CN105441367A

CN105441367A - Serratia marcescens and application thereof

Info

Publication number: CN105441367A
Application number: CN201610007451.7A
Authority: CN
Inventors: 陈济琛; 林新坚; 贾宪波; 蔡海松
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: 2016-03-30
Anticipated expiration: 2036-01-07
Also published as: CN105441367B

Abstract

The invention provides a strain of Serratia marcescens and application thereof, belonging to the field of bioengineering. The invention provides the strain of Serratia marcescens (the Preservation No.: CCTCC M 2015732) which takes saccharose as a carbon source to promote enzyme production and is subjected to liquid fermentation to obtain high-yield catalase of which the enzyme activity is up to 32752U/mLU/ml. A catalase preparation provided by the invention is simple to prepare, high in enzyme activity and low in cost and has a favorable application prospect in the fields, such as textiles and papermaking.

Description

One strain serratia marcescens and application thereof

Technical field

The invention provides strain serratia marcescens and an application thereof, belong to bioengineering field.

Background technology

Hydrogen peroxide is a kind of strong oxidizer, the bleaching of widespread use and industry, food and medical field and sterilization, but it is residual for having a strong impact on production technique and healthy.Catalase can specific decomposition of hydrogen peroxide be nontoxic water and oxygen, is a kind of hydrogen peroxide removal approach of high-efficiency environment friendly.Catalase is extensively present in animals and plants and aerobic microbiological, initial catalase is extraction purification from the animal liverss such as pig ox, but the method is limited to, and raw material is limited, cost is high and complex process, Comparatively speaking fermentable produces catalase then the plurality of advantages such as output is large, activity is high, zymologic property abundant, with low cost.External existing related microorganisms fermentation catalase commodity at present, domestic have some documents and patent report catalase bacterium producing multi enzyme preparation, most strain enzyme-producing ability is at every milliliter of fermented liquid hundreds of to several thousand enzyme activity units, and only Southern Yangtze University Chen Jian professor (document) and Southern Yangtze University patent CN101805709B reports that bacterial strain production of enzyme reaches 20,000 enzymes left and right alive.The present inventor finds that a plant height produces catalatic bacterial strain, and obtains catalase enzyme liquid by liquid fermenting, and this enzyme liquid can efficient-decomposition hydrogen peroxide.The present invention is proposed based on above discovery.

Summary of the invention

The object of this invention is to provide a strain Catalase-Producing Strain strain, and be nitrogenous source with soy peptone, sucrose is that carbon source through fermentation produces catalatic technique.To produce catalase agent activity high, low production cost, is suitable for the removal of residual hydrogen dioxide in the production technique such as weaving, papermaking.

Described serratia marcescens (Serratiamarcescens) KAT-1, this bacterial strain is kept at China typical culture collection center on December 9th, 2015, and address is Wuhan University, and deposit number is CCTCCNO:M2015732.

Technical scheme of the present invention:

1, the screening of bacterial strain and qualification

(1) screen: the bacterium pure culture of choosing separation and purification from soil shakes cultivation in liquid seed culture medium, and get 100 microlitre fermented liquids and add 100 gel hydrogen peroxide, the bacterial strain having a large amount of bubble to produce is bacterium producing multi enzyme preparation.

(2) identify: carry out pcr amplification and order-checking to this bacterial strain 16SrRNA, it is serratia marcescens that ncbi database comparison shows this bacterium.

(3) strain characteristics: bacterium colony is circular projection, oyster white; And fermented liquid is faint yellow, does not produce red prodigiosin.Light microscopic hypothallus is circular.

(4) the catalase activity property surveyed method: fermented liquid ultrasonication is until clarification, for subsequent use after diluting suitable multiple with the PBS damping fluid of pH7.0.Catalase enzyme activity determination method: 3.9ml reaction solution (02MPBSpH7.0,20mMH ₂o ₂) add rapidly in 0.1ml enzyme liquid, under 240nm wavelength, measure the change of absorbancy, every 5s counting once.H ₂o ₂molar extinction coefficient be 43.6.Catalase enzyme is lived and is defined as enzyme minute consumption 1 micromole H ₂o ₂enzyme amount used is 1U.

2, bacterial strain Kat-1 fermentative production catalase is applied.Technique is as follows:

(1) seed liquor preparation:

Seed culture medium is: soy peptone 10g/L, extractum carnis 2g/L, and NaCl1g/L, pH are 7.0.

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

(2) enzymatic production:

Culture medium: soy peptone 10 ~ 40g/L, extractum carnis 2g/L, NaCl1g/L, sucrose 30 ~ 90g/L, pH are 7.0.

Enzymatic production: with 2% inoculum size inoculation liquid Shake flask medium.Leavening temperature is 30 ~ 35 DEG C, and rotating speed is 180 ~ 220r/min.

Apply this bacterial strain and reach 32752U/mL so that the highest catalase enzyme of above-mentioned technique fermentation fermented liquid is alive.

When sucrose and maltose are carbon source, this bacterium produces catalase in a large number, and glycerine, lactose and glucose produce enzyme to this bacterium does not have promoter action, and citric acid seriously suppresses thalli growth and produces enzyme.

Beneficial effect of the present invention: the present invention provide firstly a plant height and produces catalase bacterial strain, liquid submerged culture enzyme is lived and is reached 32752U/mL.This bacteria growing speed is fast, and fermentation time is short; Medium component is simple, is that the substratum of carbon source can greatly promote this strain enzyme-producing with sucrose, and fermenting process is without the need to the induction such as hydrogen peroxide or ethanol, and technique is simple, with low cost.Apply catalase prepared by this bacterial strain and process matched therewith and can be applied to the industry such as food-processing and papermaking weaving, therefore bacterial classification provided by the invention and technique are with a wide range of applications and obvious economic benefit.

Embodiment

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

The each bacterial strain pure culture of picking one ring is transferred respectively in the triangular flask containing 50ml aforementioned liquids seed culture medium, and fermented liquid 200 microlitre getting each bacterial strain adds equal-volume hydrogen peroxide and observes aerogenesis bubble situation.There is bubble producer for catalase bacterium producing multi enzyme preparation, select the most violent bacterial strain Kat-1 of aerogenesis bubble to produce as catalase and use bacterium.Bacterial strain Kat-1 bacterium colony is circular, oyster white, and 100 times of oily Microscopic observation thalline are circular.The fermented liquid ice-bath ultrasonic of bacterial strain Kat-1 is broken, and until clarify, broken condition is 300W, broken 2s pause 3s, broken liquid PBS damping fluid is diluted 20 times of measurement enzymes and lives.Activity of catalase measuring method is: 3.9ml reaction solution (0.2MPBSpH7.0,20mMH ₂o ₂) add rapidly in 0.1ml enzyme liquid, under 240nm wavelength, measure the change of absorbancy, every 5s counting once.H ₂o ₂molar extinction coefficient be 43.6.Catalase enzyme is lived and is defined as enzyme minute consumption 1 micromole H ₂o ₂enzyme amount used is 1U.Recording now catalase activity is 2230U/ml.Get 3ml bacterium liquid bacterial genomes extraction test kit and extract this bacterium genome, with the 16SrDNA of bacterial 16 S rDNA universal primer (27F:5 '-AGAGTTTGATCCTGGCTCAG-3 ' and 1492R:5 '-TACGGCTACCTTGTTACGACTT-3 ') this bacterium of pcr amplification, amplification program is 94 DEG C of sex change 1min, 55 DEG C of annealing 1min, 72 DEG C extend 2min, and amplification cycles number is 35 circulations.Pcr amplification product send biotech firm to check order.After NCBI carries out sequence alignment, confirm that this bacterium is serratia marcescens.

Embodiment 2: strain fermentation produces catalase

From inclined-plane, picking one ring Kat-1 pure culture is in the 250ml triangular flask filling 50ml seed liquid nutrient medium, and 12h cultivated by 37 DEG C of shaking tables, and shaking speed is 180r/min.Be forwarded to culture medium with the inoculum size of 2%, the liquid amount of 250ml triangular flask is that 50ml produces enzymic fermentation substratum, and culture condition is 35 DEG C, shaking speed 180r/min, cultivates 24 hours.Take out fermented liquid ice-bath ultrasonic broken until clarification, broken condition is 300W, broken 2s pause 3s.The liquid that is clarified is catalase liquid, and now enzyme liquid activity reaches 18660U/mL.

Described seed liquor substratum: soy peptone 10g/L, extractum carnis 2g/L, NaCl1g/L, pH are 7.0; Culture medium: soy peptone 30g/L, extractum carnis 2g/L, NaCl1g/L, sucrose 60g/L, pH are 7.0.

Embodiment 3: strain fermentation produces catalase

From inclined-plane, picking one ring Kat-1 pure culture is in the 250ml triangular flask filling 50ml seed liquid nutrient medium, and 12h cultivated by 37 DEG C of shaking tables, and shaking speed is 180r/min.Be forwarded to liquid fermentation medium with the inoculum size of 2%, the liquid amount of 250ml triangular flask is that 50ml produces enzymic fermentation substratum, and culture condition is 32 DEG C, shaking speed 180r/min, cultivates 48 hours.Take out fermented liquid ice-bath ultrasonic broken until clarification, broken condition is 300W, broken 2s pause 3s.The liquid that is clarified is catalase liquid, and now enzyme liquid activity reaches 32752U/mL.

Described seed liquor substratum: soy peptone 10g/L, extractum carnis 2g/L, NaCl1g/L, pH are 7.0; Culture medium: soy peptone 40g/L, extractum carnis 2g/L, NaCl1g/L, sucrose 90g/L, pH are 7.0.

The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.

SEQUENCELISTING

<110> Fujian Province Agriculture InstituteSoil and Fertilizer Institute

<120> mono-strain serratia marcescens and application thereof

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Claims

1. a strain serratia marcescens, it is characterized in that: described bacterial strain is serratia marcescens (Serratiamarcescens) KAT-1, this bacterial strain is kept at China typical culture collection center on December 9th, 2015, and deposit number is CCTCCNO:M2015732.

2. utilize the catalatic method of serratia marcescens fermentative production described in claim 1, it is characterized in that: with described serratia marcescens KAT-1 as starting strain, through seed liquor cultivate and be that carbon source through fermentation produces catalase with sucrose.

3. the catalatic method of serratia marcescens fermentative production according to claim 2, is characterized in that: concrete technology is:

(1) seed liquor is cultivated:

Prepared by seed liquor: be placed in 50ml seed liquor substratum from solid agar dull and stereotyped picking one ring list bacterium colony, cultivate 12h for 37 DEG C, rotating speed is 180r/min, obtains seed liquor;

(2) enzymatic production:

Enzymatic production: get step (1) seed liquor with 2% inoculum size inoculation liquid culture medium, leavening temperature is 30 ~ 35 DEG C, and rotating speed is 180 ~ 220r/min, and fermentation time is 36 ~ 60h.

4. the catalatic method of serratia marcescens fermentative production according to claim 3, it is characterized in that: described seed liquor substratum: soy peptone 10g/L, extractum carnis 2g/L, NaCl1g/L, pH are 7.0; Culture medium: soy peptone 10 ~ 40g/L, extractum carnis 2g/L, NaCl1g/L, sucrose 30 ~ 90g/L, pH are 7.0.