CN103695387B - Saccharomonospora viridis maltose alpha-amylase mutant and application thereof - Google Patents
Saccharomonospora viridis maltose alpha-amylase mutant and application thereof Download PDFInfo
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- CN103695387B CN103695387B CN201310703005.6A CN201310703005A CN103695387B CN 103695387 B CN103695387 B CN 103695387B CN 201310703005 A CN201310703005 A CN 201310703005A CN 103695387 B CN103695387 B CN 103695387B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01133—Glucan 1,4-alpha-maltohydrolase (3.2.1.133), i.e. maltogenic alpha-amylase
Abstract
The invention relates to a maltose alpha-amylase mutant and application thereof. The nucleotide sequence of the maltose alpha-amylase mutant is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO:2. The mutant is obtained by performing protein engineering reconstruction and screening on maltose alpha-amylase from Saccharomonospora viridis. The application of the mutant enzyme in starch hydrolysis ensures that the maltose content in the hydrolyzed starch product is increased, thereby being beneficial to malt syrup production. By singly using the maltose alpha-amylase mutant for starch hydrolysis, the maltose content of the obtained syrup is 72% or above; and the invention has a potential of favorably lowering the production cost.
Description
Technical field
The present invention relates to enzyme engineering and genetically engineered field, specifically the mutant of the sugared sporangium maltogenic alpha-amylase enzyme of a kind of green and application thereof.
Background technology
Malt syrup utilizes purified starch for raw material, and after zymin liquefaction, saccharification, through refining, concentrated a kind of starch syrup, its composition is mainly Fructus Hordei Germinatus disaccharides (>=50%), glucose, trisaccharide maltose, maltotetrose and tetrose with first-class.In malt syrup, the boundary regulation of maltose content is not strict, in usual maltose, the content of maltose is 40 ~ 50%, have also up to 60%, in high maltose syrup, the content of maltose is 50 ~ 70%, in superhigh maltose syrup, maltose content is greater than 70%, current industrial production syrup output is comparatively large, and commonplace be high maltose syrup.
In maltose production development historical process, the product of main experience has three kinds: one to be common malt syrup, and maltose content is between 40 ~ 50%; Two is high maltose syrups, and maltose content is between 50 ~ 70%; Three is superhigh maltose syrups, and maltose content is more than 70%.In malt syrup, glucose is easy to carbonize coloured effect to the quality of malt syrup and visual appearance, along with people are to the quality of maltose, particularly the requirement of the content of maltose is more and more higher, and the maltose product that modern enzyme industrial expansion more makes superhigh maltose syrup become main at present with application.The requirement of high-quality superelevation Fructus Hordei Germinatus syrup is that maltose content is high in the world, grape content is low, especially for the malt syrup of special industry, as higher in the requirement of deep processing to the content of maltose and the content of glucose for pharmaceutical prod and maltose alcohol.
The production technique of current superhigh maltose syrup is generally first utilize common α-amylase starch controlling to be not exclusively hydrolyzed the dextrin generating certain DE value, recycle one or both debranching factors and α-amylase acting in conjunction reduces the content of oligosaccharides to obtain superhigh maltose syrup, its be exactly in fact utilize multienzyme to be hydrolyzed further to reduce to greatest extent glucose and and the generation of oligosaccharides to improve the content of maltose to greatest extent, but produce high to the requirement of equipment in reality, need to control accurately to production technique.In actual production, need the reaction conditions to producing to control closely, should ensure that starch is fully hydrolyzed into limit dextrin and avoids the excessively too much glucose of hydrolysis generation could obtain higher maltose output again, this manufacturing technique requirent is closely the gordian technique of the restriction maltose quality of production and cost, is also that domestic a lot of general enterprises is very inaccessible.The key that ultrapure high malt sugar is produced has some: the DE value 1. controlling liquefier; 2. multiple enzyme synergy; 3. go out rapidly after saccharification enzyme, and object makes a diastatic malt sugar content reach 80%, for good basis established by subsequent production crystallization or injection maltose.While producing maltose, also with more glucose, follow-up technique can be had influence on owing to using α-amylase.Fungal alpha-amylase the most frequently used at present uses and is used alone the malt syrup can only producing 40 ~ 50%, and more than 80% ultrapure malt syrup will be produced and produce crystalline-maltose syrup, need to use beta-amylase, and be auxiliary de-the maltose that could to obtain more than 80% with the efficiency increasing beta-amylase with Pullulanase.Current industrial beta-amylase mainly extracts from barley, and cost is high, expensive and vigor is lower, constrains the fast development of maltose industry.Therefore maltose finds simpler production technique in producing at present, improves the controllability of production technique, improves the content of maltose, is the key reducing maltose production cost.
