CN103484443B - A kind of Pullulan enzymatic mutant and preparation method thereof - Google Patents

A kind of Pullulan enzymatic mutant and preparation method thereof Download PDF

Info

Publication number
CN103484443B
CN103484443B CN201310471021.7A CN201310471021A CN103484443B CN 103484443 B CN103484443 B CN 103484443B CN 201310471021 A CN201310471021 A CN 201310471021A CN 103484443 B CN103484443 B CN 103484443B
Authority
CN
China
Prior art keywords
mutant
pullulanase
enzyme
thermostability
bacterium
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
Application number
CN201310471021.7A
Other languages
Chinese (zh)
Other versions
CN103484443A (en
Inventor
吴敬
陈晟
段绪果
陈坚
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangnan University
Original Assignee
Jiangnan University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Jiangnan University filed Critical Jiangnan University
Priority to CN201310471021.7A priority Critical patent/CN103484443B/en
Publication of CN103484443A publication Critical patent/CN103484443A/en
Application granted granted Critical
Publication of CN103484443B publication Critical patent/CN103484443B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/2457—Pullulanase (3.2.1.41)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01041—Pullulanase (3.2.1.41)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses one and there is high specific activity and high thermal stability Pullulan enzymatic mutant and preparation method thereof, described mutant comprise one, two or three replacements relative to de-genus bacillus Pullulanase reactive amino acid residues, relevant to the thermostability of Pullulanase; Described reactive amino acid residues comprises the aspartic acid of the 503rd, arginine, phenylalanine, tryptophane or tyrosine can be mutated into respectively, said mutation improves Pullulanase Rate activity and thermostability, and one of at least following character changes: 1) optimal reactive temperature improves; 2) improve in pH4.0-5.0 thermostability; 3) improve at pH4.0-5.0 Rate activity.These mutant are more suitable for mashing production process than wild-type Pullulanase.

