Wide-temperature-range high-activity medium-temperature alpha-amylase mutant and application thereof

Abstract

The invention discloses a medium-temperature alpha-amylase mutant with wide temperature range and high activity and application thereof. The alpha-amylase mutant is obtained by mutating serine at the 218th site of wild-type alpha-amylase with an amino acid sequence shown as SEQ ID NO: 1 into aspartic acid, asparagine at the 21st site of the wild-type alpha-amylase with the amino acid sequence shown as SEQ ID NO: 1 into phenylalanine, valine at the 317th site of the wild-type alpha-amylase with the amino acid sequence shown as SEQ ID NO: 1 into isoleucine and phenylalanine at the 321st site of the wild-type alpha-amylase with the amino acid sequence shown as SEQ ID NO: 1 into glutamic acid, and the alpha-amylase mutant is named as 4M3a. Compared with a wild type enzyme, the mutant 4M3a keeps the maximum activity of at least 95% within the temperature range of 50-70 DEG C and still keeps the activity of 82% or above at the temperature of 80 DEG C, and the specific enzyme activity is improved by 52%. The mutant can be widely applied to the fields of starch liquefaction, starch sugar preparation, food processing, alcoholic fermentation, textile desizing, feed and the like, and has important industrial application value.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering, specifically to a wide-temperature-range, highly active mesophilic α-amylase mutant and its applications. Background Technology

[0002] α-Amylase (EC 3.2.1.1) is an endonuclease capable of randomly hydrolyzing the α-1,4-glycosidic bonds in starch molecules, and it has important applications in industrial production. Based on their optimal reaction temperature, α-amylases can be classified into low-temperature α-amylases (20-45℃), medium-temperature α-amylases (45-60℃), and high-temperature α-amylases (65-98℃). Among these, medium-temperature α-amylases, due to their mild reaction conditions and specific catalytic properties, are widely used industrially in starch liquefaction, starch sugar preparation, food processing, alcoholic fermentation, and textile desizing.

[0003] Currently, the mesophilic α-amylases used in industry are mainly derived from Bacillus subtilis (Bacillus subtilis). Bacillus subtilis ), Bacillus amyloliquefaciens ( B. amyloliquefaciens Microorganisms such as Bacillus amyloliquefaciens are widely used for their excellent catalytic properties. However, existing mesophilic α-amylases still have the following shortcomings: (a) their temperature adaptability range is narrow, usually concentrated around 50-60℃, with an optimal temperature of about 55℃, which limits their application in conditions requiring higher or lower temperatures; (b) their specific enzyme activity needs to be improved, as lower specific activity increases the production cost of enzyme preparations and reduces their competitiveness in industrial applications; (c) in certain special application environments, such as high-temperature processing or processes with large temperature fluctuations, the catalytic efficiency and stability of existing mesophilic α-amylases cannot fully meet industrial needs.

[0004] Therefore, developing a mesophilic α-amylase with a wider temperature adaptability range and higher specific enzyme activity is of great significance for expanding its application areas, improving production efficiency, and reducing production costs. Summary of the Invention

[0005] The purpose of this invention is to address the problems of narrow temperature adaptability and insufficient specific enzyme activity of existing mesophilic α-amylases derived from Bacillus amyloliquefaciens. By using molecular modification technology, this invention provides a wide-temperature-range, high-activity mesophilic α-amylase mutant and its applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a wide-temperature-range, highly active mesophilic α-amylase mutant, which is obtained by mutating serine at position 218 to aspartic acid, asparagine at position 221 to phenylalanine, valine at position 317 to isoleucine, and phenylalanine at position 321 to glutamic acid in the wild-type α-amylase shown in SEQ ID NO:1.

[0007] The amino acid sequence of the above-mentioned wide-temperature-range high-activity mesophilic α-amylase mutant is shown in SEQ ID NO:2, and it maintains at least 80% of its maximum activity in a temperature range of 50-80℃.

[0008] The present invention also provides a gene encoding the above-mentioned wide-temperature-range high-activity mesophilic α-amylase mutant, the nucleotide sequence of which is shown in SEQ ID NO:4, and the gene does not contain a coding sequence encoding a signal peptide with an amino acid sequence as shown in SEQ ID NO:2.

[0009] The present invention also provides a recombinant expression vector containing the above-mentioned genes.

