CN106085994B

CN106085994B - A kind of alkaline pectin enzyme mutant that specific enzyme activity improves

Info

Publication number: CN106085994B
Application number: CN201610699824.1A
Authority: CN
Inventors: 张桂敏; 望潇文; 易犁; 蒋思婧; 马延和
Original assignee: Hubei University
Current assignee: Hubei University
Priority date: 2016-08-22
Filing date: 2016-08-22
Publication date: 2019-09-03
Anticipated expiration: 2036-08-22
Also published as: CN106085994A

Abstract

The invention discloses the alkaline pectin enzyme mutants that a kind of specific enzyme activity improves.The amino acid sequence of the mutant is as shown in following (1) or (2) or (3) or (4) or (5) or (6): (1) amino acid sequence shown in SEQ ID NO.1, (2) amino acid sequence shown in SEQ ID NO.2, (3) amino acid sequence shown in SEQ ID NO.3, (4) amino acid sequence shown in SEQ ID NO.4, (5) amino acid sequence shown in SEQ ID NO.5, (6) pass through one or several amino acid substitutions on the basis of the amino acid sequence of (1) or (2) or (3) or (4) or (5), missing or addition, and there is the active amino acid sequence of alkaline pectase PGL.Mutant enzyme of the invention compares the application for being more advantageous to it industrially with protoenzyme, has a good application prospect in weaving degumming field.

Description

A kind of alkaline pectin enzyme mutant that specific enzyme activity improves

Technical field

The invention belongs to the alkaline pectin enzyme mutants that technical field of bioengineering more particularly to a kind of specific enzyme activity improve.

Background technique

Pectin molecule is the polysaccharide chain being polymerized by the galacturonic acid of different esterification degrees with α -1-4 glycosidic bond, removes ester The pectin of change is referred to as pectic acid or polygalacturonase.Pectin is widely present in all higher plant cell interstitials In, pectin is the support substance of cell interior, and cellulose, hemicellulose and lignin be then present in primary cell wall, they with And some extensins and pectin are cross-linked with each other, and form hard eucaryotic cell structure to maintain the form of cell.Various ramies, it is sub- Fiber crops, decortication, depolymerization and the degumming process of the plant fibers such as jute require to remove the pectic substance on fiber, therefore utilize fruit Glue enzyme degumming has important industrial application value.

Multiple-microorganism can generate pectase, such as bacterium, yeast, fungi, actinomyces.Pectase is to decompose pectin class The general name of a variety of enzymes of substance, pectin can be the substances such as galacturonic acid by it.Pectase can be divided by the pH value of its effect Are as follows: acid pectase, neutral pectase and alkaline pectase.Pectase can be divided by its mode of action: pectinesterase and pectin Depolymerase.The effect of pectinesterase is that methyl is removed from pectin molecule, and the effect of Pectin depolymerase is depolymerized pectin glycosidic bond. Pectin depolymerase is divided into two classifications: pectin hydrolase and pectin lyase again.The effect of pectin hydrolase is hydrolysis of pectin sugar Glycosidic bond, the effect of pectin lyase are to be broken glycosidic bond by β elimination reaction, which attacks the glycosidic bond of substrate in neighbouring carboxylic Glycosidic bond in β elimination long-chain molecule occurs on one side for base or the carboxyl of esterification, and circumscribed depolymerase refers to from one end of chain one by one Cut off glycosidic bond.And various Pectin depolymerases have difference to the selection of substrate again, and some pectin high to esterification degree, which acts on, to be held Easily, some pectin effects low to esterification degree are easy.

