CN103571812A - Pullulanase mutant with improved secretion efficiency and heat stability and preparation method of pullulanase mutant - Google Patents
Pullulanase mutant with improved secretion efficiency and heat stability and preparation method of pullulanase mutant Download PDFInfo
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Abstract
The invention discloses a pullulanase mutant with improved secretion efficiency and heat stability and a preparation method of the pullulanase mutant, and belongs to the field of gene engineering and enzyme engineering. The secretion efficiency and the heat stability of pullulanase are improved through structural domain deletion mutation; deleted structural domains are CBM41, X45 and X25 structural domains or combinations thereof; and the mutation technical scheme capable of improving the secretion efficiency and the heat stability of pullulanase is provided. The obtained pullulanase structural domain deletion mutant has at least one of changed properties as follows: 1), the extracellular secretion efficiency is improved after recombinant bacteria are fermented; and 2), and the heat stability is improved under the conditions of pH 4.0-5.0 and 60 DEG C. Compared with natural pullulanase, structural domain deletion mutants are more suitable for production, preparation and applications of the pullulanase.
Description
Technical field
The present invention relates to mutant of a kind of Pullulanase and preparation method thereof, particularly utilize the domain deletion method of protein engineering to improve the secernment efficiency of Pullulanase and the technology of thermostability, belong to genetically engineered and enzyme engineering field.
Background technology
Pullulanase (EC3.2.1.41) is a kind of starch-debranching enzyme with significant application value, and it can be hydrolyzed α-1 in pulullan polysaccharide, amylopectin, α-limit dextrin equimolecular, 6 glucoside bonds.Pullulanase appropriate effect lower molecular weight dextrin ，Qi least action unit is malt-base maltose, is mainly used in and the composite glucose of preparing of saccharifying enzyme.Pullulanase can cut off the tapping point in starch, accelerate the reaction of follow-up enzyme, Reaction time shorten, the transformation efficiency of raising starch, reduce the usage quantity of zymin for other saccharification, thereby reach the object that increases output, improves plant factor, reduces production costs.
Contriver has carried out heterogenous expression and Fixedpoint mutation modified to deriving from the Pullulanase of a de-genus bacillus (Bacillus deramifican) early stage, has obtained the Pullulanase mutant (a kind of Pullulanase mutant and preparation method thereof .201210256804.9) that a plurality of thermostabilitys and catalytic performance are improved.And control in conjunction with the mode of adding Twen80 and trimethyl-glycine by fermenting process condition, optimized the zymotechnique (Wu Jing of recombinant bacterial strain fermentative production Pullulanase, Chen Sheng, Duan Xuguo, Chen Jian. a kind of by promoting the depolymerization of activated protein aggregate to improve the method for starch debranching production of enzyme, 201210035298.0; Wu Jing, Duan Xuguo, Chen Sheng, Chen Jian. a kind of fermentation manufacturing technique of the starch-debranching enzyme of recombinating, 201210519218.9).Yet contriver's Pullulanase vigor that the ratio in fermentation supernatant and cellular component is analyzed in precipitating in cleer and peaceful born of the same parents on finding in outside upper clear, the born of the same parents of restructuring mycetocyte to restructuring Pullulanase accounts for respectively 15.4%, 47.1% and 37.5% of total enzyme activity.The exocytosis efficiency of visible restructuring Pullulanase is very low, and most of recombinase is present in cell interior, and wherein also has greatly and be present in cell with insoluble aggregate form.In fermentation subsequent processes, need thalline to be carried out to the processing such as fragmentation, reclaim the zymoprotein of cell interior.Yet cytoclastic cost is generally higher in scale operation, and cytoclasis process conditional is often more violent, the easy inactivation of zymoprotein.If zymoprotein can mainly be secreted into born of the same parents' outer (in substratum) during the fermentation, later separation purge process just only need to be separated and concentrated through simple thalline, and the production cost of zymin just can significantly reduce.
