Amylase mutant having high specific activity and thermal stability, gene of mutant, and applications thereof

Abstract

The present invention relates to the field of agriculture biotechnology, specially relates to an amylase mutant having high specific activity and thermal stability, gene and use thereof. Said amylase mutant is obtained by performing substitution of S33A/S34E/V35H, and deletion of amino acids at the sites of 178 and 179 of the wild type amylase having amino acid sequence of SEQ ID NO:1, and having improved enzymatic activity and thermal stability than the wild type amylase.

Claims

1. An amylase mutant, having the amino acid sequence of SEQ ID NO: 2.

2. A gene comprising a nucleotide sequence encoding the amylase mutant of claim 1.

3. A recombinant vector of comprising the gene of claim 2.

4. An isolated recombinant cell comprising the gene of claim 2.

5. A method of preparing amylase having high enzymatic activity and thermal stability, comprising the steps of (1) constructing a recombinant vector comprising the gene of claim 2; (2) transforming an isolated host cell with the recombinant vector obtained by the step (1); and (3) cultivating the recombinant host cell and isolating said amylase.

6. A method of preparing a feed, medicine, or food, comprising incubating the amylase mutant of claim 1 with a feed, medicine, or food comprising an α-1,4-glycosidic bond, to hydrolyze said α-1,4-glycosidic bond.

7. A method of preparing a feed, medicine, or food, comprising incubating the gene of claim 2 with a feed, medicine, or food comprising an α-1,4-glycosidic bond, to hydrolyze said α-1,4-glycosidic bond.

Description

BRIEF DESCRIPTIONS OF THE DRAWINGS

(1) FIG. 1 shows the enzymatic properties of the wild-type amylase;

(2) FIG. 2 shows the comparison of thermal stability of the amylase mutant with that of the wild-type amylase;

(3) FIG. 3 shows the SDS-PAGE electrophoresis results of amylase expressed in Bacillus subtilis sck6.

EMBODIMENT

(4) The present invention is further illustrated with reference to the following examples and the appended drawings, which should by no means be construed as limitations of the present invention.

(5) Test Materials and Reagents

(6) 1. Strains and vectors: host: Bacillus subtilis SCK6; and vector pHYP16-BKAMY

(7) 2. Enzymes and other biochemical reagents: restriction endonucleases (Fermentas); and ligase (Promaga).

(8) 3. Medium: LB medium; starch medium

(9) Suitable biology laboratory methods not particularly mentioned in the examples as below can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other kit laboratory manuals.

Example 1 Site Directed Mutagenesis of the Gene Encoding the Amylase

(10) The optimized mutation site wasS33A/S34E/V35H/ΔR178/Δ G179. The mutation site was introduced with the primers of table 1 by Site-directed Mutagenesis Kit followed by sequencing to obtain the mutant gene.

(11) TABLE-US-00003 TABLE 1 Primers SEQ Length ID NO. Primers Sequence (5′.fwdarw. 3′).sup.a (bp) 3 Bkamy-F ttacaaaaacatcagccgtaggatccg 50 ccgcaacgaacggaacaatgatg 4 Bkamy-R gggacgtcgacttagtggtggtggtgg 54 tggtgctgctgaacccacactgagacg 5 pHYP16-F gggttcagcagcaccaccaccaccacc 51 actaagtcgacgtccccggggcag 6 pHYP16-R attgttccgttcgttgcggcggatcct 50 acggctgatgtttttgtaatcgg

(12) Sequencing results showed that the nucleotide sequence amplified by mutation had 1458 bp, comprising the encoding area of 1455 bp encoding the amino acid sequence of SEQ ID No: 2 having 485 amino acid residues. The protein with the amino acid sequence of SEQ ID No: 2 was named as amylase mutant BKAMYA.

Example 2 Preparation of Amylase Mutant BKAMYA

(13) 1. Preparation of Recombinant Plasmid pHYP16-BKAYA

(14) Firstly, the vector and the target fragment were amplified by POE-PCR followed by being recovered and mixed in a proper proportion, and then added into the PCR system to construct the recombinant plasmid containing the said amylase gene. Mutation of nucleotides was introduced by Site Mutation kit using the wild type plasmid as the temple. Then, the obtained recombinant plasmid was sequenced to test the validity of purpose sequence. The recombinant plasmid inserting the exogenous gene was named aspHYP16-BKAMYA.

(15) 2. Preparation of the Recombinant Strain SCK6/BKAMYA

(16) The above recombinant plasmid pHYP16-BKAMYA was transformed into Bacillus spk6 cells to obtain the recombinant strain SCK6/BKAMYA.

(17) 3. Preparation of the Amylase Mutant

(18) The above recombinant strainSCK6/BKAMYA was inoculated into 50 mL of medium in 100 mL of flask and cultured on a shaker operating at 220 rpm at 37° C. and for 24 h. Then, the cultured medium was transferred to 200 mL medium in a 1 L of flask to culture at 37° C. and 220 RPM again. The supernatant was collected by centrifugation to purify the amylase mutant BKAMYA by affinity chromatography for analyzing activity.

Example 3 Analysis and Comparison of the Amylase Mutant BKAMYA and Wild Type Amylase

(19) 1. Analysis and Comparison of the Enzymatic Activity

(20) The enzymatic activity of amylase was determined with UV spectrophotometer by the steps of performing the enzymatic reaction at the certain temperature and pH for 20 min, wherein 1 mL of said enzymatic reaction system included 100 μL of appropriate diluted enzyme solution and 900 μL of substrate, measuring the absorbance at 540 nm and calculating the enzymatic activity. A unit of enzymatic activity (U) is defined as the amount of enzyme to produce 1 μmol glucose per unit time under given conditions.

(21) The purified amylase mutant prepared in example 2 and the wild type amylase were performing the enzymatic reaction at pH 7.0 and 55° C. to determine their enzymatic activity.

(22) As showed in FIG. 1, the enzymatic activity of wild type is 5553.53 U/Mg, and that of amylase mutant is 17067.57 U/mg.

(23) 2. Analysis and Comparison of Thermal Stability

(24) The thermal stability of both the purified amylase mutant prepared in example 2 and the wild type was determined by treating at 60° C. for 10 min, 30 min and 60 min respectively or 70° C. for 2 min, 5 min, and 10 min respectively, in 0.1 mol/L of citric acid-disodium hydrogen phosphate buffer (pH 7.0), and their retained enzymatic activity at 55° C. was determined.

(25) As showed in FIG. 2, the mutant S33A/S34E/V35H/ΔR178/G179 retained more than 99% of enzymatic activity, but the wild type amylase retained about 10% of enzymatic activity, after being treated at 70° C. for 5 min; and, the mutant retained 32% of enzymatic activity, but the wild type almost lost enzymatic activity after being treated at 80° C. for 5 min. And, the specific activity of mutant was increased to 17067.57 U/mg compared with that of the wild type amylase.