The effect optimal pH of alkaline pectase is weaved, is made in the extraction of botanical medicine class, tea and coffee fermentation in 8.0-10.0 The industries such as paper, detergent, plant fiber processing, Industrial Wastewater Treatment containing pectin and biotechnology are applied to as people's research Hot spot.The alkaline pectase that microorganism generates industrially has good application, as in cotton fabric dye transfer scouring process Most important enzyme preparation, the research and development of alkaline pectase and its application in fabric dye transfer kiering make green, mild biology Kiering technology is possibly realized.Under alkaline condition, the fiber as cotton fiber main body is known as the trend of swollen, and pectase utilizes Its specificity effectively decomposes the pectic substance that removal plays cohesive action on cotton fiber surface to the hydrophobicitys commensal such as cotton wax, makes hydrophobic Property substance falls off from cotton fiber surface, thus reach scouring result, and fiber itself is not damaged.Therefore, concise through biology Cotton fiber can be maximally maintained fibre strength, weightless small, fabrics feel soft is thick and solid, and high resilience.And it can save About a large amount of process waters, production time, energy consumption, raw material and the processing cost to waste water, reduction contain the salt in environmental emission water Amount and COD value etc..

The alkaline pectinase gene of early-stage study of the present invention derives from Bacillus subtilis 168, contains 1263bp Open reading frame, 420 amino acid are encoded, by Signal P analysis shows that the amino acid sequence contains 21 amino acid Signal peptide, by gene pel168 expressed in Escherichia coli obtained by alkaline pectase it is purified after carry out zymologic property point Analysis studies have shown that the optimum temperature of the enzyme be 55 DEG C, optimal pH 9.4, specific enzyme activity 353.62U/mg, can effectively into Row China grass degumming, but application cost is excessively high, there are also gaps for industrial application, therefore pass through the method for molecular modification and random mutation It is significant to its popularization in industrial application to obtain the higher alkaline pectase of specific enzyme activity.

Summary of the invention

The object of the present invention is to provide a kind of alkaline pectin enzyme mutant, especially a kind of alkalinity with higher specific enzyme activity Pectin enzyme mutant.

Shown in the amino acid sequence of the mutant for example following (1) or (2) or (3) or (4) or (5) or (6):

(1) amino acid sequence shown in SEQ ID NO.1,

(2) amino acid sequence shown in SEQ ID NO.2,

(3) amino acid sequence shown in SEQ ID NO.3,

(4) amino acid sequence shown in SEQ ID NO.4,

(5) amino acid sequence shown in SEQ ID NO.5,

(6) pass through one or several amino acid on the basis of the amino acid sequence of (1) or (2) or (3) or (4) or (5) Replace, lack or add, and there is the active amino acid sequence of alkaline pectase PGL.

The present invention also provides a kind of genes for encoding above-mentioned mutant, and further, the gene includes the base of password optimization Cause.

The present invention can use following mutant code genes to realize, can also pass through the degenerate code of other forms Gene order is realized, such as gene after codon optimization.

Encode the nucleotide sequence of above-mentioned mutant preferably as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, Nucleotide sequence shown in SEQ ID NO.9 or SEQ ID NO.10.

The nucleotide sequence of the mutant of amino acid sequence shown in SEQ ID NO.1 is encoded preferably such as SEQ ID NO.6 Shown in nucleotide sequence.

The nucleotide sequence of the mutant of amino acid sequence shown in SEQ ID NO.2 is encoded preferably such as SEQ ID NO.7 Shown in nucleotide sequence.

The nucleotide sequence of the mutant of amino acid sequence shown in SEQ ID NO.3 is encoded preferably such as SEQ ID NO.8 Shown in nucleotide sequence.

The nucleotide sequence of the mutant of amino acid sequence shown in SEQ ID NO.4 is encoded preferably such as SEQ ID NO.9 Shown in nucleotide sequence.

The nucleotide sequence of the mutant of amino acid sequence shown in SEQ ID NO.5 is encoded preferably such as SEQ ID NO.10 Shown in nucleotide sequence.

The amino acid sequence such as mutant of SEQ ID NO.1 is that nucleotide sequence is wild as shown in SEQ ID NO.11 47th lysine Lys of alkaline pectase is mutated into glutamic acid Glu.Gained mutant is named as K47E.

The amino acid sequence such as mutant of SEQ ID NO.2 is by nucleotide sequence alkalinity as shown in SEQ ID NO.6 132nd valine Val of pectin enzyme mutant is mutated into phenylalanine Phe.Gained mutant is named as K47E V132F.

