CN104928269A - Maltogenic amylase mutant with low conversion byproducts and mutation method of maltogenic amylase mutant - Google Patents
Maltogenic amylase mutant with low conversion byproducts and mutation method of maltogenic amylase mutant Download PDFInfo
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Abstract
The invention relates to a maltogenic amylase mutant with low conversion byproducts and a production method of the maltogenic amylase mutant and belongs to the fields of genetic engineering and enzyme engineering. The production method includes the amino acid residues on catalytic activity center receptor binding sites of thermophilic fat Bacillus stearothermophilus-sourced maltogenic amylase are replaced. Compared with natural maltogenic amylase, the maltogenic amylase mutant has the advantages that the maltogenic amylase mutant is high in conversion efficiency and low in conversion byproducts during high-maltose syrup preparation. The B. stearothermophilus maltogenic amylase mutant has at least one of the following advantages that preparation cycle is shortened; byproduct proportion is lowered. The maltogenic amylase mutant is more suitable for high-maltose syrup preparation as compared with the natural maltogenic amylase.
Description
Technical field
The present invention relates to a kind of mutant and the mutation method with the maltogenic amylase of low conversion byproducts, belong to genetically engineered and enzyme engineering field.
Background technology
Maltose is the reducing disaccharides connected to form through α-Isosorbide-5-Nitrae glycosidic link by two glucose units, and chemical name is 4-O-D-six ring glucosyl group-D-six ring glucose.Its sugariness is soft, again because of features such as low viscosity, agent of low hygroscopicity and good thermostabilitys, can be used as food enhancing agent and replaces dextrose plus saccharose, have huge application potential in field of food industry.High maltose syrup is the syrup of a kind of based on maltose (maltose content is more than 45%).One of purposes in the food industry makes the product such as cake, candy.Syrup infusion temperature far above maltose, generally more than 140 DEG C.Maltose content is greater than 70%, even up to more than 90%, is then called as superhigh maltose syrup.Glucose compared by maltose can avoid blood sugar increasing, the preparation being applied to antibody, vaccine etc. is had to the application advantage being better than glucose.Therefore the malt syrup of ultra-high purity also result in increasing concern in the application of field of medicaments.
The production of industrial high maltose syrup is raw material with starchiness, and through α-amylase, Fructus Hordei Germinatus (or beta-amylase, fungal amylase) hydrolysis process is obtained.Current comparatively ripe technique, can prepare maltose content at least up to 70% superhigh maltose syrup, glucose, trisaccharide maltose, maltotetrose and part oligose and dextrin are main conversion byproducts.For obtaining more highly purified malt syrup or pure maltose, the malt syrup to Starch Hydrolysis obtains is needed to carry out follow-up product Isolation and purification.Dextrin in reaction product and part oligose are removed by alcohol settling, and the small molecular sugar such as trisaccharide maltose, tetrose, then to be removed by methods such as chromatographic separation.Chromatographic separation can remove glucose and tetrose and above small molecules carbohydrate substantially, but the trisaccharide maltose in product is due to maltose character is comparatively close and content is higher, often become the major impurity in separation and purification, not only directly reduce product purity, the moisture content of returning maltose crystallinity, Viscosity of Syrup and the finished product brings very large disadvantageous effect, and maltose ultimate yield is reduced greatly.Therefore need to obtain the alap original malt syrup of trisaccharide maltose content, to simplify maltose extraction purification step, reduce the production cost of high maltose syrup.
Maltogenic amylase has small molecular sugar hydrolytic activity, the small molecular sugars such as hydrolyzable trisaccharide maltose, form glucose and maltose, therefore in high maltose syrup preparation usually and the composite use such as α-amylase, beta-amylase and Pullulanase to reduce proportion of by-product.It is reported, the maltogenic amylase deriving from bacstearothermophilus (Bacillus stearothermophilus) has higher optimal reactive temperature and lower optimal pH reaction conditions, comparatively harsh industrial process conditions can be met, maltose ratio in product is significantly improved, industrially has great application advantage.But contriver finds in application in early stage, the hydrolytic activity simultaneously had due to this enzyme and turn glycosides activity, in the process of hydrolysis small molecular sugar, can turn again glycosides and generate new trisaccharide.In order to reduce the generation of by product in production, the prolongation reaction times is often needed to make hydrolysis reaction reach balance, cause on the one hand that the production cycle is long, efficiency is low, the prolongation in reaction times on the other hand, easily causes again the maltose generated to be re-used formation and turns glycosides by product.
Therefore, the present invention utilizes protein engineering and enzyme engineering means, and that reduces maltogenic amylase turns glycosides activity, to reduce the proportion of by-product in transformation system, makes this enzyme have higher maltose preparation efficiency, gives its industrially better application performance.
Summary of the invention
The invention provides a kind of mutant of maltogenic amylase, this mutant is the replacement of the amino-acid residue on B.stearothermophilus maltogenic amylase catalytic active center receptor binding site, compared with native malt saccharogenic amylase, in high maltose syrup preparation, there is transformation efficiency and less conversion byproducts faster.
B.stearothermophilus maltogenic amylase consensus amino acid sequence (NCBI numbers: 1QHO_A) in the aminoacid sequence of described maltogenic amylase and ncbi database.
Described mutant is that the tryptophane (Trp) of the 177th in native malt saccharogenic amylase gene has been mutated into phenylalanine (Phe), tyrosine (Tyr), leucine (Leu), aspartic acid (Asn) and Serine (Ser) respectively, called after W177F, W177Y, W177L, W177N and W177S respectively.
Mutant W177F, W177Y, W177L, W177N and W177S of the present invention are respectively used to high maltose syrup when preparing, under same enzyme conversion condition, in end product, maltose content is respectively 90.38%, 90.64%, 91.11%, 91.26% and 91.46%, and trisaccharide maltose content is respectively 1.10%, 1.11%, 0.62%, 0.27% and 0.19%; Mutant W177L, W177N and W177S reaction system reaches molecular balance when 9h.And natural enzyme is applied to high maltose syrup when preparing, in end product, maltose content is 89.34%, and trisaccharide maltose content is 3.15%, substantially reaches molecular balance at 10h.As can be seen here, mutant W177F, W177Y, W177L, W177N and W177S are applied to high maltose syrup when preparing, compare the content that natural enzyme significantly reduces byproduct of reaction trisaccharide maltose, improve maltose preparation efficiency, shorten reacting balance time, significant for summarized chromatogram separation and purification process, in the preparation of superhigh maltose syrup, there is potential application advantage.
Accompanying drawing explanation
Maltose content change in Fig. 1 native malt saccharogenic amylase and Mutant Preparation high maltose syrup process
Trisaccharide maltose content in Fig. 2 native malt saccharogenic amylase and Mutant Preparation high maltose syrup process
The optimum temperuture of Fig. 3 native malt saccharogenic amylase and mutant
60 DEG C of temperature stabilities of Fig. 4 native malt saccharogenic amylase and mutant
The optimal pH of Fig. 5 native malt saccharogenic amylase and mutant
Embodiment
Embodiment 1: this example illustrates the preparation of native malt saccharogenic amylase.
(1) structure of maltogenic amylase recombinant bacterium
According to maltogenic amylase enzyme amino acid sequence (the NCBI numbering: 1QHO_A) in B.stearothermophilus source in ncbi database, carry out codon optimized to the gene order of maltogenic amylase with e. coli codon Preference, adopt chemical total synthesis method to synthesize the gene amyM after optimizing.PET24a (+) for building the plasmid of coli expression carrier.Nco I and Hind III double digestion is carried out respectively by pET24a (+) plasmid with the plasmid of amyM gene, digestion products is after glue reclaims, connect with T4 ligase enzyme and spend the night, connect product conversion to escherichia coli jm109 competent cell, converted product is coated containing 30mgL
-1the LB of kantlex is dull and stereotyped, and through 37 DEG C of overnight incubation, picking 2 single bacterium colonies on flat board, access LB liquid nutrient medium, extracting plasmid checking after 8h, result is correct, obtains the amyM/pET24a plasmid of enrichment.By plasmid amyM/pET24a transformation of E. coli BL21 (DE3) competent cell, picking transformant at LB liquid nutrient medium (containing 30mgL
-1kantlex) in 37 DEG C of overnight incubation, preserve glycerine pipe, called after amyM/pET24a/BL21 (DE3).
(2) expression and purification of maltogenic amylase
From glycerine pipe inoculation amyM/pET24a/BL21 (DE3) in LB liquid nutrient medium (containing 30mgL
-1kantlex) grow 8h, by 5% inoculum size, seed is accessed TB liquid fermentation medium (containing 30mgL
-1kantlex).Intestinal bacteria are cultivated after 2h at 37 DEG C, and the IPTG adding 0.01mM final concentration induces, and after 25 DEG C of shaking tables continue cultivation and fermentation 48h, by fermented liquid in 4 DEG C, 8,000 × g centrifugal 10min except thalline, collect fermentation supernatant.
In supernatant liquor, slowly add 50% ammonium sulfate, 4 DEG C of placements are spent the night, 4 DEG C, 8,000 × g centrifugal 20min, collecting precipitation.After the 20mM phosphoric acid buffer redissolution precipitation of pH 7.5, dialysed overnight in 20mM phosphoric acid buffer.Period changes 2-3 damping fluid.By making loading sample after 0.22 μm of membrane filtration, avant protein purification instrument is adopted to carry out the purifying of recombinant protein.Anion-exchange chromatography purification step: (1) balances: with the 20mM damping fluid balance DEAE anion-exchange column of 5 times of volumes; (2) loading: the sample of anticipating is with 1mLmin
-1flow velocity loading; (3) wash-out: flow velocity 1mLmin
-1carry out gradient elution, determined wavelength is 280nm, and Fraction collection is containing the elutriant of maltogenic amylase enzyme activity.Obtain the native malt saccharogenic amylase of purifying.
Embodiment 2: this example illustrates maltogenic amylase Mutant Preparation.
(1) rite-directed mutagenesis
On the basis of cyclomaltodextrin glucanotransferase (CGTase) sequence alignment analysis that the maltogenic amylase in B.stearothermophilus source and Bacillus circulans (B.circulans strain 251) are originated, model analysis is carried out to the protein structure of maltogenic amylase, chooses the amino acid design sudden change of maltogenic amylase enzyme active center receptor binding site.The tryptophane (Trp) of maltogenic amylase the 177th is mutated into phenylalanine (Phe), tyrosine (Tyr), leucine (Leu), aspartic acid (Asn) and Serine (Ser) respectively, is labeled as W177F, W177Y, W177L, W177N and W177S respectively.
The rite-directed mutagenesis primer introducing W177F sudden change is:
Forward primer: 5 '-GCGATATTTCTAAC
tTCgACGACCGCTACGAA-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-TTCGTAGCGGTCGTC
gAAgTTAGAAATATCGC-3 ' (underscore is mutating alkali yl)
The rite-directed mutagenesis primer introducing W177Y sudden change is:
Forward primer: 5 '-GCGATATTTCTAAC
tATgACGACCGCTACGAA-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-TTCGTAGCGGTCGTC
aTAgTTAGAAATATCGC-3 ' (underscore is mutating alkali yl)
The rite-directed mutagenesis primer introducing W177L sudden change is:
Forward primer: 5 '-GCGATATTTCTAAC
cTAgACGACCGCTACGAA-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-TTCGTAGCGGTCGTC
tAGgTTAGAAATATCGC-3 ' (underscore is mutating alkali yl)
The rite-directed mutagenesis primer introducing W177N sudden change is:
Forward primer: 5 '-GCGATATTTCTAAC
aATgACGACCGCTACGAA-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-TTCGTAGCGGTCGTC
aTTgTTAGAAATATCGC-3 ' (underscore is mutating alkali yl)
The rite-directed mutagenesis primer introducing W177S sudden change is:
Forward primer: 5 '-GCGATATTTCTAAC
tCCgACGACCGCTACGAA-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-TTCGTAGCGGTCGTC
gGAgTTAGAAATATCGC-3 ' (underscore is mutating alkali yl)
Utilize above-mentioned primer, with amyM/pET24a plasmid for template, carry out PCR reaction.Reaction is all carried out in 50 μ L systems, and reaction conditions is: 94 DEG C of denaturation 4min; Carry out subsequently 30 circulations (94 DEG C, 10s; 55 DEG C, 10s; 72 DEG C, 7min 20s); 72 DEG C extend 10min; Last 4 DEG C of insulations.PCR primer is through Dpn I (Fermentas company) digestion, and transformation of E. coli JM109 competent cell respectively, converted product is coated containing 30mgL
-1the LB of kantlex is dull and stereotyped, through 37 DEG C of overnight incubation, and picking 2 single bacterium colonies on flat board, access LB liquid nutrient medium, extracting plasmid after 8h, all correct through order-checking, preserve glycerine pipe.
(2) expression and purification of mutant
Mutant Expression and purification process as described in Example 1.
Embodiment 3: this example illustrates native malt saccharogenic amylase and mutant Determination of Kinetic Parameters.
Respectively with the trisaccharide maltose solution of the phosphoric acid buffer of pH 5.5 preparation different concns (0.1,0.25,0.5,1.0,2.0,4.0,6.0,8.0,10.0 and 12.5mgmL
-1) as reaction substrate.Get the trisaccharide maltose solution of 500 μ L different concns in test tube, 60 DEG C of water-bath preheating about 10min.Add the enzyme liquid sample that 500 μ L dilute, concussion mixing, reaction 10min.Add 1mL 0.06N NaOH termination reaction.With the glucose content in glucose sensor assaying reaction system.Per minute catalysis produces enzyme amount needed for 1 μm of ol glucose as a unit of activity.The trisaccharide maltose recorded under different concentration of substrate is initially hydrolyzed vigor, adopts the non-linear regression in GraphPad Prism 5.0 software to carry out matching to data, obtains the K of Michaelis (Michaelis-Menten) equation respectively
mand V
maxvalue, then calculate k
catand k
cat/ K
mvalue.
Embodiment 4: this example illustrates that the method for high maltose syrup and product detection is prepared in an enzyme process saccharification.
10% (m/v) yam starch is suspended in 50mM phosphoric acid buffer (pH 4.5), and in boiling water bath, continuously stirring 1.5min makes starch pasting.Add 10Ug
-1dS high temperature acid alpha-amylase (purchased from Jie Neng section), in boiling water bath after continuously stirring 4min, adds 1M hydrochloric acid by below pH regulator to 4.0, to stop liquefaction.Liquefied starch is cooled to room temperature, regulates pH to 5.2, add 15Ug
-1dS beta-amylase (purchased from Jie Neng section) and 1Ug
-1dS Pullulanase (purchased from Jie Neng section), reacts 24h (rotating speed 150rpm) in 60 DEG C of shaking baths.Sample at regular intervals.
Sample boils 10min with termination reaction in boiling water bath, and 10,000 × g centrifugal 10min get supernatant and carry out HPLC analysis.Analysis condition is: Elite 2000HPLC chromatographic instrument, Elite automatic sampler, chromatographic column Thermo APS-2HYPERSIL13286 (4.6mm × 250mm), HITACHI L-2490 Composition distribution; Moving phase is 75% (v/v) acetonitrile solution, flow velocity 0.8mLmin
-1; Column temperature 40 DEG C.
Through a saccharification, in reaction system, the content of the above small molecular sugar of glucose, maltose, trisaccharide and trisaccharide is respectively 0.52%, 85.78%, 10.46% and 3.25%.
Embodiment 5: this example illustrates the method utilizing native malt saccharogenic amylase dextrine conversion to prepare high maltose syrup and product detection.
On the basis of embodiment 4, by reaction solution pH regulator to 5.5, in reaction system, add 6.67 μ gg
-1dS maltogenic amylase, reacts (rotating speed 150rpm) in 60 DEG C of shaking baths.Sample at regular intervals.
Product detection method as described in Example 4.
Reaction reaches balance substantially to about 10h, and maltose content rises to 89.34% (Fig. 1) by 85.78%.Trisaccharide content extends with the reaction times and reduces gradually, and 10h reduces to 3.15% (Fig. 2).
Embodiment 6: this example illustrates the method utilizing mutant W177F dextrine conversion to prepare high maltose syrup and product detection.
On the basis of embodiment 4, by reaction solution pH regulator to 5.5, in reaction system, add 6.67 μ gg
-1dS mutant W177F, reacts (rotating speed 150rpm) in 60 DEG C of shaking baths.Sample at regular intervals.
Product detection method as described in Example 4.
Reaction reaches balance substantially at 10h, and maltose content rises to 90.38% (Fig. 1) by 85.78%.Trisaccharide content extends with the reaction times and reduces gradually, and 10h reduces to 1.10% (Fig. 2).
Embodiment 7: this example illustrates the method utilizing mutant W177Y dextrine conversion to prepare high maltose syrup and product detection.
On the basis of embodiment 4, by reaction solution pH regulator to 5.5, in reaction system, add 6.67 μ gg
-1dS mutant W177Y, reacts (rotating speed 150rpm) in 60 DEG C of shaking baths.Sample at regular intervals.
Product detection method as described in Example 4.
Reaction reaches balance substantially at 10h, and maltose content rises to 90.64% (Fig. 1) by 85.78%.Trisaccharide content extends with the reaction times and reduces gradually, and 10h reduces to 1.11% (Fig. 2).
Embodiment 8: this example illustrates the method utilizing mutant W177L dextrine conversion to prepare high maltose syrup and product detection.
On the basis of embodiment 4, by reaction solution pH regulator to 5.5, in reaction system, add 6.67 μ gg
-1dS mutant W177L, reacts (rotating speed 150rpm) in 60 DEG C of shaking baths.Sample at regular intervals.
Product detection method as described in Example 4.
Reaction reaches balance substantially at 9h, and maltose content rises to 91.11% (Fig. 1) by 85.78%.Trisaccharide content extends with the reaction times and reduces gradually, and 10h reduces to 0.62% (Fig. 2).
Embodiment 9: this example illustrates the method utilizing mutant W177N dextrine conversion to prepare high maltose syrup and product detection.
On the basis of embodiment 5, by reaction solution pH regulator to 5.5, in reaction system, add 6.67 μ gg
-1dS mutant W177N, reacts (rotating speed 150rpm) in 60 DEG C of shaking baths.Sample at regular intervals.
Product detection method as described in Example 4.
Reaction reaches balance substantially at 9h, and maltose content rises to 91.26% (Fig. 1) by 85.78%.Trisaccharide content extends with the reaction times and reduces gradually, and 10h reduces to 0.27% (Fig. 2).
Embodiment 10: this example illustrates the method utilizing mutant W177S dextrine conversion to prepare high maltose syrup and product detection.
On the basis of embodiment 4, by reaction solution pH regulator to 5.5, in reaction system, add 6.67 μ gg
-1dS mutant W177S, reacts (rotating speed 150rpm) in 60 DEG C of shaking baths.Sample at regular intervals.
Product detection method as described in Example 4.
Reaction reaches balance substantially at 9h, and maltose content rises to 91.46% (Fig. 1) by 85.78%.Trisaccharide content extends with the reaction times and reduces gradually, and 10h reduces to 0.19% (Fig. 2).
Embodiment 11: this example illustrates native malt saccharogenic amylase and mutant characterization analysis.
(1) optimum temperuture of native malt saccharogenic amylase and mutant
Take final concentration as 10mgmL
-1trisaccharide maltose be substrate, at different temperatures (30 DEG C-80 DEG C) measure Rate activity, Rate activity under optimum temperuture is set to 100%, and Rate activity and its ratio are the vigor of comparing at each temperature, determine the optimum temperuture of natural enzyme and mutant.
(2) 60 DEG C of temperature stability analyses of native malt saccharogenic amylase and mutant
The natural enzyme obtained by purifying and mutant are incubated under pH 5.5 and 60 DEG C of conditions, and taken out by part enzyme liquid at regular intervals, cooling rapidly, take final concentration as 10mgmL
-1trisaccharide maltose be substrate measure Rate activity.Rate activity during 0h is set to 100%, and the Rate activity of other each time points and its ratio are residual Rate activity.
(3) the optimal pH analysis of maltogenic amylase and mutant
Use the phosphoric acid buffer of different pH value (pH 4.0-6.5) to dissolve trisaccharide maltose respectively, preparation final concentration is 10mgmL
-1trisaccharide maltose solution, under 60 DEG C of conditions, measure the Rate activity of natural enzyme and mutant.Rate activity under optimal pH is set to 100%, and under each pH, Rate activity and its ratio are the vigor of comparing, and determine the optimal reaction pH of natural enzyme and mutant.
Said mutation body W177F, W177Y, W177L, W177N and W177S are compared with natural enzyme, can be found by Fig. 3, except mutant W177Y, the optimum temperuture of other mutant is 60 DEG C, consistent with natural enzyme.The optimum temperuture of mutant W177Y is 50 DEG C, but at 60 DEG C, still remain the enzyme activity of 94.2%.60 DEG C of temperature stabilities of natural enzyme are better than mutant, but the transformation period of natural enzyme and mutant reaches more than 100h all, all can meet actual production demand (Fig. 4).The optimal pH of natural enzyme and mutant is 5.5, remains the maximum enzyme vigor (Fig. 5) of more than 70% within the scope of the pH of pH 5.0 to 6.0.
Claims (6)
1. the mutant of a maltogenic amylase, it is characterized in that, the parent amino acid sequence of described maltogenic amylase is consistent with the bacstearothermophilus maltogenic amylase being numbered 1QHO_A in ncbi database, described sudden change is that the 177th tryptophane is sported phenylalanine, called after W177F.
2. the mutant of a maltogenic amylase, it is characterized in that, the parent amino acid sequence of described maltogenic amylase is consistent with the bacstearothermophilus maltogenic amylase being numbered 1QHO_A in ncbi database, described sudden change is that the 177th tryptophane is sported tyrosine, called after W177Y.
3. the mutant of a maltogenic amylase, it is characterized in that, the parent amino acid sequence of described maltogenic amylase is consistent with the bacstearothermophilus maltogenic amylase being numbered 1QHO_A in ncbi database, described sudden change is that the 177th tryptophane is sported leucine, called after W177L.
4. the mutant of a maltogenic amylase, it is characterized in that, the parent amino acid sequence of described maltogenic amylase is consistent with the bacstearothermophilus maltogenic amylase being numbered 1QHO_A in ncbi database, described sudden change is that the 177th tryptophane is sported aspartic acid, called after W177N.
5. the mutant of a maltogenic amylase, it is characterized in that, the parent amino acid sequence of described maltogenic amylase is consistent with the bacstearothermophilus maltogenic amylase being numbered 1QHO_A in ncbi database, described sudden change is that the 177th tryptophane is sported Serine, called after W177S.
6. the preparation method of the mutant described in claim 1-5, comprises the steps:
1) on the basis of bacstearothermophilus maltogenic amylase enzyme amino acid sequence, mutational site is determined; The mutant primer of design rite-directed mutagenesis, carries out rite-directed mutagenesis with the carrier carrying maltogenic amylase gene for template and also builds the plasmid vector containing mutant;
2) mutant plasmid is transformed into host cell;
3) select positive colony and carry out fermentation culture, and purifying maltogenic amylase enzyme mutant;
Described plasmid vector is pUC series, pET series, or any one in pGEX; Described host cell is bacterium and fungal cell; Described bacterium is gram negative bacterium or gram positive bacterium.
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|---|---|---|---|---|
| CN109251912A (en) * | 2018-11-22 | 2019-01-22 | 湖南汇升生物科技有限公司 | A method of improving maltogenic amylase production of enzyme |
| CN109370973A (en) * | 2018-11-22 | 2019-02-22 | 湖南汇升生物科技有限公司 | A kind of maltogenic amylase production bacterial 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 |
| CN109370973B (en) * | 2018-11-22 | 2020-12-29 | 湖南汇升生物科技有限公司 | Maltogenic amylase producing strain |
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