CN118207187B - High specific activity amylase mutant - Google Patents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/84—Pichia
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Abstract
The invention relates to the technical fields of genetic engineering and protein engineering, and particularly provides an amylase mutant with high specific activity. The mutant contains a single mutation site of Y176F, the specific activity of the mutant is improved by 58.48 percent compared with that of a wild type, and the mutant reaches 1491.32U/mg, so that unexpected technical effects are achieved. The amylase mutant has greatly reduced production cost, thereby being beneficial to promoting the wide application in the fields of feed, industry and the like.
Description
Technical Field
The invention relates to the technical fields of genetic engineering and protein engineering, in particular to a high specific activity amylase mutant and application thereof in feed.
Background
Alpha-amylase (EC 3.2.1.1) specifically cleaves alpha-1, 4-glucosidic bonds within starch to yield products such as glucose, maltose and dextrins, and thus, amylase has been widely used in starch processing, feed, washing, baking and beverage industries (S. Janecek et al, 2014, cellular and Molecular LIFE SCIENCES; R. Gupta, P. Gigras et al; L. Dijkhuizen et al, 2002,Journal of Biotechnology).
Many microorganisms are capable of expressing amylase production, such as Aspergillus, mucor, bacillus, geobacillus, and the like. However, amylases of different origins also differ in substrate specificity; at the same time, their tolerance to temperature stability and pH also varies greatly. Among them, bacillus and Geobacillus production amylase can withstand 90-100deg.C and is commonly used in starch liquefaction process.
The main energy source in the livestock and poultry breeding process is starch in feed, the starch comprises amylopectin, amylose, resistant starch and the like according to different structures, and different animals have certain difference on the digestion and utilization rates of starch from different sources. Especially, the development of the digestive tract of young animals is imperfect, and amylase secretion is insufficient, so that starch nutrients in the feed are not fully utilized; the novel feed raw materials such as corn and cassava cannot be fully utilized in the digestive tracts of livestock and poultry due to the starch structure, and the exogenous amylase can be added to cooperate with the endogenous amylase in the digestive tracts of livestock and poultry to improve the utilization rate of starch nutrients in the feed, improve the production performance of livestock and poultry and reduce the feed cost.
The amylase from geobacillus stearothermophilus (G. Stearothermophilus) has the advantages of better temperature resistance performance, and almost no loss of enzyme activity after high-temperature granulation of feed; if the specific activity of the feed is improved through protein engineering transformation, the production cost can be saved for the feed preparation industry, and the feed utilization rate can be improved. Through protein engineering means, the improvement of feeding enzyme additives and the search of amylase with high specific activity are hot spots and difficulties in current research.
Disclosure of Invention
The invention provides an amylase mutant with high specific activity, which aims to solve the problems in the prior art and screens out mutation sites capable of improving the specific activity of amylase by protein engineering technology. The production cost of the mutant is greatly reduced, and the mutant can be widely applied to the fields of feed, industry and the like.
One aspect of the invention relates to an amylase mutant, which has an amino acid sequence of SEQ ID NO:1 amylase at position 176 the amino acid is changed from Tyr to Phe.
The amino acid sequence of the amylase mutant is SEQ ID NO:3.
The invention also relates to DNA molecules encoding the above amylase mutants.
The invention also relates to recombinant expression plasmids containing the DNA molecules.
The invention also relates to a host cell comprising the recombinant expression plasmid.
The plasmid is transferred into a host cell, and the specific activity of the recombinant amylase mutant is obviously improved.
In some embodiments of the invention, the host cell is Pichia pastoris (Pichia pastoris).
Compared with the wild amylase AMY1, the specific activity of the amylase mutant containing the single-point mutation of Y176F provided by the invention is improved by 58.48%, which reaches 1491.32U/mg, and unexpected technical effects are achieved. The amylase mutant has greatly reduced production cost, thereby being beneficial to promoting the wide application in the fields of feed, industry and the like.
Detailed Description
The present invention uses conventional techniques and methods used in the fields of genetic engineering and molecular biology, such as MOLEC μm LAR CLONING: a LABORATORY MANUAL,3nd Ed. (Sambrook, 2001) and CURRENT PROTOCOLS IN MOLEC μm LAR BIOLOGY (Ausubel, 2003). These general references provide definitions and methods known to those skilled in the art. However, those skilled in the art may adopt other methods, experimental schemes and reagents which are conventional in the art on the basis of the technical scheme described in the present invention, and are not limited to the specific embodiments of the present invention. For example, the invention may be used with the following experimental materials and reagents:
Strains and vectors: coli DH 5. Alpha., pichia pastoris GS115, vector pPIC9k, amp, G418 were purchased from Invitrogen corporation.
