阅读说明：本技术 一种α-淀粉酶、编码基因、载体、宿主及其应用 (Alpha-amylase, coding gene, vector, host and application thereof ) 是由 赵海 易卓林 陈兰钗 方扬 靳艳玲 何开泽 于 2019-10-12 设计创作，主要内容包括：本发明提供了一种α-淀粉酶编码基因,α-淀粉酶,包含所述α-淀粉酶编码基因的重组载体,包含所述α-淀粉酶编码基因或所述重组载体的宿主,以及制备所述α-淀粉酶的方法,并证实了所述α-淀粉酶对多种淀粉底物具有水解活性,因而所述α-淀粉酶及宿主可在水解淀粉中应用,具体可应用于洗涤行业、纺织行业及食品行业等,为可应用于洗涤、纺织及食品等行业的淀粉水解提供了新的选择。(The invention provides an alpha-amylase coding gene, alpha-amylase, a recombinant vector containing the alpha-amylase coding gene, a host containing the alpha-amylase coding gene or the recombinant vector, and a method for preparing the alpha-amylase, and proves that the alpha-amylase has hydrolytic activity on various starch substrates, so that the alpha-amylase and the host can be applied to hydrolyzing starch, can be particularly applied to washing industry, textile industry, food industry and the like, and provide a new choice for starch hydrolysis in washing industry, textile industry, food industry and the like.)

1. An alpha-amylase encoding gene, wherein the encoding gene encodes a protein having an amino acid sequence shown in SEQ ID No.2, or encodes a protein having homology of 90% or more with the amino acid sequence shown in SEQ ID No.2 and having alpha-amylase activity.

2. The alpha-amylase encoding gene of claim 1, wherein the encoding gene has a nucleotide sequence shown as SEQ ID No.1, or a nucleotide sequence having more than 90% homology with the nucleotide sequence shown as SEQ ID No. 1.

3. An alpha-amylase, characterized in that the alpha-amylase is a protein having an amino acid sequence shown in SEQ ID No.2, or a protein having 90% or more homology with the amino acid sequence shown in SEQ ID No.2 and having alpha-amylase activity.

4. A recombinant vector comprising the gene encoding the alpha-amylase of claim 1 or 2 or the gene encoding the alpha-amylase of claim 3.

5. A host comprising a gene encoding the alpha-amylase of claim 1 or 2, or a gene encoding the alpha-amylase of claim 3, or a recombinant vector of claim 4.

6. A method of preparing the α -amylase of claim 3, comprising: culturing the host of claim 5 under conditions suitable for the production of α -amylase by the host; and isolating and purifying the alpha-amylase produced by the host.

7. Use of the alpha-amylase of claim 3 and the host of claim 5 for hydrolyzing starch.

8. Use according to claim 7, wherein the alpha-amylase of claim 3 or the host of claim 5 is used for the hydrolysis of starch, wherein the alpha-amylase of claim 3 or the host of claim 5 is contacted with a reaction system comprising the starch to be hydrolyzed, and the hydrolysis is carried out under conditions suitable for the alpha-amylase to carry out the hydrolysis.

9. The use of claim 8, wherein the pH of the reaction system is controlled to be 5.0-12.5, and the temperature for hydrolysis is controlled to be 30-65 ℃.

10. Use according to claim 8, wherein Ca is added to the reaction system²⁺The hydrolysis reaction is carried out under the conditions of (1).

Technical Field

The invention belongs to the field of biological engineering and enzyme engineering, and relates to alpha-amylase, a coding gene, a vector, a host and application thereof.

Background

Alpha-amylase (e.c.3.2.1.1.) is the most important enzyme of industrially applicable enzymes, accounting for 25% of all industrial enzymes. Alpha-amylase is an endo-starch hydrolase, and acts on the alpha-1, 4-glycosidic bond of a starch substrate to release oligosaccharides such as glucose, maltose, maltotriose, and the like. As representative of the glycoside hydrolase 13 family (GH13), there are GH13_1, 5, 6, 7, 15, 24, 27, 28, 32, 36, 37 subfamilies according to sequence similarity and catalytic function. Among them, fungal-origin amylases are classified in GH13_1 and 5 subfamilies. GH13_1 contains only alpha-amylases of fungal and yeast origin, and most of the alpha-amylases are extracellular enzymes; the GH13_5 subfamily contains liquefying amylases of bacterial origin, intracellular enzymes of fungi and amylases of some archaea. More fungal amylases are currently reported to be derived from the GH13_1 subfamily, as represented by the Taka-amylase derived from Aspergillus oryzae. Whereas fungal amylases derived from the GH13_5 subfamily are less reported, there are fewer enzymatic characterization analyses for this class of amylases relative to the GH13_1 family.