Description

A kind of Pullulan enzymatic mutant and preparation method thereof
The application is the patent No.: 201210256804.9, and the applying date is: on July 23rd, 2012, patent name is: the divisional application of a kind of Pullulan enzymatic mutant and preparation method thereof.
Technical field
The present invention relates to mutant of a kind of Pullulanase and preparation method thereof, particularly utilize the directed mutagenesis method of protein engineering to the technology of the Rate activity and thermostability that improve Pullulanase, belong to genetically engineered and enzyme engineering field.
Background technology
Pullulanase (Pullulanase, EC3.2.1.41) be a kind of debranching factor, specificity can cut α-1 in pulullan polysaccharide, Zulkovsky starch, amylopectin and some oligosaccharides, 6-glycosidic link, be applied in starch processing industry, greatly can improve utilization ratio and the production efficiency of starch.Pullulanase is mainly used in compositely with saccharifying enzyme preparing glucose.Because saccharifying enzyme is very low to α in branched dextrin-1,6-glycosidic link cutting efficiency, cause that saccharification time is long, glucose yield is low, production cost is high.And Pullulanase can cut off the tapping point in starch, accelerate saccharification react, shorten the saccharification react time, improve glucose yield, reduce the usage quantity of saccharifying enzyme, thus the object reaching increase yield, improve plant factor, reduce production cost.
In order to better act synergistically with saccharifying enzyme, the optimal pH of Pullulanase, temperature and thermostability are the keys that can it be applied to mashing and produce.In order to play the maximum efficiency of saccharifying enzyme in industrial production, reduce microbiological contamination probability in saccharifying, saccharifying generally carries out under high temperature, low ph value, and saccharifying temperature and pH control at 60 DEG C and pH4.5, general about the 48h of saccharification time.Therefore, Pullulanase must possess under 60 DEG C and pH4.5 condition, to retain the needs that higher activity and satisfactory stability could meet saccharifying.Recent domestic scholar using the development research of Pullulanase as very important research theme.Foreign scholar isolates the Pullulanase having and can tolerate close to 100 DEG C of high temperature from extreme thermophilic microorganisms, but these enzymes often only just have higher activity at more than pH6.0.Domesticly mainly have studied that the Screening and Identification, the recombinant bacterium that produce Pullulanase wild mushroom build, fermentation condition optimization and the application in starch material enzymolysis.Soviet Union's Zhe etc. achieves the successful expression of Pullulanase encoding gene in subtilis deriving from Thermotogamaritima, this enzyme optimum temperuture 90 DEG C, optimal pH 6.0, fermenting enzyme work can reach 89.1U/mL(ZheSu, Fu-PingLu, QiangGao, Xiao-GuangLiu, Bin-ZheWang, TaoNiu.Cloningandexpressionofathermostablepullulanasegen efromThermotogamaritimaMSB8inBacillussubtilisWB600.2010 whole nation biomaterial conference collection of thesis, 2010,141-144.).The OptimaxL-1000 of the commercialization starch-debranching enzyme that better coordinates mainly Genencor Company of the U.S. can be carried out at present with saccharifying enzyme.Although China has carried out large quantifier elimination to Pullulanase, also do not realize the suitability for industrialized production of this enzyme, still dependence on import.
Contriver clones and have expressed the Pullulanase (NCBI accession number: AX203845) deriving from a de-genus bacillus (Bacillusderamifican) early stage, and this enzyme optimal pH 4.5, optimum temperuture 55 DEG C is 23h in pH4.5,60 DEG C of condition half-life.By testing with saccharifying enzyme composite usage, find that it can be composite and improve glucose yield with saccharifying enzyme.But this enzyme can't meet the needs of saccharifying completely in optimum temperuture and thermostability, and catalytic efficiency is on the low side.Therefore, improve further the optimum temperuture of this enzyme, thermostability and catalytic efficiency will give it and industrially better apply potential.
Summary of the invention
