CN105734034B - The method for improving Pullulanase catalytic performance using flexible residue is truncated - Google Patents
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Abstract
The invention discloses the methods for improving Pullulanase catalytic performance using flexible residue is truncated, and belong to technical field of enzyme engineering.The present invention by the N-terminal of the amino acid sequence of removal Bacillus naganoensis JNB-1 Pullulanase preceding 5,22,45,64,78, rear the 9 of 106 amino acids and C-terminal, 36 amino acids, the K of all mutant enzymes of acquisitionmValue all reduces, and illustrates to truncate the affinity for improving enzyme-to-substrate；Furthermore the enzyme activity of Pul Δ N5 and Pul Δ N106 improves 42%, 17% than wild enzyme respectively, the Rate activity of Pul Δ N5, Pul Δ N106, Pul Δ C9 improves 21%, 15% than wild enzyme respectively, the half-life period of Pul Δ N5, Pul Δ N78, Pul Δ C9 improves 10h, 12h, for 24 hours, the k of Pul Δ N5, N45, Pul Δ N78, Pul Δ N106 and Pul Δ C9 than wild type respectivelycat/KmValue improves, and illustrates that mutant enzyme possesses higher catalytic efficiency.Pullulanase of the invention is more suitable for industrial application.
Description
Technical field
It the present invention relates to the use of the method that flexible residue improves Pullulanase catalytic performance that truncates, belong to enzyme engineering technology neck
Domain.
Background technique
Pullulanase is the one kind for solving branch enzyme, and the minimum substrate that can be hydrolyzed only needs two by α -1, and 6 glucosides key connections contain
There are two the glucose segments by α -1,4 glucosides key connection.Due to the complexity of starch structure, starch depolymerization be oligosaccharide or
It must be acted synergistically by a variety of enzymes during small molecule monosaccharide.In mashing process, since carbohydrase is to α -1,6 glucosides
The effect of key is limited, therefore the maximum conversion of the starch sugar prod of two enzymes method production at present can only achieve 96%.If being saccharified
Addition Pullulanase and carbohydrase act synergistically in the process, then conversion ratio can be increased to 97% or more, and improve production efficiency,
Reduce production cost.
I type Pullulanase is one of the member of 13 family of glycoside hydrolase, the main catalytic site of the family member by
Four structural domains A, B, C, F are constituted.Structural domain A is (beta/alpha) for the center catalysis being made of 8 beta sheets, 8 alpha-helixes8Folding
Folded barrel structure, which contains catalytic triads (Asp, Asp, Glu).Lesser structural domain B is inserted into (beta/alpha)8Folding bucket
The centre of third beta sheet and third alpha-helix, active pocket are sitting at the structural domain, and (v beta/alpha)8Folding bucket bucket wall
It is made of the structural domain.The characteristic structural that Greece's key topological structure is domain C is formed by 8 beta sheets.And F structural domain
By a small a spiral and 6 beta sheets, the antiparallel lamella of β-of the β sandwich motif of formation is folded.The C-terminal of structural domain A and
N-terminal respectively connected domain C and structural domain F.
Nineteen forty-three reports Pullulanase for the first time, Tomimura in 1991 from Nagano bacillus (B.naganoensis,
ATCC No.53909) in isolated Pullulanase.Up to the present, from bacillus, Aerobacter aerogenes, actinomyces,
Find that Pullulanase, the Pullulanase of separate sources, zymologic property are also had nothing in common with each other in streptococcus, strepto- and some Thermophilic Bacterias.
Since the action condition of carbohydrase has the characteristics that high temperature (55 DEG C-65 DEG C) and faintly acid (pH 4.5-5.5), so most of
The zymologic property of natural Pullulanase limits its application in the industrial production.
Therefore, improve the zymologic property of Pullulanase to widen its application in the industrial production, be current urgent need to resolve
The problem of.
Summary of the invention
In order to overcome the above problem, Pullulanase catalytic performance is improved using truncation flexible residue the present invention provides a kind of
Method, and provide a kind of Pullulan enzymatic mutant, the amino acid sequence of the mutant is relative to Bacillus
The truncation of N-terminal or C-terminal flexible region has occurred in the amino acid sequence of the Pullulanase in the source naganoensis.
