CN104531636B - Mutant of maltogenic amylase and preparation method of mutant - Google Patents
Mutant of maltogenic amylase and preparation method of mutant Download PDFInfo
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- C12Y302/01133—Glucan 1,4-alpha-maltohydrolase (3.2.1.133), i.e. maltogenic alpha-amylase
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Abstract
The invention discloses mutant of maltogenic amylase and a preparation method of the mutant, and belongs to fields of genetic engineering and enzyme engineering. According to the mutant, amino acid residues in the glucoside conversion activity center of maltogenic amylase derived from Bacillus stearothermophilus are substituted. Compared with maltogenic amylase of a parental generation, the maltogenic amylase has a relatively high conversion rate to maltose and a relatively small amount of conversion byproducts. At least one of the following properties of B.stearothermophilus maltogenic amylase mutant is changed: 1) the production period is shortened; 2) the product ratio is increased. Compared with wild maltogenic amylase, the mutant is relatively applicable to the production process of maltose.
Description
Technical field
The present invention relates to a kind of mutant of maltogenic amylase and preparation method thereof, belong to genetic engineering and enzyme engineering neck
Domain.
Background technology
By two glucose units through the bonded reducing disaccharides forming of α-Isosorbide-5-Nitrae glucosides, chemical name is maltose
4-O-D- six ring glucosyl group-D- six ring glucose.Its sugariness is soft, and because of low viscosity, agent of low hygroscopicity and good thermally-stabilised
Property feature, can as food enhancing agent replace dextrose and saccharose, have huge application potential in field of food industry.Industry
The preparation of upper maltose, is with starchiness as raw material, through α-amylase, Fructus Hordei Germinatus (or beta amylase, fungal amylase) hydrolysis
Technique, is obtained a kind of syrup of based on maltose (40%-60%), if maltose content more than 45% (preferably 50% with
On), then it is referred to as high maltose syrup.One of purposes of high maltose syrup is to make the products such as cake, confection in the food industry.
Syrup infusion temperature be far above maltose, generally more than 140 DEG C.Maltose content be more than 70%, even as high as more than 90%, then by
Referred to as superhigh maltose syrup.Maltose is compared glucose and can be avoided blood glucose rise, has for the preparation being applied to antibody, vaccine etc.
There is the application advantage better than glucose.Therefore ultra-high purity maltose syrup field of medicaments application also result in more and more
Concern.
Current maltose production technology is more ripe, when producing maltose using α-amylase and beta amylase, product
Middle maltose content may be up to 90%, and glucose, trisaccharide, tetrose and part oligosaccharide and dextrin are main conversion by-products
Thing.Wherein dextrin and part oligosaccharide, can be removed by ethanol precipitation.The preparation of ultra-high purity maltose, then chromatograph to be passed through
The method such as separate and crystallize to obtain.Because maltose viscosity is big, difficult crystallization, usually require that in crystallization raw material, maltose purity exists
More than 90%, the therefore purity of chromatographic isolation, vital effect is played to maltose crystallization.Chromatographic isolation is substantially removable
Glucose and pentasaccharides and above small molecule saccharide are less to maltose impurities affect.But the trisaccharide in product and tetrose due to
More close with maltose property, often become the major impurity in isolating and purifying, not only directly reduce product purity, return
The moisture of maltose crystallinity, Viscosity of Syrup and final products brings great disadvantage to affect, and makes maltose ultimate yield
Substantially reduce.
Maltogenic amylase has small molecular sugar hydrolysing activity, the small molecular sugar such as hydrolyzable trisaccharide, tetrose, forms glucose
And maltose, therefore in the production of superelevation maltose generally and the compounding use such as α-amylase, beta amylase and pullulanase with
Reduce proportion of by-product, make maltose be more conducive to crystallize.It is reported that, from bacstearothermophilus (Bacillus
Stearothermophilus maltogenic amylase) has higher optimal reactive temperature and relatively low optimum pH reaction bar
Part, can meet more harsh industrial process conditions, maltose ratio in product is improved to 92%, industrially has greatly
Application advantage.But inventor finds in early stage application, the hydrolysing activity simultaneously being had due to this enzyme and turn glycosides activity, in hydrolysis
During small molecular sugar, glycosides can be turned again and produce new trisaccharide and tetrose etc..In order to reduce the generation of by-product in production, often
Need the prolongation response time to make hydrolysis reach balance, on the one hand lead to long the production cycle, efficiency low, when on the other hand reacting
Between prolongation, the maltose being again easily caused generation is re-used formation and turns glycosides by-product.