About Patents and the research report of the α-amylase for the production of maltose, the U.S. two patent U.S Patent4604355 and U.S Patent:6274355 are the patents of preparation method about maltogenic amylase (maltogenic amylase enzyme) and use, this enzyme is by Novozymes Company's commercialization, the direct product of this enzyme converted starch is not maltose but more than trisaccharide maltose high malt sugar oligosaccharides, fewer than the glucose of the easy control of fungal alpha-amylase and generation, so just Starch Conversion can be become high malt sugar syrup further combined with the acting in conjunction of other debranching factor.Petr í cek M also reports the new bud sugar alpha-amylase gene of of being cloned into from Thermomonospora curvata, can hydrolyzed starch generate more than 50% maltose and a small amount of glucose (Petr í cek M,
p, Kuncov á M.Characterization of thealpha-amylase-encoding gene from Thermomonospora curvata.Gene.1992,112 (1): 77-83), YangCH also reports and is cloned into a new bud sugar alpha-amylase gene from Thermobifida fusca, the maltose of more than 50% and a small amount of glucose can be generated for hydrolyzed starch, its homology and above Petr í cek M report have 98% homology (YangCH, Liu WH.Cloning and characterization of a maltotriose-producing alpha-amylase gene fromThermobifida fusca.J Ind Microbiol Biotechnol.Epub, 2007,34 (4): 325-330), within 2002, Ammar YB reports from the separation and purification of Streptomyces sp Pseudomonas to a kind of novel maltogenic alpha-amylase enzyme (Maltose-forming α-Amylase), Starch Conversion just can be become the maltose of 58% by this enzyme in 4 hours when converted starch, the trisaccharide maltose of 27.5%, the maltotetrose of 14.5% and pentasaccharides, and glucose (Ammar Y B can not be produced, Matsubara T, Ito K, Iizuka M, Limpaseni T, Pongsawasdi P, Minamiura N.New action pattern of a maltose-formingalpha-amylase from Streptomyces sp.and its possible application in bakery.J Biochem Mol Biol.2002, 35 (6): 568-575), its amylatic characteristic seemingly a kind of very promising maltogenic alpha-amylase enzyme, unfortunately it does not have the report of Cloning of Genes Related.The domestic report about maltose alpha-amylase gene only slanders the reports such as nanmu are cloned into maltogenic alpha-amylase gene from Bacillus licheniformis, its amylatic product mainly maltose and glucose (slanders nanmu, Shen Wei, stone Kweiyang, Wang Zhengxiang. the gene clone of Bacillus licheniformis maltogenic alpha-amylase and qualification. apply and environmental organism journal .2009,15 (1): 130 ~ 133).I am also cloned into a maltose alpha-amylase gene from streptomyces thermophilus (Streptomyces thermoviolaceus) Pseudomonas, can the hydrolyzed starch content that generates maltose reach 50% syrup (Chinese Patent Application No.: 201010045645.9).
Increasing new enzyme is applied to be produced in malt syrup, but find and separately can generate the α-amylase of the high malt syrup of maltose content for hydrolyzed starch, make that the production technique of malt syrup is simpler, the utilization ratio that improves starch and production cost lower be the current study hotspot in this area.