Enzyme and kit: the PCR enzyme and the ligase were purchased from Takara, the restriction enzyme from Fermentas, the plasmid extraction kit and the gel purification recovery kit from Omega, and the GeneMorph II random mutagenesis kit from Beijing Bomeis Biotechnology Co.
The formula of the culture medium comprises:
coli medium (LB medium): 0.5% yeast extract, 1% peptone, 1% NaCl, pH7.0;
yeast Medium (YPD Medium): 1% yeast extract, 2% peptone, 2% glucose;
Yeast screening medium (MD medium): 2% peptone, 2% agarose;
BMGY medium: 2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4X 10 -5% biotin, 1% glycerol;
BMMY medium: 2% peptone, 1% yeast extract, 100 mM% potassium phosphate buffer (pH 6.0), 1.34% YNB, 4X 10 -5% biotin, 0.5% methanol;
LB-AMP medium: 0.5% yeast extract, 1% peptone, 1% NaCl, 100. Mu.g/mL ampicillin, pH7.0;
LB-AMP plate: 0.5% yeast extract, 1% peptone, 1% NaCl,1.5% agar, 100. Mu.g/mL ampicillin, pH7.0;
upper medium: 0.1% MgSO 4,1%KH2PO4,0.6%(NH4)2SO4, 1% glucose, 18.3% sorbitol, 0.35% agarose;
Lower medium plates: 2% glucose ,0.5%(NH4)2SO4,1.5%KH2PO4,0.06%MgSO4,0.06%CaCl2,1.5% agar.
The invention is further illustrated by the following examples:
EXAMPLE 1 construction of recombinant plasmid
The amylase gene from Geobacillus stearothermophilus (Geobacillus stearothermophilus) was optimized for Pichia pastoris codon bias and increased by 6 bases GAATTC (EcoR I cleavage site) before its start codon ATG and GCGGCCGC (Not I cleavage site) after its stop codon TAA. The optimized nucleotide sequence is synthesized by Shanghai JieRui bioengineering Co. The amylase is named AMY1, and the amino acid sequence of the amylase is SEQ ID NO:1, the coding nucleotide sequence is SEQ ID NO:2.
The amylase gene was digested with restriction enzymes EcoR I and Not I (Fermentas); at the same time, plasmid pPIC9K was digested with restriction enzymes EcoR I and Not I. The cleavage products were purified using a gel purification kit and the two cleavage products were ligated with T4 DNA ligase (Fermentas). The ligation product was transformed into DH 5. Alpha. E.coli (Invitrogen) and selected with ampicillin. To ensure accuracy, several clones were sequenced (Invitrogen).
The plasmid was purified from E.coli clones with correct sequencing results using a plasmid miniprep kit (Omega) to obtain 1 recombinant plasmid, which was designated pPIC9K-AMY1.
EXAMPLE 2 screening of high specific Activity mutants
Amylase AMY1 is a glycoside hydrolase G11 family amylase, and in order to further improve the specific activity of the amylase AMY1, the applicant has carried out a large number of mutant screening on the amylase by a directed evolution technology.
Designing PCR primers, taking an AMY1 gene (SEQ ID NO: 2) as a template, carrying out PCR amplification by using a GeneMorph II random mutation PCR kit (Bomeis) by using the primers, recovering PCR products by using glue, connecting EcoRI and NotI after enzyme digestion treatment with a pET21a carrier subjected to enzyme digestion, converting into escherichia coli BL21 (DE 3), coating the escherichia coli BL21 on an LB+amp plate, carrying out inversion culture at 37 ℃, picking up the transformants into 96-well plates one by using toothpicks after the transformants appear, adding 150 mu l of LB+amp culture medium containing 0.1mM IPTG into each well, culturing at 37 ℃ and 220rpm for about 6 h, centrifuging, discarding supernatant, re-suspending thalli by using a buffer solution, and repeatedly freezing and thawing to break walls to obtain escherichia coli cell lysate containing amylase.
