CN101892169B

CN101892169B - Halophilic archaea for high production of halophilic lipase

Info

Publication number: CN101892169B
Application number: CN2008102358538A
Authority: CN
Inventors: 许正宏; 张晓梅; 窦文芳; 许泓瑜; 张萌
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2008-11-28
Filing date: 2008-11-28
Publication date: 2012-06-27
Anticipated expiration: 2028-11-28
Also published as: CN101892169A

Abstract

The invention belongs to the field of biotechnology, and in particular relates to a red halophilic archaea strain which can produce halophilic lipase and belongs to Haloterrigena sp. species. The strain is screened from salt lakes in the Xilinhot district in Inner Mongolia province. Fermentation researches discover that: the activity of the red strain for producing the lipase reaches 25 U/ml by using 1 percent of olive oil as an inducer.

Description

What a kind of high yield was had a liking for salt lypase has a liking for the salt Archimycetes

Technical field

The present invention relates to a kind of high yield and have a liking for the extreme halotolerant bacteria strain of salt lypase, belong to biological technical field.

Background technology

Lypase is widely used in fields such as washing composition, food, medicine, process hides, weaving.The lypase biotechnology applications is an emerging field in the biological prosthetic and the waste treatment of contaminated environment.The oil leakage that produces in oil production and the refining process, the refuse that contains fat refuse and catering generation that produces in the grease course of processing can effectively be handled with the lypase of different sources.Lypase has obtained widespread use in the biological prosthetic contaminated environment.An European patent has been reported and has been utilized the lypase inhibition and remove the microbial film settling in the cooling water system.

Along with seawater substitutes the development of engineering, the biological treatment of high salt sewage becomes the focus of research, and the application under high salt condition of common micro-organisms and enzyme thereof is restricted, and halophilic bacterium and the application of having a liking for salt lypase thereof will provide new instrument for high salt WWT.

Natrinema altunense sp is a quasi-microorganism that is grown in high salt extreme environment, mainly is present in the environment such as salt lake, salt alkali lake, sabkha, the Dead Sea and saltern.People such as Baratti reported first in 2006 have a liking for salt Archimycetes Natronococcus sp. and produce and have a liking for salt lypase, enzyme activity reaches 50U/L.So widen the source of having a liking for salt lypase, improve enzymatic productivity improving its using value and become the focus of research,

Summary of the invention

[problem that will solve]

The purpose of this invention is to provide a kind of Haloterrigena sp. genus that can in high salt fermention medium, accumulate lypase in a large number and have a liking for salt Archimycetes bacterial strain.

Summary of the invention

Contriver of the present invention has a liking for salt lypase generation bacterium from obtained a strain from screening the bacterial classification sample in salt lake, and this strain bacterium has been carried out fermentation research.

Lypase produces bacterium screening culture medium and fermention medium:

Screening culture medium (g/L): NaCl 200.0; KCl 2.0; MgSO ₄.7H ₂O 20.0; FeSO ₄.7H ₂O 0.04; Casamino acids 7.5; Yeast powder 10.0; Trisodium citrate 3.0; Sweet oil 10.0 rhodamine Bs 1.0; PH 7.5

Fermention medium (g/L): NaCl 200.0; KCl 2.0; MgSO ₄.7H ₂O 20.0; FeSO ₄.7H ₂O 0.04; Casamino acids 7.5; Yeast powder 10.0; Trisodium citrate 3.0; Sweet oil 10.0; PH 7.5

Through dull and stereotyped primary dcreening operation with shake that the multiple sieve of bottle obtains have a liking for salt Archimycetes well-grown on high salt culture medium, bacterium colony smooth surface, neat in edge, redness.Gram-negative, microscopy are shaft-like.For identifying its type, respectively it has been carried out Molecular Identification and Physiology and biochemistry evaluation, the aerobic or amphimicrobian of this bacterium, growth temperature range is 30～60 ℃, optimum growth temperature is 42 ℃; Suitable growth pH scope 6.0～9.0, the righttest growth pH 7.0; In 0.5～5.2mol/L NaCl scope, all can grow, the righttest growth concentration is 3.5mol/L.This bacterium only can utilize sucrose, fructose preferably, and starch hydrolysis experiment, gelatin hydrolysis test, casein degraded test, nitrate reduction test are positive; Indole test, catalase test, hydrogen sulfide production test are negative.This bacterial strain 16S rDNA full length sequence is 1581bp (GenBank accession number EU557270); With the similarity of the bacterial strain Haloterrigena thermotolerans strain 10R (GenBank accession number AY994199) with highest homology property be 99%; Combining form characteristic and physio-biochemical characteristics; It is accredited as has a liking for salt Archimycetes Haloterrigena sp., called after Haloterrigena sp.Z4.This biomaterial preservation information: the specific name of this Natrinema altunense sp is Haloterrigena thermotolerans; Depositary institution is that Chinese microorganism strain preservation center is called for short at China Committee for Culture Collection of Microorganisms common micro-organisms center; The address is Datun Road, Chaoyang District, Beijing City (institute of microbiology of the Chinese Academy of Sciences); Preservation date is on April 21st, 2008, deposit number CGMCC No2463.

Find that in the fermentation research process this bacterium can be the inductor yielding lipase with sweet oil, peanut oil, til, rapeseed oil, tea oil, adds 1% sweet oil, 42 ℃, to produce enzyme when pH 7.5, NaCl concentration 3.5mol/L the highest, reaches 22U/ml.