Summary of the invention
The object of this invention is to provide gene and the application thereof of a kind of green sugared sporangium maltogenic alpha-amylase enzyme mutant.
On the basis of a α-amylase of the present inventor's green sugared sporangium at clone identification, homology modeling is utilized to infer the amino-acid residue of this diastatic substrate binding site, then utilize fixed point saturation mutation technology to carry out saturation mutation to this site, then screen the mutant enzyme of an acquisition high yield maltose further.Green of the present invention sugared sporangium maltogenic alpha-amylase enzyme mutant, its nucleotide sequence is as shown in SEQ ID NO.1.The α-amylase protein of coded by said gene, its aminoacid sequence is as shown in sequence table SEQ ID NO.2, and wherein 193 hyte propylhomoserins are replaced by glutamine, and the aspartic acid in 273 sites is replaced by L-glutamic acid.
The application of α-amylase protein in hydrolyzed starch of coded by said gene, the product that independent hydrolyzed starch obtains is the syrup of maltose and trisaccharide maltose and a small amount of glucose composition, wherein maltose content reaches 72.1%, about 12% is improve than 59.5% of protoenzyme, this mutant enzyme is conducive to the maltose content improving malt syrup, produces in maltose play effect greatly at hydrolyzed starch.
Even if the α-amylase of different sources exists certain homology, but its molecular size range is different, and optimal reactive temperature, optimal reaction pH and hydrolyzed starch obtain that the enzymatic properties such as product composition all can have very big-difference, thus also different in purposes.The present invention passes through gene clone, exogenous gene expression, the experimental procedure of the Various Complexes such as the enzymology of expression product, confirm that this genes encoding is α-amylase, and inferred by the amino-acid residue of the substrate binding site to this enzyme, then carry out fixed point saturation mutation, screening, obtain the muton of the difference to characteristics such as starch hydrolysate composition differences, its maximum feature maltose content improves.
The present invention compared with prior art, has substantive distinguishing features and significant advantage:
1. with use at present compared with maximum commercialization fungal alpha-amylases, the amylatic product of fungi Alpha-starch is maltose and some oligose and a small amount of glucose mainly, the optimum temperature of fungal alpha-amylase is about 55 DEG C, inactivation is started more than 60 DEG C, optimal reaction pH is 4.8 ~ 5.4, fungal alpha-amylase hydrolyzed starch product can only obtain the maltose of about 50%, and α-amylase of the present invention has the higher temperature of reaction 55 ~ 65 DEG C that is suitable for, thus the requirement of industrialization hydrolyzed starch to the resistance to higher reaction temperatures of enzyme is more suitable for, optimal reaction pH is 5.5 ~ 7.5, closer to neutral reaction condition, do not need to add acid aborning and carry out adjust ph, α-amylasehydrolysis product of the present invention is also simpler, primary product maltose and trisaccharide maltose and a small amount of glucose, there is no oligose, in hydrolyzed starch product, maltose can reach about 72%, prepare on malt syrup at hydrolyzed starch and there is some superiority, the utilization ratio of starch can be improved.
2. compare with protoenzyme, the thermostability of zymetology does not obviously change, but in its amylatic malt syrup, the content of maltose improves 12%.
Accompanying drawing explanation
Fig. 1 is the graphic representation of temperature on the impact of enzymic hydrolysis starch vigor.
Fig. 2 is the graphic representation of pH on the impact of enzymic hydrolysis starch vigor.
Embodiment
By the following examples technical scheme of the present invention is described further.
Embodiment
1. the structure of mutant enzyme
Be assumed to diastatic ORF(GI:256583961 according in sugared sporangium genomic dna sequence green on GenBank) aminoacid sequence, infer that the amino-acid residue in V188, K192, H193, I2683 and D27 site is the substrate binding site of enzyme by homology modeling analysis, then design and synthesize the DNA sequence dna of totally 100 mutant enzyme genes according to the DNA sequence dna of GI:256583961.Apply round pcr synthesis after the DNA sequence dna synthesis employing synthetic primer of gene, the experimental methods of molecular biology do not elaborated in the present embodiment is the normal experiment method that molecular biology professional is familiar with.
