CN106754848B - Alkaline pectinase mutant with improved thermal stability - Google Patents
Alkaline pectinase mutant with improved thermal stability Download PDFInfo
- Publication number
- CN106754848B CN106754848B CN201611222643.6A CN201611222643A CN106754848B CN 106754848 B CN106754848 B CN 106754848B CN 201611222643 A CN201611222643 A CN 201611222643A CN 106754848 B CN106754848 B CN 106754848B
- Authority
- CN
- China
- Prior art keywords
- ser
- gly
- ala
- alkaline pectinase
- asn
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
- C12Y402/02002—Pectate lyase (4.2.2.2)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an alkaline pectinase mutant with improved thermal stability, belonging to the field of enzyme engineering. The sequence of the alkaline pectinase mutant provided by the invention is shown as SEQ ID No.1, and compared with the existing PGL, the mutant has the advantages that the specific enzyme activity is improved by 3.4 times, the half-life period at 60 ℃ is improved to 19min from the original 5.2min, and the half-life period is improved by 3.6 times. The alkaline pectinase of the invention can catalyze the alpha-1, 4 glycosidic bond cracking of polygalacturonic acid through trans-elimination under alkaline conditions, and can be widely applied to industries of food, textile, paper making and the like.
Description
Technical Field
The invention relates to an alkaline pectinase mutant with improved thermal stability, belonging to the field of enzyme engineering.
Background
Pectinase is a complex enzyme which can break down pectin polymers into unsaturated oligogalacturonans. The enzyme is widely distributed and found in partial parasitic nematodes, plants and microorganisms. Pectinase has been widely used, and has been used industrially for over 40 years. Pectinase is classified into acid pectinase and alkaline pectinase PGL according to the difference of optimal reaction pH. The acidic pectinase is mainly applied to aspects of clarifying fruit juice, fruit wine, extracting fruit and vegetable juice, peeling fruits and the like. PGL applications are mainly applied in the textile, food, paper industry and environmental fields. The enzyme method is applied to the related reactions in the field, and the method has the advantages of environmental protection, material and material saving, mild reaction conditions and the like. However, there are few studies on molecular modification of PGLs and few PGLs that are commercially available.
At present, the strains which are deeply researched on the alkaline pectinase are mainly pichia pastoris, bacillus subtilis and escherichia coli. Compared with different hosts capable of expressing alkaline pectinase comprehensively, the pichia pastoris has long fermentation period, complex process and high low-temperature induction energy consumption although the expressed protein is easy to purify and the yield is high; the bacillus subtilis is not easy to express or the expression enzyme activity is low.
Disclosure of Invention
In order to solve the problems, the invention provides an alkaline pectinase mutant, the amino acid sequence of which is shown as SEQID NO. 1.
The second purpose of the invention is to provide a gene for coding the alkaline pectinase mutant.
In one embodiment of the invention, the gene sequence is shown in SEQ ID NO. 2.
The third purpose of the invention is to provide a method for improving the thermal stability of alkaline pectinase, which is to connect the N end of the alkaline pectinase shown in SEQ ID NO.3 with the amphiphilic short peptide shown in SEQ ID NO.4 through PT-Linker.
In one embodiment of the invention, the PT-Linker sequence is PTPPTTPTPPTTPTPT.
The fourth purpose of the invention is to provide a cell line expressing the alkaline pectinase mutant.
The fifth purpose of the invention is to provide a genetic engineering bacterium for producing alkaline pectinase, which takes escherichia coli as a host and pET-22b (+) as a vector to express an alkaline pectinase mutant shown in SEQ ID NO. 1.
The sixth purpose of the invention is to provide a construction method of the genetic engineering bacteria, which takes pET-22b (+) as a vector, connects the gene shown in SEQ ID NO.2 with the vector and transforms the gene into escherichia coli.
The seventh purpose of the invention is to provide a production method of alkaline pectinase, which is to inoculate the genetically engineered bacteria to a fermentation medium and culture the genetically engineered bacteria at 37 ℃ to OD600Adding IPTG with the final concentration of 0.04-0.06 mmol/L to the mixture of 0.6-1.0, and inducing the mixture for 24-72 hours at the temperature of 30 ℃.