[0010] This invention also provides a method for preparing a wide-temperature-range, highly active mesophilic α-amylase mutant. The steps are as follows: transforming the recombinant expression vector into Escherichia coli BL21 to construct recombinant bacteria; inoculating the recombinant bacteria into LB medium at an inoculum of 3-7% for fermentation culture; collecting the supernatant by centrifugation; and purifying to obtain the α-amylase mutant.

[0011] Furthermore, the fermentation conditions are as follows: fermentation at 15~20℃ for 24~48 h, with lactose added every 8 h to a final concentration of 5 g / L.

[0012] The present invention also provides the application of the above-mentioned wide-temperature-range, highly active mesophilic α-amylase mutant in starch liquefaction.

[0013] The present invention also provides the application of the above-mentioned wide-temperature-range, highly active, mesophilic α-amylase mutant in fabric desizing.

[0014] The beneficial effects of this invention are as follows:

[0015] (1) The mutant 4M3a prepared by the present invention has a significantly widened temperature range, maintaining at least 95% of the maximum enzyme activity in the range of 50-70℃, and even maintaining more than 82% of the enzyme activity at 80℃, enabling it to efficiently catalyze starch hydrolysis reaction under more temperature conditions. (2) The mutant 4M3a prepared in this invention has a 52% higher enzyme activity than the wild type, which greatly reduces the amount of enzyme preparation used and the production cost. (3) The mutant 4M3a prepared by the present invention can not only be applied to traditional fields such as starch liquefaction, starch sugar preparation, food processing, alcohol fermentation and textile desizing, but also play a stabilizing role in processes with higher temperature requirements or large temperature fluctuations. Because it can maintain high activity over a wider temperature range, the mutant can adapt to temperature fluctuations in different industrial production processes, thereby improving production stability and product quality. Attached Figure Description

[0016] Figure 1 This is a graph comparing the relative activities of mutant 4M3a and wild-type enzyme (Zhongdian) at different temperatures. Detailed Implementation

[0017] The specific implementation methods of the present invention will be described in detail below. The specific implementation methods described herein are for illustration and explanation only and are not intended to limit the present invention.

[0018] Example 1 Construction of a mesothermal α-amylase mutant

[0019] In this embodiment, using Bacillus amyloliquefaciens mesophilic α-amylase as a template, site-directed mutations were introduced at four sites, S218, N221, V317, and F321, using the overlap extension PCR method to construct mutant 4M3a (S218D / N221F / V317I / F321E).

[0020] The Bacillus amyloliquefaciens CICIM B2125 (Liu Y., et al.) used in this invention. Current Microbiology 2010, 60 (3): 162–166.) The amino acid sequence of α-amylase is shown below (the underlined part is the signal peptide sequence): MIQKRKRTVSFRLVLMCTLLFVSLPITKTSAVNGTLMQYFEWYTPNDGQHWKRLQNDADHLSDIGITAVWIPPAYKGLSQSDNGYGPYDLYDLGEFQQKGTVRTKYGTKSELQDAIGSLHSRNVQVYGDVVLNHKAGADATEDVTAVE VNPANRNQETSEEYQIKAWTDFRFPGRGNTYSDFKWHWYHFDGADWDESRKISRIFKFRGEGKAWDWEVSSENGNYDYLMYADVDYDHPDVVAETKKWGIWYANELSLDGFRIDAAK HIKFSFLRDWVQAVRQATGKEMFTVAEYWQNNAGKLENYLNKTSFNQSVFDVPLHFNLQAASSQGGGYDMRRLLDGTVVSRHPEKAVTFVENHDTQPGQSLESTVQTWFKPLAYAFI LTRESGYPQVFYGDMYGTKGTSPKEIPSLKDNIEPILKARKEYAYGPQHDYIDHPDVIGWTREGDSSAAKSGLAALITDGPGGSKRMYAGLKNAGETWYDITGNRSDTVKIGSDGWGE FHVNDGSVSI YVQK.

[0021] In the above sequence, the sites for site-directed mutation are: serine at position 218 (S218), asparagine at position 221 (N221), valine at position 317 (V317), and phenylalanine at position 321 (F321). Specifically, serine at position 218 is mutated to aspartic acid, asparagine at position 221 is mutated to phenylalanine, valine at position 317 is mutated to isoleucine, and phenylalanine at position 321 is mutated to glutamic acid.