2. the expression of mutant enzyme gene and slightly enzyme preparation
By the DN sequence of the mutant enzyme of synthesis after sequence verification, be connected to expression plasmid expression vector pSEe380 building mutant enzyme gene, then expression plasmid imported in intestinal bacteria and carry out abduction delivering.The intestinal bacteria importing expression plasmid are cultivated in 37 DEG C of shaking tables, when OD600 reaches about 0.8, add IPTG make its final concentration reach 1mmol/L 37 DEG C continue cultivate 20h carry out abduction delivering.Abduction delivering cultured fermented liquid collected by centrifugation thalline, utilize broken born of the same parents' buffer solution thalline once, under condition of ice bath, ultrasonic wave is utilized to carry out brokenly born of the same parents after the broken resuspended thalline of born of the same parents' damping fluid of recycling, broken born of the same parents clarify to bacterium liquid, with 1200r/min centrifugal 15 minutes collected by centrifugation supernatants, collected supernatant liquor is crude enzyme liquid, can be used for next step hydrolyzed starch product test, filters out the muton that maltose content is high.The combinatorial mutagenesis of multidigit point is carried out in the site high to maltose content in hydrolyzed starch product again, then screening obtains the mutant enzyme that hydrolyzed starch maltose content is 72% further.In the aminoacid sequence of mutant enzyme, 193 hyte propylhomoserins (H) are replaced by glutamine (Q), the aspartic acid (D) in 273 sites is replaced by L-glutamic acid (E), the DNA sequence dna of mutant enzyme is SEQ ID NO.1, aminoacid sequence is SEQ ID NO.2, and then this mutant enzyme is carried out to the detailed analysis of zymologic property.
3. mutant enzyme and the suitableeest temperature of reaction analysis of protoenzyme hydrolyzed starch
The 1% Zulkovsky starch solution that the enzyme liquid getting 50 μ L and 450 μ L pH are 7.0 mixes, amylase activity is measured after reacting 10min respectively at 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, result shows that the optimum temperuture of enzyme is 60 DEG C, between 50 DEG C to 65 DEG C, this enzyme has the activity of more than 80%, after 65 DEG C, enzymic activity sharply declines, result as shown in Figure 1, shows that mutant enzyme and protoenzyme do not have obvious difference.
4. mutant enzyme and the suitableeest reaction pH of protoenzyme hydrolyzed starch analyze
Use the Zulkovsky starch solution of the buffer 1% of pH4.5, pH5.0, pH5.5, pH6.0, pH6.5, pH7.0, pH7.5, pH8.0, pH8.5, pH9.0 respectively, then 450 μ L add the enzyme liquid of 50 μ L respectively, amylase activity is measured react 10min under the condition of 65 DEG C after, result shows that the optimal reaction pH of enzyme is pH6.5, between pH5.5 to pH8.0, this enzyme has the activity of more than 80%, after pH8, enzymic activity sharply declines, as shown in Figure 2, result shows that mutant enzyme and protoenzyme do not have obvious difference to result.
5. the comparative analysis of mutant enzyme hydrolyzed starch product composition
The commodity fungal alpha-amylase sold with market and protoenzyme are contrast, the composition of comparative analysis mutant enzyme hydrolyzed starch product
The enzyme adding same protein content in the pH7.0 sodium phosphate buffer of 2w/v% soluble starch substrate is contained at 1mL, 50 DEG C of water-baths, the sampling of reaction different time, got sample is boiled in boiling water and within 10 minutes, stops its reaction, centrifugal 30 minutes of 12000rpm/min, get after supernatant dilutes 10 times and detect wild-type and saltant type amylase reaction product with HPLC, its amylatic product percentages is in table 1, result shows that in the maltose syrups that mutant enzyme hydrolyzed starch obtains, maltose content improves 12% than wild enzyme, also higher than commodity fungal alpha-amylase conventional at present.
