A thermostable alkaline amylase mutant and its application

Abstract

The present invention relates to a thermostable alkaline amylase mutant and its application. The alkaline amylase mutant of the present invention is obtained by deleting the amino acids at positions 2-11, 2-21, 2-31, 2-36, 2-41, and 2-43 at the N-terminus of the alkaline amylase with the amino acid sequence shown in SEQ ID NO.1. The obtained alkaline amylase mutant has good heat resistance, and its residual enzyme activity at 70 °C is significantly higher than that of the alkaline amylase before the deletion; at 100 °C, the alkaline amylase without N-terminal deletion is inactivated in about 5 minutes, while the alkaline amylase mutant after N-terminal deletion still has activity in the first 40 minutes. In addition, the alkaline amylase mutant of the present invention also has antioxidant properties. Therefore, the alkaline amylase mutant of the present invention can not only adapt to high-temperature environments above 70 °C but also has good antioxidant properties.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a thermotolerant alkaline amylase mutant and its application. Background Art

[0002] To increase the abrasion resistance, smoothness, and antistatic properties of fabrics during weaving, improve their strength and cohesion, and enhance their weavability, sizing processing is performed on yarns before fabric weaving. Sizing processing of yarns refers to the treatment of yarns with sizing agents during the weaving process, and common sizing agents include starch. However, after sizing, the sizing agent penetrates between the fibers and partially adheres to the surface of the yarn. The sizing agent will contaminate the dyeing and finishing processing liquid, hinder the chemical reaction between the fiber and the dye, and make it difficult to carry out the dyeing and finishing processing. Therefore, it is necessary to desize the sized fabric for subsequent dyeing and finishing processing. Fabric desizing mainly includes alkali desizing, enzyme desizing, acid desizing, etc. Traditional fabric desizing treatment mainly uses alkaline amylase to hydrolyze starch under high-temperature and strong-alkali conditions, so that the starch falls off from the yarn, thereby realizing fabric desizing. This method combines the advantages of thermal alkali desizing and enzyme desizing, can effectively remove starch and impurities on the yarn fiber, and can improve the whiteness of the fabric, so it is widely used in fabric desizing. However, during the desizing process, the working temperature is as high as 70°C, and the instantaneous working environment temperature may be higher than 100°C. Most of the reported alkaline amylases currently have an optimum temperature lower than 70°C and are easily inactivated at temperatures higher than 70°C, making it difficult to meet the process requirements. In addition, during the desizing process, oxidants are often added to assist desizing, improve the desizing effect, improve the performance of the fiber, and enhance the strength of the fiber material. However, the existing alkaline amylases are easily inactivated in an oxidative environment, thereby affecting the desizing effect.

[0003] Currently, foreign research on alkaline amylase mainly focuses on the construction of enzyme-producing recombinant bacteria and the modification of the characterization, antioxidant properties, and thermal stability of alkaline amylase. Domestic research on alkaline amylase mainly focuses on the screening of enzyme-producing microorganisms, the optimization of enzyme-producing conditions, the separation and purification of enzymes, and a small amount of modification of the thermal stability of alkaline amylase. However, the thermal stability of the modified alkaline amylase decreases under conditions higher than 50°C and still cannot meet the industrial requirements. Therefore, there is an urgent need for an alkaline amylase that still has good stability under high-temperature and oxidative conditions. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problem in the prior art that there is a lack of an alkaline amylase that still has good stability under high-temperature and oxidative conditions.

[0005] To solve the above technical problems, the present invention provides a thermostable alkaline amylase mutant and its application. The present invention respectively deletes the amino acids at positions 2-11, 2-21, 2-31, 2-36, 2-41, and 2-43 at the N-terminus of the alkaline amylase with the amino acid sequence shown in SEQ ID NO.1. The obtained alkaline amylase mutant has good heat resistance, and the residual enzyme activity at 70°C is significantly higher than that of the alkaline amylase before the deletion; at 100°C, the alkaline amylase without N-terminal deletion is inactivated in about 5 minutes, while the alkaline amylase mutant after N-terminal deletion has activity in the first 40 minutes. In addition, the alkaline amylase mutant of the present invention also has antioxidant properties. Therefore, the alkaline amylase mutant of the present invention can adapt to high-temperature environments above 70°C and has good antioxidant properties.

