High specific activity alkaline amylase mutant and high-yield strain thereof

Abstract

The present application relates to the technical field of genetic engineering and protein engineering, and particularly relates to a high specific activity alkaline amylase mutant and a high-yield strain thereof. The mutant comprises three mutant sites of Q84S, P287D and G299A, and the specific activity is significantly improved, which is beneficial to reduce the production cost of the enzyme and promote the wide application of the enzyme in the industrial field such as washing.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and protein engineering technology, specifically to a high-specific-activity alkaline amylase mutant and its high-yield strain. Background Technology

[0002] Alkaline α-amylase is an industrial enzyme that catalyzes the hydrolysis of α-1,4-glycosidic bonds in substrates such as starch and glycogen under alkaline pH conditions. Due to its excellent alkali adaptability and substrate specificity, alkaline amylase has been widely used in textile desizing, food processing, and pharmaceutical production, and has become a core enzyme component in modern detergent formulations, playing a crucial role in effectively removing starch-containing stains (such as sauces, rice paste, and chocolate).

[0003] In detergent applications, the specific activity (catalytic activity per unit mass of enzyme protein) of alkaline amylase is the primary factor determining its industrial cost-effectiveness. High specific activity means faster and more thorough removal of stubborn starchy stains with the same protein dosage, or a significant reduction in enzyme dosage while achieving the same washing effect, thereby reducing detergent production costs and product volume. However, wild-type alkaline amylases and their conventional mutants currently used on a large scale in industry still have significant deficiencies in specific activity, making it difficult to achieve efficient stain removal under gentle washing conditions with low enzyme dosages. To achieve ideal washing results, it is often necessary to increase the enzyme concentration, which undoubtedly increases the cost pressure of detergent formulations and limits its widespread application in energy-saving and cost-reducing concentrated detergents.

[0004] To improve the industrial adaptability of alkaline amylase, researchers have explored various protein engineering strategies. Through site-directed mutagenesis, directed evolution, and computer-aided design, several mutants with improvements in thermal stability, pH tolerance, or catalytic efficiency have been successfully obtained. For example, researchers have attempted to optimize the conformation of substrate-binding channels to enhance catalytic rates by replacing flexible amino acid residues near the enzyme's active site; others have indirectly maintained catalytic conformational stability by introducing disulfide bonds or metal-binding sites to enhance enzyme rigidity. However, the core objective of most of these modification strategies is to improve enzyme tolerance (stability), rather than directly enhancing specific activity. Due to the inherent "stability-activity trade-off" effect between the catalytic activity and conformational flexibility of enzyme molecules, many mutants with improved stability often experience a decrease or no change in specific activity, making it difficult to simultaneously meet the dual requirements of detergents for high catalytic efficiency and good stability.

[0005] Furthermore, in terms of industrial production, even if a few mutants exhibit good catalytic performance under laboratory conditions, their fermentation levels are often low. The expression levels of high specific activity mutants are often limited by factors such as reduced protein folding efficiency or increased metabolic burden on host cells, resulting in a decrease rather than an increase in fermentation enzyme activity (activity unit per unit volume of fermentation broth, U / mL). This significantly increases the purification and overall manufacturing costs for industrial production, severely restricting the commercialization of such mutants.

[0006] Therefore, there is an urgent need in this field to develop a novel alkaline amylase mutant that can overcome the activity-stability trade-off and significantly improve specific activity. At the same time, it must also have good industrial production characteristics and be able to achieve efficient fermentation preparation through high-yield strains, so as to truly meet the urgent needs of the detergent industry for low-cost, high-performance alkaline amylase. Summary of the Invention

[0007] To address the problems of existing technologies, this invention provides a high-specific-activity alkaline amylase mutant. The mutant exhibits significantly increased specific activity and can be widely applied in industrial fields such as detergent processing.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention relates to an alkaline amylase mutant, wherein the 84th amino acid of the alkaline amylase with the amino acid sequence SEQ ID NO:1 is mutated from Gln to Ser, the 287th amino acid is mutated from Pro to Asp, and the 299th amino acid is mutated from Gly to Ala.