In one embodiment of the invention, the truncation refers to first 5 for clipping N-terminal, 22,45,64,78
A or 106 amino acid (are respectively designated as Pul Δ N5, Pul Δ N22, Pul Δ N45, Pul Δ N64, Pul Δ N78, Pul Δ
N106), or last 9 or last 36 amino acid (being respectively designated as Pul Δ C9, Pul Δ C36) of C-terminal are clipped.
In one embodiment of the invention, the amino of the Pullulanase in the source the Bacillus naganoensis
Acid sequence is as shown in SEQ ID NO:1 (by N-terminal-C-terminal sequence).
In one embodiment of the invention, the nucleosides of the Pullulanase in the source the Bacillus naganoensis
Acid sequence is as shown in SEQ ID NO:2.
A second object of the present invention is to provide the genetic engineering bacteriums for expressing the mutant, and the genetic engineering bacterium is big
Enterobacteria, yeast or bacillus subtilis.
In one embodiment of the invention, the genetic engineering bacterium be using Escherichia coli be host, pET-28a is load
What body constructed.
Third object of the present invention is to provide a kind of raising Pullulanase substrate affinity and/or enzyme activity and/or compare enzyme
Living and/or thermal stability and/or catalytic efficiency method, is parent's Pullulanase to the source Bacillus naganoensis
Carry out amino acid truncation；Preferably, it is described truncate refer to the amino acid sequence for removing parent's Pullulanase N-terminal preceding 5,22,
45,64,78 or 106 amino acid, or last 9 or 36 amino acids of removal C-terminal.
The amino acid sequence of parent's Pullulanase is as shown in SEQ ID NO:1.
In one embodiment of the invention, the method is to remove the N-terminal of the amino acid sequence of parent's Pullulanase
Preceding 5,22,45,64,78 or 106 amino acid, or removal C-terminal rear 9 or 36 amino acids；The method improves
Pullulanase substrate affinity.
In one embodiment of the invention, the method is the enzyme activity for improving Pullulanase simultaneously, Rate activity, urges
Change efficiency and substrate affinity, is preceding 5 amino acid (Pul Δ N5) for removing the N-terminal of the amino acid sequence of parent's Pullulanase
Or preceding 106 amino acid (Pul Δ N106) of N-terminal.
In one embodiment of the invention, the method is to improve Rate activity and thermal stability and catalysis effect simultaneously
Rate is rear 9 amino acid (Pul Δ C9) for removing the C-terminal of the amino acid sequence of parent's Pullulanase.
In one embodiment of the invention, the method is to improve thermal stability and catalytic efficiency, is to remove parent
Preceding 78 amino acid (Pul Δ N78) of the N-terminal of the amino acid sequence of this Pullulanase.
In one embodiment of the invention, the method is to improve catalytic efficiency, is to remove parent's Pullulanase
Amino acid sequence N-terminal preceding 45 amino acid (Pul Δ N45).
The present invention, which is also claimed, encodes the nucleotide fragments of the mutant, containing the load for encoding the mutant gene
The application of body, the genetic engineering bacterium of the expression power mutant and the mutant in food, chemical industry or field of textiles.
Other explanations:
(1) it mutant naming method: indicates to be mutated using " position of truncation amino acid adds the quantity of truncation amino acid "
Body.Such as Pul Δ N5, the preceding five amino acid of parent's Pullulanase N-terminal is removed in expression；
(2) " last 9 of C-terminal ", referring to indicates in the way of N-terminal-C-terminal in parent amino acid sequence, removes the ammonia
Last 9 amino acid of base acid sequence.
Beneficial effects of the present invention:
The present invention carries out truncation transformation to Pullulanase, solves Propiram by the truncation using albumen flexible region
The thermal stability of enzyme is not able to satisfy the problem of saccharification reaction condition, lays a good foundation to widen the industrial application of Pullulanase.
