CN102477415A

CN102477415A - Lipase with fat degradation capacity under alkaline condition

Info

Publication number: CN102477415A
Application number: CN2011102592912A
Authority: CN
Inventors: 方井霞; 张柏松; 刘刚; 苏东风
Original assignee: Individual
Current assignee: Individual
Priority date: 2011-09-05
Filing date: 2011-09-05
Publication date: 2012-05-30

Abstract

The invention discloses a nucleotide sequence and an amino acid sequence of lipase which has fat degradation capacity under an alkaline condition through transformation. The technical scheme disclosed by the invention is characterized in that: a serratia proteamaculans lipase gene is synthesized first and is connected with plasmid; a primer is designed for carry out error-prone PCR (Polymerase Chain Reaction) amplification; after the error-prone PCR product is purified and is connected with a carrier, and then a competent cell is converted; transformants containing alkali-resistance lipase are screened; and a gene sequence of the newly-constructed lipase is determined by measurement. The alkali-resistance lipase can overcome the defects of fast enzyme activity reduction and poor practicability of lipase used under the alkaline condition during existing industrial application so as to achieve the advantages of high alkali-resistance performance, good stability, reduced cost, reduced process steps and good practicability of the lipase in the using process.

Description

The lypase that has the fatty ability of degraded under a kind of alkaline condition

Technical field

The invention belongs to molecular biology, zymetology, physiology field.Specifically, relate to a kind of through the molecular biology method transformation, can alkali-proof lypase dna sequence dna and corresponding amino acid sequence thereof.

Background technology

Lypase (lipase; EC3.1.1.3) be that one type of catalysis triglyceride decomposes, the enzyme of synthetic and transesterify; Have extremely strong selectivity and specificity, and have special performance, be widely used in industries such as food, oil and fat chemical in water and organic phase interface catalyzed reaction.Along with widening of Application Areas, related to emerging fields such as single cell protein production, cosmetics production, environment protection, novel biomaterial, biosensor, biomedicine and bioenergy, wide application prospect is arranged.

Lypase extensively is present in natural animal and plant and the microbe body.As: bacterium has 28 genus, 4 genus of actinomycetes, 10 genus of yeast, 23 genus of other fungi all can produce lypase.In view of microbial lipase has important industrial application value and potential great demand; Many industrial experiments chamber and R&D institution are being engaged in loaded down with trivial details high yielding lipase microbe to screen work for a long time; But because the complicacy of microorganism growth condition makes the lypase development of resources with special property receive the restriction of microorganism growth.