[0009] The present invention also relates to DNA molecules encoding the above-mentioned basic amylase mutant.

[0010] The present invention also relates to recombinant expression plasmids comprising the above-described DNA molecules.

[0011] This invention also relates to a Bacillus licheniformis ( Bacillus licheniformis ), which contains the above recombinant expression plasmid.

[0012] The present invention also relates to the application of the aforementioned Bacillus licheniformis in the production of alkaline amylase.

[0013] The present invention also relates to the application of the alkaline amylase mutant in detergents.

[0014] This invention, based on alkaline amylase AH, provides a mutant containing three mutation sites: Q84S, P287D, and G299A. The specific activity of this mutant is increased by 135.8%, reaching 1805 U / mg, achieving unexpected technical results.

[0015] The alkaline amylase mutant provided by this invention has high specific activity, which is beneficial to reducing the production cost of the enzyme and promoting its widespread application in the industrial field. Detailed Implementation

[0016] This invention discloses a high-specific-activity alkaline amylase mutant, its preparation method and application, the DNA molecule encoding the alkaline amylase mutant, the vector, and the host cell. Those skilled in the art can refer to the content herein and appropriately modify the process parameters to achieve the desired result. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0017] This invention utilizes conventional techniques and methods used in the fields of genetic engineering and molecular biology, such as those described in *MOLECMLAR CLONING: A LABORATORY MANUAL, 3rd Ed.* (Sambrook, 2001) and *CURRENT PROTOCOLSIN MOLECMLAR BIOLOGY* (Ausubel, 2003). These general references provide definitions and methods known to those skilled in the art. However, those skilled in the art can use other conventional methods, experimental protocols, and reagents based on the technical solutions described in this invention, without being limited to the specific embodiments of this invention. For example, the following experimental materials and reagents may be used in this invention: Strains and vectors: Escherichia coli DH5α was preserved in our company, and the expression host BL46 and expression vector pTS were constructed by ourselves.

[0018] Culture medium: LB medium (E. coli medium): 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0; LB+Amp medium: LB medium with 100 μg / mL ampicillin; 1* Salt solution: K₂HPO₄ 14g / L; KH₂PO₄ 6g / L; Ammonium sulfate 2g / L; Trisodium citrate 1g / L; MgSO₄·7H₂O 0.2g / L; GMI solution: 95.6 mL of 1* salt solution, 2.5 mL of 20% glucose, 0.4 mL of 5% hydrolyzed casein, and 1 mL of 10% yeast extract; GMII solution: 96.98 mL of 1* salt solution, 2.5 mL of 20% glucose, 0.08 mL of 5% hydrolyzed casein, 0.04 mL of 10% yeast extract, 0.25 mL of 1M MgCl2, and 0.05 mL of 1M CaCl2; Fermentation medium: corn starch 64 g / L, soybean meal 40 g / L, Na2HPO4 4 g / L, KH2PO4 0.3 g / L, high-temperature amylase 0.7 g / L.

[0019] Experimental reagents: DNA polymerase was purchased from Takara, T4 ligase and restriction endonuclease were purchased from Fermentas, plasmid extraction kit and gel purification and recovery kit were purchased from Omega, and GeneMorph II random mutagenesis kit was purchased from Beijing Bomais Biotechnology Co., Ltd.

[0020] The present invention will be further described below with reference to specific embodiments.

[0021] Example 1 Construction of recombinant expression plasmid The amino acid sequence of alkaline amylase AH is SEQ ID NO: 1, and its encoding nucleotide sequence is SEQ ID NO: 2.