A technical problem to be solved by this invention is to provide a kind of mutant of Pullulanase, comprise containing draw together one, two or three relative to the replacement of the Pullulanase active amino acid deformity of de-genus bacillus, have more higher specific activity and thermostability compared with parent's Pullulanase.
The parental gene of a described de-genus bacillus Pullulanase and the de-genus bacillus Pullulanase consistent (accession number: AX203845) in ncbi database.
Described mutant is that the aspartic acid (Asp) of the 437th in parent's Pullulanase gene has been mutated into Histidine (His), called after D437H; The aspartic acid (Asp) of the 503rd in Pullulanase gene is mutated into arginine (Arg), phenylalanine (Phe) respectively, tryptophane (Trp) or tyrosine (Tyr) called after D503R respectively, D503F, D503W and D503Y; The L-glutamic acid (Glu) of the 589th in Pullulanase gene has been mutated into tyrosine (Tyr), called after E589Y.
Described mutant is that the aspartic acid (Asp) of the 503rd in single-mutant enzyme D437H gene has been mutated into phenylalanine (Phe), tryptophane (Trp) or tyrosine (Tyr), called after D437H/D503F respectively, D437H/D503W, D437H/D503Y; The L-glutamic acid (Glu) of the 589th in single-mutant enzyme D437H gene has been mutated into tyrosine (Tyr), called after D437H/D589Y.
Described mutant is that the L-glutamic acid (Glu) of the 589th in double-mutant enzyme D437H/D503Y gene has been mutated into tyrosine (Tyr), called after D437H/D503Y/D589Y.
Another technical problem to be solved by this invention is to provide raising and derives from de-genus bacillus Pullulanase (NCBI accession number: the AX203845) specific activity of mutant and the preparation method of thermostability, comprises the steps:
1) on the basis of a de-genus bacillus Pullulanase aminoacid sequence, mutational site is determined; The mutant primer of design rite-directed mutagenesis, the mutant primer of design rite-directed mutagenesis, carries out rite-directed mutagenesis with the carrier carrying Pullulanase gene for template and also builds the plasmid vector containing mutant;
2) mutant plasmid is transformed into host cell;
3) select positive colony and carry out fermentation culture, and purifying Pullulan enzymatic mutant D437H, D503R, D503F, D503W, D503Y, E589Y, D437H/D503F, D437H/D503W, D437H/D503Y, D437H/D589Y and D437H/D503Y/D589Y.
Described plasmid vector is pUC series, pET series, or any in pGEX.
Described host cell comprises: bacterium, yeast and fungal cell, and it is also the scope of protection of present invention.
Described bacterium is gram negative bacterium or gram positive bacterium.
The present invention constructs 11 significant mutant, achieves the raising of Pullulanase Rate activity and thermostability.The Rate activity of single-mutant enzyme all increases, and wherein D437H, E589Y and D503Y Rate activity improves at most, reaches 411.3,425.6 and 452.8U/mg respectively.Double-mutant D437H/D503Y, D437H/D589Y and Trimutant D437H/D503Y/D589Y specific activity are respectively 430.8,415.9 and 421.5U/mg.At pH4.5, in the water-bath of 60 degree, the transformation period of wild-type enzyme is 20h, D437H, E589Y, D503Y transformation period is about 40h, and double-mutant D437H/D503Y, D437H/D589Y and Trimutant D437H/D503Y/D589Y transformation period are all more than 70h.Wherein D437H/D503Y and D437H/D503Y/D589Y thermostability is best.Mutant is more suitable for being applied to industrialized mashing process than wild-type Pullulanase.
Accompanying drawing explanation
The thermostability of Fig. 1 wild-type Pullulanase and mutant enzyme.
Embodiment
Embodiment 1: recombinant bacterium builds
According to the gene order (NCBI accession number: AX203845) of the pulA that NCBI logs in, adopt chemical total synthesis method synthetic starch debranching factor gene order pulA.PET20b (+) for building the plasmid of coli expression carrier, with T7 promotor.PET20b (+) plasmid and the plasmid containing pulA gene are carried out Nco I and Hind III double digestion respectively, after digestion products rubber tapping is reclaimed, connect with T4 ligase enzyme again, connect product conversion E.coliJM109 competent cell, 8h is cultivated through 37 DEG C, choose transformant shaking culture in the LB containing 100mg/L ampicillin liquid, extract plasmid, digestion verification obtains expression plasmid pulA/pET20b (+).