Description of drawings:

The different paramorphogens of Fig. 1 are to producing the influence of enzyme

Fig. 2 NaCl concentration, pH and temperature are to producing the influence of enzyme

[beneficial effect]

Provide a kind of high yield to have a liking for the strain excellent of salt lypase with industrial application potentiality.

Embodiment

Embodiment 1: have a liking for the screening of salt lypase superior strain

With the bacterial classification activation of transferring, 37 ℃ of cultivations, picking list bacterium colony and dibbling were cultivated 7～14 days for dull and stereotyped 37 ℃ in sweet oil-rhodamine B, and the bacterium colony of selecting fluorescent ring separates the back through line and obtains single bacterium colony, is inoculated in the slant medium preservation and carries out shake flask fermentation and sieve again.

Embodiment 2: the mensuration that the lypase enzyme is lived

The mensuration that the lypase enzyme is lived adopts colourimetry; Its principle is: p-NPB decomposes generation a part p-NP under the catalysis of lypase; P-NP has maximum absorption band at the 410nm place; Through measuring the light absorption value of reaction solution at 410nm place, but quantitatively determined p-NPB amount of oxidation, thus calculate the enzyme work unit of lypase.

The measuring method reference carries out, and is specific as follows:

Solution A: 176 μ l p-NPB are dissolved in the 10mL Virahol, 4 ℃ of preservations

Solution B: pH 8.0,50mM Tris-HCl damping fluid

During use solution A is mixed by the volume ratio of 1:9 with solution B, be made into 3.6mL substrate reactions liquid, add 400 μ l crude enzyme liquids, 37 ℃ of reaction 10min survey light absorption value at 410nm.

The definition of 1 enzyme of lypase unit alive: PM discharges the required enzyme amount of 1 μ mol p-NP.

Enzyme (U/ml)=(A alive ₄₁₀-0.1084) * 2.5 ÷ 0.0511

Embodiment 3:16s rDNA strain identification

Get fresh bacterium liquid exponential phase of growth, centrifugal collection thalline is pressed genome extraction agent box and is extracted genomic dna.Amplification primers is the Archimycetes universal primer, as follows:

P ₁Forward primer 5 ＇-ATTCCGGTTGATCCTGC-3 ＇

P ₂Reverse primer 5 ＇-AGGAGGTGATCCAGCCGCAG-3 ＇

The system of 50 μ l is adopted in the PCR reaction, and is specific as follows:

10×buffer 5ul

dNTPs(2.5mM) 4ul

P ₁(10pM) 2ul

P ₂(10pM) 2ul

Taq archaeal dna polymerase (5U/ul) 0.6ul

Template?DNA 1ul

ddH ₂O 35.4ul

The pcr amplification condition: 95 ℃ of sex change 1min, 50 ℃ of annealing 45s, 72 ℃ are extended 90s, 50 μ L systems, 30 circulations.The a small amount of glue that the purifying of pcr amplification product is given birth to worker biotech company by Shanghai reclaims the explanation of PCR product purification test kit, and order-checking is given birth to worker biotech company by Shanghai and accomplished.

The 16S rDNA sequence that after order-checking, obtains is carried out the BLAST comparison and is confirmed its kind in GenBank.

Embodiment 4: fermenting process research

Shake-flask culture method: insert seed culture medium behind the inclined-plane seed activation, cultivate 12h, insert fermention medium by 10% inoculum size and produce the enzyme cultivation.

Different inductors are to producing the influence of enzyme: add the inductor of different concns in the seed culture medium, 37 ℃ of shake flask fermentation 84h measure the fermented liquid lipase activity, with do not add inductor as contrast.

Temperature is to producing the influence of enzyme: respectively at 30,37,42,50,60 ℃ of shake flask fermentation 84h, measure the fermented liquid lipase activity, draw and produce the enzyme curve.

Initial pH is to producing the influence of enzyme: the initial pH of fermention medium is respectively 4,5,6,7,7.5,8,9,10,11,37 ℃ of shake flask fermentation 84h, measures the fermented liquid lipase activity, draws and produces the enzyme curve.

Salt concn is to producing the influence of enzyme: fermention medium NaCl concentration is respectively 0.5,1.5,2.5,3.5,4.5,5.2mol/L, and 37 ℃ of shake flask fermentation 84h measure the fermented liquid lipase activity, draw and produce the enzyme curve.

< 110>Southern Yangtze University

What < 120>a kind of high yield was had a liking for salt lypase has a liking for the salt Archimycetes

<140>2008102358538

<141>2008-11-28

<212>DNA

< 213>have a liking for salt Archimycetes (Haloterrigena thermotolerans)

<400>1

P ₁Forward primer 5 '-ATTCCGGTTGATCCTGC-3 '

P ₂Reverse primer 5 '-AGGAGGTGATCCAGCCGCAG-3 '

Claims

1. strain ability high yield is had a liking for the extreme halotolerant bacteria strain Z4 of the Haloterrigena sp. genus of salt lypase, is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number: CGMCC No2463, preservation date: on April 21st, 2008.

2. the extreme halotolerant bacteria strain Z4 that Haloterrigena sp. as claimed in claim 1 belongs to has following characteristic:

A, in 0.5-5.2mol/L NaCl concentration substratum, grow;

B, salt lypase is had a liking in a large amount of accumulation in the high salt fermention medium that with 1% sweet oil is inductor;

C, institute's yielding lipase have has a liking for the salt characteristics significantly, in 0.5-5.2mol/L NaCl environment, all keeps high reactivity.