Taking 10 μl of lysate out to two new 96-well plates; adding 190 μl of substrate into one 96-well plate, reacting at 60deg.C for 5min, taking 20 μl of reaction system to a new 96-well plate, adding 160 μl of iodine solution and 20 μl of HCl (0.1 mol/L) to terminate and develop color, measuring residual starch content by iodine color development method, and calculating hydrolyzed starch content; another plate was added with 190. Mu.l of Coomassie Brilliant blue solution, allowed to stand for 10min, and the protein content was determined by Coomassie Brilliant blue (Bradford) binding, and the levels of mutant enzyme activity and protein content were calculated, respectively. Finally, the applicant screened out mutation sites that significantly improved amylase AMY1 specific activity: Y176F.
Based on the wild-type amylase AMY1, the invention provides a mutant containing a single mutation site of Y176F, and the amino acid sequence of the mutant is SEQ ID NO:3.
Example 3 expression of amylase in Pichia pastoris
3.1 Construction of expression vectors
The gene sequences of amylase AMY1 and mutants thereof are respectively optimized according to the password preference of pichia pastoris, and are synthesized by Shanghai JieRui bioengineering Co., ltd, and EcoRI and NotI cleavage sites are respectively added at the 5 'and 3' ends of the synthesized sequences.
The gene sequences of the synthesized amylase AMY1 and its mutants were digested with EcoRI and NotI, respectively, and then ligated overnight at 16℃with the pPIC-9K vector digested in the same manner, and E.coli DH5a was transformed, spread on LB+Amp plates, cultured upside down at 37℃and, after the appearance of the transformants, colony PCR (reaction system: template-picked monoclonal, rTaqDNA polymerase 0.5. Mu.l, 10 XBuffer 2.0. Mu.l, dNTPs (2.5 mM) 2.0. Mu.l, 5 'X primer (10 mM): 0.5. Mu.l, 3' AOX primer: 0.5. Mu.l, ddH 2 O14.5. Mu.l, reaction procedure: 5min at 95℃for pre-denaturation, 30 cycles: 94℃30sec,55℃30sec,72℃2min,72℃for 10 min) was performed as described in example 1. And (3) verifying positive clones, and obtaining the correct recombinant expression plasmid after sequencing verification.
3.2 Construction of Pichia pastoris engineering strains
3.2.1 Yeast competent preparation
Performing YPD plate activation on Pichia pastoris GS115 strain, culturing at 30 ℃ for 48 h, inoculating activated GS115 monoclonal in a 6mL YPD liquid culture medium, culturing at 30 ℃ for 220 rpm, transferring bacterial liquid after culturing at about 12 h into a triangular flask filled with 30mL of YPD liquid culture medium, culturing at 30 ℃ for about 5 hours at 220 rpm, detecting the bacterial density by an ultraviolet spectrophotometer, respectively collecting 4mL of bacterial bodies after OD600 value is in the range of 1.1-1.3, centrifuging at 4 ℃ 9000 rpm for 2 min into a sterilized EP tube, slightly discarding supernatant, sucking residual supernatant with sterilized filter paper, re-suspending the bacterial bodies with precooled 1mL sterilized water, centrifuging at 4 ℃ 9000 rpm for 2 min, slightly discarding supernatant, re-washing with 1mL sterilized water once, centrifuging at 4 ℃ 9000 rpm for 2 min, slightly discarding supernatant, and re-suspending the pre-cooled 1mL sorbitol (1 mol/L); centrifuge 2 min at 4℃with 9000 rpm, gently discard supernatant, gently resuspend cells with 100-150. Mu.l sorbitol (1 mol/L) pre-chilled.
3.2.2 Transformation and screening
Linearizing the recombinant expression plasmid obtained by constructing 3.1 by Sac I, purifying and recovering linearization fragments, respectively converting Pichia pastoris GS115 by electroporation, screening on an MD plate to obtain Pichia pastoris recombinant strain, and screening multiple copies of transformants on YPD plates (0.5 mg/mL-8 mg/mL) containing geneticin at different concentrations.
Transferring the obtained transformants into BMGY culture medium respectively, and culturing at 30 ℃ and 250rpm in a shaking way for 1d; then transferring the strain into a BMMY culture medium, and carrying out shaking culture at 30 ℃ and 250 rpm; adding 0.5% methanol every day, and inducing expression 4 d; and (5) centrifuging at 9000rpm for 10min to remove thalli, thus obtaining fermentation supernatant respectively containing amylase AMY1 and amylase mutants.