By plasmid pulA/pET20b (+) Transformed E .coliBL21 (DE3) Host Strains, coating is containing on the LB flat board of penbritin (100mg/L), cultivate 8h, called after pulA/pET20b (+)/BL21 (DE3) for 37 DEG C.Choose single bacterium colony in liquid LB, 37 DEG C of overnight incubation, preserve glycerine pipe.
Embodiment 2: the preparation of mutant.
(1) single mutation
Derive from six kinds of single-mutant enzyme D437H of the Pullulanase of B.deramificans, D503R, D503F, D503W, D503Y and E589Y:
On the basis of different sources Pullulanase sequence alignment analysis, a de-genus bacillus Pullulanase protein structure is simulated and rational analysis, again in conjunction with the result of Pullulanase thermokinetic analysis, three amino acid sites (Asp437, Asp503 and Glu589) this enzyme heat stability to potential impact are found in a de-genus bacillus Pullulanase molecule.
The aspartic acid (Asp) of the 437th in Pullulanase gene has been mutated into Histidine (His), called after D437H; The aspartic acid (Asp) of the 503rd in Pullulanase gene is mutated into arginine (Arg), phenylalanine (Phe) respectively, tryptophane (Trp) or tyrosine (Tyr) called after D503R respectively, D503F, D503W and D503Y; The L-glutamic acid (Glu) of the 589th in Pullulanase gene has been mutated into tyrosine (Tyr); Mutant has higher Rate activity and thermostability compared to wild-type Pullulanase.
Six kinds of single-mutant enzyme D437H, D503R, D503F, the preparation method of D503W, D503Y and E589Y, according to B.deramificans Pullulanase gene order, design and synthesize respectively and introduce D437H, D503R, D503F, D503W, the primer of D503Y or E589Y sudden change, rite-directed mutagenesis is carried out to Pullulanase gene, measures DNA encoding sequence, identify the 437th Asp codon respectively and become His codon, 503rd Asp codon becomes Arg respectively, the mutant of Phe, Trp or Tyr codon, the 589th Glu codon becomes Tyr codon.Mutant gene is placed in suitable expression vector and imports subtilis, bacillus licheniformis or intestinal bacteria and express, obtain single mutation Pullulanase.
The rite-directed mutagenesis of single mutation D437H, D503R, D503F, D503W, D503Y and E589Y: utilize fast PCR technology, with expression vector pulA/pET-20b (+) for template,
The rite-directed mutagenesis primer introducing D437H sudden change is:
Forward primer: 5 '-ATCTATGAAATG cATgTCCGTGACTTT-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-AAAGTCACGGAC aTGcATTTCATAGAT-3 ' (underscore is mutating alkali yl)
The rite-directed mutagenesis primer introducing D503R sudden change is:
Forward primer: 5 '-GATCCAACCCAA cGTaATTGGGGTTAT-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-GTAACCCCAGTT aCGtTGGGTCGGATC-3 ' (underscore is mutating alkali yl)
The rite-directed mutagenesis primer introducing D503F sudden change is:
Forward primer: 5 '-GATCCAACCCAA tTTaATTGGGGTTAT-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-GTAACCCCAGTT aAAtTGGGTCGGATC-3 ' (underscore is mutating alkali yl)
The rite-directed mutagenesis primer introducing D503W sudden change is:
Forward primer: 5 '-GATCCAACCCAA tGGaATTGGGGTTAT-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-GTAACCCCAGTT cCAtTGGGTCGGATC-3 ' (underscore is mutating alkali yl)
The rite-directed mutagenesis primer introducing D503Y sudden change is:
Forward primer: 5 '-GATCCAACCCAA tATaATTGGGGTTAT-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-GTAACCCCAGTT aTAtTGGGTCGGATC-3 ' (underscore is mutating alkali yl)
The rite-directed mutagenesis primer introducing D589Y sudden change is:
Forward primer: 5 '-GGTACTGGAAAT gAAaTTGCAGCCGAA-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-TTCGGCTGCAAT tTCaTTTCCAGTACC-3 ' (underscore is mutating alkali yl)
PCR reaction system is: 5 × PSbuffer10 μ L, dNTPsMix (2.5mM) 4 μ L, forward primer (10 μMs) 1 μ L, reverse primer (10 μMs) 1 μ L, template DNA 1 μ L, PrimeStarHS (5U/ μ L) 0.5 μ L, adds distilled water to 50 μ L.Pcr amplification condition is: 94 DEG C of denaturation 4min; Carry out 30 circulations (98 DEG C of 10s, 58 DEG C of 5s, 72 DEG C of 6min) subsequently; 72 DEG C are continued to extend 10min.
PCR primer is through DpnI(Fermentas company) digestion, transformation of E. coli JM109 competent cell, competent cell is after LB solid medium (containing 100 μ g/mL penbritins) overnight incubation, choose mono-clonal to cultivate in LB liquid nutrient medium (containing 100 μ g/mL penbritins), rear extraction plasmid, transformed by mutant plasmid and express host e. coli BL21 (DE3) competent cell, all mutant plasmids all check order correctly.