[0022] Primer sequences were designed based on the alkaline amylase AH gene and synthesized by BGI Genomics. Using the alkaline amylase AH gene sequence (SEQ ID NO: 2) as a template, PCR amplification was performed using the above primers with the GeneMorph II random mutagenesis PCR kit (Bomais). PCR products were recovered from the gel. Sal I, Mlu After enzyme digestion, I was ligated into the pTS vector, which had also been digested with the same enzymes, and transformed into E. coli DH5α. The transformed samples were plated on LB+Amp plates and incubated upside down at 37°C. Once transformants appeared, they were picked up with a toothpick for PCR verification. Positive transformants were transferred to LB+Amp liquid medium and incubated overnight. Plasmids were extracted using a plasmid extraction kit.

[0023] Example 2: Transformation screening of high specific activity alkaline amylase mutants The correctly sequenced recombinant plasmids were transformed into competent Bacillus licheniformis cells. The specific transformation process was as follows: A single BL4 colony growing on an LB agar plate was picked with a pipette tip and placed in 2 mL of GMⅠ, and cultured for 12 h; the overnight culture was added to 98 mL of GMⅠ and cultured at 37℃ and 200 rpm for approximately 4 h; 10 mL of the culture was added to 90 mL of GMⅡ and cultured at 37℃ and 200 rpm for approximately 1.5 h; the cells were then incubated in an ice-water bath for 30 min, centrifuged at 4000 rpm and 4℃ for 30 min, and the supernatant was discarded; 10 mL of GMⅢ was added and mixed well to obtain competent cells. Then, 5 μL of recombinant plasmid was added to 500 μL of competent cells, and the cells were directly cultured on a shaker at 37℃ and 200 rpm for 1.5 h, centrifuged at low speed for 3 min, some supernatant was discarded, and the cells were evenly spread onto agar plates containing 10 μg / mL chloramphenicol in skim milk powder and incubated at 37℃ for 12 h. The single colony on the plate the next day is the recombinant strain.

[0024] The recombinant engineered bacteria were inoculated into 5 mL of LB liquid medium (1% peptone, 1% NaCl, 0.5% yeast extract), and 10 μg / mL of chloramphenicol was added. The culture was carried out at 37°C with shaking at 200 rpm for 12 h. The bacterial solution was then transferred to fermentation enzyme production medium in 24-well deep-well plates at a 2% inoculation rate and cultured at 37°C with shaking at 200 rpm for 48 h to prepare alkaline amylase solution.

[0025] The alkaline amylase activity of the above enzyme solutions was measured and compared with that of alkaline amylase AH. Finally, a recombinant strain with significantly increased alkaline amylase activity was screened.

[0026] Sequencing analysis revealed that the alkaline amylase mutant expressed by the recombinant strain contained three mutation sites: Q84S, P287D, and G299A.

[0027] Example 3: Shake-flask fermentation verification The recombinant strain was inoculated into 5 mL of LB liquid medium (containing kanamycin, 10 μg / mL) and cultured overnight at 37°C and 220 r / min. It was then transferred to 50 mL of fermentation medium at a 2% inoculation rate and cultured at 37°C and 220 r / min for another 48 h. The supernatant was taken as the crude enzyme solution, and the alkaline amylase activity and protein content of the obtained enzyme solution were determined. The specific activity was calculated.

[0028] The results showed that, compared with alkaline amylase AH, the alkaline amylase mutant containing three point mutations of Q84S, P287D, and G299A provided by the present invention had a specific activity increased by 135.8%, reaching as high as 1805 U / mg, achieving unexpected technical effects.

[0029] The alkaline amylase mutant exhibits significantly increased specific activity, which helps reduce its production costs and promotes its widespread application in the industrial field.

[0030] (I) Methods for detecting amylase activity Refer to the method in GB / T24401-2009.