[0006] The first object of the present invention is to provide an alkaline amylase mutant, and the alkaline amylase mutant is any one of the following modifications to the alkaline amylase with the amino acid sequence shown in SEQ ID NO.1:

[0007] (1) Deletion of amino acids at positions 2-11 at the N-terminus;

[0008] (2) Deletion of amino acids at positions 2-21 at the N-terminus;

[0009] (3) Deletion of amino acids at positions 2-31 at the N-terminus;

[0010] (4) Deletion of amino acids at positions 2-36 at the N-terminus;

[0011] (5) Deletion of amino acids at positions 2-41 at the N-terminus;

[0012] (6) Deletion of amino acids at positions 2-43 at the N-terminus.

[0013] Furthermore, the sequence of SEQ ID NO.1 is shown as follows:

[0014] MNASYVKKVSLYLLLSFVITLPLFSPTFSINSVDASQGGEQQSFSWDNATVYFAITDRFHDGNPSNNQSYGRPQEDAWGQNIGTFHGGDLQGLTDKLNEGYFTELGINAIWITAPYEQVHGWVGGGSDGDFAHYAYHGYYALDYTMIDQNMGTVEDMREFVHTAHEQGIRVVLDVVMNHPGYNTIKDMHEYGFGNPGVSEHWTPGQGQNWHDVHQQINYEDAGAWRHWWGPWIRAGIAGYESCGNSEITMCLAGLPDFRTELTHSVGLPPLLETKWNQERQGGYEDWIVPAANDLRRDLGVAPADYIVKWLSAWVEEFGIDGFRVDTAKHVELSRWQQLKDAANEALWKWREENPEAPGANWTDDFWMVGEVWGHGVGRSEYFDHGFDSVINFTFQGEHGNGPAYRLDTMESTFSRYADAINTDSTFNVLSYLSQHDTQLYPRERLIDGGTYLMLLPGGVQVFYGDETARPFGPTGSDPHQGTRSSMNWDSVNEEVLHHWQKMGQFRSNHLAVGAGEHRQIAASPYTFSRTYVEGDVDDRVVVVVGASGETTIDVSSVFSDGQQVRDFYTGNVTVVENGHVTFPAHRNGVILIERAGDLLPSVSASPAGGEFDREQQEVTLYVQHAEWGAYTIDGSDPKEEGTAFANGDVITVGRELNIDESLTLRLYAENETGEAKAEFTFTKVPAYPQVFADPPGGTFAGDTLKVTLHTRNVEGGTYSIDGSDEQAFTDGDEIMIGKGIEAGSETTLQLKAENEFGTIEETYTYTKSTGLTIYFKKPDSWGTPHLYYYDTNPKVDEPTWSEAPEMEHYEGDWYTHTIEGVESVRLLFKDRGTNQWPGPGEPGFFRDQDGWFDGEWHVDRPGDATDITIYYKTGWTHPHIHYSLNQGAWTTLPGVPLTKSEYEGYVKVTIEAEEGSQLRAAFNNGSGQWDNNQGRDYDFSSGVHTLADGRILSGTPK

[0015] The second object of the present invention is to provide a gene encoding the above-mentioned alkaline amylase mutant.

[0016] The third object of the present invention is to provide a recombinant plasmid carrying the above-mentioned gene.

[0017] The fourth object of the present invention is to provide a recombinant cell expressing the above-mentioned alkaline amylase mutant.

[0018] Further, the recombinant cell is a bacterium or a fungus.

[0019] The fifth object of the present invention is to provide an application of the above-mentioned alkaline amylase mutant, the above-mentioned gene, the above-mentioned recombinant plasmid or the above-mentioned recombinant cell in hydrolyzing starch.

[0020] Further, the application is to add the alkaline amylase mutant or an expression system containing the alkaline amylase mutant to a system containing starch for reaction.

[0021] The sixth object of the present invention is to provide a method for desizing a fabric, wherein the desizing method is to apply the above-mentioned alkaline amylase mutant to the fabric for desizing, wherein the fabric is sized with starch, and the pH of the desizing is 9-11.

[0022] Further, the temperature of the desizing is 70-100 °C.

[0023] Further, the desizing method is to apply the above-mentioned alkaline amylase mutant and an oxidant to the fabric for desizing, wherein the fabric is sized with starch, the pH of the desizing is 9-11, and the oxidant includes hydrogen peroxide.