(2) two sudden change
Four kinds of double-mutant enzyme D437H/D503F of B.deramificans Pullulanase, D437H/D503W, D437H/D503Y or D437H/D589Y: the aspartic acid (Asp) of the 503rd in single-mutant enzyme D437H gene has been mutated into phenylalanine (Phe), tryptophane (Trp) or tyrosine (Tyr), called after D437H/D503F respectively, D437H/D503W, D437H/D503Y; The L-glutamic acid (Glu) of the 589th in single-mutant enzyme D437H gene has been mutated into tyrosine (Tyr), called after D437H/D589Y, and mutant has higher Rate activity and thermostability compared to wild-type Pullulanase.
Four kinds of double-mutant enzyme D437H/D503F, the preparation method of D437H/D503W, D437H/D503Y or D437H/D589Y, with single-mutant enzyme D437H encoding gene for template, design and synthesize respectively and introduce D437H, D503R, D503F, the primer of D503W or D503Y sudden change, carries out rite-directed mutagenesis to single-mutant enzyme D437H encoding gene, measures sequence, the Asp identifying 503 is mutated into Arg, Phe, Trp or, the mutant of Tyr.Mutant gene is placed in suitable expression vector and imports subtilis, bacillus licheniformis or intestinal bacteria and express, obtain two sudden change Pullulan enzymatic mutant.
Two sudden change D437H/D503F, D437H/D503W, D437H/D503Y and D437H/D589Y, rite-directed mutagenesis: utilize fast PCR technology, with expression vector D437H/pET-20b (+) for template,
The rite-directed mutagenesis primer introducing D503F sudden change is:
Forward primer: 5 '-GATCCAACCCAA tTTaATTGGGGTTAT-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-GTAACCCCAGTT aAAtTGGGTCGGATC-3 ' (underscore is mutating alkali yl)
The rite-directed mutagenesis primer introducing D503W sudden change is:
Forward primer: 5 '-GATCCAACCCAA tGGaATTGGGGTTAT-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-GTAACCCCAGTT cCAtTGGGTCGGATC-3 ' (underscore is mutating alkali yl)
The rite-directed mutagenesis primer introducing D503Y sudden change is:
Forward primer: 5 '-GATCCAACCCAA tATaATTGGGGTTAT-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-GTAACCCCAGTT aTAtTGGGTCGGATC-3 ' (underscore is mutating alkali yl)
The rite-directed mutagenesis primer introducing D589Y sudden change is:
Forward primer: 5 '-GGTACTGGAAAT gAAaTTGCAGCCGAA-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-TTCGGCTGCAAT tTCaTTTCCAGTACC-3 ' (underscore is mutating alkali yl)
The sequence measurement of PCR reaction system, reaction conditions and mutator gene is with the method for single mutant.
(3) three sudden changes
The Trimutant enzyme D437H/D503Y/D589Y of B.deramificans Pullulanase: the L-glutamic acid (Glu) of the 589th in double-mutant enzyme D437H/D503Y gene has been mutated into tyrosine (Tyr), called after D437H/D503Y/D589Y, mutant has higher Rate activity and thermostability compared to wild-type Pullulanase.
The preparation method of Trimutant enzyme D437H/D503Y/D589Y, with double-mutant enzyme D437H/D503Y encoding gene for template, with the primer introducing D589Y sudden change, rite-directed mutagenesis is carried out to double-mutant enzyme D437H/D503Y encoding gene, measure sequence, the L-glutamic acid (Glu) identifying 589 has been mutated into the mutant of tyrosine (Tyr).Mutant gene is placed in suitable expression vector and imports subtilis, bacillus licheniformis or intestinal bacteria and express, obtain three sudden change Pullulan enzymatic mutant.
The rite-directed mutagenesis of three sudden change D437H/D589Y/D589Y: utilize fast PCR technology, with expression vector D437H/D503Y/pET-20b (+) for template,
The rite-directed mutagenesis primer introducing D589Y sudden change is:
Forward primer: 5 '-GGTACTGGAAAT gAAaTTGCAGCCGAA-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-TTCGGCTGCAAT tTCaTTTCCAGTACC-3 ' (underscore is mutating alkali yl)
The sequence measurement of PCR reaction system, reaction conditions and mutator gene is with the method for single mutant.
(4) expression and purification of mutant enzyme:
The positive monoclonal that picking proceeds to expressive host e. coli bl21 (DE3) grows 8 ~ 10h in LB liquid nutrient medium (containing 100 μ g/mL penbritins), by 5% inoculum size, seed fermentation liquid is received TB liquid nutrient medium (containing 100 μ g/mL penbritins); Intestinal bacteria are cultured to OD at 30 DEG C of shaking tables 600=0.6, the IPTG adding 0.01mM final concentration induces extracellular expression, and continues cultivation and fermentation after 50 hours at 25 DEG C of shaking tables, by fermented liquid in 4 DEG C, the centrifugal 10min of 10000g except thalline, collect centrifuged supernatant.