1. Method for measuring amylase activity
(1) Definition of amylase enzyme activity units
1G of solid enzyme powder is liquefied for 1h at 60 ℃ and pH value of 6.0 to obtain 1g of soluble starch, namely 1 enzyme activity unit expressed as u/g.
(2) Method for measuring amylase activity
1G of enzyme powder (accurate to 0.0001 g) is weighed, fully dissolved by a small amount of phosphate buffer solution, the supernatant is carefully poured into a volumetric flask, if residues remain, a small amount of phosphate buffer solution is added for full grinding, and finally, all samples are transferred into the volumetric flask, and the volume is fixed to a scale by the phosphate buffer solution and is uniformly shaken. Filtering with four layers of gauze, and standing filtrate.
Sucking 20.0 mL soluble starch solution into a test tube, adding 5.00 phosphate buffer solution mL, shaking, and preheating 8min in a constant-temperature water bath at 60+ -0.2 deg.C.
Adding 1.00 mL diluted enzyme solution to be detected, immediately timing, shaking uniformly, and accurately reacting 5 min.
Immediately, 1.00mL of the reaction solution was pipetted with an automatic pipette, added to a test tube previously containing 0.5mL of hydrochloric acid solution and 5.00mL of diluted iodine solution, shaken well, and the absorbance (A) was rapidly measured with a10 mm cuvette at 660 nm wavelength using 0.5mL of hydrochloric acid solution and 5.00mL of diluted iodine solution as a blank. And obtaining the concentration of the test enzyme solution according to the absorbance.
The enzyme activity calculation formula: x1=c×n.
Wherein:
x1-enzyme activity of sample, u/mL (or u/g);
c-measuring the concentration of the enzyme sample, u/mL (or u/g);
n-dilution of the sample.
The result is expressed as an integer.
(3) Measurement results
The enzyme activity detection is carried out according to the method, and the result shows that: the enzyme activity of the recombinant strain fermentation supernatant of the recombinant expression wild amylase AMY1 and the mutant thereof is 360-420U/mL.
2. Protein content determination method
The determination of protein content by coomassie brilliant blue (Bradford) binding is a complex method of colorimetry combined with the pigment method. Coomassie brilliant blue G-250 appears brownish red in acidic solution, turns blue when bound to protein, and accords with beer's law in a certain concentration range of protein, and can be colorimetrically measured at 595 nm. A large amount of absorption is obtained in 3-5 minutes, and the absorption is stable for at least 1 hour. In the range of 10-1000. Mu.g/mL, absorbance is proportional to protein concentration.
According to the volume ratio of the enzyme solution to the coomassie brilliant blue solution of 1:5, and standing for 10mm, and determining protein content by Coomassie Brilliant blue (Bradford) binding method.
The protein content was measured as described above. The results show that: the protein content of the recombinant strain fermentation supernatant of the pichia pastoris for recombinant expression of the wild amylase AMY1 and the mutant thereof obtained by the construction is 0.372-0.456 mg/mL.
3. Specific activity calculation
"Specific activity (SPECIFIC ACTIVITY)" means: the number of units of enzyme activity per unit weight of protein is generally expressed as U/mg protein.
The specific activity calculation formula: specific activity (U/mg) =enzyme activity (U/mL)/protein content (mg/mL).
The specific calculation results are shown in Table 1.
TABLE 1 comparison of specific Activity of amylase mutants
| Amylase and single point mutant thereof | Specific activity (U/mg) |
| Wild type AMY1 | 941.02 |
| Y176F | 1491.32 |
As can be seen from the results in Table 1, compared with the wild-type amylase AMY1, the specific activity of the amylase mutant containing the single-point mutation of Y176F provided by the invention is improved by 58.48%, and unexpected technical effects are obtained.
In conclusion, the specific activity of the amylase mutant provided by the invention is obviously improved, so that the production cost of the amylase is reduced, and the amylase mutant is widely applied to the fields of feed and industry.
Claims (4)
1. An amylase mutant, which is characterized in that the amino acid sequence of the mutant is SEQ ID NO. 3.
2. A DNA molecule encoding the amylase mutant of claim 1.
3. A recombinant expression plasmid comprising the DNA molecule of claim 2.
4. A host cell comprising the recombinant expression plasmid of claim 3, wherein the host cell is Pichia pastoris.
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