CN105441367B

CN105441367B - One plant of serratia marcescens and its application

Info

Publication number: CN105441367B
Application number: CN201610007451.7A
Authority: CN
Inventors: 陈济琛; 林新坚; 贾宪波; 蔡海松; 方宇
Original assignee: Institute of Soil and Fertilizer Fujian Academy of Agricultural Sciences
Current assignee: Institute of Soil and Fertilizer Fujian Academy of Agricultural Sciences
Priority date: 2016-01-07
Filing date: 2016-01-07
Publication date: 2019-09-03
Anticipated expiration: 2036-01-07
Also published as: CN105441367A

Abstract

The present invention provides one plant of serratia marcescens and its application, belongs to bioengineering field.The present invention provides one plant of serratia marcescens, deposit number is (the CCTCC M 2015732) bacterium using sucrose as carbon source promotion producing enzyme, the catalase of high yield can be obtained through liquid fermentation, enzyme activity is up to 32752U/mLU/ml.Hydrogen peroxide enzyme preparation preparation of the present invention is simple, enzymatic activity is high, and cheap, which has good application prospect in fields such as weaving, papermaking.

Description

One plant of serratia marcescens and its application

Technical field

The present invention provides one plant of serratia marcescens and its application, belongs to bioengineering field.

Background technique

Hydrogen peroxide is a kind of strong oxidizer, extensive use and industry, the bleaching and disinfection of food and medical field, still It is remained for seriously affecting production technology and health.Catalase can specificity peroxynitrite decomposition hydrogen be it is nontoxic Water and oxygen, be a kind of hydrogen peroxide removal approach of high-efficiency environment friendly.Catalase is widely present in animals and plants and aerobic In microorganism, initial catalase is the extraction purification from the animal's livers such as pigs and cattle, but this method be limited to raw material it is limited, At high cost and complex process, in comparison microbial fermentation production catalase then has big yield, activity height, zymologic property rich Many advantages, such as rich, low in cost.Existing related microorganisms fermentation catalase commodity external at present, there are some documents in the country And patent report catalase bacterium producing multi enzyme preparation, most strain enzyme-producing abilities are in every milliliter of fermentation liquid hundreds to thousands enzyme activity Unit, only Southern Yangtze University Chen Jian teaches (document) and 101805709 B of Southern Yangtze University patent CN report bacterial strain production of enzyme reaches 2 Ten thousand enzyme activity or so.The inventors discovered that a plant height produces the bacterial strain of catalase, and hydrogen peroxide is obtained by liquid fermentation Enzyme enzyme solution, the enzyme solution can be with efficient-decomposition hydrogen peroxide.The present invention is proposed based on the above discovery.

Summary of the invention

The object of the present invention is to provide one plant of Catalase-Producing Strain strains, and using soy peptone as nitrogen source, sucrose is The technique of carbon source through fermentation production catalase.Produced catalase agent activity is high, low production cost, be suitable for weaving, The removal of residual hydrogen dioxide in the production technologies such as papermaking.

Serratia marcescens (Serratia marcescens) KAT-1, the bacterial strain are protected on December 9th, 2015 There are China typical culture collection center, address is Wuhan University, and deposit number is CCTCC NO:M 2015732.

Technical solution of the present invention:

1, the screening and identification of bacterial strain

(1) it screens: choosing from the bacterium pure culture isolated and purified in soil the shake culture in liquid seed culture medium, take 100 microlitres of hydrogen peroxide are added in 100 microlitres of fermentation liquids, and the bacterial strain for having a large amount of bubbles to generate is bacterium producing multi enzyme preparation.

(2) it identifies: PCR amplification and sequencing being carried out to bacterial strain 16S rRNA, ncbi database, which compares, shows that the bacterium is viscous Matter Serratieae.

(3) strain characteristics: bacterium colony is round protrusion, milky；And fermentation liquid be it is faint yellow, do not produce red prodigiosin.Light Mirror hypothallus is circle.

(4) the catalase activity property surveyed method: fermentation liquid ultrasonication is dilute with the PBS buffer solution of pH7.0 until clarification It releases spare after suitable multiple.Catalase enzyme activity determination method: 3.9ml reaction solution (02M PBS pH7.0,20mM H₂O₂) It is rapidly added in 0.1ml enzyme solution, the variation of absorbance is measured under 240nm wavelength, counted every 5s primary.H₂O₂Mole disappear Backscatter extinction logarithmic ratio is 43.6.The definition of catalase enzyme activity is enzyme minute to consume 1 micromole H₂O₂Enzyme amount used is 1U.