Regulate the fermented supernatant fluid pH of Pullulan enzymatic mutant to 4.5, be placed in water-bath 55 DEG C of thermal treatments 1 hour, 4 DEG C, the centrifugal 20min of 10000g, collect supernatant liquor.(the NH of 70% is slowly added in supernatant liquor 4) 2sO 4, 4 DEG C of placements are saltoutd and are spent the night.4 DEG C, the centrifugal 20min of 10000g, collecting precipitation.After 20mmol/L phosphoric acid buffer redissolution precipitation, dialysed overnight in 20mmol/L phosphoric acid buffer, period changes 2-3 dialysis buffer liquid, by making loading sample after 0.22 μm of membrane filtration.Adopt AKTA protein purification instrument to carry out the purifying of recombinant protein, whole purge process is carried out in chromatography cabinet, and control temperature is 4 DEG C.Anion-exchange chromatography purification step: (1) balances: with the 20mmol/L phosphoric acid buffer balance DEAE anion-exchange column of 5 times of volumes; (2) loading: the sample of anticipating is with the flow velocity loading of 1mL/min; (4) wash-out, flow velocity 1.0mL/min, carries out gradient elution, and determined wavelength is 280nm, the elutriant that fraction collection is lived containing Pullulanase enzyme; Vigor component after dialysed overnight, obtains purified mutant body enzyme D437H respectively, D503R, D503F, D503W, D503Y, D437H/D503R, D437H/D503F, D437H/D503W and D437H/D503Y in 50mMpH4.5 acetate buffer solution.
Embodiment 3: the present embodiment illustrates that enzyme is lived and analyzes.
1) enzyme activity determination method
The mensuration of Pullulanase enzymic activity adopts 3,5-dinitrosalicylic acid to send out (DNS method).Pullulanase under certain condition, catalytic hydrolysis pulullan generates reducing sugar, 3, aobvious henna amido complex compound is reduced to after 5-dinitrosalicylic acid and reducing sugar solution are total to heat, the depth of its color is directly proportional to the amount of reducing sugar within the specific limits, therefore colorimetric can be carried out under the wavelength of 540nm, calculate enzyme and live.Enzyme unit definition alive: under these conditions, per minute catalysis produces the enzyme amount of 1 μm of ol glucose as a unit of activity.
Enzyme activity determination step:
A. preheating: the 0.5% Propiram solution (50mMpH4.5 acetic acid bufffer) getting 2ml, in test tube, is placed in 60 DEG C of water-bath preheating about 10min,
B. react: add 0.1ml sample enzyme liquid, vibration mixing, accurate timing 10min, add 3mlDNS mixing, put into frozen water termination reaction, boiling water bath 7min, cooling.
C. measure: in above-mentioned reaction system, add distilled water and be settled to 15ml, mixing.Under 540nm, measure its light absorption value and calculate enzyme activity.
2) compare than enzyme work:
Experimental result lists in table 1, by pure for mutant enzyme compared with the pure enzyme of wild-type, can find: the Rate activity of single-mutant enzyme all increases, and wherein D437H, E589Y and D503Y Rate activity improves at most, reaches 411.3,425.6 and 452.8U/mg respectively.Double-mutant D437H/D503Y, D437H/D589Y and Trimutant D437H/D503Y/D589Y specific activity are respectively 430.8,415.9 and 421.5U/mg.
Table 1
Embodiment 4: the present embodiment illustrates the thermostability of Pullulanase.
It is 0.4mg/mL that the wild-type of purifying and mutant Pullulanase 100mMpH4.5 acetate buffer solution are diluted to protein content, and pH is 4.5, is placed in 60 DEG C of waters bath with thermostatic control, every 5h sampling once, surveys its residual enzyme and lives, compare its stability.
The pure enzyme preparation of mutant and the pure enzyme preparation thermostability of wild-type that said mutation body surface are reached acquisition compare, experimental result as shown in Figure 1, can find, single mutant D437H, E589Y, D503Y, double-mutant D437H/D503Y, D437H/D589Y and Trimutant D437H/D503Y/D589Y thermostability all improve.The transformation period of wild-type enzyme is 20h, D437H, E589Y, D503Y transformation period is about 40h, and double-mutant D437H/D503Y, D437H/D589Y and Trimutant D437H/D503Y/D589Y transformation period are all more than 70h.Wherein D437H/D503Y and D437H/D503Y/D589Y thermostability is best.

Claims (2)

1. a Pullulan enzymatic mutant, it is characterized in that the replacement having relative to a de-genus bacillus Pullulanase reactive amino acid residues, described amino acid is relevant to the thermostability of Pullulanase; It is characterized in that being that the aspartic acid of the 503rd of the Pullulanase of AX203845 is mutated into arginine, phenylalanine, tryptophane or tyrosine respectively by accession number in ncbi database, called after D503R, D503F, D503W and D503Y respectively.
2. the preparation method of mutant according to claim 1, comprises the steps:
1) on the basis of a de-genus bacillus Pullulanase aminoacid sequence, mutational site is determined; The mutant primer of design rite-directed mutagenesis, carries out rite-directed mutagenesis with the carrier carrying Pullulanase gene for template and also builds the plasmid vector containing mutant;
2) mutant plasmid is transformed into host cell;
3) select positive colony and carry out fermentation culture, and purifying Pullulan enzymatic mutant;
Described plasmid vector is pUC series, pET series, or any one in pGEX; Described host cell is bacterium or fungal cell; Described bacterium is gram negative bacterium or gram positive bacterium.
CN201310471021.7A 2012-07-23 2012-07-23 A kind of Pullulan enzymatic mutant and preparation method thereof Active CN103484443B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201310471021.7A CN103484443B (en) 2012-07-23 2012-07-23 A kind of Pullulan enzymatic mutant and preparation method thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201310471021.7A CN103484443B (en) 2012-07-23 2012-07-23 A kind of Pullulan enzymatic mutant and preparation method thereof
CN201210256804.9A CN102876650B (en) 2012-07-23 2012-07-23 Pullulan enzymatic mutant and preparation method thereof

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
CN201210256804.9A Division CN102876650B (en) 2012-07-23 2012-07-23 Pullulan enzymatic mutant and preparation method thereof

Publications (2)

Publication Number Publication Date
CN103484443A CN103484443A (en) 2014-01-01
CN103484443B true CN103484443B (en) 2015-12-09

Family

ID=47478169

Family Applications (2)

Application Number Title Priority Date Filing Date
CN201210256804.9A Active CN102876650B (en) 2012-07-23 2012-07-23 Pullulan enzymatic mutant and preparation method thereof
CN201310471021.7A Active CN103484443B (en) 2012-07-23 2012-07-23 A kind of Pullulan enzymatic mutant and preparation method thereof

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN201210256804.9A Active CN102876650B (en) 2012-07-23 2012-07-23 Pullulan enzymatic mutant and preparation method thereof

Country Status (1)

Country Link
CN (2) CN102876650B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106084016A (en) * 2016-03-07 2016-11-09 南宁邦尔克生物技术有限责任公司 A kind of mutant of signal peptide that can improve restructuring pullulanase expression and application thereof

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103571812B (en) * 2013-11-26 2015-06-24 江南大学 Pullulanase mutant with improved secretion efficiency and heat stability and preparation method of pullulanase mutant
HUE056192T2 (en) 2014-01-22 2022-01-28 Novozymes As Pullulanase variants and polynucleotides encoding same
CN104558144B (en) * 2014-12-30 2017-08-08 深圳市第二人民医院 Mouse Bpifb5 mutant, albumen and its application
WO2016126294A1 (en) * 2015-02-04 2016-08-11 Nanjing Bestzyme Bio-Engineering Co. Ltd. Truncated pullulanases, methods of production, and methods of use thereof
CN105368805A (en) * 2015-12-16 2016-03-02 江南大学 Pullulanase mutants capable of improving heat stability and catalytic efficiency
CN105441415B (en) * 2016-01-22 2018-10-09 南宁邦尔克生物技术有限责任公司 A kind of preparation method and applications of Pullulan enzymatic mutant PulB-d99-D436H
CN106801046B (en) * 2016-09-13 2020-09-15 中国农业科学院生物技术研究所 Acid pullulanase mutant with improved thermal stability as well as encoding gene and application thereof
CN108384770B (en) * 2018-03-01 2019-11-22 江南大学 A method of cyclodextrin is reduced to Pullulanase inhibiting effect
CN108374004A (en) * 2018-05-29 2018-08-07 天津中天精科科技有限公司 A kind of Pullulanase and its application
CN110184259B (en) * 2019-07-09 2020-12-29 江南大学 Pullulanase mutant derived from anaerobic bacillus and application thereof
CN111808836B (en) * 2020-07-23 2021-12-07 中国农业科学院农产品加工研究所 Heat-resistant mutant enzyme of pullulanase I and preparation method and application thereof
CN114807099B (en) * 2022-03-18 2024-05-03 浙江工业大学 Pullulanase mutant, engineering bacterium and application thereof
CN115975989B (en) * 2023-01-17 2023-07-04 江西省科学院微生物研究所(江西省流域生态研究所) Type III pullulan hydrolase mutant for preparing corn resistant starch, and preparation method and application thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2216401A1 (en) * 1998-07-02 2010-08-11 Novozymes A/S Starch debranching enzymes
EP1250423B1 (en) * 2000-01-12 2008-09-03 Novozymes A/S Pullulanase variants and methods for preparing such variants with predetermined properties

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106084016A (en) * 2016-03-07 2016-11-09 南宁邦尔克生物技术有限责任公司 A kind of mutant of signal peptide that can improve restructuring pullulanase expression and application thereof
CN106084016B (en) * 2016-03-07 2020-03-20 南宁邦尔克生物技术有限责任公司 Signal peptide mutant capable of improving expression quantity of recombinant pullulanase and application thereof

Also Published As

Publication number Publication date
CN102876650A (en) 2013-01-16
CN103484443A (en) 2014-01-01
CN102876650B (en) 2014-06-18

Similar Documents

Publication Publication Date Title
CN103484443B (en) A kind of Pullulan enzymatic mutant and preparation method thereof
CN103571812B (en) Pullulanase mutant with improved secretion efficiency and heat stability and preparation method of pullulanase mutant
CN102676480B (en) Method for producing extracellular pullulanase by applying auto-induction culture medium and dual-temperature control strategy
CN108034645A (en) The preparation and its application of a kind of yclodextrin glycosyltransferase mutant
CN110592059B (en) Maltooligosyl trehalose synthase mutant
CN107739734A (en) The glutamine transaminage mutant that a kind of enzyme activity improves
CN108384770B (en) A method of cyclodextrin is reduced to Pullulanase inhibiting effect
CN104531629A (en) Cyclodextrin glucosyltransferase mutant for improving AA-2G conversion rate
CN104789539A (en) Mutant for trehalose synthase as well as preparation method and application of mutant
CN109486791A (en) A kind of preparation and its application of maltogenic amylase enzyme mutant
CN109929822A (en) A kind of Aspergillus oryzae lipase mutant and its application
CN103614303B (en) A kind of Li's Trichoderma strains of expressing saccharifying enzyme
CN110343687B (en) Pullulanase mutant with high secretion capacity and application thereof
CN116286762A (en) Glutamate decarboxylase and application thereof
CN110184258A (en) A kind of Pullulan enzymatic mutant
CN110172468A (en) One kind is from the novel Pullulanase of series bacillus and its gene and application
CN104877983A (en) Trehalose synthase mutant and preparation method and application thereof
CN109576240A (en) A kind of amylosucrase mutant and the preparation method and application thereof
CN103966185A (en) Double-enzyme system for efficiently synthesizing S-adenosylhomocysteine and application method thereof
CN104928269A (en) Maltogenic amylase mutant with low conversion byproducts and mutation method of maltogenic amylase mutant
CN102712932B (en) Modified nucleotide molecules of xylanase and application thereof
CN108165540B (en) Rhizomucor miehei alpha-amylase and coding gene and application thereof
CN110804620A (en) Efficient expression and application of recombinant isoamylase for maltose production
CN109370973A (en) A kind of maltogenic amylase production bacterial strain
CN103255079A (en) Heatproof acidic pullulanase production strain and its screening method

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant