CN105734034A - Method for improving catalytic performance of pullulanase with truncated flexible residues - Google Patents
Method for improving catalytic performance of pullulanase with truncated flexible residues Download PDFInfo
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- CN105734034A CN105734034A CN201610259979.3A CN201610259979A CN105734034A CN 105734034 A CN105734034 A CN 105734034A CN 201610259979 A CN201610259979 A CN 201610259979A CN 105734034 A CN105734034 A CN 105734034A
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- 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)
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- 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)
Abstract
The invention discloses a method for improving catalytic performance of pullulanase with truncated flexible residues, and belongs to the technical field of enzyme engineering.The method includes the steps that the first 5, 22, 45, 64, 78 and 106 amino acid at the N terminal and the latter 9 and 36 amino acid at the C terminal of the amino acid sequence of Bacillus naganoensis JNB-1 pullulanase are removed, the Km values of all obtained mutant enzymes are reduced, and it is proved that affinity of the enzymes to a substrate is improved through truncating; in addition, enzyme activity of Pul delta N5 and enzyme activity of Pul delta N106 are improved by 42% and 17% respectively compared with that of wild enzymes, specific activity of Pul delta N5, specific activity of Pul delta N106 and specific activity of Pul delta C9 are improved by 21% and 15% respectively compared with that of wild enzymes, the half-life period of Pul delta N5, the half-life period of Pul delta N78 and the half-life period of Pul delta C9 are prolonged by 10 h, 12 h and 24 h compared with that of wild enzymes, and the kcat/Km values of Pul delta N5, Pul delta N45, Pul delta N78, Pul delta N106 and Pul delta C9 are increased, which explains that the mutant enzymes have higher catalysis efficiency.Pullulanase is more suitable for industrial application.
Description
Technical field
The present invention relates to the use of the method that truncate flexible residue improves pullulanase catalytic performance, belong to technical field of enzyme engineering.
Background technology
Pullulanase is the one solving enzyme, the minimum substrate that can hydrolyze only need two by α-1,6 glycosidic bonds connections containing two by
The glucose segment that α-1,4 glycosidic bond connects.Due to the complexity of starch structure, it is oligosaccharide or little molecule monosaccharide in starch depolymerization
During must be by multiple enzyme synergism.In mashing process, owing to saccharifying enzyme is to α-1, the limited use of 6 glycosidic bonds,
Therefore the maximum conversion of the starch sugar goods of two enzymes method production at present can only achieve 96%.If adding Pullulan in saccharifying
Enzyme and saccharifying enzyme synergism, then can bring up to conversion ratio more than 97%, and improve production efficiency, reduces production cost.
I type pullulanase is one of member of glycoside hydrolase 13 family, and the main catalytic site of this family member is by four knots
Structure territory A, B, C, F are constituted.Domain A is central (beta/alpha) being catalyzed being made up of 8 beta sheets, 8 alpha-helixs8Fold
Barrel structure, this region contains catalytic triads (Asp, Asp, Glu).Less domain B inserts (beta/alpha)8The of Folding bucket
Three beta sheets and the centre of the 3rd alpha-helix, active pocket is sitting at this domain, and (v beta/alpha)8Folding bucket bucket wall is also by this
Domain is constituted.The characteristic structural that Greece's key topological structure is domain C is formed by 8 beta sheets.And F domain by
One little a spiral and 6 beta sheets, fold the β-antiparallel lamella of the β sandwich motif formed.The C end of domain A and
N end respectively connected domain C and domain F.
Nineteen forty-three reported first pullulanase, within 1991, Tomimura is from Nagano bacillus (B.naganoensis, ATCC No.
53909) pullulanase has been isolated in.Up to the present, from bacillus cereus, Aerobacter aerogenes, actinomycetes, streptococcus,
Finding pullulanase in strepto-and some Thermophilic Bacteria, the pullulanase of separate sources, zymologic property is also had nothing in common with each other.Due to saccharifying
The action condition of enzyme has high temperature (55 DEG C 65 DEG C) and the feature of faintly acid (pH 4.5 5.5), thus most of natural
The zymologic property of pullulanase limits its application in the industrial production.
Therefore, improve the zymologic property of pullulanase to widen its application in the industrial production, be the problem needing solution at present badly.
Summary of the invention
In order to overcome the problems referred to above, the invention provides a kind of method utilizing truncate flexible residue to improve pullulanase catalytic performance,
And providing a kind of Pullulan enzymatic mutant, the aminoacid sequence of described mutant is originated relative to Bacillus naganoensis
The aminoacid sequence of pullulanase, there occurs N end or the truncate of C end flexible region.