Therefore, the present invention utilizes protein engineering and enzyme engineering means, reduces this enzyme and turns glycosides activity, reduces in transformation system
Proportion of by-product, make that there is higher maltose production efficiency, give its industrially more preferable application performance.
Content of the invention
The invention provides a kind of mutant of maltogenic amylase, this mutant is B.stearothermophilus
Maltogenic amylase turns the replacement of the related amino acid residue in glycosides active center, compared with the maltogenic amylase of its parental generation, its
There are faster transformation efficiency and less conversion byproducts to maltose.
In the parent amino acid sequence of described B.stearothermophilus maltogenic amylase and ncbi database
B.stearothermophilus maltogenic amylase consensus amino acid sequence (accession number：AAA22233.1).
Described mutant is to be mutated into the tryptophan (Trp) of the 177th in parent's maltogenic amylase gene respectively
Phenylalanine (Phe) and tyrosine (Tyr), are respectively designated as W177F and W177Y.
When mutant enzyme W177F and W177Y of the present invention is respectively used to maltose production, under same enzyme conversion condition, add
Plus mutant enzyme W177F reaches reaction balance in 12h, maltose content rises to 95.79%, in reaction system by 90.49%
Trisaccharide and tetrose are not contained；Add mutant enzyme W177Y and reach reaction balance in 8h, maltose content is risen to by 90.49%
96.10%, do not contain trisaccharide and tetrose in reaction system.And add wild enzymatic conversion and produce maltose, maltose content during 8h
For 93.20%, trisaccharide and four sugared contents are respectively 4.10% and 0.50%；During 12h maltose content be 94.61%, trisaccharide and
Four sugared contents are respectively 2.57% and 0.22%；React and still not up to balance to during 30h, now maltose content is 95.45%,
Trisaccharide and four sugared contents are respectively 0.67% and 0.35%.As can be seen here, add mutant enzyme and produce maltose, can be by reactant
Trisaccharide in system and tetrose are degradable.Add in the reaction system of wild enzyme, three sugared contents with reaction be gradually lowered for
0.67%；Tetrose reaches minimum 0.22% in 12h, but extends with the response time, presents the trend regenerating again.Cause
This mutant enzyme W177F and W177Y, in maltose production application, not only can greatly shorten reacting balance time, improves wheat
Bud sugar production efficiency, the major impurity trisaccharide in simultaneously degradable by-product and tetrose, simplify chromatographic separation and purification mistake
Journey, has potential application advantage in the production of high-purity crystallized maltose.
Brief description
Fig. 1 wild type maltogenic amylase and mutant (W177F and W177Y) produce maltose content during maltose
Change
During Fig. 2 wild type maltogenic amylase and mutant (W177F and W177Y) production maltose, maltotriose contains
Amount change
During Fig. 3 wild type maltogenic amylase and mutant (W177F and W177Y) production maltose, maltotetraose contains
Amount change
Fig. 4 wild type maltogenic amylase and the optimum temperature of mutant (W177F and W177Y)
Fig. 5 wild type maltogenic amylase and 60 DEG C of temperature stabilities of mutant (W177F and W177Y)
Fig. 6 wild type maltogenic amylase and the optimum pH of mutant (W177F and W177Y)
Fig. 7 wild type maltogenic amylase and the pH stability of mutant (W177F and W177Y)
The impact to wild type maltogenic amylase and mutant (W177F and W177Y) Rate activity for Fig. 8 metal ion
Specific embodiment
Embodiment 1：This example illustrates the preparation of wild maltogenic amylase.