In addition, the enzyme activity of Pul Δ N5 and Pul Δ N106 is by 540UmL-1768UmL is respectively increased-1And 633U
mL-1, 42%, 17% is improved than wild enzyme；Pul Δ N5, Pul Δ N106 and Pul Δ C9 Rate activity is by 267Umg-1Respectively
It is increased to 323,381 and 306Umg-1, 21%, 15% is improved than wild enzyme；By being found to study on the stability, mutant enzyme
Half-life period of Pul Δ N5, Pul Δ N78 and Pul Δ C9 at 60 DEG C improves 10h, 12h, for 24 hours than wild type respectively；It is all prominent
Become the K of enzymemValue all reduces, and illustrates to truncate and improves the affinity of enzyme-to-substrate, and Pul Δ N5, Pul Δ N45, Pul Δ
The k of N78, Pul Δ N106 and Pul Δ C9cat/KmValue improves, and shows that truncating transformation produces product to the catalytic efficiency of Pullulanase
Pole influences.
Specific embodiment
Pullulanase measuring method:
200 μ L are taken to be dissolved in 100mM, pH with isometric with the suitably diluted enzyme solution of acetate buffer of 100mM, pH 4.5
20g/L pulullan polysaccharide in 4.5 acetate buffers, which is mixed, keeps the temperature 20 minutes in 60 DEG C of water-baths.600 μ L of DNS reagent is added to shake
It is even, it is placed in boiling water and boils 5 minutes, after taking out cooling rapidly and 3mL deionized water being added, at 540nm wavelength, measurement reaction
The absorbance value of liquid.
Enzyme-activity unit definition: under conditions of above-mentioned specified, pulullan polysaccharide generation is catalytically decomposed per minute and is equivalent to 1 μM
Enzyme needed for the reduced sugar of glucose is an enzyme-activity unit (U).
Embodiment 1: mutant plasmid building
Using plasmid extraction kit Plasmid Mini Kit (being purchased from OMEGA BIO-TEK company) from laboratory preservation
E.coli BL21 (DE3)/pET-28a-PelB-pul (containing amino acid sequence be SEQ ID NO:1 shown in, nucleotides sequence
Be classified as Pullulanase gene shown in SEQ ID NO:2) in extract 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)) it is used as truncated template, the primer sequence such as table 1.
Utilize PremixHS PCR enzyme (being purchased from TaKaRa company) carries out BnPul using the method for PCR
Truncate transformation.PCR reaction system are as follows: 50 μ L, 25 μ L PremixHS, 0.2 μM 1,0.2 μM of Primer
Primer2,10ng-100ng Template.
PCR reaction condition: 98 DEG C of 10s, 58 DEG C of 15s, 72 DEG C of 180s, totally 30 recycle.
PCR product uses T4DNA ligase (TaKaRa) after being handled with BamHI and XhoI restriction endonuclease (being purchased from TaKaRa company)
Connect the expression vector pET-28a-PelB (laboratory preservation) containing signal peptide.Enzyme disjunctor system are as follows: 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 of connections overnight.Connection product conversion
Escherichia coli JM109 competent cell.
1 primer of table
Embodiment 2: the building of recombinant bacterium
The preparation of competence: according to Competent Cell Preparation kit (being purchased from TaKaRa company) specification
Preparation.With the single colonie of oese picking E.coli JM109, single colonie is separated in the flat lining out of LB newly prepared, in 37 DEG C
Under be incubated overnight.After bacterium colony grows up to, chooses single colonie and be inoculated into 50mL LB liquid medium, at 37 DEG C, under the conditions of 200r/min
It cultivates to OD600Reach 0.35-0.5, is put into ice at once and stops culture.Shift the sterilized 50mL of bacterium solution 30mL to pre-cooling
In centrifuge tube, 4000r/min is centrifuged 4min at 4 DEG C, supernatant is abandoned as far as possible clean.The Solution A of 3mL pre-cooling is added, gently
Light to mix, 4 DEG C, 4000r/min centrifugation 4min as far as possible abandon supernatant clean.The Solution B of 3mL pre-cooling is added, gently
It mixes, dispenses 80 μ L in the 1.5mL EP pipe of each pre-cooling, be put into -70 DEG C and save.
Conversion operation: competent cell is placed in ice and is melted.The product of 10 μ L PCR is added, mixes gently and is placed on ice
Middle 30min.42 DEG C of water-bath thermal shock 90s are transferred to, then 890 μ L LB culture mediums are added after placing 2min in ice, in 37 DEG C, 150r/
Recovery 1h under the conditions of min.Bacterium solution after recovery is centrifuged 4min at 4000r/min, abandons 800 μ L supernatants, the thallus pressure-vaccum of precipitating
It is resuspended after mixing and is coated on the LB plating medium containing kanamycins, be inverted overnight incubation at 37 DEG C.Picking single colonie upgrading
Grain sequencing.
Embodiment 3: the acquisition of mutant enzyme
The successful plasmid Transformed E .coli BL21 of picking sequence verification, step of converting are same as above.Picking transformant, which is inoculated in, to be contained
In the 50mL self-induction fluid nutrient medium of 50 μ g/mL kanamycins of final concentration, 2~3h is cultivated at 37 DEG C of 200r/min, is transferred to
17 DEG C, Fiber differentiation 70h under 200r/min, are centrifuged 10min under the conditions of 4 DEG C, 12000r/min.Thallus is collected, precipitating is used
The acetic acid-sodium acetate buffer solution of 0.1mol/LpH 4.5 is resuspended, and is centrifuged under the conditions of 4 DEG C, 12000r/min after ultrasonication
20min, obtained supernatant are crude enzyme liquid, arrive pure enzyme solution after ni-sepharose purification, are used for follow-up study.
Self-induction culture medium: alpha-lactose 10g/L, DEXTROSE ANHYDROUS 0.5g/L, glycerol 5g/L, KH2PO46.8g/L
MgSO42mmol/L, tryptone 10g/L, yeast extract 5g/L, Na2HPO47.1g/L, Na2SO40.71g/L, NH4Cl
2.67g/L。pH 7.5-8.0。
Embodiment 4: mutation front and back enzyme activity and Rate activity compare
The present invention compares the variation of mutation front and back enzyme activity and Rate activity, wherein wild enzyme refers to
The source B.naganoensis JNB-1 Pullulanase BnPul (amino acid sequence be SEQ ID NO:1 shown in, nucleotide sequence
For SEQ ID NO:2), GENBANK accession no.AEV53626.1.
With the method shake flask fermentation of self-induction culture produce wild enzyme BnPul, Pul Δ N5, Pul Δ N22, Pul Δ N45,
The crude enzyme liquid of Pul Δ N64, Pul Δ N78, Pul Δ N106, Pul Δ C9, Pul Δ C36.By measuring enzyme respectively after ni-sepharose purification
Living and protein concentration calculates Rate activity.768UmL is respectively increased in the enzyme activity of Pul Δ N5 and Pul Δ N106-1And 633U
mL-1, and the enzyme activity of wild enzyme is 540UmL-1.The Rate activity of Pul Δ N5, Pul Δ N106 is increased to 323Umg-1、
381U·mg-1, and the Rate activity of wild enzyme is 267Umg-1。
The enzyme activity and specific enzyme activity of 2 wild type of table and mutant compare
Embodiment 5: the thermal stability of mutation front and back enzyme compares
(1) optimum temperature compares
The Acetic acid-sodium acetate buffer for taking pH 4.5 is diluted to the Pullulanase enzyme solution of suitable concentration, respectively 45 DEG C,
Enzyme activity is measured at 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C.With highest enzyme activity for 100%, the opposite enzyme at each temperature is calculated
It is living, to 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 period 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, keeps the temperature in the buffer of pH 4.5, measures remaining enzyme activity respectively, calculates half-life period,
Half-life period of mutant enzyme Pul Δ N5, Pul Δ N78, Pul Δ C9 at 60 DEG C improves 10h, 12h, 34h than wild type respectively.
Embodiment 6: the optimal pH of mutation front and back enzyme compares
The enzyme activity of wild enzyme and mutant enzyme is measured under the conditions of 60 DEG C, pH 3.0,3.5,4.0,4.5,5.0 and 5.5 respectively,
It was found that mutant of the invention does not change the optimal pH of Pullulanase while improving Pullulanase thermal stability substantially.
With high thermal stability and substantially the Pullulanase for having not been changed optimal pH is more suitable for applying in saccharification.
Embodiment 7: the variation of mutation front and back enzyme kinetics is compared
Prepare various concentration 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), initial velocity of reaction is measured under 60 DEG C, the different concentration of substrate of pH 4.5 respectively, benefit
Nonlinear fitting, which is carried out, with GraphPad Prism software obtains every kinetic parameter.The results are shown in Table 3.
The kinetic parameter of 3 wild type of table and mutant compares
As the result is shown:
(1) K of all mutant enzymesmValue all reduces, and illustrates the binding force enhancing of enzyme-to-substrate.
(2) k of Pul Δ N5, Pul Δ N45, Pul Δ N78, Pul Δ N106 and Pul Δ C9catValue improves, and Pul Δ
The k of N5, N45, Pul Δ N78, Pul Δ N106 and Pul Δ C9cat/KmValue improves, and shows to truncate catalysis of the transformation to Pullulanase
Efficiency produces positive influence.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (12)
1. a kind of Pullulan enzymatic mutant, which is characterized in that the amino acid sequence of the mutant is relative to Bacillus
The amino acid sequence of the Pullulanase in the source naganoensis, have occurred before N-terminal 5,22,45,64,78 or
The truncation of 106 amino acid or the truncation of C-terminal last 9 or last 36 amino acid；The Bacillus naganoensis
The amino acid sequence of the Pullulanase in source is as shown in SEQ ID NO:1.
2. mutant according to claim 1, which is characterized in that the source the Bacillus naganoensis it is general
The nucleotide sequence of Shandong orchid enzyme is as shown in SEQ ID NO:2.
3. encoding the nucleotide fragments of any mutant of claim 1-2.
4. the carrier of the gene containing the coding any mutant of claim 1-2.
5. expressing the genetic engineering bacterium of any mutant of claim 1-2.
6. genetic engineering bacterium according to claim 5, which is characterized in that the genetic engineering bacterium is Escherichia coli, yeast
Or bacillus subtilis.
7. any mutant of claim 1-2 is in the application of food, chemical industry or field of textiles.
8. a kind of method for improving Pullulanase substrate affinity, which is characterized in that the method is to Bacillus
The Pullulanase in the source naganoensis carries out amino acid truncation；The method, which refers to, removes Bacillus naganoensis
Preceding 5,22,45,64,78 or 106 amino acid of the N-terminal of the amino acid sequence of the Pullulanase in source, or removal C-terminal
Rear 9 or 36 amino acids；The amino acid sequence of the Pullulanase in the source the Bacillus naganoensis such as SEQ ID
Shown in NO:1.
9. a kind of enzyme activity, method of Rate activity, catalytic efficiency and substrate affinity for improving Pullulanase simultaneously, feature exist
In the method is to carry out amino acid truncation to the Pullulanase in the source Bacillus naganoensis；The method is
Before falling preceding 5 amino acid or N-terminal of the N-terminal of the amino acid sequence of the Pullulanase in the source Bacillus naganoensis
106 amino acid；The amino acid sequence of the Pullulanase in the source the Bacillus naganoensis such as SEQ ID NO:1
It is shown.
10. a kind of method for the thermal stability for improving Pullulanase, which is characterized in that the method is to Bacillus
The Pullulanase in the source naganoensis carries out amino acid truncation；The method is to remove Bacillus naganoensis
Rear 9 amino acid of the C-terminal of the amino acid sequence of the Pullulanase in source；The source the Bacillus naganoensis it is general
The amino acid sequence of Shandong orchid enzyme is as shown in SEQ ID NO:1.
11. a kind of method for the thermal stability and catalytic efficiency for improving Pullulanase simultaneously, which is characterized in that the method is pair
The Pullulanase in the source Bacillus naganoensis carries out amino acid truncation；The method is to remove Bacillus
Preceding 78 amino acid of the N-terminal of the amino acid sequence of the Pullulanase in the source naganoensis；The Bacillus
The amino acid sequence of the Pullulanase in the source naganoensis is as shown in SEQ ID NO:1.
12. a kind of method for the catalytic efficiency for improving Pullulanase, which is characterized in that the method is to Bacillus
The Pullulanase in the source naganoensis carries out amino acid truncation；The method is to remove Bacillus naganoensis
Preceding 45 amino acid of the N-terminal of the amino acid sequence of the Pullulanase in source；The source the Bacillus naganoensis it is general
The amino acid sequence of Shandong orchid enzyme is as shown in SEQ ID NO:1.
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