[0022] The α-amylase mutant obtained after mutation was named mutant 4M3a, and its amino acid sequence is shown below (the underlined part is the signal peptide sequence): MIQKRKRTVSFRLVLMCTLLFVSLPITKTSAVNGTLMQYFEWYTPNDGQHWKRLQNDADHLSDIGITAVWIPPAYKGLSQSDNGYGPYDLYDLGEFQQKGTVRTKYGTKSELQDAIGSLHSRNVQVYGDVVLNHKAGADATEDVTAVEVNPANRNQETSEEYQIKAWTDFRFPGRGNTYSDFKWHWYHFDGADWDESRKISRIFK FRGEGKAWDWEVDSEFGNYDYLMYADVDYDHPDVVAETKKWGIWYANELSLDGFRIDAAKHIKFSFLRDWVQAVRQATGKEMFTVAEYWSNNAGKLENYLNKTSFNQSVFDIPLHENLQAASSQGGGYDMRRLLDGTVVSRHPEKAVTFVENHDTQPGQSLESTVQTWFKPLAYA FILTRESGYP QVFYGDMYGTKGTSPKEIPS LKDNIEPILKEYAYGPQHDYIDHPDVIGWTREGDSSAAKSGLAALITDGPGGSKRMYAGLKNAGETWYDITGNRSDTVKIGSDGWGEFHVNDGSVSIYVQK.

[0023] The specific construction steps of the above mutant 4M3a are as follows: Primers were designed based on the mesophilic α-amylase gene sequence of Bacillus amyloliquefaciens, including internal primers containing mutation sites and amplification primers at both ends, as shown in Table 1. Table 1 Primer design and sequences for site-directed mutagenesis of mesophilic α-amylase

[0024] Note: The underlined areas are the added restriction enzyme sites. DNA fragments containing each mutation site were obtained by PCR amplification using internal primers containing the mutation sites. Taking the S218D and N221F mutations as an example, the upstream and downstream gene fragments of Bacillus amyloliquefaciens mesophilic α-amylase were first amplified using primers BAA-1F and S218N221F2 and primers S218N221F1 and BAA-2R, and the S218D and N221F mutation sites were then introduced using primers. The PCR amplification conditions were: 96℃ for 30 s, 56℃ for 90 s, and 72℃ for 2 min.

[0025] 3. Using overlap extension PCR technology, the above DNA fragments were spliced ​​into a complete mutant gene. Taking the S218D and N221F mutations as an example, the upstream and downstream gene fragments obtained above were mixed in an equimolar ratio, and the combination of primers BAA-1F and BAA-2R was added to amplify the full-length mesophilic α-amylase gene with the S218D and N221F mutation sites introduced by the primers.

[0026] 4. Repeat steps 1-3 above. Based on the above mutations, use primers V317I1 and V317I2, and primers F321E1 and F321E2 respectively to further introduce V317I and F321E mutations, and finally obtain the α-amylase mutant gene as shown in SEQ ID NO:4 (the gene does not contain the coding sequence for the signal peptide with the amino acid sequence shown in SEQ ID NO:2). 5. The α-amylase mutant gene obtained above was then processed using... Bam After HI digestion, the cloned gene was inserted into the expression vector pET28a(+). Bam HI+ Sma At site I, the recombinant expression vector pET-4M3a is formed; 6. The above recombinant expression vector was transformed into Escherichia coli BL21(DE3) to construct recombinant bacteria BL21-4M3a. The recombinant bacteria were inoculated at a 5% inoculum in 250 mL Erlenmeyer flasks containing 50 mL LB medium for the preparation of recombinase. The fermentation temperature was set at 16℃ and the fermentation time was 48 h. During the process, lactose was added every 8 h to a final concentration of 5 g / L. 7. After fermentation, collect the cells by centrifugation, add 0.1 mg / mL lysozyme, centrifuge again to collect the supernatant; purify the supernatant by nickel column affinity chromatography to obtain the target mutant protein 4M3a.

[0027] Example 2: Determination of the enzymatic properties of mutant 4M3a

[0028] In this embodiment, the optimal operating temperature and specific enzyme activity of mutant 4M3a and wild-type mesophilic α-amylase were determined and compared.