The amino acid sequence such as mutant of SEQ ID NO.3 is by nucleotide sequence alkalinity as shown in SEQ ID NO.7 272nd arginine Arg of pectin enzyme mutant is mutated into tryptophan Trp.Gained mutant is named as K47E V132F R272W。

The amino acid sequence such as mutant of SEQ ID NO.4 is by nucleotide sequence alkalinity as shown in SEQ ID NO.7 47th lysine Lys of pectin enzyme mutant is mutated into asparatate Asp.Gained mutant is named as K47D V132F。

The amino acid sequence such as mutant of SEQ ID NO.5 is by nucleotide sequence alkalinity as shown in SEQ ID NO.9 272nd arginine Arg of pectin enzyme mutant is mutated into tryptophan Trp.Gained mutant is named as K47D V132F R272W。

The present invention also provides a kind of carrier, recombinant cell or recombinant bacteriums for carrying said gene.

The present invention also provides a kind of methods for obtaining the alkaline pectin enzyme mutant, are with wild type alkaline pectase Gene is template, and it is prominent to carry out rite-directed mutagenesis acquisition coding using tendency mistake PCR, fixed point saturation mutation and/or Overlap extension PCR The gene loci of encoding mutant body is then carried out recombinant expression and obtains mutant by the gene of variant.

It specifically, is wild type alkaline pectinase gene (the SEQ ID that will derive from Bacillus subtilis 168 NO.11) it is template, random mutation is first carried out using error-prone PCR, is screened to obtain the volume of specific enzyme activity raising with 96 orifice plates The nucleotide sequence of the gene of code alkaline pectin enzyme mutant is as shown in SEQ ID NO.6；It is obtained using the method for Overlap extension PCR Nucleotide sequence such as SEQ ID NO.7, SEQ ID NO.8 and the SEQ ID of the gene of alkaline pectin enzyme mutant must be encoded Shown in NO.10；The nucleotide sequence of the gene of coding alkaline pectin enzyme mutant is obtained such as using the method for fixed point saturation mutagenesis Shown in SEQ ID NO.9.It identifies that all mutational sites have succeeded by sequencing to be mutated according to goal-selling.

Further, for the building of amino acid sequence alkaline pectase single mutant K47E as shown in SEQ ID NO.1, Being template using the gene of wild type alkaline pectase carries out random mutagenesis by tendency mistake PCR, and screening obtains bad by 47 Histidine mutations are the mutant strain of glutamic acid；For amino acid sequence alkaline pectase double-mutant as shown in SEQ ID NO.2 The building of K47E V132F is obtained by Overlap extension PCR and screening by 132 figured silk fabrics ammonia using mutant enzyme K47E gene as template Acid mutation is the mutant strain of phenylalanine；For amino acid sequence alkaline pectase double-mutant as shown in SEQ ID NO.3 The building of K47D V132F screens to obtain using mutant enzyme K47E V132F gene as template by the fixed point saturation mutagenesis in 47 sites It is asparatate by the lysine mutation in 47 sites；For amino acid sequence alkaline pectase as shown in SEQ ID NO.4 The building of Trimutant K47E V132F R272W, will by Overlap extension PCR by template of mutant enzyme K47E V132F gene The arginine in 272 sites sports tryptophan；Amino acid sequence alkaline pectase three as shown in SEQ ID NO.5 is mutated The building of body K47D V132F R272W passes through Overlap extension PCR for 272 sites by template of mutant enzyme K47D V132F gene Arginine sport tryptophan.

The recombinant expression is preferably using pET28a as expression vector, with Escherichia coli Rosetta (DE3) for expressive host structure Build recombinant bacterium.

The primer for error-prone PCR is:

Eppel FP:5 '-GCGTCGACGAGCTGAT-3'；

Eppel RP:5 '-ATAAGAATGCGGCCGCTTAAT-3'；

The primer for rite-directed mutagenesis is:

V132F FP:5 '-CTGCAAACACGACGATCTTCGGTTCAG-3'；

V132F RP:5 '-CTTTAGCGTTAGTCCCTGAACCGAAGATC-3'；

R272W FP:5 '-AAATTACGCTGCATCATAACTGGTATAAA-3'；

R272W RP:5 '-CGCGCTGGACAATATTTTTATACCAGTTA-3'；

The degenerate primer for pinpointing saturation mutation is:

