 80% of activity after being preserved for 1h, has serious enzyme activity loss when exceeding 55 ℃, and has 28.6% of enzyme activity after being preserved for 1h under the condition of 55 ℃.

(3) Influence of Metal ions on protease Activity

And adding different metal ions into the purified protease liquid for testing, controlling the concentration of the metal ions to be 20mM in each experimental group respectively, mixing uniformly, then carrying out enzyme activity testing, setting three groups in parallel, and detecting the influence of different metal ions on the protease activity.

The detection result shows that the protease X-21 is specific to Cr ⁶⁺ Has certain tolerance to Fe ²⁺ 、Ca ² 、Mg ²⁺ These divalent ions are more tolerant.

In industrial washing water, if Fe ²⁺ 、Ca ² 、Mg ²⁺ The higher divalent ion content seriously affects the activity of the enzyme used for washing, and thus it is generally required to treat the washing water. The protease produced by the X-21 strain of the invention can effectively tolerate Fe ²⁺ 、Ca ² 、Mg ²⁺ The divalent ions greatly reduce the requirement of enzyme on washing water, and the optimal pH reaches 10, so that the method is very suitable for being applied to the washing industry.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

<110> Wiihai Galaxy biotechnology Co., ltd

<120> Pseudomonas fish-killing and use thereof

<130> 1916003

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1263

<212> DNA

<213> Pseudomonas fish (Pseudoalteromonas piscicida)

<400> 1

aggcccggga acgtattcac cgcaacattc tgatttgcga ttactagcga ttccgacttc 60

atggagtcga gttgcagact ccaatccgga ctacgacagg ctttaaggga ttcgctcact 120

atcgctagct cgctgctctc tgtacctgcc attgtagcac gtgtgtagcc ctacacgtaa 180

gggccatgat gacttgacgt cgtccccacc ttcctccggt ttatcaccgg cagtctcctt 240

agagttcccg accgaatcgc tggcaactaa ggataggggt tgcgctcgtt gcgggactta 300

acccaacatc tcacaacacg agctgacgac agccatgcag cacctgtatc agagctcccg 360

aaggcaccaa accatctctg gtaagttctc tgtatgtcaa gtgtaggtaa ggttcttcgc 420

gttgcatcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca attcatttga 480

gttttaacct tgcggccgta ctccccaggc ggtctactta atgcgttagc tttggaaaag 540

ttgtccgaag accccagctc ctagtagaca tcgtttacgg cgtggactac cagggtatct 600

aatcctgttt gctccccacg ctttcgtaca tgagcgtcag tgttgaccca ggtggctgcc 660

ttcgccatcg gtattccttc agatctctac gcatttcacc gctacacctg aaattctacc 720

accctctatc acactctagt ttgccagttc gaaatgcagt tcccaggtta agcccggggc 780

tttcacatct cgcttaacaa accgcctgcg tacgctttac gcccagtaat tccgattaac 840

gctcgcaccc tccgtattac cgcggctgct ggcacggagt tagccggtgc ttcttctgtc 900

agtaacgtca cagctagcag gtattaacta ctaacctttc ctcctgactg aaagtgcttt 960

acaacccgaa ggccttcttc acacacgcgg catggctgca tcaggcttgc gcccattgtg 1020

caatattccc cactgctgcc tcccgtagga gtctggaccg tgtctcagtt ccagtgtggc 1080

tgatcatcct ctcaaaccag ctagggatcg tcgccttggt gagcctttac ctcaccaact 1140

agctaatccc acttgggcca atctaaaggc gagagccgaa gccccctttg gtccgtagac 1200

attatgcggt attagccatc gtttccaatg gttgtccccc acctcaaggc atgttcccaa 1260

gca 1263

Claims

1. A protease-producing microbial strain, characterized in that the microbial strain is:

pseudoalteromonas fish (Pseudoalteromonas piscicida) X-21 is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.17513.

2. A protease produced by fermentation of pseudoalteromonas fish (Pseudoalteromonas piscicida) X-21 according to claim 1.

3. A method of preparing a protease, the method comprising the steps of:

(1) Fermenting the microbial strain of claim 1;

(2) Separating protease from the fermentation broth.

4. A method according to claim 3, wherein in step (1) the fermentation salinity is 5-20% (w/w).

5. A method according to claim 3, wherein in step (1), the carbon source used for fermentation is sucrose, soluble starch, or maltose.

6. A process according to claim 3, wherein in step (1) the fermentation temperature is 15-30 ℃.

7. A process according to claim 3, wherein in step (1) the fermentation onset pH is 7.0-8.0.

8. A biological enzyme preparation, characterized in that it comprises the protease of claim 2.

9. The biological enzyme preparation of claim 8, further comprising one or more biological enzymes selected from the group consisting of:

xylanase, pectinase, lipase, cellulase, and esterase.

10. Use of pseudoalteromonas fish (Pseudoalteromonas piscicida) for the preparation of a protease, wherein the pseudoalteromonas fish (Pseudoalteromonas piscicida) is the microbial strain of claim 1.