[0031] (II) Coomassie Brilliant Blue Assay for Protein Content Determination 1. Reagents (1) Coomassie Brilliant Blue G-250 staining solution: Dissolve 100mg of Coomassie Brilliant Blue G-250 in 50ml of 95% ethanol, add 100ml of 85% phosphoric acid, dilute with water to 1 liter, and it can be used at room temperature for 1 month; (2) Standard protein solution: Bovine serum albumin was used to determine the protein content in advance by micro-Kjeldahl nitrogen determination method, and a protein standard solution of 1 mg / ml was prepared according to its purity. (3) Preparation of standard stock solution: Accurately weigh 0.05 g of crystalline bovine serum albumin into a small beaker, add a small amount of distilled water to dissolve it, and then transfer it to a 50 ml volumetric flask. Rinse the remaining liquid in the beaker several times with a small amount of distilled water, and pour the rinsing solution into the volumetric flask. Finally, dilute to the mark with distilled water to prepare the standard stock solution, in which the bovine serum albumin concentration is 1000 μg / ml.

[0032] 2. Drawing the standard curve (1) Take 6 test tubes, number them, add the reagents according to the table below, and mix well.

[0033] Sample (ml) 0 0.1 0.2 0.3 0.4 0.5 Water (ml) 2.0 1.9 1.8 1.7 1.6 1.5 Protein content (mg / ml) 0 0.05 0.1 0.15 0.2 0.25 Accurately pipette 2.5 ml of Coomassie Brilliant Blue solution into 6 clean test tubes. Accurately pipette 0.1 ml of the solution from each of the above tubes into their respective numbered test tubes. Vortex to mix well, let stand at room temperature for 5 min, zero the test tube with test tube number 1, and measure the absorbance at 595 nm. Record the absorbance value.

[0034] Plot the standard curve: Record the absorbance values ​​of tubes 1-6, and plot the standard curve with protein content (μg) on ​​the x-axis and absorbance on the y-axis. Note that because Coomassie Brilliant Blue has a strong staining ability, the cuvettes must be thoroughly cleaned. Quartz cuvettes cannot be used.

[0035] 3. Sample determination Sample preparation: (1) Liquid samples: Dilute the sample to be tested to a protein content of 0.1-0.3 mg / ml, and control the absorbance value after removing the blank (after subtracting the blank) to be between 0.2-0.4; (2) Solid sample: Accurately weigh 1.0000g of sample into a 100ml Erlenmeyer flask, add 20ml of deionized water with a pipette, stir magnetically for 10min, centrifuge at 4000rpm for 10min, take the supernatant for further dilution to determine the protein content, and refer to the liquid sample for dilution method.

[0036] Sample testing: Take a clean test tube, add 2.5 ml of Coomassie Brilliant Blue solution, then add the sample to be tested, vortex and shake well, let stand at room temperature for 5 min, use the standard curve blank as a control, measure the absorbance at 595 nm using a micro cuvette with a 1 cm optical path, and calculate the protein content according to the standard curve.

[0037] 4. Protein content calculation Protein content = X × dilution factor × standard conversion factor.

[0038] X: Protein content (mg / ml) calculated from the standard curve; Standard sample conversion value: The standard sample is 47 mg / ml, and a coefficient is calculated based on the measured value.

[0039] (III) Calculation of specific vitality Specific activity refers to the number of enzyme activity units per unit weight of protein, usually expressed as U / mg protein. Generally speaking, the higher the specific activity of an enzyme, the purer the enzyme.

[0040] Specific activity calculation formula: Specific activity (U / mg) = enzyme activity (U / mL) / protein content (mg / mL).

Claims

1. An alkaline amylase mutant, characterized in that, The mutant is an alkaline amylase with the amino acid sequence SEQ ID NO:1, in which the 84th amino acid is mutated from Gln to Ser, the 287th amino acid is mutated from Pro to Asp, and the 299th amino acid is mutated from Gly to Ala.

2. A DNA molecule encoding the alkaline amylase mutant of claim 1.

3. A recombinant expression plasmid comprising the DNA molecule of claim 2.

4. A type of Bacillus licheniformis ( Bacillus licheniformis ), characterized in that, The Bacillus licheniformis comprises the recombinant expression plasmid of claim 3.

5. The application of Bacillus licheniformis as described in claim 4 in the production of alkaline amylase.

6. The use of the alkaline amylase mutant of claim 1 in detergents.