BH1 FP:5 '-GCTTGTCTCGGCATTAGGGVMAGAAACG-3'；

BH1 RP:5 '-GCGTTGTGTTCGTTTCTKBCCCTAATG-3'；

BH2 FP:5 '-AGCTTGTCTCGGCATTAGGGNDTGAAAC-3'；

BH2 RP:5 '-GGCGTTGTGTTCGTTTCAHNCCCTAATG-3'；

BH3 FP:5 '-GCTTGTCTCGGCATTAGGGATGGAAACG-3'；

BH3 RP:5 '-GGCGTTGTGTTCGTTTCCATCCCTAATG-3'；

BH4 FP:5 '-GTCTCGGCATTAGGGTGGGAAACGAAC-3'；

BH4 RP:5 '-TTGGCGTTGTGTTCGTTTCCCACCCTAA-3’。

The present invention provides the method for producing the alkaline pectin enzyme mutant using engineering bacteria fermentation, and being will be containing volume Code mutation alkaline pectase gene recombinant bacterium (such as: the Rosetta of the gene containing encoding mutant alkaline pectase (DE3)) it accesses 100ml after activation culture to contain in the LB fermentation medium of 50 μ g/ml kanamycins, in 37 DEG C, 220rpm is trained It supports, thalli growth to certain phase (OD₆₀₀=0.6-0.8), final concentration of 0.5mM IPTG is added and is induced, while will be warm Degree is adjusted to 18 DEG C, induction fermentation 16h.Thallus ultrasonic wave is collected after centrifugation in gained bacterium solution and breaks bacterium, bacteria break supernatant liquid is alkali Property pectase crude enzyme liquid, wherein containing mutant.

The activation culture is that recombinant bacterium is inoculated in the LB that 20ml contains 50 μ g/ml kanamycins from glycerol tube to cultivate In base, 37 DEG C of cultivation temperature, shaken cultivation 10h on 220rpm shaking table.

The supernatant of above-mentioned acquisition can also be sloughed by Ni-NTA affinity chromatography column purification and GE-desalting column Imidazoles obtains alkaline pectin enzyme mutant after purification.

Alkaline pectinase gene is transformed by the method for random mutation, rite-directed mutagenesis and saturation mutation in the present invention, makes to be compiled The PGL activity of code improves, specific enzyme activity enhancing.Alkaline pectin enzyme mutant specific enzyme activity after mutation is respectively increased as 640.81, 873.30,1388.37,1256.17 and 1544.42U/mg is 1.81,2.47,3.93,3.55 and the 4.37 of wild type respectively Times.The specific enzyme activity of PGL provided by the invention has raising by a relatively large margin, is more suitable the needs of industrialized production, meets society The demand of production.

The present invention also provides a kind of application of mutant in industrial textile degumming.

Specific embodiment

The principles and features of the present invention are described below, and the given examples are served only to explain the present invention, is not intended to limit Determine the scope of the present invention.

The invention discloses the alkaline pectin enzyme mutants that a kind of specific enzyme activity improves, and belong to genetic engineering and enzyme engineering neck Domain.The present invention will carry out tendency mistake from the alkaline pectin enzyme coding gene pel168 of Bacillus subtilis 168 PCR carries out random mutagenesis, and screening obtains single mutant K47E；It is whole later in conjunction with the mutational site V132F for having delivered patent before It closes mutation and obtains double-mutant K47E V132F；Then Trimutant is obtained by the method rite-directed mutagenesis of Overlap extension PCR K47E V132F R272W, while 47 sites progress saturation mutation is obtained into K47D V132F；It is obtained finally by rite-directed mutagenesis Trimutant K47D V132F R272W.Five plant mutant enzyme K47E, K47E V132F, K47D V132F, K47E V132F R272W and K47D V132F R272W shows higher specific enzyme activity, respectively protoenzyme 1.81,2.47,3.93, 3.55 and 4.37 times.Mutant enzyme of the invention compares the application for being more advantageous to it industrially with protoenzyme, in weaving degumming neck Domain has a good application prospect.

It is introduced below by specific embodiment.