[0024] Advantages of the present invention:

[0025] The present invention provides a heat-resistant alkaline amylase mutant and its application. The present invention respectively deletes the amino acids at positions 2-11, 2-21, 2-31, 2-36, 2-41, and 2-43 at the N-terminus of the alkaline amylase shown in the amino acid sequence SEQ ID NO.1. The obtained alkaline amylase mutant has good heat resistance, and the residual enzyme activity at 70 °C is significantly higher than that of the alkaline amylase before deletion; at 100 °C, the alkaline amylase without N-terminal deletion is inactivated in about 5 minutes, while the alkaline amylase mutant after N-terminal deletion still has activity in the first 40 minutes. In addition, the alkaline amylase mutant of the present invention also has antioxidant properties. Therefore, the alkaline amylase mutant of the present invention can adapt to high-temperature environments above 70 °C and has good antioxidant properties. Description of the Drawings

[0026] To make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments of the present invention and in conjunction with the accompanying drawings, wherein

[0027] Figure 1 is the pET-28a-AmyⅠ map;

[0028] Figure 2 is a schematic diagram of the mutation sites of alkaline amylase, where Amy I represents alkaline amylase, T-11 represents a mutant of alkaline amylase with amino acids 2-11 deleted at the N-terminus, T-21 represents a mutant of alkaline amylase with amino acids 2-21 deleted at the N-terminus, T-31 represents a mutant of alkaline amylase with amino acids 2-31 deleted at the N-terminus, T-36 represents a mutant of alkaline amylase with amino acids 2-36 deleted at the N-terminus, T-41 represents a mutant of alkaline amylase with amino acids 2-41 deleted at the N-terminus, and T-43 represents a mutant of alkaline amylase with amino acids 2-43 deleted at the N-terminus;

[0029] Figure 3 is the residual enzyme activity of alkaline amylase AmyⅠ and mutants of alkaline amylase after incubation at 70°C for 60 min, where WT represents alkaline amylase AmyⅠ;

[0030] Figure 4 is the thermal stability result of alkaline amylase AmyⅠ and mutants of alkaline amylase at 100°C;

[0031] Figure 5 is the antioxidant property of alkaline amylase AmyⅠ and mutants of alkaline amylase. Specific Embodiments

[0032] The following further illustrates the present invention in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the examples given are not intended to limit the present invention.

[0033] Example 1: Construction of a Mutant Library

[0034] (1) Construction of the pET-28a-AmyⅠ Expression Vector

[0035] Using pET-28a as the vector, a pET-28a-AmyⅠ expression vector containing the alkaline amylase AmyⅠ gene was synthesized: the pET-28a vector was amplified with primers pET-28a-F / pET-28a-R, and the target gene fragment was amplified with primers AmyⅠ-F / AmyⅠ-R.

[0036] The pET-28a vector and the target gene fragment obtained by amplification in the previous step were homologously recombined using the one-step cloning method to obtain the pET-28a-AmyⅠ plasmid (Figure 1 )。

[0037] Table 1 Primers involved in Example 1(1)

[0038]

[0039] (2) Construction of N-terminal truncated alkaline amylase mutants

[0040] Circular PCR was performed on plasmid pET-28a-AmyⅠ using primers T-11-F / T-11-R to obtain plasmid pET-28a-AmyⅠ-T11.

[0041] Circular PCR was performed on plasmid pET-28a-AmyⅠ using primers T-21-F / T-21-R to obtain plasmid pET-28a-AmyⅠ-T21.

[0042] Circular PCR was performed on plasmid pET-28a-AmyⅠ using primers T-31-F / T-31-R to obtain plasmid pET-28a-AmyⅠ-T31.

[0043] Circular PCR was performed on plasmid pET-28a-AmyⅠ using primers T-36-F / T-36-R to obtain plasmid pET-28a-AmyⅠ-T36.

[0044] Circular PCR was performed on plasmid pET-28a-AmyⅠ using primers T-41-F / T-41-R to obtain plasmid pET-28a-AmyⅠ-T41.

[0045] Circular PCR was performed on plasmid pET-28a-AmyⅠ using primers T-43-F / T-43-R to obtain plasmid pET-28a-AmyⅠ-T43.