The product composition of table 1. mutant enzyme and protoenzyme and fungal alpha-amylase hydrolyzed starch different time
The commodity fungal alpha-amylase sold with market equally and protoenzyme are for contrast, comparative analysis mutant enzyme is adding the composition of Pullulanase cohydrolysis starch products, contain at 1mL in the pH7.0 sodium phosphate buffer of 20w/v% soluble starch substrate and add excessive enzyme, and add Pullulanase and react 24 hours, then boil in boiling water and within 10 minutes, stop its reaction, centrifugal 30 minutes of 12000rpm/min, get after supernatant dilutes 100 times and detect wild-type and saltant type amylase reaction product with HPLC, its amylatic product percentages is in table 2.Result shows under the condition of excessive enzyme, the starch of mutant enzyme hydrolysis 20% can obtain the maltose syrups that maltose content is 75%, after adding Pullulanase, maltose content brings up to 78%, and Reducing sugar brings up to 93% from 78, and this is conducive to the utilization ratio improving starch.Also illustrate that mutant enzyme is under two kinds of conditions of not adding Pullulanase and interpolation Pullulanase, the output degree of maltose, higher than wild enzyme and commodity fungal alpha-amylase, which illustrates mutant enzyme and have good potentiality on production superhigh maltose syrup simultaneously.
Table 2 mutant enzyme and protoenzyme and fungal alpha-amylase add the product composition of Pullulanase hydrolyzed starch different time
Claims (3)
1. the sugared sporangium of green (Saccharomonospora viridis) maltogenic alpha-amylase enzyme mutant gene, is characterized in that, its nucleotide sequence is as shown in SEQ ID NO.1.
2. the α-amylase protein of coded by said gene according to claim 1, is characterized in that, its aminoacid sequence is as shown in sequence table SEQ ID NO.2, and wherein 193 hyte propylhomoserins are replaced by glutamine, and the aspartic acid in 273 sites is replaced by L-glutamic acid.
3. the application of α-amylase protein in hydrolyzed starch of coded by said gene according to claim 1, it is characterized in that, the product that independent hydrolyzed starch obtains is the syrup of maltose and trisaccharide maltose and a small amount of glucose composition, and wherein maltose content reaches 75%.
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| CN105802940B (en) * | 2016-04-18 | 2019-04-16 | 广西大学 | A kind of bacillus licheniformis high-temperatureα-amylase mutant and its application |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4604355A (en) * | 1983-03-25 | 1986-08-05 | Novo Industri A/S | Maltogenic amylase enzyme, preparation and use thereof |
| US6274355B1 (en) * | 1998-12-29 | 2001-08-14 | Roquette Freres | Immobilized maltogenic α-amylase and its use in the manufacture of a maltose-rich syrup |
| CN102382849A (en) * | 2011-11-01 | 2012-03-21 | 广西大学 | Gene of saccharomonospora viridis alpha-diastase and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4604355A (en) * | 1983-03-25 | 1986-08-05 | Novo Industri A/S | Maltogenic amylase enzyme, preparation and use thereof |
| US6274355B1 (en) * | 1998-12-29 | 2001-08-14 | Roquette Freres | Immobilized maltogenic α-amylase and its use in the manufacture of a maltose-rich syrup |
| CN102382849A (en) * | 2011-11-01 | 2012-03-21 | 广西大学 | Gene of saccharomonospora viridis alpha-diastase and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| glycosidase [Saccharomonospora viridis];匿名;《NCBI Reference Seqence: WP_012796032.1》;20130517;第1页 * |
| Saccharomonospora viridis DSM 43017, complete genome;Pati A., 等;《GenBank: CP001683.1》;20090826;第1页 * |
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