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

The acquisition of embodiment 1, alkaline pectin enzyme mutant

One, random mutation obtains K47E

Alkaline pectinase gene pel mutant library is constructed by error-prone PCR, according to alkaline pectinase gene The nucleotide sequence design point mutation primer of pel is to as follows:

Forward primer F1:5 '-GCGTCGACGAGCTGAT-3′；

Reverse primer R1:5 '-ATAAGAATGCGGCCGCTTAAT-3′；

The underscore part of forward primer is the restriction enzyme site of SalI, and the underscore part of reverse primer is NotI digestion position Point.

Using original gene pel as template (sequence information is as shown in SEQ ID NO.11), carried out with the primer pair of design Error-prone PCR amplification.

PCR reaction system:

10 × buffer	10μl
		dATP	2.2μl
dCTP	2μl
		dGTP	3.4μl
dTTP	2.36μl
		BSA	5μl
MgCl₂	6.64μl
		MnCl₂	10μl
Taq archaeal dna polymerase	0.5μl
		Forward primer F1 (concentration is 10 μM)	2.5μl
Reverse primer R1 (concentration is 10 μM)	2.5μl
		Template	1μl
Water	50.4μl

PCR reaction condition: 94 DEG C of initial denaturation 5min, then 91 DEG C of denaturation 1min, 55 DEG C of annealing 30s, 72 DEG C of extension 90s, 25 circulations, last 72 DEG C of extensions 5min.PCR product detects yield and specificity with 1% agarose gel electrophoresis, and uses DNA Purification Kit.

By PCR product segment and pET28a carrier (being purchased from Stratagene) NotI and SalI double digestion, agarose electricity Swimming recycling digestion products, electrotransformation enters Escherichia coli Rosetta (DE3) cell (purchased from Transgen) after enzyme connects, and is coated on and contains There is the LB plate of 50 μ g/ml kanamycins, 37 DEG C are incubated overnight.The bacterial strain obtained on plate is alkaline pectinase gene mutation Single colonie is inoculated in 37 DEG C of culture 12h in 96 orifice plates containing 200 μ l (containing 50 μ g/ml kanamycins) LB, with end by body library Concentration is that 0.5mM IPTG induces 2h, takes supernatant to survey enzyme activity after breaking bacterium with broken bacterium buffer (being purchased from GBCBIO technology), It is the bacterial strain for producing alkaline pectin enzyme mutant K47E that preliminary screening, which goes out the highest bacterial strain of enzyme activity, is named as Rosetta/ pET28a-pelK47E.The plasmid obtained in the mutant strain is pET28a-pelK47E, which carries SEQ ID Sequence shown in NO.1.

Two, rite-directed mutagenesis obtains K47E V132F

With reference to delivered in the past patent (application title: a kind of pH stablize the high recombinant basic pectase of specific enzyme activity and its Construction method, publication number: CN103881996A), the 132nd V is mutated into aromatic amino acid phenylalanine F, Ke Yizeng Strong-hydrophobicity.Point mutation primer is designed to as follows according to the nucleotide sequence of alkaline pectinase gene pel:

Forward primer F2:5 '-CTGCAAACACGACGATCTTCGGTTCAG-3 '

Reverse primer R2:5 '-CTTTAGCGTTAGTCCCTGAACCGAAGATC-3 '

Using plasmid pET28a-pel K47E as template, Overlap extension PCR amplification is carried out with the primer pair of design.

PCR reaction system:

10 × buffer	5μl
		dNTP	5μl
Pyrobest archaeal dna polymerase	0.5μl
		Forward primer (concentration is 10 μM)	1μl
Reverse primer (concentration is 10 μM)	1μl
		Template	0.5μl
Water	37μl

PCR reaction condition: 94 DEG C of initial denaturation 5min, then 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 7min, 16 circulations, last 72 DEG C of extensions 10min.

PCR product detects yield and specificity with 1% agarose gel electrophoresis, and with DNA Purification Kit.It will be pure PCR product after change carries out demethylation with DpnI, is transformed into Escherichia coli XL-Gold clone's competent cell and (is purchased from Stratagene in), two transformants is selected and are sequenced to obtain the recombinant vector pET28a-pel of correct point mutation K47EV132F carries nucleotide sequence shown in SEQ ID NO.7, nucleosides shown in SEQ ID NO.7 on the recombinant vector Mutant shown in sequences code SEQ ID NO.2.