[0046] The linearized fragments amplified in the previous step were purified by one-step cloning, and after transformation, plasmids pET-28a-AmyⅠ-T11, pET-28a-AmyⅠ-T21, pET-28a-AmyⅠ-T31, pET-28a-AmyⅠ-T36, pET-28a-AmyⅠ-T41, pET-28a-AmyⅠ-T43( Figure 2 )。

[0047] Table 2 Sequences involved in Example 1(2)

[0048]

[0049]

[0050] Example 2: Enzyme activity verification of N-terminal truncated alkaline amylase mutants

[0051] (1) Determination of the residual enzyme activity of alkaline amylase and alkaline amylase mutants

[0052] Using the E. coli BL21(DE3) strain containing the pET-28a-AmyⅠ plasmid as the control group, single colonies of the E. coli BL21(DE3) strains carrying the pET-28a-AmyⅠ, pET-28a-AmyⅠ-T11, pET-28a-AmyⅠ-T21, pET-28a-AmyⅠ-T31, pET-28a-AmyⅠ-T36, pET-28a-AmyⅠ-T41, pET-28a-AmyⅠ-T43 plasmids were picked into 24 deep-well plates (1 mL of liquid LB) and cultured overnight at 37 °C; inoculated at an inoculation amount of 2% into 24 deep-well plates (2 mL of liquid TB) and cultured with shaking at 37 °C until OD 600 reached 0.6 - 0.8, IPTG with a final concentration of 0.5 mmol / L was added for induction, and cultured in a shaker at 30 °C for about 18 h. The cells were collected by centrifugation at 10000 r / min, and the supernatant was discarded; the cells were suspended with the same volume of phosphate buffer and sonicated, and the broken supernatant was collected at 10000 r / min, and after purification, SDS-PAGE detection was carried out to obtain alkaline amylase and the alkaline amylase mutants T-11, T-21, T-31, T-36, T-41, T-43 with N-terminal deletion respectively.

[0053] The obtained alkaline amylase and the alkaline amylase mutants with N-terminal deletion were diluted to appropriate concentrations respectively. 100 μL of the diluted enzyme solution was incubated at 70 °C for 60 min respectively, then added to 400 μL of the pre-warmed starch substrate, reacted at 50 °C for 10 min, then 500 μL of DNS was added to terminate the reaction, boiled in boiling water for 10 min, after cooling, 3 ml of deionized water was added, and the absorbance was measured at 540 nm. The amount of enzyme required to produce 1 μmol / L of reducing sugar per minute was used as one enzyme activity unit (U). The initial enzyme activity measured without the 70 °C incubation treatment was used as the highest enzyme activity, and the enzyme activity measured after the 70 °C incubation treatment was used as the residual enzyme activity.

[0054] As Figure 3 shown, the residual enzyme activities of the alkaline amylase mutants at 70 °C were all improved compared with alkaline amylase. Among them, the residual enzyme activities of T-11, T-21, T-36, T-41, T-43 were greatly improved, and their residual enzyme activities were increased by 70%, 71%, 70%, 77%, 70% respectively compared with the control group, indicating that the mutants T-11, T-21, T-36, T-41, T-43 have good thermal stability at 70 °C and can be used as a heat-resistant alkaline amylase for textile industrial production.

[0055] (2) Determination of thermal stability at 100 °C

[0056] Using the E. coli BL21(DE3) strain containing the pET-28a-AmyⅠ plasmid as the control group, single colonies of the E. coli BL21(DE3) strains carrying pET-28a-AmyⅠ, pET-28a-AmyⅠ-T11, pET-28a-AmyⅠ-T21, pET-28a-AmyⅠ-T31, pET-28a-AmyⅠ-T36, pET-28a-AmyⅠ-T41, and pET-28a-AmyⅠ-T43 plasmids were picked into a 24-deep well plate (1 mL of liquid LB) and cultured overnight at 37 °C; inoculated at an inoculum size of 2% into a 24-deep well plate (2 mL of liquid TB) and cultured with shaking at 37 °C until the OD 600 reached 0.6 - 0.8. IPTG with a final concentration of 0.5 mmol / L was added for induction, and the cells were cultured at 30 °C for about 18 h. The cells were collected by centrifugation at 10000 r / min, and the supernatant was discarded; the cells were suspended with the same volume of phosphate buffer and sonicated. The broken supernatant was collected at 10000 r / min, and after purification, SDS-PAGE was performed for detection to obtain alkaline amylase and the N-terminal deleted alkaline amylase mutants T-11, T-21, T-31, T-36, T-41, and T-43, respectively.

[0057] The obtained alkaline amylase and the N-terminal deleted alkaline amylase mutants were diluted to appropriate concentrations. 100 μL of the diluted enzyme solution was taken and added to 400 μL of the preheated starch substrate, and the reaction was carried out in a water bath at 100 °C for 80 min. Samples were taken every 20 min and their residual enzyme activities were measured. The enzyme activity of the unheated enzyme was used as the highest enzyme activity to determine its thermal stability.