In one embodiment of the invention, described truncate refer to clip first 5 of N end, 22,45,64,
78 or 106 aminoacid (be respectively designated as Pul Δ N5, Pul Δ N22, Pul Δ N45, Pul Δ N64, Pul Δ N78,
Pul Δ N106), or clip last 9 or last 36 aminoacid (being respectively designated as Pul Δ C9, Pul Δ C36) of C end.
In one embodiment of the invention, the aminoacid sequence of the pullulanase in described Bacillus naganoensis source is such as
Shown in SEQ ID NO:1 (by the order of N end-C end).
In one embodiment of the invention, the nucleotide sequence of the pullulanase in described Bacillus naganoensis source is such as
Shown in SEQ ID NO:2.
Second object of the present invention is to provide the genetic engineering bacterium expressing described mutant, described genetic engineering bacterium be escherichia coli,
Yeast or bacillus subtilis.
In one embodiment of the invention, described genetic engineering bacterium be with escherichia coli as host, pET-28a be carrier structure
Build and obtain.
Third object of the present invention is to provide a kind of raising pullulanase substrate affinity and/or enzyme work and/or specific enzyme activity and/or heat
Stability and/or the method for catalytic efficiency, be that the parent's pullulanase to Bacillus naganoensis source carries out aminoacid truncate；
Preferably, described truncate refers to remove front 5,22,45,64,78 or the 106 of the N end of the aminoacid sequence of parent's pullulanase
The aminoacid of position, or remove last 9 or 36 amino acids of C end.
The aminoacid sequence of described parent's pullulanase is as shown in SEQ ID NO:1.
In one embodiment of the invention, described method be the aminoacid sequence removing parent's pullulanase N end front 5,
22, the aminoacid of 45,64,78 or 106, or remove rear 9 or 36 amino acids of C end；Described method improves
Pullulanase substrate affinity.
In one embodiment of the invention, described method is to improve the enzyme work of pullulanase, Rate activity, catalysis effect simultaneously
Rate and substrate affinity, be the N end of the aminoacid sequence removing parent's pullulanase front 5 aminoacid (Pul Δ N5) or
Front 106 aminoacid (Pul Δ N106) of N end.
In one embodiment of the invention, described method is to improve Rate activity and heat stability and catalytic efficiency simultaneously, is
Remove rear 9 aminoacid (Pul Δ C9) of the C end of the aminoacid sequence of parent's pullulanase.
In one embodiment of the invention, described method is to improve heat stability and catalytic efficiency, is to remove parent Pu Lu
Front 78 aminoacid (Pul Δ N78) of the N end of the aminoacid sequence of blue enzyme.
In one embodiment of the invention, described method is to improve catalytic efficiency, is the amino removing parent's pullulanase
Front 45 aminoacid (Pul Δ N45) of the N end of acid sequence.
The present invention is also claimed and encodes the nucleotide fragments of described mutant, containing the coding carrier of described mutant gene, table
Reach the genetic engineering bacterium weighing described mutant, and described mutant is in the application of food, chemical industry or field of textiles.
Other explanations:
(1) mutant naming method: use " block amino acid whose position add block amino acid whose quantity " to represent mutant.
Such as Pul Δ N5, represent and the front five amino acid of parent's pullulanase N end is removed；
(2) " last 9 of C end ", refer to represent according to the mode of N end-C end at parent amino acid sequence, remove this ammonia
Last 9 aminoacid of base acid sequence.
Beneficial effects of the present invention:
The present invention, by utilizing the truncate of albumen flexible region, carries out truncate transformation to pullulanase, solves the heat of pullulanase
Stability can not meet the problem of saccharification react condition, lays a good foundation for widening the commercial Application of pullulanase.
Additionally, the enzyme work of Pul Δ N5 and Pul Δ N106 is by 540U mL-1It is respectively increased 768U mL-1With 633U mL-1,
42%, 17% is improve than wild enzyme；Pul Δ N5, Pul Δ N106 and Pul Δ C9 Rate activity are by 267U mg-1It is respectively increased
To 323,381 and 306U mg-1, improve 21%, 15% than wild enzyme；By study on the stability is found, mutant enzyme
Pul Δ N5, Pul Δ N78 and Pul Δ C9 half-life at 60 DEG C improve 10h, 12h, 24h than wild type respectively；All
The K of mutant enzymemValue all reduces, and illustrates that truncate improves the affinity of enzyme-to-substrate, and Pul Δ N5, Pul Δ N45, Pul Δ N78,
Pul Δ N106 and the k of Pul Δ C9cat/KmValue improves, and shows that truncate transformation creates actively impact to the catalytic efficiency of pullulanase.