Three, fixed point saturation mutation obtains K47D V132F

Overlap extension PCR is carried out by design degenerate primer to No. 47 site saturation mutations, so that screening is preferably prominent Variant.Degenerate primer is designed to as follows according to the nucleotide sequence of alkaline pectinase gene pel:

BH1FP:5 '-GCTTGTCTCGGCATTAGGGVMAGAAACG-3 '；

BH1RP:5 '-GCGTTGTGTTCGTTTCTKBCCCTAATG-3 '；

BH2FP:5 '-AGCTTGTCTCGGCATTAGGGNDTGAAAC-3 '；

BH2RP:5 '-GGCGTTGTGTTCGTTTCAHNCCCTAATG-3 '；

BH3FP:5 '-GCTTGTCTCGGCATTAGGGATGGAAACG-3 '；

BH3RP:5 '-GGCGTTGTGTTCGTTTCCATCCCTAATG-3 '；

BH4FP:5 '-GTCTCGGCATTAGGGTGGGAAACGAAC-3 '；

BH4RP:5 '-TTGGCGTTGTGTTCGTTTCCCACCCTAA-3 '；

Using plasmid pET28a-pel K47E V132F as template, Overlap extension PCR amplification is carried out with the primer pair of design, PCR reaction system and condition are as described in step 2.PCR product detects yield and specificity with 1% agarose gel electrophoresis, and With DNA Purification Kit.PCR product after purification is transformed into Escherichia coli Rosetta (DE3) competent cell, is obtained To transformant picking and put functionality screening is carried out on pectin substrate plate, using mutant K47E V132F as compare, Screening hydrolysis circle carries out shake flask fermentation than compareing bigger transformant.Finally it is by the transformant that sequence verification is screened Rosetta/pET28a-pelK47D V132F, the plasmid which contains are pET28a-pel K47D V132F, the plasmid Containing nucleotide sequence shown in SEQ ID NO.9, nucleotide sequence coded SEQ ID NO.4 institute shown in SEQ ID NO.9 The mutant shown.

Four, rite-directed mutagenesis obtains K47E V132F R272W, K47D V132F R272W

The arginine R in 272 sites is sported into tryptophan W, is designed according to the nucleotide sequence of alkaline pectinase gene pel Point mutation primer is to as follows:

R272W FP:5 '-AAATTACGCTGCATCATAACTGGTATAAA-3 '；

R272W RP:5 '-CGCGCTGGACAATATTTTTATACCAGTTA-3 '；

Respectively using plasmid pET28a-pel K47E V132F and plasmid pET28a-pel K47D V132F as template, with setting The primer pair of meter carries out Overlap extension PCR amplification, and PCR reaction system and condition are as described in step 2.1% fine jade of PCR product Sepharose electrophoresis detection yield and specificity, and with DNA Purification Kit.By PCR product after purification with DpnI into Row demethylation is transformed into Escherichia coli XL-Gold clone's competent cell, selects two transformants and be sequenced to obtain just The recombinant vector pET28a-pel K47E V132F R272W and pET28a-pel K47D V132F R272W of true point mutation.

Recombinant vector pET28a-pel K47E V132F R272W contains nucleotide sequence shown in SEQ ID NO.8, Mutant shown in nucleotide sequence coded SEQ ID NO.3 shown in SEQ ID NO.8.

Recombinant vector pET28a-pel K47D V132F R272W contains nucleotide sequence shown in SEQ ID NO.10, Mutant shown in nucleotide sequence coded SEQ ID NO.5 shown in SEQ ID NO.10.

The expression and purification of embodiment 2, alkaline pectase

By plasmid pET28a-pel, pET28a-pelK47E V132F, pET28a-pelK47E V132F R272W and PET28a-pelK47D V132F R272W is transferred in Rosetta respectively, acquisition recombinant bacterium Rosetta/pET28a-pel, Rosetta/pET28a-pelK47E V132F, Rosetta/pET28a-pelK47E V132F R272W and Rosetta/ pET28a-pelK47D V132F R272W。

By six kinds of recombinant bacteriums Rosetta/pET28a-pel, Rosetta/pET28a-pelK47E, Rosetta/pET28a- pelK47E V132F、Rosetta/pET28a-pelK47D V132F、Rosetta/pET28a-pelK47E V132F R272W It is incubated at the LB culture medium containing 50 μ g/ml kanamycins respectively with Rosetta/pET28a-pelK47D V132F R272W In, 37 DEG C of culture 3h；OD₆₀₀When=0.6-0.8, IPTG is added to its final concentration 0.5mM in LB culture medium, goes to 18 DEG C Continue to cultivate 16h.

Thalline were collected by centrifugation by 5000rpm, 10min, be suspended in solution A (20mM Tris-HCl, pH9.4,0.5M NaCl, 10mM imidazoles) in, ultrasonication (60w, the 10min in ice bath；Ultrasonic 2s stops 4s), 12000rpm is centrifuged 10min and removes later Cell fragment is removed, supernatant is taken；Supernatant is crossed into Ni-IDA HisBind Superflow purification column, is rushed with 5ml solution A It washes, then is rinsed with 10ml solution B (20mM Tris-HCl, pH9.4,0.5M NaCl, 60m M imidazoles), finally use 5ml solution C (20mM Tris-HCl, pH9.4,0.5M NaCl, 500mM imidazoles) elution, collects eluent.Then by eluent GE- Desalting column sloughs imidazoles, obtains the solution of six kinds of alkaline pectases of purifying.

PEL168, PEL168K47E, PEL168K47E V132F, PEL168K47D of SDS-PAGE electrophoresis showed purifying V132F, PEL168K47E V132F R272W and PEL168K47D V132F R272W albumen molecular weight be each about 46kDa.

The specific enzyme activity of embodiment 3, six kind of alkaline pectase is compared

One, the measuring method of pectase enzyme activity

Respectively by enzyme dilution and PGA solution in 55 DEG C of preheating 5min；20 μ l enzyme dilutions are taken, 2ml is added and contains 0.2% The buffer of PGA starts enzymatic reaction；Reaction condition is 55 DEG C, 15min, then the phosphate buffer with 3ml 0.03mol/L Reaction is terminated, its absorbance value is measured at 235nm.

Blank control: inactive enzyme solution reaction (that is: first take 3ml phosphate buffer and enzyme solution to be measured to mix, it is such as above-mentioned anti- Conditioned response is answered, Substrate Stop reaction is added).

Enzyme activity calculates:

In formula: 4600 (Lmol^-1cm^-1Molar absorption coefficient of the)-unsaturation PGA at 235nm；

T (min)-time of enzymatic reacting (in the range of linearity of enzyme reaction)；

B (cm)-quartz cuvette thickness；

Simplified: enzyme activity (U/ml)=3.6232x extension rate xOD₂₃₅。

Two, determination of protein concentration method

Using the determination of protein concentration kit for being purchased from the green skies, compound of reaction has maximum light to inhale at 465-595nm Receipts value, the depth of compound colors and the height of protein concentration are proportional.Pure BSA Bovine serum albumin is taken, is used 0.05mol/L Gly-NaOH buffer is at 0.5mg/ml protein solution.Draw standard protein solution 0,1,2,4,8,12, 16 and 20 μ l are settled to 20 μ l with Gly-NaOH buffer, the 200 μ l of G250 dyeing liquor in kit are added, mix, react 3- 5 minutes, OD value is measured at 595nm, draws the standard curve of protein concentration and OD595.Sample to be tested albumen and Gly-NaOH Buffer is mixed in a certain proportion, and total volume is 20 μ l, same that 200 μ l of G250 dyeing liquor is added, and is mixed, is reacted 3-5 minutes, OD value is measured at 595nm, and sample to be tested protein concentration is calculated according to standard curve.