[0029] Enzyme activity assay: α-amylase activity was determined using the 3,5-dinitrosalicylic acid (DNS) method. The reaction system contained 1% soluble starch, 50 mM phosphate buffer (pH 6.0), and 0.1 mL of diluted enzyme solution. The reaction was carried out at different temperatures for 10 min, and the DNS reagent was added to terminate the reaction. After boiling in a water bath for 5 min, the mixture was cooled to room temperature, and the absorbance at 540 nm was measured to calculate the enzyme activity. Each experiment was repeated three times.

[0030] Optimal operating temperature determination: Enzyme activity was measured at 5°C intervals within the range of 30-85°C. Results are as follows:Figure 1 As shown, the optimal activity temperature for wild-type α-amylase is 55℃, at which temperature the enzyme activity is 100%. At 50℃ and 60℃, the activity decreases to 85% and 82%, respectively. At 70℃, the activity drops sharply to 36%, and at 80℃, only 15% of the activity remains. In contrast, mutant 4M3a maintains over 95% of its maximum activity across a wide temperature range of 50-70℃, exhibiting a significant wide temperature range characteristic. At 50℃, 55℃, 60℃, 65℃, and 70℃, mutant 4M3a maintains 96%, 100%, 98%, 96%, and 95% of its maximum activity, respectively. At a high temperature of 80℃, mutant 4M3a still maintains 82% of its maximum activity, while the activity of wild-type α-amylase at this temperature has decreased to 15%.

[0031] The specific enzyme activity (U / mg) of mutant 4M3a and wild-type α-amylase was determined under optimal conditions of 55℃ and pH 6.0. The results are shown in Table 2. The specific enzyme activity of mutant 4M3a reached 155.6 U / mg, which was 52% higher than that of wild-type α-amylase (102.4 U / mg), significantly improving the enzyme's catalytic efficiency.

[0032] Table 2 Comparison of specific enzyme activities between wild-type α-amylase and mutant 4M3a

[0033] Example 3: Application of mutant 4M3a in starch liquefaction This embodiment examines the application effect of mutant 4M3a in the starch liquefaction process under different temperature conditions and compares it with wild-type α-amylase.

[0034] Liquefaction conditions: The pH of a 30% (w / v) corn starch slurry was adjusted to 6.0. Equal amounts of wild-type α-amylase (10 U / g starch) and mutant 4M3a (10 U / g starch) were added, and liquefaction reactions were carried out at different temperatures (55℃, 60℃, 70℃, and 80℃). Samples were taken every 30 minutes to determine the degree of liquefaction (DE value). Each experiment was repeated three times.

[0035] Effect of temperature on DE value: The DE values ​​measured after liquefaction for 2 hours under different temperature conditions are shown in Table 3.

[0036] Table 3 Comparison of starch liquefaction DE values ​​between wild-type enzyme and mutant 4M3a at different temperatures.

[0037] The results showed that both mutant 4M3a and wild-type α-amylase exhibited high starch liquefaction capacity at 55℃ and 60℃, but the DE value of mutant 4M3a was slightly higher than that of wild-type. With increasing temperature, the difference between the two increased significantly: at 70℃, the DE value of wild-type α-amylase had decreased significantly to 22.6, while mutant 4M3a maintained high liquefaction efficiency with a DE value of 34.2, an increase of 51.3%; at 80℃, the DE value of wild-type α-amylase dropped sharply to 10.4, while mutant 4M3a still maintained high liquefaction efficiency with a DE value of 29.6, an increase of 184.6% compared to wild-type α-amylase.

[0038] Liquefaction kinetics study: The DE values ​​of wild-type α-amylase and mutant 4M3a were measured at different time points under 60℃ conditions, and the results are shown in Table 4.

[0039] Table 4. Changes in DE value over time during starch liquefaction at 60℃

[0040] As can be seen from the liquefaction kinetics curves, mutant 4M3a exhibits a higher liquefaction rate than wild-type α-amylase throughout the entire liquefaction process, especially in the initial stage of liquefaction (30-60 min), where the DE value increases significantly faster than that of wild-type α-amylase. This characteristic makes mutant 4M3a particularly suitable for industrial applications requiring rapid liquefaction.

[0041] In summary, mutant 4M3a not only exhibits higher liquefaction efficiency than wild-type α-amylase within its optimal temperature range, but more importantly, it maintains highly efficient starch liquefaction capacity even at high temperatures (70-80℃), while the activity of wild-type α-amylase significantly decreases at these temperatures. This wide temperature range characteristic allows mutant 4M3a to adapt to more diverse industrial production conditions, especially processes with large temperature fluctuations or those requiring higher temperatures.