(1) structure of maltogenic amylase recombinant bacterium
According to the amyM aminoacid sequence on NCBI, (NCBI numbers：AAA22233.1), by the amyM gene sequence on NCBI
(NCBI numbers row：M36539) carry out codon optimization, synthesize the gene sequence of maltogenic amylase using chemical total synthesis method
Row amyM.Plasmid for building coli expression carrier be pET24a (+).By pET24a (+) plasmid and carry amyM base
The plasmid of cause carries out Nco I and Hind III double digestion respectively, and digestion products, after glue reclaim, connect overnight with T4 ligase, even
Thing of practicing midwifery converts to escherichia coli jm109 competent cell, and converted product coats the LB flat board containing 100mg/L kanamycin,
Through 37 DEG C of overnight incubation, 2 single bacterium colonies of picking on flat board, access LB fluid medium, extracting plasmid checking after 8h, result is just
Really, obtain the amyM/pET24a plasmid being enriched with.Will be thin for plasmid amyM/pET24a conversion e. coli bl21 (DE3) competence
Born of the same parents, picking transformant 37 DEG C of overnight incubation in LB fluid medium (kanamycin containing 100mg/L), preserve glycerol tube, name
For amyM/pET24a/BL21 (DE3).
(2) expression and purification of maltogenic amylase
Raw in LB fluid medium (kanamycin containing 100mg/L) from glycerol tube inoculation amyM/pET24a/BL21 (DE3)
Seed is accessed TB liquid fermentation medium (kanamycin containing 100mg/L) by 5% inoculum concentration by long 8h.Escherichia coli are at 37 DEG C
After culture 2h, add the IPTG of 0.01mM final concentration to be induced, and after 25 DEG C of shaking tables continue cultivation and fermentation 48h, will ferment
Liquid collects fermentation supernatant in 4 DEG C, 8000rpm centrifugation 10min except thalline.
It is slowly added to 50% (NH in supernatant4)2SO4, 4 DEG C stand overnight, 4 DEG C, 8000rpm centrifugation 20min, collect
Precipitation.Redissolve after precipitation with the 20mM citrate buffer solution of pH7.5, dialysed overnight in 20mM citrate buffer solution.Period is more
Change 2-3 buffer.Make loading sample after 0.22 μm of membrane filtration, recombiant protein is carried out using avant protein purification instrument
Purification.Anion-exchange chromatography purification step：(1) balance：Balance DEAE anion exchange with the 20mM buffer of 5 times of volumes
Chromatographic column；(2) loading：The sample anticipated is with the flow velocity loading of 1mL/min；(3) eluting：Flow velocity 1mL/min carries out ladder
Degree eluting, Detection wavelength is 280nm, and Fraction collection contains the eluent of maltogenic amylase enzyme activity.Obtain the wild Fructus Hordei Germinatus of purification
Saccharogenic amylase.
Embodiment 2：This example explanation maltogenic amylase enzyme mutant preparation.
(1) rite-directed mutagenesises
Maltogenic amylase and Bacillus circulans (Bacillus in B.stearothermophilus source
Circulans strain 251) on the basis of cyclodextrin glycosyltransferase (CGTase) sequence alignment analysis originated,
Analysis is simulated to the protein structure of maltogenic amylase it was found that related with turning glycosides activity possibility in maltogenic amylase
Amino acid sites Trp177, Phe188, Ala229, Tyr258.By the tryptophan (Trp) of the 177th in maltogenic amylase
It is mutated into histidine (His) and N (Asn) respectively, be respectively labeled as W177H, W177N；By in maltogenic amylase
The Phenylalanine (Phe) of 188 is mutated into tyrosine (Tyr), is labeled as F188Y；By the third of the 229th in maltogenic amylase
Propylhomoserin (Ala) is mutated into L-Valine (Val), threonine (Thr) and leucine (Leu) respectively, is respectively labeled as A229V, A229T
And A229L；258th tyrosine (Tyr) in maltogenic amylase is mutated into N (Asn) and glutaminase respectively
(Gln), it is respectively labeled as Y258N and Y258Q.
Introduce W177H mutation rite-directed mutagenesis primer be：
Forward primer：5’-GCGATATTTCTAACCATGACGACCGCTACGAA-3 ' (underscore is mutating alkali yl)
Reverse primer：5’-TTCGTAGCGGTCGTCATGGTTAGAAATATCGC-3 ' (underscore is mutating alkali yl)
Introduce W177N mutation rite-directed mutagenesis primer be：
Forward primer：5’-GCGATATTTCTAACAATGACGACCGCTACGAA-3 ' (underscore is mutating alkali yl)
Reverse primer：5’-TTCGTAGCGGTCGTCATTGTTAGAAATATCGC-3 ' (underscore is mutating alkali yl)
Introduce F188Y mutation rite-directed mutagenesis primer be：
Forward primer：5’-CACAGTGGAAAAACTATACCGACCCGGCAG-3 ' (underscore is mutating alkali yl)
Reverse primer：5’-CTGCCGGGTCGGTATAGTTTTTCCACTGTG-3 ' (underscore is mutating alkali yl)
Introduce A229V mutation rite-directed mutagenesis primer be：
Forward primer：5’-GCCTGCGTATCGACGTAGTTAAACATTTCAAC-3 ' (underscore is mutating alkali yl)
Reverse primer：5’-GTTGAAATGTTTAACTACGTCGATACGCAGGC-3 ' (underscore is mutating alkali yl)
Introduce A229T mutation rite-directed mutagenesis primer be：
Forward primer：5’-GCCTGCGTATCGACACAGTTAAACATTTCAAC-3 ' (underscore is mutating alkali yl)
Reverse primer：5’-GTTGAAATGTTTAACTGTGTCGATACGCAGGC-3 ' (underscore is mutating alkali yl)
Introduce A229L mutation rite-directed mutagenesis primer be：
Forward primer：5’-GCCTGCGTATCGACCTGGTTAAACATTTCAAC-3 ' (underscore is mutating alkali yl)
Reverse primer：5’-GTTGAAATGTTTAACCAGGTCGATACGCAGGC-3 ' (underscore is mutating alkali yl)
Introduce Y258N mutation rite-directed mutagenesis primer be：
Forward primer：5’-TTGGCGAATGGAATGGCGATGATCCGGGCA-3 ' (underscore is mutating alkali yl)
Reverse primer：5’-TGCCCGGATCATCGCCATTCCATTCGCCAA-3 ' (underscore is mutating alkali yl)
Introduce Y258Q mutation rite-directed mutagenesis primer be：
Forward primer：5’-TTGGCGAATGGCAAGGCGATGATCCGGGCA-3 ' (underscore is mutating alkali yl)
Reverse primer：5’-TGCCCGGATCATCGCCTTGCCATTCGCCAA-3 ' (underscore is mutating alkali yl)
Using above-mentioned primer, with amyM/pET24a plasmid as template, enter performing PCR reaction.Reaction is all entered in 50 μ L systems
OK, reaction condition is：94 DEG C of denaturations 4min；Subsequently carry out 30 circulations (94 DEG C of 10s, 55 DEG C of 10s, 72 DEG C of 7min20s)；72
DEG C extend 10min；Last 4 DEG C of insulations.PCR primer digests through Dpn I (Fermentas company), converts escherichia coli respectively
JM109 competent cell, converted product coats the LB flat board containing 100mg/L kanamycin, through 37 DEG C of overnight incubation, on flat board
2 single bacterium colonies of picking, access LB fluid medium, extract plasmid after 8h, all correct through sequencing, preserve glycerol tube.
(2) expression and purification of mutant enzyme
Mutant expression and purge process are as described in Example 1.
Embodiment 3：This example explanation maltogenic amylase enzyme activity analysis.
(1) enzyme-activity unit definition
When measuring maltogenic amylase enzyme activity using 3,5- dinitro bigcatkin willow acid system (DNS method), catalysis per minute produces 1 μm of ol
Enzyme amount needed for reducing sugar is as a unit of activity.
(2) enzyme activity determination step
Preheating：The 0.5% soluble starch solution (50mM pH5.5 citrate buffer solution) taking 2mL, in test tube, is placed in
10min is preheated in 60 DEG C of water-baths.
Reaction：Add 0.1mL sample enzyme liquid, shaken well, accurate timing 10min, add 3mL DNS shaken well, put
Enter terminating reaction in frozen water, boiling water bath boils 7min.Cooling.
Measurement：Add distilled water and be settled to 15mL in above-mentioned reaction system, mix.Measure under 540nm wavelength and inhale
Light value simultaneously calculates enzyme activity.
The Rate activity of wild type maltogenic amylase and mutant enzyme is listed in the table below the (ratio by wild type maltogenic amylase
Vigor is set to 100%, and each mutant Rate activity is relative Rate activity with respect to the ratio of wild specific activity of enzyme)：
N.D. represent and be not detected by
Select the mutant (W177H, W177N and F188Y) of Rate activity more than 30% relatively, be applied to maltose production
Preliminary study, investigates its application performance.
Embodiment 4：The method that this example illustrates Production by Enzymes maltose and product detection.
Prepare 2L 20% (w/v) potato starch solution, adjust pH to 5.5, add the acid α-shallow lake of 30U/g dried starch
Powder enzyme (purchased from Jie Neng section), adds pullulanase (purchased from Jie Neng section) and the 10U/g of 24U/g dried starch through injection liquefaction after processing
The beta amylase (extracting from Radix Ipomoeae) of dried starch, 60 DEG C of stir process 24h, carry out a saccharifying.
Then, it is separately added into the wild type maltogenic amylase of 28U/g dried starch in a saccharification react system and dash forward
Variant enzyme W177H, W177N, F188Y.60 DEG C of stirrings, carry out dextrine conversion.React and product composition is measured by sampling to 30h.
In converted product, the content of glucose, maltose, trisaccharide and tetrose adopts HPLC to analyze.The chromatographic condition measuring
It is：Elite 2000HPLC chromatograph, Elite automatic sampler, chromatographic column Thermo APS-2HYPERSIL 13286
(4.6mm × 250mm), HITACHI L-2490 Composition distribution；Mobile phase is 70% (v/v) acetonitrile solution, flow velocity
0.8mL/min；40 DEG C of column temperature.
In reaction system, each constituent content is as shown in the table：
Embodiment 5：This example explanation maltogenic amylase Tyr177 site mutant preparation.
Found by embodiment 4, when mutant enzyme W177H, W177N and F188Y are respectively applied to maltose production, be only mutated
The application effect of body W177H is slightly better than wild enzyme.Mutant enzyme W177H, W177N protein structure is contrasted and molecule
Docking analysis, according to the heterogeneity of aminoacid, designs mutant W177F and W177Y, by the 177th in maltogenic amylase
Tryptophan (Trp) be mutated into Phenylalanine (Phe) and tyrosine (Tyr) respectively, be respectively labeled as W177F and W177Y.
Introduce W177F mutation rite-directed mutagenesis primer be：
Forward primer：5’-GCGATATTTCTAACTTCGACGACCGCTACGAA-3 ' (underscore is mutating alkali yl)
Reverse primer：5’-TTCGTAGCGGTCGTCGAAGTTAGAAATATCGC-3 ' (underscore is mutating alkali yl)
Introduce W177Y mutation rite-directed mutagenesis primer be：
Forward primer：5’-GCGATATTTCTAACTATGACGACCGCTACGAA-3 ' (underscore is mutating alkali yl)
Reverse primer：5’-TTCGTAGCGGTCGTCATAGTTAGAAATATCGC-3 ' (underscore is mutating alkali yl)
Mutation construction, expression and purge process are as described in Example 2.
Embodiment 6：This example explanation maltogenic amylase enzyme activity analysis.
Enzyme activity determination method is as described in Example 3.
Mutant enzyme W177F Rate activity is 1489U/mg, and Rate activity is 39.1% relatively；Mutant enzyme W177F Rate activity
For 2080U/mg, Rate activity relatively is 54.6%.
Embodiment 7：This example illustrates the method that saccharifying of enzyme process produces maltose and product detection.
Prepare 2L 20% (w/v) potato starch solution, adjust pH to 5.5, add the acid α-shallow lake of 30U/g dried starch
Powder enzyme (purchased from Jie Neng section), adds pullulanase (purchased from Jie Neng section) and the 10U/g of 24U/g dried starch through injection liquefaction after processing
The beta amylase (extracting from Radix Ipomoeae) of dried starch, 60 DEG C of stir process 24h.
Product detection method is as described in Example 4.
Through a saccharifying, in reaction system, glucose, maltose, trisaccharide and four sugared contents are respectively 0.43%,
90.49%, 8.09% and 0.99%.
Embodiment 8：This example explanation produces the side of maltose and product detection using wild maltogenic amylase dextrine conversion
Method.
On the basis of embodiment 7, the wild maltogenic amylase of addition 28U/g dried starch in reaction system, 60 DEG C
Stirring.Sample at regular intervals.Draw production process product curve.
Product detection method is as described in Example 4.
React and still not up to balance to 30h, maltose content rises to 95.45% (Fig. 1) by 90.49%.Three sugared contents
Extend with the response time and be gradually lowered, during 30h, be reduced to 0.67% (Fig. 2) by 8.09%.Four sugared contents are reduced to minimum in 12h
0.22%, extend with the response time, tetrose is recombined again, increases in 30h as 0.35% (Fig. 3).
Embodiment 9：The method that this example explanation produces maltose and product detection using mutant enzyme W177F dextrine conversion.
To in reaction system, on the basis of embodiment 7, add the mutant enzyme W177F of 28U/g dried starch, 60 DEG C of stirrings.
Sample at regular intervals.Draw production process product curve.
Product detection method is as described in Example 4.
Reaction has reached balance in 12h, and maltose content rises to 95.79% (Fig. 1) by 90.49%.Saccharifying is produced
In thing, 8.09% trisaccharide and 0.99% tetrose are all by whole degradeds (Fig. 2, Fig. 3).Extend with the response time, each composition in product
Content does not have significant changes.
Embodiment 10：The method that this example explanation produces maltose and product detection using mutant enzyme W177Y dextrine conversion.
To in reaction system, on the basis of embodiment 7, add the mutant enzyme W177Y of 28U/g dried starch, 60 DEG C of stirrings.
Sample at regular intervals.Draw production process product curve.
Product detection method is as described in Example 4.
Reaction has reached balance in 8h, and maltose content rises to 96.10% (Fig. 1) by 90.49%.Saccharification product
In 8.09% trisaccharide and 0.99% tetrose all by whole degradeds (Fig. 2, Fig. 3).Extend with the response time, in product, each composition contains
Amount does not have significant changes.
Embodiment 11：This example illustrates wild maltogenic amylase and mutant (W177F and W177Y) characterization analysis.
(1) optimum temperature of maltogenic amylase and mutant (W177F and W177Y)
With starch as substrate, the citrate buffer solution of 50mM pH5.5, (30 DEG C -80 DEG C) measure than work at different temperatures
Power, Rate activity under optimum temperature is set to 100%, and Rate activity is relative Rate activity with its ratio at each temperature, determines wild enzyme
Optimum temperature with mutant enzyme.
(2) the temperature stability analysis of maltogenic amylase and mutant (W177F and W177Y)
The wild enzyme that obtain purification and mutant enzyme are incubated, at regular intervals by portion under the conditions of pH5.5 and 60 DEG C
Enzyme liquid is divided to take out, rapid cooling, measure Rate activity.Rate activity during 0h is set to 100%, Rate activity and its ratio are that residual ratio is lived
Power.
(3) the optimum pH analysis of maltogenic amylase and mutant (W177F and W177Y)
Prepare the buffer in the buffer replacement enzyme activity determination method of different pH value (pH 4.0-6.5), at 60 DEG C
Measure the mutant Rate activity of wild enzyme.Rate activity under optimum pH is set to 100%, under each pH, Rate activity is relative with its ratio
Rate activity, determines the optimal reaction pH of wild enzyme and mutant enzyme.Buffer system used is respectively：Acetate buffer system (pH
4.0-5.0)；Phosphoric acid buffer system (pH 5.0-6.5)；
(4) the pH stability analyses of maltogenic amylase and mutant (W177F and W177Y)
Enzyme liquid is placed 24h in the buffer of 60 DEG C of different pH value (pH 4.0-6.5), surveys by enzyme activity determination method
Fixed residual ratio is lived.Rate activity under the conditions of pH corresponding during 0h is set to 100%.
(5) impact to maltogenic amylase and mutant (W177F and W177Y) activity for the metal ion
To in 0.1mg/mL enzyme liquid, (citrate buffer solution of 50mM pH5.5) adds the different metal ion of 1mM
(MnCl2, LiCl, CaCl2, BaCl2, MgCl2, CoCl2, KCl, CuSO4, FeSO4, ZnCl2) and EDTA, 60 DEG C of insulation 10min
Afterwards, measure ratio by enzyme activity determination method to live.The Rate activity not added metal ion is set to 100%.
Above-mentioned mutant enzyme W177F and W177Y is compared with wild enzyme, by Fig. 4 and Fig. 5 it is found that mutant enzyme
The optimum temperature of W177F and W177Y and temperature stability have all declined.Mutant enzyme optimum temperature be 50 DEG C, 60 DEG C when ratio
Vigor is more than 80% under optimum temperature.60 DEG C of temperature stabilities of wild enzyme are better than mutant enzyme, but wild enzyme and mutation
Body half life of enzyme is reached more than 75h, all can meet actual production demand.Mutant enzyme body W177F's and W177Y is the most suitable
The impact to Rate activity of pH, pH stability and metal ion is all with wild enzyme like (Fig. 6, Fig. 7 and Fig. 8).
Claims (3)
1. a kind of mutant of maltogenic amylase it is characterised in that the parent amino acid sequence of described maltogenic amylase with
In ncbi database accession number be AAA22233.1 bacstearothermophilus maltogenic amylase consistent, described mutation be by
177th tryptophan sports Phenylalanine, is named as W177F.
2. a kind of mutant of maltogenic amylase it is characterised in that the parent amino acid sequence of described maltogenic amylase with
In ncbi database accession number be AAA22233.1 bacstearothermophilus maltogenic amylase consistent, described mutation be by
177th tryptophan sports tyrosine, is named as W177Y.
3. the preparation method of the mutant described in claim 1 or claim 2, comprises the steps：
1) determine mutational site on the basis of bacstearothermophilus maltogenic amylase enzyme amino acid sequence；Design fixed point is prominent
The mutant primer becoming, is carried out rite-directed mutagenesises for template and is built the matter containing mutant with the carrier carrying maltogenic amylase gene
Grain carrier；
2) mutant plasmid is transformed into host cell；
3) select positive colony and carry out fermentation culture, and purification maltogenic amylase enzyme mutant；
Described plasmid vector is pUC series, pET series, or any one in pGEX；Described host cell is antibacterial or funguses
Cell；Described antibacterial is gram negative bacteria or gram-positive bacterium.
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| CN109371078B (en) * | 2018-10-18 | 2022-03-11 | 山东福田药业有限公司 | Preparation process of high-purity maltose |
| CN109234339B (en) * | 2018-11-22 | 2020-12-29 | 湖南汇升生物科技有限公司 | Production method of high-purity crystalline maltose |
| CN109439641B (en) * | 2018-11-22 | 2020-12-29 | 湖南汇升生物科技有限公司 | Application of maltogenic amylase production strain |
| CN109486791B (en) * | 2018-11-22 | 2020-11-03 | 湖南汇升生物科技有限公司 | Preparation and application of maltogenic amylase mutant |
| CN109321481B (en) * | 2018-11-22 | 2020-12-29 | 湖南汇升生物科技有限公司 | Bacterial strain for producing maltogenic amylase |
| CN109486792B (en) * | 2018-11-22 | 2019-12-10 | 湖南汇升生物科技有限公司 | Preparation and application of maltogenic amylase mutant |
| CN109370973B (en) * | 2018-11-22 | 2020-12-29 | 湖南汇升生物科技有限公司 | Maltogenic amylase producing strain |
| CN109439607B (en) * | 2018-11-22 | 2020-09-04 | 湖南汇升生物科技有限公司 | Application of maltogenic amylase production strain |
| CN109251912B (en) * | 2018-11-22 | 2020-09-04 | 湖南汇升生物科技有限公司 | Method for increasing yield of maltogenic amylase |
| CN110229800B (en) * | 2019-06-11 | 2022-03-15 | 江南大学 | Linear maltooligosaccharide-producing enzyme mutant with improved maltohexaose production capacity |
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