2, using bacterial strain Kat-1 fermenting and producing catalase.Technique is as follows:

(1) prepared by seed liquor:

Seed culture medium are as follows: soy peptone 10g/L, beef extract 2g/L, NaCl1g/L, pH 7.0.

Seed liquid and preparation method thereof: the pure culture of one ring bacterial strain Kat-1 of picking is in 50ml seed culture medium, 180r/min training Feeding 12h is seed liquor.

(2) enzymatic production:

Culture medium: 10 ~ 40g/L of soy peptone, beef extract 2g/L, NaCl 1g/L, sucrose 30 ~ 90g/L, pH are 7.0。

Enzymatic production: liquid Shake flask medium is inoculated with 2% inoculum concentration.Fermentation temperature is 30 ~ 35 DEG C, and revolving speed is 180 ~ 220 R/min,.

32752U/mL is reached with above-mentioned technique fermentation fermentation liquid highest catalase enzyme activity using the bacterial strain.

The bacterium largely generates catalase when sucrose and maltose are carbon source, and glycerol, lactose and glucose produce the bacterium Enzyme does not have facilitation, and citric acid seriously inhibits thalli growth and producing enzyme.

Beneficial effects of the present invention: present invention firstly provides a plant heights to produce catalase bacterial strain, liquid submerged culture Enzyme activity reaches 32752U/mL.The bacterium speed of growth is fast, and fermentation time is short；Medium component is simple, using sucrose as the culture medium of carbon source It can be greatly facilitated the strain enzyme-producing, fermentation process is without the induction such as hydrogen peroxide or ethyl alcohol, simple process and low cost.It answers It is industrial to can be applied to food processing and papermaking weaving etc. with catalase prepared by the bacterial strain and process matched therewith, therefore this hair The strain and technique of bright offer are with a wide range of applications and apparent economic benefit.

Specific embodiment

Embodiment 1: the screening and identification of bacterium producing multi enzyme preparation Kat-1

Each bacterial strain pure culture of one ring of picking is transferred respectively in the triangular flask containing 50ml aforementioned liquids seed culture medium, is taken 200 microlitres of fermentation liquid of each bacterial strain are added isometric hydrogen peroxide observation and produce bubble situation.Having bubble producer is peroxidating Hydrogen enzyme bacterium producing multi enzyme preparation, the bacterial strain Kat-1 for selecting production bubble most violent use bacterium as catalase production.Bacterial strain Kat-1 bacterium colony Circle, milky, 100 times of oily microscopic observation thallus are circle.The fermentation liquid ice-bath ultrasonic of bacterial strain Kat-1 is broken until clear Clearly, broken condition 300W is crushed 2s pause 3s, and broken liquid PBS buffer solution is diluted 20 times of measurement enzyme activity.Catalase Vigour-testing method are as follows: 3.9ml reaction solution (0.2M PBS pH7.0,20mM H₂O₂) be rapidly added in 0.1ml enzyme solution, The variation that absorbance is measured under 240nm wavelength counts primary every 5s.H₂O₂Molar extinction coefficient be 43.6.Catalase The definition of enzyme activity is enzyme minute to consume 1 micromole H₂O₂Enzyme amount used is 1U.Measuring catalase activity at this time is 2230U/ ml.Take 3ml bacterium solution to extract the bacterium genome with bacterial genomes extracts kit, with bacterial 16 S rDNA universal primer (27F: 5 '-AGAGTTTGATCCTGGCTCAG-3 ' and 1492R:5 '-TACGGCTACCTTGTTACGACTT-3 ') the PCR amplification bacterium 16S rDNA, amplification program are 94 DEG C of denaturation 1min, and 55 DEG C of annealing 1min, 72 DEG C of extension 2min, amplification cycles number follow for 35 Ring.Pcr amplification product send biotech firm to be sequenced.Confirm that the bacterium is serratia marcescens after NCBI carries out sequence alignment.

Embodiment 2: strain fermentation produces catalase

From on inclined-plane, one ring Kat-1 pure culture of picking is in the 250ml triangular flask for filling 50ml seed fluid nutrient mediums of saccharomycete, and 37 12h, shaking speed 180r/min are cultivated on DEG C shaking table.It is forwarded to culture medium with 2% inoculum concentration, 250ml triangular flask Liquid amount is 50ml producing enzyme fermentation medium, and condition of culture is 35 DEG C, shaking speed 180r/min, is cultivated 24 hours.Take out hair Zymotic fluid ice-bath ultrasonic is broken until clarification, broken condition 300W are crushed 2s pause 3s.Be clarified liquid is hydrogen peroxide Enzyme solution, enzyme solution activity reaches 18660U/mL at this time.