[0042] Example 4: Application of mutant 4M3a in textile desizing

[0043] This embodiment investigated the application effect of mutant 4M3a in the desizing process of textiles and compared it with wild-type α-amylase and commercially available α-amylase preparations.

[0044] The materials and methods used in this embodiment are as follows: (1) Test cloth: Industrial standard starch-sized cotton cloth (sizing rate 12±1%) with a specification of 10×10 cm was used.

[0045] (2) Desizing conditions: The enzyme dosage was 0.5 g / L (based on enzyme protein), the material-to-liquid ratio was 1:20, the pH was 6.5, and the treatment was carried out at 55℃, 65℃ and 75℃ for 30 minutes respectively.

[0046] (3) Evaluation of desizing effect: The desizing effect was evaluated by measuring the desizing rate, whiteness and tensile strength. Each group of experiments was repeated 5 times.

[0047] The desizing effect at different temperatures is shown in Table 5.

[0048] Table 5 Comparison of desizing effects of three enzymes at different temperatures

[0049] The results showed that at the conventional desizing temperature of 55℃, all three enzymes exhibited good desizing effects, but the desizing rate of mutant 4M3a (92.5%) was slightly higher than that of wild-type α-amylase (85.3%) and the commercial enzyme preparation (89.6%). With increasing temperature, at 65℃, the desizing rate of wild-type α-amylase decreased to 78.6%, and the commercial enzyme preparation achieved a desizing rate of 85.2%, while mutant 4M3a maintained a high desizing capacity of 93.1%. At a high temperature of 75℃, the desizing rate of wild-type α-amylase significantly decreased to 42.5%, and the commercial enzyme preparation also decreased to 65.3%, while mutant 4M3a still maintained a high desizing rate of 90.8%, which was 113.6% higher than wild-type α-amylase and 39.1% higher than the commercial enzyme preparation. Furthermore, fabrics treated with mutant 4M3a showed better whiteness enhancement and good strength retention under all temperature conditions. As can be seen from the above, mutant 4M3a exhibits significant wide temperature range characteristics and high efficiency in textile desizing applications. In particular, it can maintain excellent desizing effect at higher temperatures (65-75℃). This characteristic makes it particularly suitable for high-efficiency and energy-saving desizing processes that require high-temperature treatment, as well as industrial environments where temperature control is not precise enough.

[0050] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A wide-temperature-range, highly active mesophilic α-amylase mutant, characterized in that: The α-amylase mutant was obtained by mutating serine at position 218 to aspartic acid, asparagine at position 221 to phenylalanine, valine at position 317 to isoleucine, and phenylalanine at position 321 to glutamic acid in the wild-type α-amylase as shown in SEQ ID NO:

1.

2. The wide-temperature-range, high-activity mesophilic α-amylase mutant according to claim 1, characterized in that: The amino acid sequence of the α-amylase mutant is shown in SEQ ID NO:

2.

3. The wide-temperature-range, high-activity mesophilic α-amylase mutant according to claim 1, characterized in that: It maintains at least 80% of its maximum activity within a temperature range of 50-80℃.

4. A gene encoding a wide-temperature-range, highly active mesothermal α-amylase mutant as described in any one of claims 1 to 3, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

4.

5. A recombinant expression vector containing the gene of claim 4.

6. A method for preparing a wide-temperature-range, highly active mesophilic α-amylase mutant, characterized in that: The recombinant expression vector described in claim 5 was transformed into Escherichia coli BL21 to construct a recombinant bacterium. The recombinant bacterium was inoculated into LB medium at an inoculum of 3-7% for fermentation culture. The supernatant was collected by centrifugation and purified to obtain an α-amylase mutant.

7. The preparation method according to claim 6, characterized in that: The fermentation conditions are as follows: fermentation at 15~20℃ for 24~48 h, with lactose added every 8 h to a final concentration of 5 g / L.

8. The application of the wide-temperature-range, highly active mesophilic α-amylase mutant as described in claim 1 in starch liquefaction.

9. The application of the wide-temperature-range, highly active mesophilic α-amylase mutant as described in claim 1 in fabric desizing.

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