Summary of the invention

A kind of development of new type alkali-resistant fibre property lypase and structure gene thereof have been the purpose of this invention is to provide.This gene expression product lypase shows active under the highly basic condition, is applied to contain in the alkaline waste water, and the processing of oil substances has wide purposes in the environmental protection field.In order to reach the object of the invention; Realize that concrete technological step of the present invention is following: obtain Serratia proteamaculans S.proteamaculans lipase gene sequence accompanying drawing 1 by the GenBank DB; Synthetic this gene order and link to each other with plasmid pUC18 (being not limited only to this cloning vector); With the DNA of fatty enzyme gene as template; A (5 ' one CTT GAA TTC GAT TAG AGTCGT ATA AGA TG 1, EcoR I) and b (5 one CTT GGT ACC TTA ATT CGT ATT CTG TCC TC-3 KpnI) carry out the fallibility pcr amplification for primer.Every 100ul reaction system is: 10ul 10x fallibility PCR damping fluid (500mmol/L KCl, 70mmol/L MgCl ₂, 100mmol/L Tris-HCI pH8.3,0.1% (w/v gelatin); 10ul 10XdNTP mixture (2mmol/L dGTP, 2mmol/L dATP, 10mmol/LdCTP and 10mmol/LdTTP); Each 30pmol of primer a and b; The 5mmol/L MnCl2 of 10ul; DNA template 20pmol; The Taq archaeal dna polymerase is 5U, and ultrapure water to the TV that adds sterilization again is 100ul.The PCR program is: 95 ℃, and 5min; 94 ℃ of 30S, 52 ℃ of 1min, 72 ℃ of 1min, 35 circulations; 72 ℃ of 10min.With the agarose gel electrophoresis of PCR product, carry out purifying and recovering with PCR purifying and recovering test kit with 1% (w/v).Fallibility PCR product behind the purifying is connected (being not limited thereto carrier) with the pMD18-T carrier; The super competent cell of transformed into escherichia coli DH5a (is not limited thereto acceptor; But acceptor should be corresponding in plasmid vector); Coating LB (Amp that contains 100ug/mL) flat board is selected and is had the segmental clone of insertion, constitutive mutation body storehouse.All transformant pickings to RB (adding 20mL 0.2% (W/V) rhodamine B and 40mL sweet oil emulsion in the 1000mL LB substratum, the pH nature) flat board, behind 37 ℃ of cultivation 24 h, are chosen the transformant that metachromatism is arranged.Be inoculated into 3mL LB substratum (containing the 100ug/mL penbritin) respectively, 37 ℃ of shaken are about 0.6 to OD600, and it is dull and stereotyped that each draws 3ul bacterium drop to rhodamine B, and 37 ℃ of C cultivate visible obviously metachromatism behind 12～24h.Choose that variable color is fast carries out liquid fermenting with the big clone of variable color circle, prepare enough bacterium liquid measures, under the low temperature behind the IPTG abduction delivering ultrasonic disruption cell, centrifugal collection supernatant is with pNPP method mensuration lipase activity.Divide into groups; Regulating pH is 10, and each kept 2 hours, during measured a lipase activity in per 20 minutes and see Fig. 2; Select a enzyme activity to reduce the sample of less than 10%; (after this adopt same procedure that the lypase enzyme liquid of this transformant is measured when pH=9, the pH=8, enzyme activity is stable) is with its corresponding transformant dna sequencing (its nucleotide sequence and aminoacid sequence are seen SEQ NO.1 and SEQ NO.2).Sequencing result shows, has 5 bases in the structure gene sudden change taken place, and be respectively a101g, c176g, t335g, g182c, t154g.5 amino acid that cause thus change (Y34C, P59R, V112A, S61T, L52V), form the lypase that has the fatty ability of degraded under a kind of new alkaline condition that the present invention relates to.

The lypase that the present invention relates to is a kind of novel lipase; Its primary structure since 5 amino acid whose sudden changes and known lypase character differ widely; Thereby cause ability with higher tolerance alkalescence; Be suitable for containing in the alkaline waste water, the processing of oil substances has wide purposes in the environmental protection field.

Description of drawings

The former lipase gene sequence of Fig. 1 Serratia proteamaculans S.proteamaculans;

Lipase activity changes relatively under the high pH of Fig. 2;

Embodiment

Following examples only are used to the present invention is described but not are used to limit scope of the present invention.

The experimental technique of unreceipted actual conditions in the instance, usually according to normal condition, condition is carried out described in " molecular cloning " (1989).

E.coli BL21 that uses in an embodiment of the present invention and carrier pET28 are available from Novagen company.

Embodiment 1:

The acquisition of alkaline lipase enzyme liquid

According to the synthetic alkaline lipase gene of SEQ NO.1, the pcr amplification synthetic product connects after enzyme is cut and Transformed E .coli BL2 1 (being not limited only to this plasmid and corresponding acceptor) with plasmid pET28-a.37 ℃ of liquid fermentation and culture, ultrasonic disruption cell behind the IPTG abduction delivering, centrifugal collection supernatant is and contains this alkaline lipase enzyme liquid.

Embodiment 2:

The alkali resistance lipase hydrolyzation of oil and fat

In 40% vegetable oil solution, in (pH10), add an amount of enzyme liquid, change with PM 100 and stirred 2 hours, chromatography records oil phase and contains lipid acid, and water contains glycerine, explains that lypase hydrolyzable grease produces glycerine and lipid acid.

Claims

1. alkali-proof lypase, its aminoacid sequence is made up of the aminoacid sequence shown in the SEQ NO.2.

2. gene of right 1 described alkaline lipase of encoding.

3. gene according to claim 2, its nucleotide sequence is made up of the nucleotide sequence shown in the SEQ NO.1.

4. the recombinant plasmid that contains the 3 described genes of having the right.

5. contain the described reorganization of claim 4 bacterium.

6. utilize the lypase of claim 1,2 or 3 preparations.

7. the alkaline lipase that obtains through claim 1,2,3 or 5 is in the application of industrial aspect such as chemical industry, weaving, papermaking, clothes and environmental protection