CN103215244B

CN103215244B - Alkaline pectinase PelN, as well as encoded gene and application thereof

Info

Publication number: CN103215244B
Application number: CN201310155628.4A
Authority: CN
Inventors: 李小曼; 王辉林; 宋江宁; 马延和
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2013-04-28
Filing date: 2013-04-28
Publication date: 2014-11-05
Anticipated expiration: 2033-04-28
Also published as: CN103215244A

Abstract

The invention discloses alkaline pectinase PelN, as well as encoded gene and an application of the alkaline pectinase PelN. The PelN is protein formed by an amino acid sequence shown as sequence 1 in a sequence table, and has alkaline pectinase activity, and the preservation number of a recombinant bacterium (escherichia coli) BL21 (DE3) PelN containing the encoded gene of the protein in China General Microbiological Culture Collection Center (CGMCC) is CGMCC No. 7166. Experiments prove that the enzyme activity of the protein PelN for degrading polygalacturonic acid is 4590-4950U/mg; the heat stability is better; the relative enzyme activity is above 90% through heat preservation for 120 minutes at 45 DEG C; the optimum pH value of the alkaline pectinase is 9.8; the optimum temperature is 65 DEG C; and production methods of the protein PelN, the recombinant bacterium and the alkaline pectinase provide efficient paths for industrial large-scale production of the alkaline pectinase.

Description

A kind of alkaline pectase PelN and encoding gene and application

Technical field

The present invention relates to a kind of alkaline pectase PelN and encoding gene and application.

Background technology

Pectin is the important composition composition of plant cell wall, comprises protopectin-, pectic acid and pectinic acid.The existence of pectin substance can increase the flintiness of plant cell wall, is conducive to maintain the given configuration structure of plant, but has increased the difficulty of plant being carried out to the industrial operation of deep processing.

Polygalacturonase refers to class of enzymes that can decompose pectin matter, is prevalent in natural higher plant and microorganism, and be important industrial enzyme preparation.

Alkaline pectase (EC:4.2.2.2) be a class under alkaline condition, with trans-elimination, disconnect pectin substance main chain, produce the enzyme of undersaturated oligogalacturonans, in the crudefiber crop of textile industry, come unstuck, cotton goods refining etc. has a wide range of applications in field.Than traditional high-alkali, pyroprocessing means, use alkaline pectase not only pectin substance to be had to good removal effect, less to native cellulose fibre damage, and can reduce materials consumption and environmental pressure, for a road that green is clean has been opened up in textile industry.

At present, the widespread use of alkaline pectase has been subject to the restriction of the factors such as production cost and enzyme stability.The production level of the wild bacterium producing multi enzyme preparation of alkaline pectase that domestic screening obtains is uneven, and the throughput of wild mushroom is also far apart from the demand of industrial application.Therefore, screening stability high alkaline pectase, and to build the engineering bacteria that production technique is simple, output is high be the emphasis that improves alkaline pectase industrialized level.

Summary of the invention

The object of this invention is to provide a kind of alkaline pectase PelN and encoding gene and application.

Protein PelN provided by the present invention, from series bacillus (Paenibacillus sp.PL0602), has the function of alkaline pectase, is following (a) or protein (b):

(a) protein that the aminoacid sequence shown in sequence 1 forms in sequence table;

(b) by the aminoacid sequence of sequence 1 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have alkaline pectase function by the derivative protein of sequence table sequence 1.

For make (a) or (b) in protein PelN be convenient to purifying, the N-terminal of the protein that can form at the aminoacid sequence shown in sequence in sequence table 1 or C-terminal connect label as shown in table 1.

The sequence of table 1 label

Label	Residue number	Sequence
			Poly-Arg	5-6(is generally 5)	RRRRR

Poly-His	2-10(is generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag?II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned protein PelN can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.The encoding gene of above-mentioned protein PelN can be by lacking the codon of one or several amino-acid residue in the DNA sequence dna shown in the 7th to the 1434th of sequence 2 in sequence table, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence that connects the label shown in table 1 at its 5 ' end and/or 3 ' end obtains.