Three, specific enzyme activity is compared

Specific enzyme activity is obtained divided by protein concentration using enzyme activity is measured, experimental result is listed in table 1, by wild enzyme and mutant enzyme phase Than, it can be found that the specific enzyme activity of five plant mutant enzymes is compared with wild enzyme and is greatly improved, the alkaline pectase after mutation Mutant specific enzyme activity is respectively increased to 640.81,873.30,1388.37,1256.17 and 1544.42U/mg, is wild type respectively 1.81,2.47,3.93,3.55 and 4.37 times.

The wild enzyme of table 1 is compared with the specific enzyme activity of five plants of alkaline pectin enzyme mutants

The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.

Claims

1. the alkaline pectin enzyme mutant that a kind of specific enzyme activity improves, which is characterized in that the amino acid sequence of the mutant such as with Under shown in (1) or (2) or (3) or (4) or (5):

(1) amino acid sequence shown in SEQ ID NO.1,

(2) amino acid sequence shown in SEQ ID NO.2,

(3) amino acid sequence shown in SEQ ID NO.3,

(4) amino acid sequence shown in SEQ ID NO.4,

(5) amino acid sequence shown in SEQ ID NO.5.

2. encoding the gene of mutant described in claim 1.

3. gene according to claim 2, which is characterized in that the gene including password optimization.

4. carrying carrier, recombinant cell or the recombinant bacterium of gene described in Claims 2 or 3.

5. a kind of method for obtaining alkaline pectin enzyme mutant described in claim 1, which is characterized in that be with wild type alkalinity fruit The gene of glue enzyme is template, carries out rite-directed mutagenesis using fixed point saturation mutation and/or Overlap extension PCR and obtains encoding mutant body The gene loci of encoding mutant body is then carried out recombinant expression and obtains mutant by gene.

6. acquisition alkaline pectin enzyme mutant method according to claim 5, which is characterized in that such as amino acid sequence The building of alkaline pectase single mutant K47E shown in SEQ ID NO.1, using the gene of wild type alkaline pectase as template By site-directed mutagenesis technique by 47 lysine mutations be glutamic acid mutant strain；For amino acid sequence such as SEQ ID The building of alkaline pectase double-mutant K47E V132F shown in NO.2 is prolonged using mutant enzyme K47E gene as template by overlapping It stretches PCR and obtains the mutant strain by 132 valine mutations for phenylalanine；For amino acid sequence such as SEQ ID NO.3 institute The building of the alkaline pectase Trimutant K47E V132F R272W shown passes through using mutant enzyme K47E V132F gene as template The arginine in 272 sites is sported tryptophan by Overlap extension PCR；For amino acid sequence alkali as shown in SEQ ID NO.4 Property pectase double-mutant K47D V132F building, it is full by 47 sites fixed point by template of mutant enzyme K47E V132F gene It is asparatate with being mutated the glutamic acid mutation in 47 sites；For amino acid sequence alkalinity as shown in SEQ ID NO.5 The building of pectase Trimutant K47D V132F R272W passes through overlap-extension PCR using mutant enzyme K47D V132F gene as template The arginine in 272 sites is sported tryptophan by PCR.

7. according to the acquisition alkaline pectin enzyme mutant method of claim 5 or 6, which is characterized in that with pET28a be expression Carrier is simultaneously that expressive host constructs the recombinant bacterium containing mutant described in claim 1 with Escherichia coli Rosetta (DE3).

8. according to claim 7 obtain alkaline pectin enzyme mutant method, which is characterized in that with LB liquid medium be hair Ferment culture medium, recombinant bacterium is in 37 DEG C of cultures to OD₆₀₀=0.6-0.8 is added after IPTG in 18 DEG C of progress inducing expression 16h, by institute It obtains bacterium solution and the broken bacterium of thallus ultrasonic wave is collected after centrifugation, bacteria break supernatant liquid is alkaline pectase crude enzyme liquid, and the alkaline pectase is thick Contain alkaline pectin enzyme mutant in enzyme solution.

9. obtaining alkaline pectin enzyme mutant method according to claim 8, which is characterized in that supernatant is affine by Ni-NTA Column chromatography and GE-desalting column slough imidazoles, obtain alkaline pectin enzyme mutant after purification.

10. application of the mutant described in claim 1 in industrial textile degumming.