The brewing process of Chinese liquor is different from the foreign pure-bacteria fermentation liquor production, and is completely open solid-state fermentation with the participation of multiple microorganism directionality. Daqu is a leaven for solid-state white spirit fermentation, and has the ancient 'bone of spirit'. The diversity of the microbial flora of the yeast determines the abundant enzyme system network. In the study of enzyme systems in Daqu, it has been reported most frequently that crude enzyme solutions thereof are extracted to measure liquefaction capacity, saccharification capacity, esterification capacity, and the like. For the excavation of enzyme resources in the yeast, microorganisms producing enzymes with specific functions are screened mainly based on a traditional culture method, and clones with specific functions are randomly screened to obtain target enzyme genes based on metagenome method library construction. The culture method cannot effectively mine the enzyme genes in the non-culturable microorganisms, and the metagenome method cannot faithfully reflect the functions of the functional enzyme genes in the environment.

Disclosure of Invention

The invention aims to provide alpha-amylase, a coding gene, a vector, a host and application thereof.

The alpha-amylase coding gene provided by the invention is derived from a strong aromatic Daqu uncultured microorganism, and codes a protein with an amino acid sequence shown as SEQ ID NO.2 or codes a protein which has more than 90% homology with the amino acid sequence shown as SEQ ID NO.2 and has alpha-amylase activity.

Furthermore, the coding gene codes a protein with an amino acid sequence shown in SEQ ID NO.2, or codes a protein which has more than 95 percent of homology, preferably more than 98 percent of homology with the amino acid sequence shown in SEQ ID NO.2 and has alpha-amylase activity.

Furthermore, the coding gene has a nucleotide sequence shown in SEQ ID NO.1, or a nucleotide sequence which has more than 90%, preferably more than 95%, more preferably more than 98% homology with the nucleotide sequence shown in SEQ ID NO. 1. A nucleotide sequence having more than 90%, preferably more than 95%, more preferably more than 98% homology with the nucleotide sequence shown in SEQ ID NO.1, which can be generated by performing one or more base substitutions or/and deletions or/and additions to the nucleotide sequence shown in SEQ ID NO. 1.

The alpha-amylase provided by the invention is a protein with an amino acid sequence shown in SEQ ID NO.2, or a protein which has more than 90% of homology with the amino acid sequence shown in SEQ ID NO.2 and has alpha-amylase activity.

Further, the α -amylase is a protein having an amino acid sequence shown in SEQ ID NO.2, or a protein having 90% or more, preferably 95% or more, more preferably 98% or more homology with the amino acid sequence shown in SEQ ID NO.2 and having α -amylase activity. A protein having 90% or more, preferably 95% or more, more preferably 98% or more homology with the amino acid sequence shown in SEQ ID NO.2 and having alpha-amylase activity, which can be derived by substituting or/and deleting or/and adding one or more amino acids to the amino acid sequence shown in SEQ ID NO. 2.

The invention also provides a recombinant vector containing the alpha-amylase coding gene or a recombinant vector containing the alpha-amylase coding gene.

The invention also provides a host containing the alpha-amylase coding gene, or a host containing the recombinant vector.

Further, the recombinant vector is formed by inserting the above-mentioned gene encoding alpha-amylase or the above-mentioned gene encoding alpha-amylase into a vector, and the vector refers to a bacterial plasmid, a bacteriophage, a yeast plasmid, an animal cell virus, a retrovirus or other vectors well known in the art. Any vector can be used as long as it can replicate and stably remain in the host, and there is no particular requirement. Illustratively, the present invention uses the pDE2 plasmid. There is also no particular requirement for the host, which may be prokaryotic cells such as Escherichia coli, Bacillus, etc., or eukaryotic cells such as Pichia pastoris, Saccharomyces cerevisiae, Aspergillus niger, etc.