The seed liquid culture medium: soy peptone 10g/L, beef extract 2g/L, NaCl 1g/L, pH 7.0；Producing enzyme Culture medium: soy peptone 30g/L, beef extract 2g/L, NaCl 1g/L, sucrose 60g/L, pH 7.0.

Embodiment 3: strain fermentation produces catalase

From on inclined-plane, one ring Kat-1 pure culture of picking is in the 250ml triangular flask for filling 50ml seed fluid nutrient mediums of saccharomycete, and 37 12h, shaking speed 180r/min are cultivated on DEG C shaking table.Liquid fermentation medium, 250ml triangle are forwarded to 2% inoculum concentration The liquid amount of bottle is 50ml producing enzyme fermentation medium, and condition of culture is 32 DEG C, shaking speed 180r/min, is cultivated 48 hours.It takes Fermentation liquid ice-bath ultrasonic is broken until clarification, broken condition 300W are crushed 2s pause 3s out.Be clarified liquid is peroxide Change hydrogen enzyme solution, enzyme solution activity reaches 32752U/mL at this time.

The seed liquid culture medium: soy peptone 10g/L, beef extract 2g/L, NaCl 1g/L, pH 7.0；Producing enzyme Culture medium: soy peptone 40g/L, beef extract 2g/L, NaCl 1g/L, sucrose 90g/L, pH 7.0.

The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, is all covered by the present invention.

SEQUENCE LISTING

<110>Fujian Province Agriculture InstituteSoil and Fertilizer Institute

<120>one plants of serratia marcescens and its applications

<130> 2

<160> 3

<170> PatentIn version 3.3

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agagtttgat cctggctcag

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tacggctacc ttgttacgac tt

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tgcagtcgag cggtagcaca ggggagcttg ctccctgggt gacgagcggc ggacgggtga

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gcataacgtc gcaagaccaa agagggggac cttcgggcct cttgccatca gatgtgccca

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gatgggatta gctagtaggt ggggtaatgg ctcacctagg cgacgatccc tagctggtct

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gagaggatga ccagccacac tggaactgag acacggtcca gactcctacg ggaggcagca

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gtggggaata ttgcacaatg ggcgcaagcc tgatgcagcc atgccgcgtg tgtgaagaag

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gccttcgggt tgtaaagcac tttcagcgag gaggaaggtg gtgaacttaa tacgttcatc

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aattgacgtt actcgcagaa gaagcaccgg ctaactccgt gccagcagcc gcggtaatac

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ggagggtgca agcgttacca ggaattactg ggcgtaaagc gcacgcaggc ggtttgttaa

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gtcagatgtg aaatccccgg gctcaacctg ggaactgcat ttgaaactgg caagctagag

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tctcgtagag gggggtagaa ttccaggtgt agcggtgaaa tgcgtagaga tctggaggaa

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taccggtggc gaaggcggcc ccctggacga agactgacgc tcaggtgcga aagcgtgggg

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agcaaacagg attagatacc ctggtagtcc acgctgtaaa cgatgtcgat ttggaggttg

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cggccgggaa ctcaaaggag actgccagtg ataaactgga ggaaggtggg gatgacgtca

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Claims

1. one plant of serratia marcescens, it is characterised in that: the bacterial strain be serratia marcescens (Serratia marcescens) KAT-1, the bacterial strain are stored in China typical culture collection center on December 9th, 2015, and deposit number is CCTCC NO: M 2015732。

2. utilizing the method for serratia marcescens fermenting and producing catalase described in claim 1, it is characterised in that: described in use Serratia marcescens KAT-1 produces catalase as starting strain, through seed liquor culture and by carbon source through fermentation of sucrose.

3. the method for serratia marcescens fermenting and producing catalase according to claim 2, it is characterised in that: specific Technique are as follows:

(1) seed liquor culture:

Seed liquor preparation: being placed in 50ml seed liquid culture medium from one ring single colonie of solid agar plate picking, 37 DEG C of cultures 12h, revolving speed 180r/min obtain seed liquor；

(2) enzymatic production:

Enzymatic production: taking step (1) seed liquor to be inoculated with liquid culture medium with 2% inoculum concentration, and fermentation temperature is 30 ~ 35 DEG C, turns Speed is 180 ~ 220 r/min, and fermentation time is 36 ~ 60h.

4. the method for serratia marcescens fermenting and producing catalase according to claim 3, it is characterised in that: described Seed liquid culture medium: soy peptone 10g/L, beef extract 2g/L, NaCl 1g/L, pH 7.0；Culture medium: soybean egg White 10 ~ 40g/L of peptone, beef extract 2g/L, NaCl 1g/L, sucrose 30 ~ 90g/L, pH 7.0.