[0058] As Figure 4 shown, the residual enzyme activities of the alkaline amylase mutants T-11, T-21, T-31, T-36, T-41, and T-43 at 100 °C were all increased compared with the control group. Among them, the residual enzyme activity of T-40 was increased by 13.16 times compared with the control group.

[0059] (3) Determination of antioxidant properties of alkaline amylase mutants

[0060] Using the E. coli BL21(DE3) strain containing the pET-28a-AmyⅠ plasmid as the control group, single colonies of the E. coli BL21(DE3) strains carrying the pET-28a-AmyⅠ, pET-28a-AmyⅠ-T10, pET-28a-AmyⅠ-T20, pET-28a-AmyⅠ-T30, pET-28a-AmyⅠ-T35, pET-28a-AmyⅠ-T40, pET-28a-AmyⅠ-T42 plasmids were picked into 24 deep-well plates (1 mL of liquid LB) and cultured overnight at 37°C; inoculated into 24 deep-well plates (2 mL of liquid TB) at an inoculation amount of 2% and cultured with shaking at 37°C until the OD 600 reached 0.6 - 0.8, IPTG with a final concentration of 0.5 mmol / L was added for induction, and cultured at 30°C for about 18 h. The cells were collected by centrifugation at 10000 r / min, and the supernatant was discarded; the cells were suspended with the same volume of phosphate buffer and sonicated, and the broken supernatant was collected at 10000 r / min. After purification, SDS-PAGE detection was carried out to obtain alkaline amylase and the N-terminal deleted alkaline amylase mutants T-11, T-21, T-31, T-36, T-41, T-43 respectively.

[0061] The obtained alkaline amylase and the N-terminal deleted alkaline amylase mutants were incubated with different concentrations of H2O2 (0 mM, 50 mM, 100 mM, 200 mM, 400 mM, 500 mM) at 35°C for 30 min, and then catalase with a final concentration of 2000 U / mL was added to obtain the enzyme solutions treated with different concentrations of H2O2. The treated enzyme solutions were diluted to an appropriate concentration. 100 μL of the diluted enzyme solution was taken and added to 400 μL of the pre-warmed starch substrate, and the reaction was carried out at 50°C for 10 min. Then 500 μL of DNS was added to terminate the reaction, boiled in boiling water for 10 min, and after cooling, 3 ml of deionized water was added, and the absorbance was measured at 540 nm. The enzyme activity at 0 mM was taken as the highest enzyme activity ( Figure 5 ).

[0062] As Figure 5 shown, the residual enzyme activities of the alkaline amylase mutants at different concentrations of H2O2 were significantly improved compared with the control group. Among them, the residual enzyme activity of T-40 at 200 mM H2O2 was increased by about 43% compared with the control group.

[0063] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. An alkaline amylase mutant, characterized in that, The alkaline amylase mutant is any of the following modifications to the alkaline amylase with the amino acid sequence shown in SEQ ID NO. 1: (1) Deletion of amino acids at positions 2-11 at the N-terminus; (2) Deletion of amino acids at positions 2-21 at the N-terminus; (3) Deletion of amino acids at positions 2-31 at the N-terminus; (4) Deletion of amino acids at positions 2-36 at the N-terminus; (5) Deletion of amino acids at positions 2-41 at the N-terminus; (6) Deletion of amino acids at positions 2-43 at the N-terminus.

2. A gene encoding the alkaline amylase mutant according to claim 1.

3. A recombinant plasmid carrying the gene according to claim 2.

4. A recombinant cell expressing the alkaline amylase mutant according to claim 1, characterized in that, The recombinant cell is a bacterium or a fungus.

5. Use of the alkaline amylase mutant according to claim 1, the gene according to claim 2, the recombinant plasmid according to claim 3 or the recombinant cell according to claim 4 in the hydrolysis of starch.

6. The application according to claim 5, wherein: The use is to add the alkaline amylase mutant or an expression system containing the alkaline amylase mutant to a system containing starch for reaction.

7. A desizing method for a fabric, characterized in that, The desizing method is to apply the alkaline amylase mutant according to claim 1 to the fabric for desizing, wherein the fabric is sized with starch and the pH of the desizing is 9-11.

8. The desizing method according to claim 7, characterized in that, The temperature of the desizing is 70-100 °C.

9. The desizing method according to claim 7, characterized in that The desizing method is to apply the alkaline amylase mutant according to claim 1 and an oxidizing agent to the fabric for desizing, wherein the fabric is sized with starch and the pH of the desizing is 9-11, and the oxidizing agent includes hydrogen peroxide.

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