The inefficient phenomenon of restructuring Pullulanase exocytosis may be determined by this enzyme self aminoacid sequence and space structure.Research shows, protein molecular weight and complex structure degree all have a great impact its solubility expression and secernment efficiency, and the albumen that molecular weight is larger, structure is more complicated more easily forms insoluble protein aggregation body, and exocytosis efficiency is lower.B.deramificans Pullulanase is comprised of 928 amino acid, and molecular weight is about 101kDa.Its protein structure model analysis is found, it has 6 structural domains, is respectively domain C BM41, X25, X45, CBM48, A and C.CBM41 and CBM48 structural domain belong to respectively carbohydrate binding domains 41 He48 families of family, all have and starch substrates combined function, and X25 and X45 structural domain Unknown Function.Tri-structural domain contacts of CBM48, A and C are closely linked and form an integral body, may be to keep Pullulanase protein structure and active necessary structural domain; And by flexible linker, connect between CBM41 and X25 structural domain, thering is stronger swing, the Pullulanase albumen truncated mutant after it is excised may still can retain debranching activity.On the basis that contriver analyzes at B.deramificans Pullulanase albumen model configuration, by the exocytosis efficiency that CBM41, the X25 of its N end and X45 structural domain are excised respectively to improve Pullulanase.
Summary of the invention
The technical problem that the present invention will solve is to provide the mutant that delete in a kind of natural Pullulanase N end structure territory, comprise a N end structural domain (CBM41), two structural domains (CBM41 and X25, CBM41 and X45) to delete or mutant that three structural domains (CBM41, X25 and X45) are deleted, mutant has been compared higher exocytosis efficiency and thermostability with parent's Pullulanase.
Described parental gene is consistent with a de-genus bacillus Pullulanase, and the described plasmid template as sudden change use is the carrier pulA/pET20b (+) (a kind of Pullulanase mutant and preparation method thereof .201210256804.9) that carries natural Pullulanase encoding gene.
Another technical problem that the present invention will solve be to provide that the N end structure territory of a kind of Pullulanase directed mutants (D437H/D503Y) deletes stack mutant, comprise a N end structural domain (CBM41) or two structural domains (CBM41 and X25, CBM41 and X45) to delete or stack mutant that three structural domains (CBM41, X25 and X45) are deleted, stack mutant has been compared higher exocytosis efficiency and thermostability with parent's Pullulanase.
The encoding gene of described Pullulanase directed mutants (D437H/D503Y) is consistent with the sequence of Pullulanase mutant D437H/D503Y in Chinese patent 201210256804.9, and the described plasmid template as sudden change is the carrier D437H/D503Y/pET-20b (+) (a kind of Pullulanase mutant and preparation method thereof .201210256804.9) that carries natural Pullulanase encoding gene.
The 3rd technical problem to be solved by this invention is to provide the preparation method of the Pullulanase mutant of N end structure territory that exocytosis efficiency and thermostability be all improved deleting; the present invention be take Pullulanase directed mutants (D437H/D503Y) and is described as example; protection domain is not limited to this, comprises the steps:
1) on the basis of a de-genus bacillus Pullulanase aminoacid sequence, determine structural domain and the corresponding aminoacid sequence thereof that will delete;
2) mutant primer of sudden change is deleted in design N end structure territory, and the carrier that carries natural Pullulanase encoding gene of take carries out gene amplification and builds the plasmid vector containing domain deletion mutant as template;
3) plasmid that contains truncated mutant encoding gene and the plasmid that contains stack sudden change encoding gene are transformed into host cell;
4) select positive colony and carry out fermentation culture, and purifying Pullulanase mutant Puld1, Puld2, D437H/D503Y/d1 and D437H/D503Y/d2.
By domain deletion of CBM41 of Pullulanase N end, called after Puld1; By CBM41 and two domain deletions of X25 of Pullulanase N end, called after Puld2; By CBM41, X25 and tri-domain deletions of X45 of Pullulanase N end, called after Puld3.
Described plasmid vector is pET series, pGEX series, or or pUB series in any one.
Described host cell comprises: bacterium, yeast and fungal cell, it is also the scope of protection of present invention.
The present invention has built four significant mutant, has realized the raising of Pullulanase exocytosis efficiency and thermostability.The exocytosis efficiency of Pullulanase truncated mutant all increases, the ratio that wherein extracellular enzyme of Puld1, Puld2, D437H/D503Y/d1 and D437H/D503Y/d2 accounts for total enzyme activity is respectively 48.5%, 58.7%, 57.3% and 60.8%, is respectively 3.1,3.8,5.3 and 5.6 times of corresponding parent's Pullulanase.At pH4.5, in the water-bath of 60 degree, the transformation period of natural Pullulanase is 20h, and the transformation period that mutant Puld1, Puld2 are deleted in N end structure territory is respectively 57h and 34h left and right; The transformation period of D437H/D503Y is 120h, and the transformation period of stack mutant D437H/D503Y/d1 and D437H/D503Y/d2 is respectively 203h and 160h.Wherein D437H/D503Y/d1 and D437H/D503Y/d2 thermostability are best.Domain deletion mutant and stack mutant are more suitable for exocytosis production and the application in mashing process of Pullulanase than parent Pullulanase.