The gene (PelN gene) of above-mentioned albumen of encoding also belongs to protection scope of the present invention.

Described gene can be following 1) or 2) or 3) DNA molecular:

1) in sequence table the 7th of sequence 2 the to the DNA molecular shown in the 1434th;

2) under stringent condition with 1) the DNA sequence dna hybridization that limits and the coding DNA molecular with the albumen of alkaline pectin enzymic activity;

3) with 1) or 2) DNA sequence dna that limits has the DNA molecular that 90% above homology and coding have the albumen of alkaline pectin enzymic activity.

Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5%SDS, under 65 ° of C, hybridizes, and then uses 2 * SSC, 0.1%SDS and 1 * SSC, and 0.1%SDS respectively washes film once.

The recombinant expression vector that contains above arbitrary described gene, expression cassette, transgenic cell line or recombinant bacterium all belong to protection scope of the present invention.

Described recombinant expression vector is between the NcoI of described gene insertion vector pET22b and XhoI site, to obtain;

Described recombinant bacterium imports colon bacillus (Escherichia coli) by described recombinant expression vector and obtains; Be specially colon bacillus (Escherichia coli) BL21(DE3) PelN, the preserving number at China Committee for Culture Collection of Microorganisms's common micro-organisms center is CGMCC No.7166.

Described protein provided by the present invention can be used for depolymerized pectin or prepares the product of tool alkaline pectin enzymic activity.

In above-mentioned application, described pectin specifically can be polygalacturonic acid.

In above-mentioned application, the pH value of described degraded is 7.1-11.0, or 7.6-10.4, or 8.1-10.4, or 8.6-10.4, or 9.0-10.4, or 9.4-10.4; Specifically can be 7.6,8.1,8.6,9.0,9.4,9.8 or 10.4;

The temperature of described degraded is 35 ℃-75 ℃, or 40 ℃-70 ℃, or 50 ℃-70 ℃, or 55 ℃-70 ℃, or 60 ℃-70 ℃; Specifically can be 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃ or 75 ℃.

The present invention also provides a kind of method of producing alkaline pectase, comprises the steps: described colon bacillus BL21 (DE3) PelN to be seeded to fermention medium, and 35-37 ℃ of (as 35,36 or 37 ℃) shaking culture is to OD _600nm=0.4-0.6(is as 0.4,0.5 or 0.6), then adding IPTG is that 100-200 μ M(is as 100,150 or 200 μ M to final concentration), 28-32 ℃ (as 28,30 or 32 ℃) continue shaking culture 40-45 hour (as 40,43 or 45 hours), obtain alkaline pectase.

In aforesaid method, the solvent of described fermention medium is water, solute and the final concentration in described fermention medium thereof are respectively: Tryptones 10-15g/L(as 10,12 or 15g/L), yeast extract 15-24g/L(as 15,20 or 24g/L), glycerine 8-12g/L(as 8,10 or 12g/L), potassium primary phosphate 10-17mmol/L(as 10,14 or 17mmol/L), dipotassium hydrogen phosphate 65-75mmol/L(as 65,70 or 75mmol/L).

In aforesaid method, described colon bacillus BL21 (DE3) PelN is seeded to described fermention medium in the mode of seed liquor; Inoculum size is that 1%-5%(is as 1%, 3% or 5%);

The preparation method of described seed liquor is as follows: described colon bacillus BL21 (DE3) PelN is seeded to seed culture medium, and 35-37 ℃ of (as 35,36 or 37 ℃) shaking culture is to OD _600nm=6.0-8.0(is as 6.0,7.0 or 8.0), be described seed liquor;

The solvent of described seed culture medium is water, solute and the final concentration in described seed culture medium thereof are respectively: Tryptones 10-15g/L(as 10,12,15g/L), yeast extract 5-8g/L(as 5,7,8g/L), sodium-chlor 4-6g/L(as 4,5,6g/L); The pH value of described seed culture medium is that 7.0-7.2(is as 7.0,7.1,7.2).

Experiment showed, protein PelN provided by the present invention, 65 ℃ degraded polygalacturonic acids enzyme work be 4590-4950U/mg; Better heat stability, lives still more than 90% at the relative enzyme of 45 ℃ of insulation 120min; Optimal pH as alkaline pectase is 9.8, and optimum temperuture is 65 ℃.Protein PelN provided by the present invention, recombinant bacterium and alkaline pectinase production method are produced alkaline pectase for large-scale industrialization effective way are provided.

preservation explanation

Strain name: colon bacillus

Latin name: Escherichia coli

Strain name: BL21(DE3) PelN

Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center

Preservation mechanism is called for short: CGMCC

Address: No. 3, No. 1, Chaoyang District Beijing North Star West Road institute, Institute of Microorganism, Academia Sinica

Preservation date: on January 18th, 2013

The preservation center numbering of registering on the books: CGMCC No.7166

Accompanying drawing explanation

Fig. 1 is the SDS-PAGE electrophorogram of protein of the present invention.Wherein, M road is Marker, and 1 road is the albumen in supernatant C K, and 2 roads are the albumen in supernatant A, and 3 roads are the albumen in the solution A after purifying.

Fig. 2 is that protein of the present invention is as the optimal pH curve of alkaline pectase.

Fig. 3 is that protein of the present invention is as the optimum temperuture curve of alkaline pectase.

Fig. 4 protein of the present invention is the thermally-stabilised curve at 45 ℃ as alkaline pectase.

Embodiment

The experimental technique using in following embodiment if no special instructions, is ordinary method.

In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.

The test kit that in following embodiment, total protein content mensuration is used is Bole's protein determination kit Quick Start Bradford Protein Assay Kit, and products catalogue is numbered 500-0201.

The acquisition of embodiment 1, protein PelN and encoding gene thereof

The genomic dna of series bacillus (Paenibacillus sp.) SJN-PL0602 that the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center of take is CGMCC NO.5696 is template, with primers F and R, carry out pcr amplification, amplified production is carried out to agarose gel electrophoresis, reclaim the fragment of purifying 1.4kb, order-checking shows, this clip size is 1440bp, its sequence is as shown in sequence table sequence 2, wherein, protein shown in the 7th to the 1434th code sequence list sequence 1 of sequence 2, by this protein called after PelN, its encoding gene called after PelN.

The sequence of above-mentioned primers F and R is as follows:

5 '- cCATGGaTGAAAAGAACAGTACGAAGCT-3 ' (underscore base is NcoI restriction endonuclease recognition sequence);

5 '- cTCGAGtTAGTAGGAAGTCTTCAGCCAC-3 ' (underscore base is XhoI restriction endonuclease recognition sequence).

The structure of embodiment 2, recombinant expression vector and recombinant bacterium

1, by the DNA fragmentation of sequence table sequence 2, with NcoI and XhoI double digestion, with the carrier pET22b(through NcoI and XhoI double digestion purchased from Novagen, products catalogue is numbered 69744-3) skeleton fragment connect, obtain recombinant vectors pET22b-PelN, through order-checking, confirm, recombinant vectors pET22b-PelN is for having inserted the DNA fragmentation shown in the 7th to the 1434th of sequence table sequence 2 between the NcoI at carrier pET22b and XhoI site.

2, recombinant vectors pET22b-PelN step 1 being obtained transforms e. coli bl21 (DE3) (purchased from Beijing Quanshijin Biotechnology Co., Ltd, products catalogue is numbered CD601-01), by the recombinant bacterium called after BL21(DE3 that contains recombinant vectors pET22b-PelN obtaining) PelN, this recombinant bacterium is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 18th, 2013 and (is called for short CGMCC, address: No. 3, No. 1, Chaoyang District Beijing North Star West Road institute, Institute of Microorganism, Academia Sinica; Postcode: 100101), its Classification And Nomenclature is colon bacillus (Escherichia coli), and deposit number is CGMCC No.7166.

3, empty carrier pET22b is transformed to e. coli bl21 (DE3), obtain the recombinant bacterium (referred to as contrast bacterium) that contains empty carrier pET22b.

The preparation of embodiment 3, protein PelN

1, preparation seed liquor

The recombinant bacterium BL21(DE3 that embodiment 2 is obtained) PelN is seeded in the 250mL triangular flask that 25mL seed culture medium is housed, 37 ℃, 200r/min, shaking culture 6-12h to OD _600nm=6.0, obtain seed liquor;

The solvent of described seed culture medium is water, and solute and the final concentration in described seed culture medium thereof are respectively: Tryptones 12g/L, yeast extract 7g/L, sodium-chlor 5g/L; The pH value of described seed culture medium is 7.2.

2, fermentation culture

Get the seed liquor that step 1 obtains, the inoculum size with 5% is seeded in the 250mL triangular flask that 20-30mL fermention medium is housed; First 37 ℃ of shaking culture are to OD _600nm=0.6, then add IPTG(inductor, the expression of induction target protein matter PelN) to final concentration be 200 μ M, 30 ℃ are continued shaking culture 40 hours, obtain fermented liquid;

The solvent of described fermention medium is water, and solute and the final concentration in described fermention medium thereof are respectively: Tryptones 12g/L, yeast extract 24g/L, glycerine 10g/L, potassium primary phosphate 17mmol/L, dipotassium hydrogen phosphate 70mmol/L.

3, the acquisition of protein PelN

Centrifugal 10 minutes of the fermented liquid 8000r/min that step 2 is obtained, collects supernatant liquor (called after supernatant A); Separation and purification protein PelN from this supernatant A, the solution A of acquisition protein PelN, concrete grammar is as follows:

Supernatant A is carried out to ion exchange chromatography, and using the volume of chromatography column (be purchased from BIO-RAD company, products catalogue is numbered 720-0003) is 6mL, (wide * long) 12mm * 53mm; The elution buffer (pH is 9.1, and solvent is water) using is as follows:

Solution I: be 20mmol/L PBS;

Solution II: containing 500mmol/L sodium-chlor, 20mmol/L PBS;

Elution process: (1), by 30mL solution I balance for chromatography column, flow velocity is 0.5ml/min, later all same; (2) loading 6mL supernatant A; (3) by solution I and solution II gradient, mix wash-out, the gradient of solution II is from 0-80%, and used time 50min, collects A ₂₈₀absorption value surpasses the peak of 0.1mAU, and every pipe is collected the liquid of 1mL; (4) every pipe liquid detects (method is as embodiment 6) through alkaline pectin enzymic activity, has alkaline pectin enzymic activity and has the electrophoretically pure liquid of single 50kDa, is the solution A of protein PelN.

4, with contrast bacterium, replacing recombinant bacterium BL21(DE3) PelN carries out above-mentioned steps 1 to 3, obtains the supernatant liquor (called after supernatant C K) of contrast fermented liquid and the solution (called after solution C K) obtaining through separation and purification.

5, supernatant A and supernatant C K and solution A are carried out to SDS-PAGE, result as shown in Figure 1.Result shows, recombinant bacterium BL21(DE3) solution A after the supernatant A that obtains of PelN is purified obtains electrophoretically pure single band, the predicted molecular weight that molecular weight is about 50kDa(and protein PelN is close); The supernatant C K that contrast bacterium obtains does not obtain the albumen of target sizes.

The preparation of embodiment 4, protein PelN

1, preparation seed liquor

The recombinant bacterium BL21(DE3 that embodiment 2 is obtained) PelN is seeded in the 250mL triangular flask that 25mL seed culture medium is housed, 35 ℃, 200r/min, shaking culture 6-12h to OD _600nm=8.0, obtain seed liquor;

The solvent of described seed culture medium is water, and solute and the final concentration in described seed culture medium thereof are respectively: Tryptones 10g/L, yeast extract 8g/L, sodium-chlor 4g/L; The pH value of described seed culture medium is 7.0.

2, fermentation culture

Get the seed liquor that step 1 obtains, the inoculum size with 1% is seeded in the 250mL triangular flask that 20-30mL fermention medium is housed; First 35 ℃ of shaking culture are to OD _600nm=0.4, then add IPTG(inductor, the expression of induction target protein matter PelN) to final concentration be 100 μ M, 32 ℃ are continued shaking culture 45 hours, obtain fermented liquid;

The solvent of described fermention medium is water, and solute and the final concentration in described fermention medium thereof are respectively: Tryptones 10g/L, yeast extract 20g/L, glycerine 8g/L, potassium primary phosphate 14mmol/L, dipotassium hydrogen phosphate 65mmol/L.

3, the acquisition of protein PelN

Centrifugal 10 minutes of the fermented liquid 8000r/min that step 2 is obtained, collects supernatant liquor; Separation and purification protein PelN from this supernatant liquor, the solution A of acquisition protein PelN, concrete grammar is identical with the step 3 in embodiment 3.

4, with contrast bacterium, replacing recombinant bacterium BL21(DE3) PelN carries out above-mentioned steps 1 to 3, obtains solution C K.

5, by recombinant bacterium BL21(DE3) PelN and contrast the bacterium supernatant liquor, solution A and the solution C K that obtain carry out SDS-PAGE, and result is identical with the step 5 in embodiment 3.

The preparation of embodiment 5, protein PelN

1, preparation seed liquor

The recombinant bacterium BL21(DE3 that embodiment 2 is obtained) PelN is seeded in the 250mL triangular flask that 25mL seed culture medium is housed, 36 ℃, 200r/min, shaking culture 6-12h to OD _600nm=7.0, obtain seed liquor;

The solvent of described seed culture medium is water, and solute and the final concentration in described seed culture medium thereof are respectively: Tryptones 15g/L, yeast extract 5g/L, sodium-chlor 6g/L; The pH value of described seed culture medium is 7.1.

2, fermentation culture

Get the seed liquor that step 1 obtains, the inoculum size with 3% is seeded in the 250mL triangular flask that 20-30mL fermention medium is housed; First 36 ℃ of shaking culture are to OD _600nm=0.5, then add IPTG(inductor, the expression of induction target protein matter PelN) to final concentration be 150 μ M, 28 ℃ are continued shaking culture 43 hours, obtain fermented liquid;

The solvent of described fermention medium is water, and solute and the final concentration in described fermention medium thereof are respectively: Tryptones 15g/L, yeast extract 15g/L, glycerine 12g/L, potassium primary phosphate 10mmol/L, dipotassium hydrogen phosphate 75mmol/L.

3, the acquisition of protein PelN

Centrifugal 10 minutes of the fermented liquid 8000r/min that step 2 is obtained, collects supernatant liquor; Separation and purification protein PelN from this supernatant liquor, the solution A of acquisition protein PelN, concrete grammar is as follows:

The enzyme activity determination of embodiment 6, alkaline pectase

By embodiment 3-5 recombinant bacterium BL21(DE3) supernatant C K and the solution C K of the fermented liquid that obtains of the supernatant A of the fermented liquid that obtains of PelN and solution A and contrast bacterium use respectively glycine-NaOH damping fluid (0.2mol/L, pH9.8) after dilution, as solution to be measured, carry out enzyme activity determination, result is as shown in table 2.

The enzyme activity determination result of table 2, alkaline pectase

The result of table 2 shows: the alkaline pectase enzyme work of protein PelN is 4590-4950U/mg, and the alkaline pectase enzyme work in the fermented liquid supernatant of embodiment 3-5 fermentative production gained is 4365-4950U/mL.

The concrete grammar of the enzyme activity determination of above-mentioned alkaline pectase is as follows:

Experimental group: add 20 μ L solution to be measured in 2ml solution first, 65 ℃ of reaction 15min, add 3mL0.03mol/L H ₃pO ₄the aqueous solution (stop buffer), the absorbance of mensuration 235nm;

Control group: add successively 3mL0.03mol/L H in 2ml solution first ₃pO ₄the aqueous solution and 20 μ L solution to be measured, the absorbance of mensuration 235nm.

The formula of solution first: containing 0.2g/100ml polygalacturonic acid (purchased from Sigma-Aldrich company, article No. 81325-50G, is called for short PGA) and 0.44mmol/L CaCl ₂glycine-NaOH damping fluid (0.2mol/L, pH9.8).

Alkaline pectase standard enzyme unit alive (1U) is defined as: under these conditions, 1min makes PGA produce the required enzyme amount of unsaturated polyester galacturonic acid of 1 μ mol in the reaction times.

Alkaline pectase enzyme (U/mL) calculation formula alive is as follows:

△ OD ₂₃₅the absorbance of absorbance-control group 235nm of=experimental group 235nm;

4600 be unsaturated polyester galacturonic acid at the molar absorptivity at 235nm place, unit is Lmol ^-1cm ^-1;

T is time of enzymatic reacting (in the linearity range of enzyme reaction), and unit is min, is 15min in this experiment;

B is cuvette thickness, and unit is cm, is 1cm in this experiment;

V ₀for the overall product of system, unit is mL, is 5.02mL in this experiment;

V ₁for enzyme liquid product, unit is mL, is 0.02mL in this experiment.

Concentration (mg/mL) by the alkaline pectase enzyme work (U/mL) of the solution to be measured recording divided by total protein in this solution, obtains the alkaline pectase enzyme work (U/mg) of albumen in this solution.

Embodiment 7, protein PelN are as the Property Identification of alkaline pectase

One, optimal pH

Method: the solution A that embodiment 3 is obtained is solution to be measured, according to the method for embodiment 6, at 45 ℃, carry out the detection of alkaline pectin enzymic activity, difference is the glycine-NaOH damping fluid (0.2mol/L, pH9.8) in reaction system to adopt respectively the damping fluid of following different pH values:

50mM Tris-HCl damping fluid (pH7.1-9.5);

50mM glycine-NaOH damping fluid (pH9.1-10.9);

50mM Na ₂hPO ₄-NaOH damping fluid (pH10.5-12.0);

The highest enzyme work is designated as to 100%, calculate adopts the relative enzyme of the different damping fluids of other various different pH value to live (%), result as shown in Figure 2:

Adopt glycine-NaOH damping fluid, pH is that the relative enzyme work of 9.8 o'clock is 100%, and enzyme is lived value for 2510U/mL;

Adopt 50mM Tris-HCl damping fluid, pH is that the relative enzyme work of 7.1 o'clock is that 0%, pH is that the relative enzyme work of 7.6 o'clock is that 4.76%, pH is that the relative enzyme work of 8.1 o'clock is that 9.26%, pH is that the relative enzyme work of 8.6 o'clock is 26.67%;

Adopt 50mM glycine-NaOH damping fluid, pH is that the relative enzyme work of 9.0 o'clock is that 12.77%, pH is that the relative enzyme work of 9.4 o'clock is that 28.18%, pH is that the relative enzyme work of 10.4 o'clock is 29.3%;

Adopt 50mM Na ₂hPO ₄-NaOH damping fluid, pH is that the relative enzyme work of 11 o'clock is that 0%, pH is that the relative enzyme work of 12 o'clock is 0%.

Result shows, protein PelN is that 9.8, pH is lower than 7.6 or almost lose all enzymes higher than 11.0 and live as the optimal pH of alkaline pectase.

Two, optimum temperuture

Method: the solution A that embodiment 3 is obtained is as solution to be measured, according to the method for embodiment 6, carry out the detection of alkaline pectin enzymic activity, difference is only that in reaction conditions, using different temperature is 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃ or 75 ℃.

The highest enzyme work is designated as to 100%, calculates the relative enzyme (%) alive that adopts other differing temps.Result: as shown in Figure 3.Relative enzyme in the time of 65 ℃ live for the 100%(enzyme value of living be 4593U/mL), the relative enzyme work of 35 ℃ is that the relative enzyme work of 14.75%, 40 ℃ is that the relative enzyme work of 24.29%, 45 ℃ is that the relative enzyme work of 44.44%, 50 ℃ is that the relative enzyme work of 54.73%, 55 ℃ is that the relative enzyme work of 72.41%, 60 ℃ is that the relative enzyme work of 84.61%, 70 ℃ is that the relative enzyme work of 38.92%, 75 ℃ is 17.97%.

Result shows: protein PelN is 65 ℃ as the optimum temperuture of alkaline pectase, at 55-65 ℃, keeps more than 70% enzyme to live.

Three, thermostability

Method: using 5 solution A that obtain in embodiment 2 step 3 after 45 ℃ of insulations different times (0-360min) as solution to be measured, according to the method for embodiment 6, carry out that N-acyl-homoserine lactonase is active to be detected.

The highest enzyme work is designated as to 100%, calculates the relative enzyme (%) alive that adopts other 45 ℃ insulations to process different time.Result as shown in Figure 4, the enzyme of the solution to be measured of 45 ℃ of insulation 0min is lived and is lived and be worth for 2012U/mL for 100%(enzyme), the enzyme work of the solution to be measured of 45 ℃ of insulation 30min is 98.1%, the enzyme work of the solution to be measured of 45 ℃ of insulation 60min is 95.5%, the enzyme work of the solution to be measured of 45 ℃ of insulation 90min is 92.3%, the enzyme work of the solution to be measured of 45 ℃ of insulation 120min is 90.3%, the enzyme work of the solution to be measured of 45 ℃ of insulation 180min is 90.0%, the enzyme work of the solution to be measured of 45 ℃ of insulation 240min is 88.0%, the enzyme work of the solution to be measured of 45 ℃ of insulation 300min is 87.0%, the enzyme work of the solution to be measured of 45 ℃ of insulation 360min is 86.1%.

Result shows: the better heat stability of protein PelN, the relative enzyme work that 10min was lived and be still greater than 90%, was incubated to the relative enzyme of 45 ℃ of insulation 120min is 99%.

Claims

1. a protein, is characterized in that: described protein aminoacid sequence shown in sequence 1 in sequence table forms.

2. the encoding gene of protein described in claim 1, is characterized in that: described encoding gene is the DNA molecular shown in the 7th to the 1434th of sequence 2 in sequence table.

3. the recombinant expression vector, expression cassette, transgenic cell line or the recombinant bacterium that contain gene described in claim 2.

4. recombinant expression vector according to claim 3 or recombinant bacterium, is characterized in that:

Described recombinant bacterium imports colon bacillus (Escherichia coli) by described recombinant expression vector and obtains.

5. according to the recombinant bacterium described in claim 3 or 4, it is characterized in that: described recombinant bacterium is colon bacillus (Escherichia coli) BL21 (DE3) PelN, and the preserving number at China Committee for Culture Collection of Microorganisms's common micro-organisms center is CGMCC No.7166.

Described in claim 1 protein at depolymerized pectin or prepare the application in the product of tool alkaline pectin enzymic activity.

7. application according to claim 6, is characterized in that: the pH value of described degraded is 7.6-10.4.

8. application according to claim 7, is characterized in that: the pH value of described degraded is 8.1-10.4.

9. application according to claim 8, is characterized in that: the pH value of described degraded is 8.6-10.4.

10. application according to claim 9, is characterized in that: the pH value of described degraded is 9.0-10.4.

11. application according to claim 10, is characterized in that: the pH value of described degraded is 9.4-10.4.

12. application according to claim 6, is characterized in that: the pH value of described degraded is 7.6.

13. application according to claim 6, is characterized in that: the pH value of described degraded is 8.1.

14. application according to claim 6, is characterized in that: the pH value of described degraded is 8.6.

15. application according to claim 6, is characterized in that: the pH value of described degraded is 9.0.

16. application according to claim 6, is characterized in that: the pH value of described degraded is 9.4.

17. application according to claim 6, is characterized in that: the pH value of described degraded is 9.8.

18. application according to claim 6, is characterized in that: the pH value of described degraded is 10.4.

19. according to the arbitrary described application of claim 6-18, it is characterized in that: the temperature of described degraded is 35 ℃-75 ℃.

20. application according to claim 19, is characterized in that: the temperature of described degraded is 40 ℃-70 ℃.

21. application according to claim 20, is characterized in that: the temperature of described degraded is 50 ℃-70 ℃.

22. application according to claim 21, is characterized in that: the temperature of described degraded is 55 ℃-70 ℃.

23. application according to claim 22, is characterized in that: the temperature of described degraded is 60 ℃-70 ℃.

24. application according to claim 19, is characterized in that: the temperature of described degraded is 35 ℃.

25. application according to claim 19, is characterized in that: the temperature of described degraded is 40 ℃.

26. application according to claim 19, is characterized in that: the temperature of described degraded is 45 ℃.

27. application according to claim 19, is characterized in that: the temperature of described degraded is 50 ℃.

28. application according to claim 19, is characterized in that: the temperature of described degraded is 55 ℃.

29. application according to claim 19, is characterized in that: the temperature of described degraded is 60 ℃.

30. application according to claim 19, is characterized in that: the temperature of described degraded is 65 ℃.

31. application according to claim 19, is characterized in that: the temperature of described degraded is 70 ℃.

32. application according to claim 19, is characterized in that: the temperature of described degraded is 75 ℃.

33. 1 kinds of methods of producing alkaline pectase, comprise the steps: recombinant bacterium colon bacillus BL21 (DE3) PelN described in claim 5 to be seeded to fermention medium, and 35-37 ℃ of shaking culture is to OD _600nm=0.4-0.6, then adding IPTG is 100-200 μ M to final concentration, 28-32 ℃ is continued shaking culture 40-45 hour, obtains alkaline pectase.

34. methods according to claim 33, it is characterized in that: the solvent of described fermention medium is water, solute and the final concentration in described fermention medium thereof are respectively: Tryptones 10-15g/L, yeast extract 15-24g/L, glycerine 8-12g/L, potassium primary phosphate 10-17mmol/L, dipotassium hydrogen phosphate 65-75mmol/L.

35. according to the method described in claim 33 or 34, it is characterized in that: described colon bacillus BL21 (DE3) PelN is seeded to described fermention medium in the mode of seed liquor; Inoculum size is 1%-5%;

The preparation method of described seed liquor is as follows: described colon bacillus BL21 (DE3) PelN is seeded to seed culture medium, and 35-37 ℃ of shaking culture is to OD _600nm=6.0-8.0, is described seed liquor;

The solvent of described seed culture medium is water, and solute and the final concentration in described seed culture medium thereof are respectively: Tryptones 10-15g/L, yeast extract 5-8g/L, sodium-chlor 4-6g/L; The pH value of described seed culture medium is 7.0-7.2.