The invention also provides a method for preparing the alpha-amylase, which comprises the following steps: culturing the host under conditions suitable for the production of alpha-amylase by the host; and isolating and purifying the alpha-amylase produced by the host. The host as used herein refers to a host comprising the above-mentioned gene encoding an alpha-amylase, or a host comprising the above-mentioned recombinant vector.

The preparation method has no specific requirements on the construction method of the recombinant vector and the method for transforming, transfecting or transducing the constructed recombinant vector into a host; the method has no specific requirements for the separation and purification method of the alpha-amylase produced by the host, such as a Ni column purification method, an ion exchange column method, a molecular sieve method and the like, and is feasible.

The alpha-amylase provided by the invention is a medium-temperature fungal amylase, and experiments prove that the alpha-amylase has hydrolytic activity on various starch substrates. Based on the alpha-amylase, the invention also provides the application of the alpha-amylase and the host in starch hydrolysis. The host herein refers to a host comprising the above-mentioned gene encoding an alpha-amylase, or a host comprising the above-mentioned recombinant vector.

Further, when the above-mentioned α -amylase or the above-mentioned host is used for hydrolyzing starch, the above-mentioned α -amylase or the above-mentioned host is brought into contact with a reaction system containing starch to be hydrolyzed, and the hydrolysis reaction is carried out under conditions suitable for the hydrolysis reaction by the α -amylase.

Further, the pH of the reaction system is controlled to be 5.0 to 12.5, preferably 5.0 to 8.5, and more preferably 5.5 to 6.0, and the temperature for hydrolysis reaction is controlled to be 30 to 65 ℃, preferably 50 to 55 ℃, and more preferably 54 ℃.

Experiments show that the alpha-amylase provided by the invention is Ca²⁺Increased thermal stability in the presence of, inter alia, Ca²⁺The alpha-amylase has higher thermal stability under the condition that the concentration is 0.0625-2.5 mM. Based on this, it is considered that Ca is added to the reaction system according to the details of the reaction system in practical use²⁺The hydrolysis reaction is carried out under the conditions of (1).

Experiments show that the alpha-amylase provided by the invention has good hydrolytic activity on soluble starch, amylopectin, amylose, wheat starch, potato starch, corn starch and dextrin, and particularly has higher hydrolytic activity on amylopectin, corn starch and potato starch. Based on the above, the alpha-amylase provided by the invention can be applied to the starch hydrolysis, and the alpha-amylase can be applied to the washing industry, the textile industry, the food industry and the like.

The technical scheme of the invention has the following beneficial technical effects: the invention provides an alpha-amylase coding gene, alpha-amylase coded by the coding gene, a recombinant vector containing the alpha-amylase coding gene, a host containing the coding gene or the recombinant vector, and the alpha-amylase is proved to have hydrolytic activity on various starch substrates, can be applied to the washing industry, the textile industry, the food industry and the like, and provides a new choice for starch hydrolysis in the washing, textile and food industries and the like.

Drawings

FIG. 1 is a SDS-PAGE gel of the pure α -amylase NFAmy 13B.

Fig. 2 is a hydrolysis diagram of pullulan and amylose by the α -amylase NFAmy 13B.

FIG. 3 is a graph showing the effect of different pH values on the enzymatic activity of the α -amylase NFAmy 13B.

FIG. 4 is a graph showing the effect of different reaction temperatures on the enzymatic activity of the alpha-amylase NFAmy 13B.

FIG. 5 is a pH stability curve of the α -amylase NFAmy 13B.

FIG. 6 is a thermostability curve for the alpha-amylase NFAmy 13B.

FIG. 7 shows different concentrations of CaCl₂Bar graph is affected on the thermostability of the alpha-amylase NFAmy 13B.

Detailed Description

The alpha-amylase, encoding gene, vector, host and application thereof provided by the present invention are further illustrated by the following examples. The test methods used in the following examples are, unless otherwise specified, conventional in the art. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified, and the aromatic Daqu is collected from Yibin City of Sichuan province in China.