Accompanying drawing explanation
The natural Pullulanase of Fig. 1 and truncated mutant brachymemma mode thereof
The natural Pullulanase of Fig. 2 and truncated mutant fermentation supernatant SDS-PAGE gel electrophoresis thereof
M, molecular weight of albumen standard; 1, natural Pullulanase; 2, Puld1; 3, Puld2; 4, Puld3
Fig. 3 D437H/D503Y and stack mutant fermentation supernatant SDS-PAGE gel electrophoresis
M, molecular weight of albumen standard; 1, D437H/D503Y; 2, D437H/D503Y/d1; 3, D437H/D503Y/d2
The optimum temperuture of Fig. 4 Pullulanase truncated mutant
The thermostability of Fig. 5 Pullulanase truncated mutant
Embodiment
Embodiment 1: the preparation of natural Pullulanase N end truncated mutant
(1) structure of natural Pullulanase N end truncated mutant
Mutant Puld1 and Puld2 are deleted in 2 kinds of N end structure territories that derive from the Pullulanase of B.deramificans:
On the basis of analyzing at different sources Pullulanase sequence alignment, a de-genus bacillus Pullulanase protein structure is simulated and analyzed, find that a de-genus bacillus Pullulanase has 6 structural domain: CBM41, X25, X45, CBM48, A and C.Wherein between the CBM41 of protein N terminal, X25 and X45 structural domain, by flexible linker, connect, there is stronger swing; CBM41 belongs to carbohydrate binding domains 41 families, has and starch substrates combined function, and X25 and X45 structural domain Unknown Function.And tri-structural domains of CBM48, A and C link together in mode very closely and form a complete integral body, this structure has Binding Capacity and catalytic activity institute household function.Pullulanase mutant after N-terminal structural domain is deleted may still can retain debranching activity.
By domain deletion of CBM41 of Pullulanase N end, called after Puld1; By CBM41 and two domain deletions of X25 of Pullulanase N end, called after Puld2; By CBM41, X25 and tri-domain deletions of X45 of Pullulanase N end, called after Puld3(Fig. 1).
Mutant Puld1 is deleted in 3 kinds of N end structure territories, the preparation method of Puld2 and Puld3, according to the coding gene sequence of B.deramificans Pullulanase, design and synthesize respectively and introduce Puld1, the primer of Puld2 and the sudden change of Puld3 domain deletion, Pullulanase encoding gene is deleted to sudden change, measure DNA encoding sequence, the encoding gene (the 1st to the 270th bit base is deleted) of Pullulanase mutant Puld1 is confirmed in order-checking respectively; The encoding gene of the encoding gene of Pullulanase mutant Puld2 (the 1st to the 270th bit base and the 480th to 783 s' base is deleted) and Pullulanase mutant Puld3 (the 1st to the 939th bit base deletion).Mutant gene is connected in suitable expression vector and is imported in intestinal bacteria, subtilis or bacillus licheniformis and express, obtain the Pullulanase mutant that delete in N end structure territory.
The pcr amplification of mutant Puld1 and Puld3 encoding gene: utilize fast PCR technology, take expression vector pulA/pET-20b (+) as template,
The mutant primer of introducing Puld1 sudden change is:
Pul-d1-F：5’-CATGCCATGGGTAACAGCCAGATCTTC-3’
Pul-R：5’-CCCAAGCTTACTTTTTACCGTGGTCCGGG-3’
The mutant primer of introducing Puld3 sudden change is:
Pul-d3-F：5’-CATGCCATGGACGATCTGGGTAATAACTAC-3’
Pul-R：5’-CCCAAGCTTACTTTTTACCGTGGTCCGGG-3’
Pcr amplification program setting is: first, and 94 ℃ of denaturation 4min; Then enter 30 circulations: 98 ℃ of sex change 10s, 58 ℃ of annealing 5s, 72 ℃ are extended 3min; Last 72 ℃ are extended 10min, 4 ℃ of insulations.PCR product detects with 1% agarose gel electrophoresis.
After PCR product purification, connect pMD18T-simple Transformed E .coli JM109, picking positive colony, after double digestion is identified, order-checking.Check order correct mutant plasmid after double digestion, glue reclaims, and connects expression vector pET20b (+), Transformed E .coli JM109, extracts plasmid, double digestion is identified positive colony, obtains pET20b (+)/d1 and pET20b (+)/d3.Respectively plasmid is transformed and expresses host e. coli BL21 (DE3) competent cell, obtain expressing two kinds of recombinant bacterial strains of mutant Puld1 and Puld3.
The mutant primer of introducing Puld2 sudden change is:
Pul-d1-F：5’-CATGCCATGGGTAACAGCCAGATCTTC-3’
Pul-d2-overlap-R：5’-CACACCGCTTTCTACCTGGCCCAGAGA-3’
Pul-d2-overlap-F：5’-GCGTCTCTGGGCCAGGTAGAAAGCGGTGTGAAAACGG’
Pul-R：5’-CCCAAGCTTACTTTTTACCGTGGTCCGGG-3’
Take Pul-d1-F/Pul-d2-overlap-R and Pul-d2-overlap-F/Pul-R respectively as primer, amplify object Segment A and B.The PCR product of Segment A and B is as the template of overlapping PCR for the second time.Segment A and B to adding equimolar amount in PCR reaction system, do not add primer, 10 circulations of staggered extension, and pcr amplification condition is: 94 ℃ of 4min; 98 ℃ of 10s, 58 ℃ of 5s, 72 ℃ of 3min, 10 circulation latter 72 ℃ extend again 10min.Then add primer Pul-d1-F and Pul-R, increase 30 and circulate, 94 ℃ of denaturation 4min; 98 ℃ of 10s, 58 ℃ of 5s, 72 ℃ of 2.5min, totally 30 circulations; 72 ℃ are extended 10min.
After PCR product purification, connect pMD18T-simple Transformed E .coli JM109, picking positive colony, after double digestion is identified, order-checking.Check order correct mutant plasmid after double digestion, glue reclaims, and connects expression vector pET20b (+), Transformed E .coli JM109, extracts plasmid, double digestion is identified positive colony, obtains pET20b (+)/d2.Plasmid is transformed and expresses host e. coli BL21 (DE3) competent cell, obtain expressing the recombinant bacterial strain of mutant Puld2.
(2) expression and purification of natural Pullulanase N end truncated mutant:
Picking proceeds to the positive monoclonal of expressive host e. coli bl21 (DE3) in LB liquid nutrient medium (containing the 100 μ g/mL penbritins) 8～10h that grows, and by 5% inoculum size, seed fermentation liquid is received to TB liquid nutrient medium (containing 100 μ g/mL penbritins); Intestinal bacteria are cultured to OD at 30 ℃ of shaking tables
600=0.6, the IPTG induction extracellular expression that adds 0.01mM final concentration, and continue cultivation and fermentation after 50 hours at 25 ℃ of shaking tables, by fermented liquid in 4 ℃, the centrifugal 10min of 10000g except thalline, collect centrifugal fermented supernatant fluid, and fermented supernatant fluid is carried out to the analysis of SDS-PAGE protein electrophoresis.As shown in Figure 2, compare with natural enzyme, it is many that Puld1 and Puld2 protein band obviously become, and illustrates that Puld1 and Puld2 extracellular protein secretory volume are significantly improved.And the content of Puld3 in fermented supernatant fluid is seldom, further to analyze and find mainly with occlusion body form, to exist in Puld3 expression process, soluble proteins is extremely low.Therefore, in natural enzyme truncated mutant, only select two kinds of mutant of Puld1 and Puld2 to carry out purifying and follow-up analysis.
The fermented supernatant fluid pH to 4.5 that regulates Pullulanase mutant, is placed in 55 ℃ of thermal treatments of water-bath 1 hour, and 4 ℃, the centrifugal 20min of 10000g collect supernatant liquor.Toward (the NH that slowly adds 70% in supernatant liquor
4)
2sO
4, 4 ℃ of placements are saltoutd and are spent the night.4 ℃, the centrifugal 20min of 10000g, collecting precipitation.With 20mmol/L phosphoric acid buffer, redissolve after precipitation, dialysed overnight in 20mmol/L phosphoric acid buffer, during change dialysis buffer liquid 2-3 time, by making loading sample after 0.22 μ m 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 controlling temperature is 4 ℃.Anion-exchange chromatography purification step: (1) balance: 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 detection wavelength is 280nm, the elutriant that fraction collection is lived containing Pullulanase enzyme; Vigor component after dialysed overnight, obtains respectively purified mutant body Puld1 and Puld2 in 50mM pH4.5 acetate buffer solution.
Embodiment 2: the preparation of stack mutant.
(1) structure of stack mutant
Mutant D437H/D503Y/d1 and D437H/D503Y/d2 are deleted in 2 kinds of N end structure territories that derive from the Pullulanase double-mutant D437H/D503Y of B.deramificans:
By domain deletion of CBM41 of Pullulanase double-mutant D437H/D503Y N end, called after D437H/D503Y/d1; By CBM41 and two domain deletions of X25 of Pullulanase double-mutant D437H/D503Y N end, called after D437H/D503Y/d2.
The preparation method of mutant D437H/D503Y/d1 and D437H/D503Y/d2 is deleted in 2 kinds of N end structure territories, with the encoding gene of Pullulanase double-mutant D437H/D503Y, substitute the natural Pullulanase encoding gene of B.deramificans, other method is identical with the method for natural Pullulanase N end truncated mutant.
The preparation of mutant D437H/D503Y/d1 and D437H/D503Y/d2 encoding gene: utilize fast PCR technology, take expression vector D437H/D503Y/pET-20b (+) as template,
The mutant primer of introducing D437H/D503Y/d1 sudden change is:
Pul-d1-F：5’-CATGCCATGGGTAACAGCCAGATCTTC-3’
Pul-R：5’-CCCAAGCTTACTTTTTACCGTGGTCCGGG-3’
The recombinant bacterium construction process of pcr amplification program, expression vector and product mutant is identical with the method for mutant Puld1.
The rite-directed mutagenesis primer of introducing D437H/D503Y/d2 sudden change is:
Pul-d1-F：5’-CATGCCATGGGTAACAGCCAGATCTTC-3’
Pul-d2-overlap-R：5’-CACACCGCTTTCTACCTGGCCCAGAGA-3’
Pul-d2-overlap-F：5’-GCGTCTCTGGGCCAGGTAGAAAGCGGTGTGAAAACGG’
Pul-R：5’-CCCAAGCTTACTTTTTACCGTGGTCCGGG-3’
The recombinant bacterium construction process of pcr amplification program, expression vector and product mutant is identical with the method for mutant Puld2.
(2) expression and purification of stack mutant:
The fermentation and the purification condition that produce stack mutant recombinant bacterial strain are identical with the method for natural Pullulanase N end truncated mutant.
Recombinant bacterial strain is through shake flask fermentation and fermented supernatant fluid is carried out to the analysis of SDS-PAGE protein electrophoresis.As shown in Figure 3, compare with D437H/D503Y, it is many that D437H/D503Y/d1 and D437H/D503Y/d2 protein band obviously become, and illustrates that D437H/D503Y/d1 and D437H/D503Y/d2 extracellular protein secretory volume are significantly improved.
Pullulanase, after separation and purification, obtains respectively stack mutant D437H/D503Y/d1 and the D437H/D503Y/d2 of purifying.
Embodiment 3: the present embodiment explanation enzyme is lived and analyzed.
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, 3,5-dinitrosalicylic acid and reducing sugar solution are reduced to aobvious henna amido complex compound after heat altogether, the depth of its color and the amount of reducing sugar are directly proportional within the specific limits, therefore can carry out colorimetric 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 μ mol glucose as a unit of activity.
Enzyme activity determination step:
A. preheating: get the 1.5% Propiram solution (50mM pH4.5 acetic acid bufffer) of 2ml in test tube, be placed in 60 ℃ of water-bath preheating 10min left and right,
B. reaction: add 0.1ml sample enzyme liquid, vibration mixes, accurately timing 10min, adds 3ml DNS to mix, and puts into frozen water termination reaction, and boiling water bath 7min is cooling.
C. measure: to adding distilled water in above-mentioned reaction system and being settled to 15ml, mix.Under 540nm, measure its light absorption value and calculate enzyme activity.
2) in fermented liquid supernatant and different cellular component, enzyme activity distributes relatively:
Recombinant bacterial strain is through shake flask fermentation, and fermented liquid, at the centrifugal 20min of 10000g, is collected fermented supernatant fluid and thalline.Thalline carries out fragmentation with ultrasonic disruption instrument after suspending with 20mmol/L pH4.5 acetate buffer solution.Cytoclasis liquid is at the centrifugal 20min of 10000g, and collecting supernatant liquor is supernatant in born of the same parents, and precipitation suspends and vibrates with damping fluid and mixes as precipitating suspension in born of the same parents.Pullulanase vigor in upper cleer and peaceful cell precipitation suspension in cell free fermentation supernatant, born of the same parents is measured.
Experimental result is listed in table 1, and the extracellular enzyme vigor of natural enzyme only accounts for 15.4% of total enzyme activity, and mutant Puld1 and Puld2 extracellular enzyme proportion obviously improve, and reach 52.9% and 58.7%, is respectively 3.4 and 3.8 times (table 5-2) of natural enzyme.The truncated mutant ratio that the enzyme activity in precipitation accounts for total enzyme activity in cleer and peaceful born of the same parents in born of the same parents all decreases, and wherein in born of the same parents, supernatant proportion declines the most obvious.
In addition, the outer supernatant enzyme proportion of mutant D437H/D503Y/d1 and D437H/D503Y/d2 born of the same parents is respectively 57.3% and 60.8%, is respectively 5.3 and 5.6 times (table 1) of contrast D437H/D503Y.Analyze to find, the lyoenzyme vigor of two mutant (supernatant in the outside upper cleer and peaceful born of the same parents of born of the same parents) accounts for 80% left and right of total enzyme activity, and the enzyme activity that only has 20% left and right exists with the form of activated protein aggregate.And the lyoenzyme of control enzyme D437H/D503Y and activated protein aggregate account for respectively 42.4% and 57.6% of total enzyme activity.As can be seen here, stack mutant has the ability of better solubility expression with respect to contrast, have the potentiality that are better applied to fermentative production.
Table 1 enzyme activity natural and brachymemma Pullulanase mutant distributes
Embodiment 4: optimum temperuture and the thermostability of the present embodiment explanation Pullulanase.
Acetic acid-the sodium-acetate (50mM) of pH4.5 of take is damping fluid, measures the optimum temperuture of Pullulanase mutant 40 to 70 ℃ of temperature ranges.As Fig. 4, the optimum temperuture of Puld1 and Puld2 is all 55 ℃, identical with the optimum temperuture of natural enzyme.Puld1 and Puld2 retain respectively 97.0% and 89.3% activity at 60 ℃, all higher in the activity of 60 ℃ of reservations than natural enzyme.The optimum temperuture of stack mutant enzyme D437H/D503Y/d1 and D437H/D503Y/d2 is all 60 ℃, than natural enzyme, has improved 5 ℃, identical with the optimum temperuture of double-mutant D437H/D503Y.Puld1 and D437H/D503Y/d1 retain approximately 85% activity at 70 ℃, and D437H/D503Y is identical with double-mutant; And Puld2 and D437H/D503Y/d2 only retain 39% and 51% vigor 70 ℃ of difference.
It is 0.4mg/mL that the natural Pullulanase of purifying and mutant are diluted to protein content with 50mM pH4.5 acetate buffer solution, and pH is 4.5, is placed in 60 ℃ of waters bath with thermostatic control, every 5h sampling once, surveys its residual enzyme and lives, relatively its stability.As shown in Figure 5, Puld1 and Puld2 transformation period are respectively 57h and 34h, are respectively 2.6 and 1.5 times of natural enzyme transformation period.Stack mutant enzyme D437H/D503Y/d1 and the D437H/D503Y/d2 transformation period is respectively 203h and 160h, is respectively 9.0 and 7.1 times of natural enzyme transformation period, is double-mutant D437H/D503Y(120h) 1.7 and 1.3 times.Visible, the stability of Pullulanase N end truncated mutant has obtained raising in various degree; After truncated mutant and point mutation stack, the effect of two kinds of sudden changes superposes mutually, and the thermostability of stack mutant is further enhanced.
Embodiment 5: the Determination of Kinetic Parameters of the mutant of the present embodiment explanation Pullulanase.
The kinetic parameter of natural enzyme, double-mutant D437H/D503Y and N end brachymemma enzyme (Puld1, Puld2, D437H/D503Y/d1 and D437H/D503Y/d2) is as shown in table 2.
Than natural enzyme, N end truncated mutant has caused mutant Puld1 and the reduction of Puld2 to the binding ability of pulullan polysaccharide substrate.The K of Puld1 and Puld2
mvalue is respectively 1.6 times and 4.4 times of natural enzyme, and it is also the poorest that wherein N holds maximum its Binding Capacity ability of Puld2 of brachymemma.The V of mutant Puld1 and Puld2
maxvalue is respectively 1.1 times and 1.2 times of natural enzyme.In addition, the brachymemma in N end structure territory has obviously also brought the disadvantageous effect of some, the especially impact on catalytic efficiency, the as can be seen from Table 2 K of Puld1 and Puld2
cat/ K
mvalue only has 68.8% and 19.1% of natural enzyme.This is mainly because N end truncated mutant reduces or Binding Capacity ability and K Binding Capacity ability
catcoefficient reduces simultaneously and to cause.Although the stability of truncated mutant has improved, catalytic kinetic parameter declines to some extent, therefore how to reduce the brachymemma of N end the negative impact of enzyme kinetics parameter is seemed to particularly important.
Two sudden changes and N end is deleted after sudden change superposes, can be made up the decline that the Pullulanase kinetic parameter that sudden change causes is deleted in simple N end structure territory.The kinetic parameter of stack mutant as shown in Table 2, compare all and improve a lot with Puld2, their K with the corresponding mutant Puld1 that do not superpose by the Binding Capacity ability of D437H/D503Y/d1 and D437H/D503Y/d2
mvalue is respectively 50% and 34% of Puld1 and Puld2.The K of stack mutant D437H/D503Y/d1 and D437H/D503Y/d2
catvalue is respectively 1.4 times and 1.5 times of natural enzyme.Their catalytic efficiency (K
cat/ K
mvalue) be respectively 1.8 times and 1.0 times of natural enzyme.The above results has illustrated that Binding Capacity ability and the catalytic capability of stack mutant have all obtained obvious lifting, has made up the disadvantageous effect that simple N end brachymemma causes.Stack mutant not only has the secernment efficiency of better stability and Geng Gao, and catalytic kinetic parameter is also improved, and may have better using value.
Table 2 Pullulanase truncated mutant kinetic parameter
Although the present invention with preferred embodiment openly as above; but it is not in order to limit 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, so protection scope of the present invention should be with being as the criterion that claims were defined.
Claims (10)
1. a Pullulanase mutant, is characterized in that Pullulanase N end structure territory after Pullulanase or transformation to delete, and described structural domain is relevant to secernment efficiency or the thermostability of Pullulanase.
2. mutant claimed in claim 1, is characterized in that described structural domain is CBM41, X45, X25 structural domain or its combination.
3. mutant according to claim 2, is characterized in that described structural domain is CBM41.
4. mutant according to claim 2, is characterized in that described structural domain is X45.
5. mutant according to claim 2, is characterized in that described structural domain is CBM41 and X45 or CBM41 and X25.
6. mutant according to claim 2, is characterized in that described structural domain is the combination of CBM41, X45 and X25.
7. according to the arbitrary described mutant of claim 1-6, it is characterized in that after described transformation that Pullulanase is a de-genus bacillus Pullulanase double-mutant D437H/D503Y.
8. obtain the method for the arbitrary described mutant of claim 1-6, comprise the steps:
1) Pullulanase is analyzed, determined the structural domain that will delete;
2) project organization territory deletion primer, the carrier that carries Pullulanase encoding gene of take suddenlys change and builds the plasmid vector containing domain deletion mutant as template;
3) mutant plasmid is transformed into host cell;
4) select positive colony and carry out fermentation culture, purifying obtains Pullulanase mutant.
9. method according to claim 8, is characterized in that described plasmid vector is pET series, pGEX series, or any in pUB.
10. method according to claim 9, is characterized in that described host cell is bacterium, yeast and fungal cell.
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