Detailed description of the invention
Pullulanase assay method:
Take 200 μ L 100mM, enzyme liquid that the acetate buffer of pH 4.5 suitably dilutes is dissolved in 100mM, pH 4.5 with isopyknic
20g/L pulullan polysaccharide in acetate buffer is mixed in 60 DEG C of water-baths insulation 20 minutes mutually.Add DNS reagent 600 μ L
Shake up, be placed in boiling water and boil 5 minutes, take out after cooling down rapidly and adding 3mL deionized water, at 540nm wavelength,
Measure the absorbance of reactant liquor.
Enzyme unit definition alive: under conditions of above-mentioned appointment, catalytic decomposition pulullan polysaccharide per minute generates and is equivalent to 1 μM of Portugal
Enzyme needed for the reducing sugar of grape sugar is enzyme unit (U) alive.
Embodiment 1: mutant plasmid builds
Utilize plasmid extraction kit Plasmid Mini Kit (be purchased from OMEGA BIO-TEK company) from Laboratories Accession
(containing aminoacid sequence be shown in SEQ ID NO:1, nucleotides sequence is classified as SEQ to E.coli BL21 (DE3)/pET-28a-PelB-pul
Pullulanase gene shown in ID NO:2) middle extraction plasmid (Nie, Y., Yan, W., Xu, Y., Chen, W.B.&Mu, X.Q.
High-level expression of Bacillus naganoensis pullulanase from recombinant Escherichia coli with
Auto-induction:effect of lac operator.PLoS One 8, e78416 (2013) .) as the template of truncate, the primer sequence
Row are such as table 1.
Utilize PremixHS PCR enzyme (being purchased from TaKaRa company) uses the method for PCR to carry out BnPul cutting
Short transformation.PCR reaction system is: 50 μ L, 25 μ L Premix1,0.2 μM of Primer of HS, 0.2 μM of Primer
2,10ng-100ng Template.
PCR reaction condition: 98 DEG C of 10s, 58 DEG C of 15s, 72 DEG C of 180s, totally 30 circulations.
PCR primer BamHI and XhoI restriction endonuclease (being purchased from TaKaRa company) use T4DNA ligase (TaKaRa) after processing
Connect the expression vector pET-28a-PelB (Laboratories Accession) containing signal peptide.Enzyme disjunctor system is: 1 μ L T4 DNA Ligase, 1
μ L Solution I, 2 μ L pET-28a-PelB, 6 μ L digestion products.16 DEG C overnight connect.Connect product Transformed E scherichia
Coli JM109 competent cell.
Table 1 primer
Embodiment 2: the structure of recombinant bacterium
Competent preparation: prepare according to Competent Cell Preparation kit (being purchased from TaKaRa company) description.With
Single bacterium colony of inoculating loop picking E.coli JM109, the flat lining out of LB in new preparation separates single bacterium colony, overnight trains at 37 DEG C
Support.After bacterium colony grows up to, choose single colony inoculation in 50mL LB fluid medium, at 37 DEG C, cultivate under the conditions of 200r/min to
OD600Reach 0.35 0.5, put at once and ice stops cultivate.Transfer bacterium solution 30mL is centrifuged to the sterilized 50mL of pre-cooling
Guan Zhong, at 4 DEG C, 4000r/min is centrifuged 4min, supernatant is abandoned totally as far as possible.Add the Solution A of 3mL pre-cooling, gently
Mixing, 4 DEG C, 4000r/min be centrifuged 4min, supernatant is abandoned totally as far as possible.Add the Solution B of 3mL pre-cooling,
Mix gently, subpackage 80 μ L in the 1.5mL EP pipe of each pre-cooling, put in 70 DEG C and preserve.
Conversion operation: competent cell is placed in ice thawing.Adding the product of 10 μ L PCR, mixing is placed in ice 30 gently
min.Proceed to 42 DEG C of water-bath thermal shock 90s, then add 890 μ L LB culture medium, in 37 DEG C, 150r/min after placement 2min in ice
Under the conditions of recovery 1h.Bacterium solution after recovery is centrifugal 4min under 4000r/min, abandons 800 μ L of supernatant, the thalline pressure-vaccum weight of precipitation
Coat on the LB plating medium containing kanamycin after outstanding mixing, at 37 DEG C, be inverted overnight incubation.Picking list bacterium colony upgrading grain is surveyed
Sequence.
Embodiment 3: the acquisition of mutant enzyme
Picking sequence verification successful Plastid transformation E.coli BL21, step of converting is ibid.Picking transformant is inoculated in containing final concentration
In the 50mL self-induction fluid medium of 50 μ g/mL kanamycin, under 37 DEG C of 200r/min cultivate 2～3h, proceed to 17 DEG C,
Inducing culture 70h under 200r/min, in 4 DEG C, centrifugal 10min under the conditions of 12000r/min.Collect thalline, precipitation 0.1mol/L
The acetic acid-sodium acetate buffer solution of pH 4.5 is resuspended, in 4 DEG C, centrifugal 20min under the conditions of 12000r/min after ultrasonication, obtains
Supernatant be crude enzyme liquid, after ni-sepharose purification to pure enzyme liquid, for follow-up study.
Self-induction culture medium: alpha-lactose 10g/L, anhydrous glucose 0.5g/L, glycerol 5g/L, KH2PO46.8g/L, MgSO4
2mmol/L, tryptone 10g/L, yeast extract 5g/L, Na2HPO47.1g/L, Na2SO40.71g/L, NH4Cl 2.67
g/L。pH 7.5-8.0。
Embodiment 4: before and after sudden change, enzyme activity and Rate activity compare
The present invention compares enzyme activity and the change of Rate activity before and after sudden change, and the wildest enzyme refers to B.naganoensis JNB-1
The pullulanase BnPul in source (aminoacid sequence is shown in SEQ ID NO:1, nucleotides sequence is classified as SEQ ID NO:2),
GENBANK accession no.AEV53626.1。
With self-induction cultivate method shake flask fermentation produce wild enzyme BnPul, Pul Δ N5, Pul Δ N22, Pul Δ N45, Pul Δ N64,
The crude enzyme liquid of Pul Δ N78, Pul Δ N106, Pul Δ C9, Pul Δ C36.Live dense with albumen by measuring enzyme after ni-sepharose purification respectively
Degree, calculates Rate activity.The enzyme of Pul Δ N5 and Pul Δ N106 is lived and is respectively increased 768U mL-1With 633U mL-1, and wild
The enzyme work of raw enzyme is 540U mL-1.The Rate activity of Pul Δ N5, Pul Δ N106 brings up to 323U mg-1、381U·mg-1,
And the Rate activity of wild enzyme is 267U mg-1。
The enzyme of table 2 wild type and mutant is lived and specific enzyme activity is compared
Embodiment 5: before and after sudden change, the heat stability of enzyme compares
(1) optimum temperature compares
The Acetic acid-sodium acetate buffer taking pH 4.5 is diluted to the pullulanase enzyme liquid of suitable concn, respectively 45 DEG C, 50 DEG C,
55 DEG C, 60 DEG C, 65 DEG C, measure enzyme at 70 DEG C and live.Live as 100% with the highest enzyme, calculate the relative enzyme at each temperature and live,
Thus obtain optimal reactive temperature.Compared with wild enzyme, the optimum temperature of mutant enzyme is held essentially constant.
(2) 60 DEG C of half-life compare
By wild enzyme BnPul, Pul Δ N5, Pul Δ N22, Pul Δ N45, Pul Δ N64, Pul Δ N78, Pul Δ N106, Pul Δ C9,
Pul Δ C36 is placed at 60 DEG C, is incubated in the buffer of pH 4.5, measures residual enzyme respectively and lives, calculates half-life, mutant enzyme
Pul Δ N5, Pul Δ N78, Pul Δ C9 half-life at 60 DEG C improves 10h, 12h, 34h than wild type respectively.
Embodiment 6: before and after sudden change, the optimum pH of enzyme compares
Respectively at 60 DEG C, the enzyme measuring wild enzyme and mutant enzyme under the conditions of pH 3.0,3.5,4.0,4.5,5.0 and 5.5 is lived, and sends out
The optimum pH of pullulanase is not the most changed while improving pullulanase heat stability by the mutant of the existing present invention.Tool
There is high thermal stability and substantially do not change the pullulanase of optimum pH and be more suitable in saccharifying application.
Embodiment 7: before and after sudden change, the change of enzyme kinetics is compared
Preparation variable concentrations pH 4.5 substrate pulullan polysaccharide (0.1,0.25,0.5,1.0,1.5,2.0,3.0,4.0,5.0,
6.0、8.0、10.0mg·mg-1), respectively 60 DEG C, measure initial velocity of reaction under the different concentration of substrate of pH 4.5, utilize GraphPad
Prism software carries out nonlinear fitting and obtains every kinetic parameter.Result is as shown in table 3.
The kinetic parameter of table 3 wild type and mutant compares
Result shows:
(1) K of all mutant enzymesmValue all reduces, and illustrates that the adhesion of enzyme-to-substrate strengthens.
(2) Pul Δ N5, Pul Δ N45, Pul Δ N78, Pul Δ N106 and the k of Pul Δ C9catValue improves, and Pul Δ N5,
N45, Pul Δ N78, Pul Δ N106 and the k of Pul Δ C9cat/KmValue improves, and shows that the catalysis of pullulanase is imitated by truncate transformation
Rate creates actively impact.
Although the present invention is open the most as above with preferred embodiment, but it is not limited to the present invention, any person skilled in the art,
Without departing from the spirit and scope of the present invention, all can do various changes and modification, therefore protection scope of the present invention should be with
What claims were defined is as the criterion.
Claims (10)
1. a Pullulan enzymatic mutant, it is characterised in that the aminoacid sequence of described mutant is relative to Bacillus naganoensis
The aminoacid sequence of the pullulanase in source, there occurs N end or the truncate of C end flexible region.
Mutant the most according to claim 1, it is characterised in that described truncate refer to clip first 5 of N end, 22,
45,64,78 or 106 aminoacid, or clip last 9 or last 36 aminoacid of C end.
Mutant the most according to claim 1, it is characterised in that the pullulanase in described Bacillus naganoensis source
Aminoacid sequence as shown in SEQ ID NO:1.
Mutant the most according to claim 1, it is characterised in that the pullulanase in described Bacillus naganoensis source
Nucleotide sequence as shown in SEQ ID NO:2.
5. encode the nucleotide fragments of the arbitrary described mutant of claim 1-4.
6. contain the carrier of the gene of the coding arbitrary described mutant of claim 1-4.
7. express the genetic engineering bacterium of the arbitrary described mutant of claim 1-4.
Genetic engineering bacterium the most according to claim 7, it is characterised in that described genetic engineering bacterium is escherichia coli, yeast or withered
Grass bacillus cereus.
9. the arbitrary described mutant of claim 1-4 is in the application of food, chemical industry or field of textiles.
10. one kind is improved pullulanase substrate affinity and/or enzyme work and/or specific enzyme activity and/or heat stability and/or the side of catalytic efficiency
Method, it is characterised in that described method is that the pullulanase to Bacillus naganoensis source carries out aminoacid truncate；Preferably
Ground, described truncate refers to remove the ammonia of first 5,22,45,64,78 or 106 of the N end of the aminoacid sequence of pullulanase
Base acid, or remove rear 9 or 36 amino acids of C end.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106801046A (en) * | 2016-09-13 | 2017-06-06 | 中国农业科学院生物技术研究所 | Acidic pullulanase mutant and its encoding gene and application that heat endurance is improved |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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| US20030022348A1 (en) * | 1998-03-04 | 2003-01-30 | Miller Brian S. | Modified forms of pullulanase |
| CN102796751A (en) * | 2012-08-21 | 2012-11-28 | 广西科学院 | Mutant Pul 324 of pullulanibacillus naganoensis pullulanase and use thereof |
| CN105441415A (en) * | 2016-01-22 | 2016-03-30 | 南宁邦尔克生物技术有限责任公司 | Preparation method and application of pullulanase mutant PulB-d99-D436H |
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| US20030022348A1 (en) * | 1998-03-04 | 2003-01-30 | Miller Brian S. | Modified forms of pullulanase |
| CN102796751A (en) * | 2012-08-21 | 2012-11-28 | 广西科学院 | Mutant Pul 324 of pullulanibacillus naganoensis pullulanase and use thereof |
| CN105441415A (en) * | 2016-01-22 | 2016-03-30 | 南宁邦尔克生物技术有限责任公司 | Preparation method and application of pullulanase mutant PulB-d99-D436H |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106801046A (en) * | 2016-09-13 | 2017-06-06 | 中国农业科学院生物技术研究所 | Acidic pullulanase mutant and its encoding gene and application that heat endurance is improved |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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