In one embodiment of the invention, the method is to inoculate the genetically engineered bacteria to the fermentation medium and culture the genetically engineered bacteria to OD at 37 ℃600Adding 0.04-0.06 mmol/L of the total amount of the componentsIPTG, induction at 30 ℃ for 48 h.
In one embodiment of the invention, the inoculation is to inoculate 3-5% of the seed solution by volume.
In one embodiment of the invention, the seed solution is obtained by inoculating the recombinant bacterium in an LB culture medium and culturing at 37 ℃ and 200-250 rpm for 10-12 h.
In one embodiment of the invention, the method comprises the steps of firstly culturing seeds and then fermenting; the seed culture is to take a proper amount of the constructed recombinant bacterium E.coli BL21(DE3) from a glycerol tube to inoculate the recombinant bacterium in a medium containing 100 mu g/mL-1Ampicillin and 2% glucose were added to LB medium in a volume of 20mL/250 mL. 37 ℃ and 200r min-1Shaking and culturing on a shaking table for 10 h; the shake flask fermentation is to inoculate seed liquid cultured for 10h with the inoculation amount of 3% (V/V) to the culture medium containing 100 mug. multidot.mL-1Ampicillin in TB Medium in a volume of 20mL/250mL at 37 ℃ at 200 r.min-1Culturing until the bacterial concentration OD600Induction was performed with the addition of 0.04mM IPTG at a final concentration of 0.6 and at 30 ℃ for 48 h.
The invention also claims the application of the mutant and the genetically engineered bacteria in the fields of food, textile or paper making.
Has the advantages that: compared with the existing PGL, the specific enzyme activity of the mutant PGL-3S1 constructed by the invention is improved by 3.4 times, and the half-life period at 60 ℃ is improved from 5.2min to 19min, which is improved by 3.6 times. The alkaline pectinase of the invention can catalyze the alpha-1, 4 glycosidic bond cracking of polygalacturonic acid through trans-elimination under alkaline conditions, and can be widely applied to industries of food, textile, paper making and the like.
The specific implementation mode is as follows:
culture medium:
seed culture medium: 10g/L of tryptone, 5g/L, NaCl 10g/L of yeast powder and 2g/L of glucose.
Fermentation medium: peptone 12g/L, yeast powder 24g/L, glycerin 10g/L, KH2PO42.32g/L、K2HPO416.43g/L。
And (3) measuring the enzyme activity of alkaline pectinase:
the measurement is carried out by spectrophotometry. Definition of unit enzyme activity: an amount of enzyme used to cleave polygalacturonic acid per unit time to produce 1. mu. mol of unsaturated polygalacturonic acid. The enzyme activity determination conditions are as follows: and (3) enzyme activity detection: and (4) centrifuging the fermentation liquor at 8000rpm for 10min, so that the extracellular PGL is contained in the fermentation supernatant, and taking a certain amount for detection. PGL reaction system: Glycine-NaOH buffer (0.2 mol. L.) containing 0.2% polygalacturonic acid (substrate)-1,0.44mmol·L-1CaCl2, pH9.4)2mL, 20 μ L of the sample to be tested, and a blank with inactive enzyme solution. The PGL reaction conditions were: the reaction system was placed in a water bath at 45 ℃ for 15min, and 3mL of a phosphoric acid solution (0.03 mol. L) was added-1) The reaction was stopped and the absorbance was measured at 235 nm.
And (3) measuring the thermal stability:
and (3) subpackaging the diluted enzyme solution, placing the enzyme solution in a metal bath at 60 ℃, sampling every 3min, determining the residual enzyme activity, and calculating the half-life period.
Example 1: obtaining of mutant strains
An alkaline pectinase gene with a sequence shown as SEQ ID NO.2 is obtained by adopting a PCR amplification or chemical synthesis method, then the gene is connected to pET-22b (+), and then is transformed into Escherichia coli E.coli BL21(DE3), and a correct transformant is selected and named as a recombinant bacterium E.coli BL21(DE3) (pET-22b (+)/PGL-3S 1).
Example 2: verification of mutant strains
Seed culture: the recombinant strain E.coli BL21(DE3) (pET-22b (+)/PGL-3S1) was inoculated in LB medium (100. mu.g.mL) in a suitable amount from a glycerol tube-1Ampicillin, 2% glucose), the liquid loading was 20mL/250 mL. 37 ℃ and 200r min-1Shaking and culturing on a shaking table for 10 h.
And (3) shaking flask fermentation: the seed solution cultured for 10 hours was inoculated into fermentation medium TB (100. mu.g.mL) in an inoculum size of 3% (V/V)-1Ampicillin) in the container, the liquid loading amount was 20mL/250mL, 37 ℃,200 r.min-1Culturing until the bacterial concentration OD600Induction was performed with the addition of 0.04mM IPTG at a final concentration of 0.6 and at 30 ℃ for 48 h.
Example 3: purification of alkaline pectinase
Centrifuging the recombinant bacterium fermentation liquor at 8000r/min for 20min, taking supernatant, adding ammonium sulfate for gradient salting-out, centrifuging at low temperature to collect 30-50% ammonium sulfate precipitate, dissolving the enzyme subjected to salting-out precipitation in glycine-sodium hydroxide buffer solution (pH7.5), and dialyzing with 20mmol/L glycine-sodium hydroxide buffer solution for 24 h. And (3) further separating and purifying the supernatant obtained by centrifugation through cation exchange chromatography, desalting the obtained protein with 95% electrophoretic purity through a desalting column, and performing property determination on the obtained pure enzyme solution.
Example 4: determination of specific activity and half-life period of pure alkaline pectinase
Diluting the purified alkaline pectinase, measuring the enzyme activity, and measuring the protein concentration by using a BSA protein concentration measuring kit. The result shows that the alkaline pectinase mutant PGL-3S1 constructed by the invention has obviously improved thermal stability, and the half-life period at 60 ℃ is improved to 19min from the original 5.2. And the determination result of the enzyme activity shows that the specific enzyme activity of the starting PGL is 264.17 +/-5.48U/mg, while the specific enzyme activity of the mutant PGL-3S1 is as high as 902.25 +/-20.22U/mg, and the enzyme activity is improved by 3.42 times compared with the starting PGL.
In addition, the present invention also compares the thermal stability of the remaining fusion proteins fused with self-assembled amphiphilic short peptides of different lengths, and the results are shown in table 1.
TABLE 1 Effect of different lengths of amphiphilic short peptides on enzyme thermostability
The alkaline pectinase mutant constructed by the sequence shown in SEQ ID NO.4 has obviously improved thermal stability and the half-life period of 19.06 min.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> alkaline pectinase mutant with improved thermostability
<160>6
<170>PatentIn version 3.3
<210>1
<211>474
<212>PRT
<213> Artificial sequence
<400>1
Met His His His His His His Ala Glu Ala Glu Ala Lys Ala Lys Ala
1 5 10 15
Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys Ala
20 25 30
Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys Ala
35 40 45
Glu Ala Glu Ala Lys Ala Lys Pro Thr Pro Pro Thr Thr Pro Thr Pro
50 55 60
Pro Thr Thr Pro Thr Pro Thr Pro Ala Met Asp Ala Asp Leu Gly His
65 70 75 80
Gln Thr Leu Gly Ser Asn Asp Gly Trp Gly Ala Tyr Ser Thr Gly Thr
85 90 95
Thr Gly Gly Ser Lys Ala Ser Ser Leu Asn Val Tyr Thr Val Ser Asn
100 105 110
Arg Asn Gln Leu Val Ser Ala Leu Gly Lys Glu Thr Asn Thr Thr Pro
115 120 125
Lys Ile Ile Tyr Ile Lys Gly Thr Ile Asp Met Asn Val Asp Asp Asn
130 135 140
Leu Lys Pro Leu Gly Leu Asn Asp Tyr Lys Asp Pro Glu Tyr Asp Leu
145 150 155 160
Asp Lys Tyr Leu Lys Ala Tyr Asp Pro Ser Thr Trp Gly Lys Lys Glu
165 170 175
Pro Ser Gly Thr Gln Glu Glu Ala Arg Ala Arg Ser Gln Lys Asn Gln
180 185 190
Lys Ala Arg Val Met Val Asp Ile Pro Ala Asn Thr Thr Ile Val Gly
195 200 205
Ser Gly Thr Asn Ala Lys Val Val Gly Gly Asn Phe Gln Ile Lys Ser
210 215 220
Asp Asn Val Ile Ile Arg Asn Ile Glu Phe Gln Asp Ala Tyr Asp Tyr
225 230 235 240
Phe Pro Gln Trp Asp Pro Thr Asp Gly Ser Ser Gly Asn Trp Asn Ser
245 250 255
Gln Tyr Asp Asn Ile Thr Ile Asn Gly Gly Thr His Ile Trp Ile Asp
260 265 270
His Cys Thr Phe Asn Asp Gly Ser Arg Pro Asp Ser Thr Ser Pro Lys
275 280 285
Tyr Tyr Gly Arg Lys Tyr Gln His His Asp Gly Gln Thr Asp Ala Ser
290 295 300
Asn Gly Ala Asn Tyr Ile Thr Met Ser Tyr Asn Tyr Tyr His Asp His
305 310 315 320
Asp Lys Ser Ser Ile Phe Gly Ser Ser Asp Ser Lys Thr Ser Asp Asp
325 330 335
Gly Lys Leu Lys Ile Thr Leu His His Asn Arg Tyr Lys Asn Ile Val
340 345 350
Gln Arg Ala Pro Arg Val Arg Phe Gly Gln Val His Val Tyr Asn Asn
355 360 365
Tyr Tyr Glu Gly Ser Thr Ser Ser Ser Ser Tyr Pro Phe Ser Tyr Ala
370 375 380
Trp Gly Ile Gly Lys Ser Ser Lys Ile Tyr Ala Gln Asn Asn Val Ile
385 390 395 400
Asp Val Pro Gly Leu Ser Ala Ala Lys Thr Ile Ser Val Phe Ser Gly
405 410 415
Gly Thr Ala Leu Tyr Asp Ser Gly Thr Leu Leu Asn Gly Thr Gln Ile
420 425 430
Asn Ala Ser Ala Ala Asn Gly Leu Ser Ser Ser Val Gly Trp Thr Pro
435 440 445
Ser Leu His Gly Ser Ile Asp Ala Ser Ala Asn Val Lys Ser Asn Val
450 455 460
Ile Asn Gln Ala Gly Ala Gly Lys Leu Asn
465 470
<210>2
<211>1431
<212>DNA
<213> Artificial sequence
<400>2
atgcatcatc atcatcatca tgcagaagca gaagcgaaag ccaaagcaga agcagaagcg 60
aaagccaaag cagaagcaga agcgaaagcc aaagcagaag cagaagcgaa agccaaagca 120
gaagcagaag cgaaagccaa agcagaagca gaagcgaaag ccaaatggat atcgcctact 180
ccgccgacga ccccgacccc gccgaccacc ccgactccga ccccagccat ggatgctgat 240
ttaggccacc agacgttgggatccaatgat ggctggggcg cgtactcgac cggcacgaca 300
ggcgggtcaa aagcatcgtc cttaaatgtg tataccgtca gcaacagaaa ccagcttgtc 360
tcggcattag ggaaggaaac gaacacaacg ccaaaaatca tttatatcaa gggaacgatt 420
gacatgaacg tagatgacaa tctgaagccg cttggtctaa atgactataa agatccggag 480
tatgatttgg acaaatattt gaaagcctat gatcctagca catggggcaa aaaagagccg 540
tcgggaacac aagaagaagc gagagcacgc tctcagaaaa accaaaaagc acgggttatg 600
gtggatatcc ctgcaaacac gacgatcgtc ggttcaggga ctaacgctaa agtcgtggga 660
ggaaacttcc aaatcaagag tgataacgtc attattcgca acattgaatt ccaggatgcc 720
tatgattatt ttccgcaatg ggatccgact gacggaagct caggaaactg gaactcacaa 780
tacgacaaca tcacgataaa cggcggcaca cacatctgga ttgatcactg tacatttaac 840
gacggttcgc gtccggacag cacatcaccg aaatattatg gaagaaaata tcagcaccat 900
gacggccaaa cggatgcgtc caacggcgct aactatatca cgatgtccta caactattat 960
cacgatcatg ataaaagctc cattttcgga tcaagtgaca gcaaaacctc cgatgacggc 1020
aaattaaaaa ttacgctcca tcataaccgc tataaaaata ttgtccagcg cgcgccgaga 1080
gtccgcttcg ggcaagtgca cgtatacaac aactattatg aaggaagcac aagctcttca 1140
agttatcctt tcagctatgc atggggaatc ggaaagtcat ctaaaatcta tgcccaaaac 1200
aatgtcattg acgtaccggg actgtcagct gctaaaacga tcagcgtatt cagcggggga 1260
acggctttat atgactccgg cacgttgctg aacggcacac agatcaacgc atcggctgca 1320
aacgggctga gctcttctgt cggctggacg ccgtctctgc atggatcgat tgatgcttct 1380
gctaatgtga aatcaaatgt cataaatcaa gcgggtgcgg gtaaattaaa t 1431
<210>3
<211>401
<212>PRT
<213> Artificial sequence
<400>3
Met Asp Ala Asp Leu Gly His Gln Thr Leu Gly Ser Asn Asp Gly Trp
1 5 10 15
Gly Ala Tyr Ser Thr Gly Thr Thr Gly Gly Ser Lys Ala Ser Ser Leu
20 25 30
Asn Val Tyr Thr Val Ser Asn Arg Asn Gln Leu Val Ser Ala Leu Gly
35 40 45
Lys Glu Thr Asn Thr Thr Pro Lys Ile Ile Tyr Ile Lys Gly Thr Ile
50 55 60
Asp Met Asn Val Asp Asp Asn Leu Lys Pro Leu Gly Leu Asn Asp Tyr
65 70 75 80
Lys Asp Pro Glu Tyr Asp Leu Asp Lys Tyr Leu Lys Ala Tyr Asp Pro
85 90 95
Ser Thr Trp Gly Lys Lys Glu Pro Ser Gly Thr Gln Glu Glu Ala Arg
100 105 110
Ala Arg Ser Gln Lys Asn Gln Lys Ala Arg Val Met Val Asp Ile Pro
115 120 125
Ala Asn Thr Thr Ile Val Gly Ser Gly Thr Asn Ala Lys Val Val Gly
130 135 140
Gly Asn Phe Gln Ile Lys Ser Asp Asn Val Ile Ile Arg Asn Ile Glu
145 150 155 160
Phe Gln Asp Ala Tyr Asp Tyr Phe Pro Gln Trp Asp Pro Thr Asp Gly
165 170 175
Ser Ser Gly Asn Trp Asn Ser Gln Tyr Asp Asn Ile Thr Ile Asn Gly
180 185 190
Gly Thr His Ile Trp Ile Asp His Cys Thr Phe Asn Asp Gly Ser Arg
195 200 205
Pro Asp Ser Thr Ser Pro Lys Tyr Tyr Gly Arg Lys Tyr Gln His His
210 215 220
Asp Gly Gln Thr Asp Ala Ser Asn Gly Ala Asn Tyr Ile Thr Met Ser
225 230 235 240
Tyr Asn Tyr Tyr His Asp His Asp Lys Ser Ser Ile Phe Gly Ser Ser
245 250 255
Asp Ser Lys Thr Ser Asp Asp Gly Lys Leu Lys Ile Thr Leu His His
260 265 270
Asn Arg Tyr Lys Asn Ile Val Gln Arg Ala Pro Arg Val Arg Phe Gly
275 280 285
Gln Val His Val Tyr Asn Asn Tyr Tyr Glu Gly Ser Thr Ser Ser Ser
290 295 300
Ser Tyr Pro Phe Ser Tyr Ala Trp Gly Ile Gly Lys Ser Ser Lys Ile
305 310 315 320
Tyr Ala Gln Asn Asn Val Ile Asp Val Pro Gly Leu Ser Ala Ala Lys
325 330 335
Thr Ile Ser Val Phe Ser Gly Gly Thr Ala Leu Tyr Asp Ser Gly Thr
340 345 350
Leu Leu Asn Gly Thr Gln Ile Asn Ala Ser Ala Ala Asn Gly Leu Ser
355 360 365
Ser Ser Val Gly Trp Thr Pro Ser Leu His Gly Ser Ile Asp Ala Ser
370 375 380
Ala Asn Val Lys Ser Asn Val Ile Asn Gln Ala Gly Ala Gly Lys Leu
385 390 395 400
Asn
<210>4
<211>48
<212>PRT
<213> Artificial sequence
<400>4
Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys
1 5 10 15
Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys
20 25 30
Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys
35 40 45
<210>5
<211>16
<212>PRT
<213> Artificial sequence
<400>5
Pro Thr Pro Pro Thr Thr Pro Thr Pro Pro Thr Thr Pro Thr Pro Thr
1 5 10 15
<210>6
<211>8
<212>PRT
<213> Artificial sequence
<400>6
Ala Glu Ala Glu Ala Lys Ala Lys
1 5
Claims (11)
1. An alkaline pectinase mutant is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
2. A gene encoding the alkaline pectinase mutant according to claim 1.
3. A cell line comprising the alkaline pectinase mutant of claim 1.
4. A vector carrying the gene of claim 2.
5. A method for improving the thermal stability of alkaline pectinase is characterized in that the N end of the alkaline pectinase shown in SEQ ID No.3 is connected with an amphiphilic short peptide shown in SEQ ID No.4 through PT-Linker.
6. A gene engineering bacterium for producing alkaline pectinase is characterized in that an alkaline pectinase mutant shown in SEQ ID NO.1 is expressed by taking escherichia coli as a host and pET-22b (+) as a vector.
7. The method for constructing the genetically engineered bacterium of claim 6, wherein pET-22b (+) is used as a vector, and the gene shown in SEQ ID NO.2 is connected with the vector and transformed into Escherichia coli.
8. A method for producing alkaline pectinase, which comprises inoculating the genetically engineered bacterium of claim 5 to a fermentation medium, and culturing at 30-37 deg.C to OD600Adding IPTG with the final concentration of 0.04-0.06 mmol/L into the mixture of 0.6-1.0, and inducing the mixture for 24-72 hours at the temperature of 28-30 ℃.
9. The method according to claim 8, wherein the inoculation is a 3-5% seed solution by volume inoculation.
10. The method according to claim 8 or 9, characterized in that the fermentation medium is TB medium.
11. The use of the genetically engineered bacterium of claim 6 in the preparation of alkaline pectinase in the fields of textile, food, paper making and environment.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201611222643.6A CN106754848B (en) | 2016-12-27 | 2016-12-27 | Alkaline pectinase mutant with improved thermal stability |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201611222643.6A CN106754848B (en) | 2016-12-27 | 2016-12-27 | Alkaline pectinase mutant with improved thermal stability |
Publications (2)
Publication Number | Publication Date |
---|---|
CN106754848A CN106754848A (en) | 2017-05-31 |
CN106754848B true CN106754848B (en) | 2020-11-03 |
Family
ID=58927093
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201611222643.6A Active CN106754848B (en) | 2016-12-27 | 2016-12-27 | Alkaline pectinase mutant with improved thermal stability |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN106754848B (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109750014B (en) * | 2019-03-27 | 2023-01-13 | 云南师范大学 | Fusion type rhizopus chinensis lipase with improved heat stability and application thereof |
CN113862247B (en) * | 2021-05-24 | 2022-06-21 | 中国农业科学院麻类研究所 | Pectin lyase mutant delta Pel419 and encoding gene, preparation method and application thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105316310A (en) * | 2015-11-25 | 2016-02-10 | 江南大学 | Alkaline pectinase mutant with improved specific enzyme activity and heat stability |
CN105316311A (en) * | 2015-11-27 | 2016-02-10 | 江南大学 | Alkaline pectinase mutant with improved specific enzyme activity |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7399831B2 (en) * | 2004-10-25 | 2008-07-15 | The Brigham And Women's Hospital, Inc. | Targeted delivery of biological factors using self-assembling peptide nanofibers |
-
2016
- 2016-12-27 CN CN201611222643.6A patent/CN106754848B/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105316310A (en) * | 2015-11-25 | 2016-02-10 | 江南大学 | Alkaline pectinase mutant with improved specific enzyme activity and heat stability |
CN105316311A (en) * | 2015-11-27 | 2016-02-10 | 江南大学 | Alkaline pectinase mutant with improved specific enzyme activity |
Non-Patent Citations (1)
Title |
---|
《融合自组装双亲短肽提高碱性果胶酶热稳定性》;刘松等;《食品与发酵工业》;20151231;第41卷(第11期);参见摘要,材料与方法,第1页左栏第1段,第5页左栏第2段 * |
Also Published As
Publication number | Publication date |
---|---|
CN106754848A (en) | 2017-05-31 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN106755209A (en) | A kind of method that enzyme process prepares β nicotinamide mononucleotides | |
CN106986922B (en) | Self-assembled amphiphilic short peptide and application thereof | |
CN110628741B (en) | Maltooligosyl trehalose synthase mutant and application thereof | |
CN106754447B (en) | Recombinant saccharomyces cerevisiae and application thereof in synthesis of glutamine dipeptide | |
CN105316310B (en) | A kind of alkaline pectin enzyme mutant of specific enzyme activity and thermal stability raising | |
CN112725319B (en) | Alginate lyase FaAly7 with polyG substrate specificity and application thereof | |
CN114410605B (en) | Method for promoting extracellular expression of recombinant protein by utilizing cutinase mutant | |
CN112266908B (en) | Recombinant carnosine hydrolase mutant and application thereof | |
CN111893125A (en) | Chitosan enzyme gene, chitosanase, preparation method and application thereof | |
CN106754848B (en) | Alkaline pectinase mutant with improved thermal stability | |
CN112980815B (en) | alpha-L-fucosidase OUCJdch-16 and application thereof | |
CN110055233B (en) | MTSase mutant with improved thermal stability and application thereof | |
CN111748535B (en) | Alanine dehydrogenase mutant and application thereof in fermentation production of L-alanine | |
CN114107353B (en) | Plasmid for efficiently expressing polypeptide toxin and preparation method and application thereof | |
CN113337495B (en) | Method for improving sialic acid yield and application | |
CN111304186B (en) | Construction method of high-catalytic-activity heparin C5 isomerase strain | |
CN104450762B (en) | The biological synthesis method of alpha lipoic acid, engineered strain and preparation method thereof | |
CN106636049B (en) | A kind of alkaline pectin enzyme mutant that secernment property improves | |
CN105969713B (en) | Genetic engineering bacterium for high yield of maltooligosyl trehalose hydrolase and application thereof | |
CN116286762A (en) | Glutamate decarboxylase and application thereof | |
CN108753746B (en) | Maltooligosyl trehalose synthase mutant with improved thermal stability | |
CN111172089A (en) | Method for synthesizing trehalose by using recombinant trehalose synthase | |
CN115058408B (en) | Metagenome-derived high-specific-activity acid-resistant D-psicose 3-epimerase and encoding gene and application thereof | |
CN115011622B (en) | Screening method and application of D-psicose 3-epimerase mutant | |
CN110804602B (en) | L-aspartic acid beta-decarboxylase mutant and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |