CN102965360B - Alkaline amylase mutant with high catalytic efficiency as well as preparation method and application thereof - Google Patents
Alkaline amylase mutant with high catalytic efficiency as well as preparation method and application thereof Download PDFInfo
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- CN102965360B CN102965360B CN201210533264.4A CN201210533264A CN102965360B CN 102965360 B CN102965360 B CN 102965360B CN 201210533264 A CN201210533264 A CN 201210533264A CN 102965360 B CN102965360 B CN 102965360B
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Abstract
The invention discloses an alkaline amylase mutant with a high catalytic efficiency as well as a preparation method and an application thereof, belonging to the field of genetic engineering. According to the invention, Bacillus alcalophilus JN21 (CCTCC NO: M2011231) amylase is taken as a female parent, and the sequence of the Bacillus alcalophilus amylase is subjected to oligopeptide fusion expression by using a molecular biological technology. Under such modification condition, the Bacillus alcalophilus amylase is improved by 4.1 times compared with a control case (before mutation) in terms of specific enzyme activity, and is improved by 3.5 times compared with the control case (before mutation) in terms of catalytic efficiency. With the adoption of the strategy, the catalytic efficiency of the amylase can be improved greatly, and a foundation is laid for the application of the amylase in industrial production fields such as textile desizing, detergents and additives. The strategy has an important guiding significance in improving the catalytic efficiency of the amylase and other enzymes.
Description
Technical field
The present invention relates to a kind of high catalytic efficiency amylase mutant and preparation method thereof.
Background technology
α-amylase (EC3.2.1.1) is a kind of important industrial enzymes of degraded starch, is mainly used in the fields such as food, weaving, medicine, washing composition.Scale resistance amylase can be used for textiles destarch, detergent additives and with starch, makes viscosity modifier of binding agent etc.First Horikoshi has reported the alkali starch enzyme that originates from basophilic bacterium A-40-2 in 1971.The people such as Saxena in 2007 screen the bacterial strain that a strain can be produced alkali starch enzyme.(the Pancha I such as Pancha in 2010, Jain D, ShrivastavA, Mishra S, Shethia B, Mishra S, VP M, Jha B.A thermoactive[alpha]-amylase from a Bacillus sp.isolated fromCSMCRI salt farm.International Journal of Biological Macromolecules, 2010,47 (2): 288-291.) filter out a strain and produce heatproof amylase strain.At present, domestic pre-treatment zymin market is monopolized by Denmark Novozymes Company and U.S. Genencor Company substantially.A kind of amylase has been introduced by Novozymes Company, has wider pH and temperature range.The amylase with chemically-resistant oxidisability is mainly used in the industry such as detergent additives and weaving destarch single bath process.But the diastatic suitability for industrialized production of scale resistance is report not.The acquisition that scale resistance amylase is produced bacterial strain mainly obtains by screening, mutagenesis and enzyme molecular modification.The blindness of screening is larger, is not easy to obtain object bacterial strain.Mutagenesis comprises: spontaneous mutation and mutagenesis, and the probability of spontaneous mutation is quite little, and the workload probability larger and the negative sudden change of uncontrollable appearance of mutagenesis is larger.Enzyme molecular modification purpose is strong, and for enzyme molecule concrete structure, analysis is transformed, and reaches the object of scale resistance.
Summary of the invention
The invention provides a kind of high catalytic efficiency amylase mutant, is that diastatic N end regions is connected the mutant obtaining by PT-linker with small peptide.
Described small peptide aminoacid sequence is as shown in SEQ ID NO.1.
Described amylase aminoacid sequence is as shown in SEQ ID NO.2.
Described PT-linker sequence is as shown in SEQ ID NO.3.
The present invention is also to provide a kind of method of preparing described amylase sudden change, and its technical scheme is as follows:
1), according to short peptide sequence, adopt the complete synthesis complete synthesis rear clone of method of chemistry in plasmid pET-22b (+), construction recombination plasmid pET-22b (+)-P;
2), according to the amylase sequence of Alkaliphilic bacillus (Bacillus alcalophilus), adopt the complete synthesis method of chemistry complete synthesis;
3) for the design of analytical sequence primer, complete synthesis amylase sequence is cloned in recombinant plasmid pET-22b (+)-P, the recombinant plasmid pAAQ-P that structure contains small peptide and amylase fragment, small peptide is fused to the N-end of alkali starch enzyme, obtains the recombinant vectors that contains sudden change amylase sequence and short peptide sequence fusion state;
4) will suddenly change after recombinant vectors transform e. coli bl21, abduction delivering, obtains mutant strain.
5) measure mutant strain and express diastatic catalytic efficiency.
Amylase mutant catalytic efficiency provided by the invention is high, and under this transformation condition, Alkaliphilic bacillus amylase is lived and improved 4.1 times than the enzyme ratio enzyme than contrast (before sudden change) example alive; Catalytic efficiency improves 3.5 times than the catalytic efficiency of contrast (before sudden change) example.With respect to adopting the means such as sieve bacterium or mutagenesis, shortened the zymologic property transformation time.By this high catalytic efficiency amylase mutant fields such as destarch, washing composition interpolation that are applied to weave, can, at efficient degradation starch, have broad application prospects.
Accompanying drawing explanation
The plasmid map of Fig. 1: pAAQ-P.
Fig. 2: amylase 3d space structure.
Embodiment
Embodiment 1: the high catalytic efficiency of amylase improves mutation analysis and method
By amylase 3d space structure (Fig. 2) is analyzed, definite area A(catalysis region), region B, region C.Meanwhile, catalysis region (catalysis region) is analyzed and found 3 avtive spots (Asp238, Glu268, Asp330).According to diastatic aminoacid sequence and structural dependence, select small peptide and alkali starch enzyme sequence to carry out amalgamation and expression, improve its catalytic efficiency.
According to short peptide sequence, adopt the complete synthesis complete synthesis rear clone of method of chemistry in plasmid pET-22b (+), construction recombination plasmid pET-22b (+)-P.And then according to the amylase sequence of Alkaliphilic bacillus (Bacillus alcalophilus), adopt the complete synthesis method of chemistry complete synthesis.For the design of analytical sequence primer, complete synthesis amylase sequence is cloned in recombinant plasmid pET-22b (+)-P, the recombinant plasmid pAAQ-P that structure contains small peptide and amylase fragment, small peptide is fused to the N-end of alkali starch enzyme, obtains the recombinant vectors that contains sudden change amylase sequence and short peptide sequence fusion state.After sudden change, recombinant vectors transforms e. coli bl21, and abduction delivering obtains mutant strain.By recombinant plasmid transformed escherichia coli host BL21, carry out abduction delivering, obtain the restructuring amylase after scale resistance sudden change.
Embodiment 2: before and after merging, amylase compares enzyme activity determination
DNS method is measured alkali starch enzyme enzyme and is lived
1) configuration of DNS reagent: take 2.5g3,5-dinitrosalicylic acid is dissolved in a small amount of water, add 0.5g phenol, dissolve again 0.075g S-WAT, 2.5g sodium hydroxide, 50g Seignette salt, proceeded in 500mL volumetric flask and shaken up constant volume, be stored in brown bottle and be placed in 4 ° of C refrigerators stand-by.
2) making of maltose typical curve: the maltose solution of preparation 0.2g/L-1.0g/L different concns.Get the maltose of 1mL different concns and the DNS solution of same volume mixes, put into boiling water bath, water-bath 10min.Cooling with cold water, be settled to 10mL, A
540measure light absorption value.The concentration of maltose of take is X-coordinate, take light absorption value as ordinate zou, production standard curve.
3) Zulkovsky starch of 2mL1% is joined in test tube, add the damping fluid of 1mL, mix, 55 ° of C preheating 5min, the enzyme liquid that adds 0.4mL to dilute, reaction 5min.The DNS reagent of getting 1mL reaction solution and same volume mixes, and boiling water bath boils 10min, cooling with cold water, is settled to 10mL, after mixing, there is no enzyme-added liquid but add equivalent deionized water reaction system in contrast, measure A
540light absorption value.
Amylase compares enzyme activity determination
By after alkali starch enzyme purification, diastatic protein concentration before and after utilizing Bradford method to measure to merge after purifying.By measuring the amylase of protein concentration, utilize 3) method mensuration enzyme activity.Utilize enzyme activity unit to compare with amylase protein concentration, obtain diastatic more alive than enzyme.After fusion, Alkaliphilic bacillus amylase is lived and is improved 4.1 times than the enzyme ratio enzyme than contrast (before sudden change) example alive.This catalyzed by amylase efficiency obtains greatly and improves, at efficient degradation starch more.
Enzyme activity unit definition: at pH10.0,55 ° of C of temperature, produce the needed enzyme amount of 1 μ g reducing substance (calculating with maltose) at 1min degraded Zulkovsky starch, are 1 Ge Meihuo unit (U).
Embodiment 3: catalyzed by amylase efficiency test before and after merging
By after the restructuring amylase purifying merging after small peptide, adopt pH10.0 glycine-sodium hydrate buffer solution, damping fluid and Zulkovsky starch are mixed, adopt 3) method mensuration amylase enzyme activity.By after alkali starch enzyme purification, diastatic protein concentration before and after utilizing Bradford method to measure to merge after purifying.Utilize Eadie-hofstee curve method, determine the maximum speed of reaction V of alkali starch enzyme
max.To compare in protein concentration corresponding to enzyme, obtain diastatic catalytic efficiency constant (k
cat).After small peptide merges, catalytic efficiency is than the catalytic efficiency (k of contrast (before sudden change) example
cat) improve 3.5 times.This catalyzed by amylase efficiency obtains greatly and improves, can efficient degradation starch.
Claims (1)
1. a high catalytic efficiency alkali starch enzyme mutant, is characterized in that, the amylase N end regions of aminoacid sequence as shown in SEQ ID NO.2 is connected with the small peptide of aminoacid sequence as shown in SEQ ID NO.1 the mutant obtaining by PT-linker; Described PT-linker sequence is as shown in SEQ ID NO.3.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4935351A (en) * | 1984-07-27 | 1990-06-19 | Kunio Yamane | Process for preparing oligopeptide |
| CN102533697A (en) * | 2011-12-16 | 2012-07-04 | 江南大学 | Oxidation-resistant amylase mutant and preparation method and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4935351A (en) * | 1984-07-27 | 1990-06-19 | Kunio Yamane | Process for preparing oligopeptide |
| CN102533697A (en) * | 2011-12-16 | 2012-07-04 | 江南大学 | Oxidation-resistant amylase mutant and preparation method and application thereof |
Non-Patent Citations (8)
| Title |
|---|
| Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli;Wu et al;《Microbial Cell Factories》;20111230;第10卷;全文 * |
| Lei et al.Streamlined protein expression and purification using cleavable self-aggregating tags.《Microbial Cell Factories》.2011,第10卷全文. |
| Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane;Zhang et al;《PNAS》;19931231;第90卷;全文 * |
| Streamlined protein expression and purification using cleavable self-aggregating tags;Lei et al;《Microbial Cell Factories》;20111230;第10卷;全文 * |
| Wu et al.Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli.《Microbial Cell Factories》.2011,第10卷全文. |
| Zhang et al.Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane.《PNAS》.1993,第90卷全文. |
| 杨海泉等.碱性淀粉酶的发酵生产及其应用研究进展.《生物工程学报》.2012,第28卷(第4期),全文. |
| 碱性淀粉酶的发酵生产及其应用研究进展;杨海泉等;《生物工程学报》;20120425;第28卷(第4期);全文 * |
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