Alkyl sulfatase mutant with improved enzymatic activity and stability and application thereof

Abstract

The invention discloses an alkyl sulfatase mutant with improved enzymatic activity and stability and application of the alkyl sulfatase mutant. The alkyl sulfatase sdsAp is an alkyl sulfatase which acts on primary alkyl sulfate and hydrolyzes the primary alkyl sulfate into alcohol and sulfate; and the method has huge bioremediation application potential on the environment polluted by SDS (Sodium Dodecyl Sulfate). According to the research, the enzyme activity and thermal stability of sdsAp are modified to improve the efficiency, calculation is carried out in combination with the crystal structure of sdsAp, amino acid mutation sites are semi-rationally designed by utilizing three optimization strategies, and three mutants with improved activity and stability, namely K633H, K633N and T556H, are successfully obtained; wherein the activity of the mutant T556H is improved to 1.7 times compared with that of a wild type, the thermal stability of the mutant T556H is also remarkably improved, and T1 / 2 is 2.5 times of that of the wild type. According to the research, the hydrolysis efficiency of the sdsAp can be effectively improved, the production method is optimized, and the industrial application process of the sdsAp is promoted.

Description

Technical Field

[0001] The invention discloses an alkylsulfatase mutant with improved enzyme activity and stability and application thereof, belonging to the technical field of bioenzyme engineering. Background Art

[0002] Sodium dodecyl sulfate (SDS) is an anionic surfactant. With the large-scale discharge of domestic and industrial wastewater, it is difficult to be degraded due to its long C chain, making it the main surfactant to become a major water pollutant. As this pollution phenomenon becomes increasingly serious, people focus on how to bioremediate the environment. Alkylsulfatase SdsAp is a metal β-lactamase that can hydrolyze sodium dodecyl sulfate (SDS) into high-value-added dodecanol and sulfate ions. It has great application value in the fields of sulfate pollution control and chemical production. Industrial production is often carried out under high temperature conditions. For example, the decomposition of SDS, the main ingredient of detergent, often requires more than 80°C, and natural enzymes are difficult to maintain high activity in such a high temperature environment. This places higher requirements on the activity and heat resistance of industrial enzymes. Therefore, it is particularly important to modify their performance to enhance their environmental adaptability. Summary of the invention

[0003] The object of the present invention is to provide an alkylsulfatase mutant with improved enzyme activity and stability and application thereof, wherein the enzyme activity and stability are higher than those of the wild type.

[0004] The technical solutions adopted by the present invention are summarized as follows:

[0005] The alkylsulfatase mutant is a protein described in any one of the following a1-a3:

[0006] a1: According to the sequence information of the alkylsulfatase SdsAP gene from Pseudomonas sp. S9 (GenBank: HQ189533.1) published in the NCBI database, the protein was obtained by mutation based on the wild type shown in its amino acid sequence. The mutation sites are shown in Table 1.

[0007] Table 1. Active mutant library of SdsAp

[0008]

[0009]

[0010] a2: Screening out proteins with improved enzyme activity and thermal stability obtained by subjecting the amino acid sequence shown in a1 to the aforementioned mutation;

[0011] a3: The activity-enhancing mutations (T556H, K633H and K633N) are superimposed to obtain proteins such as the combination of T556H\K633H and T556H\K633N.

[0012] Specifically, the alkylsulfatase mutant with improved enzyme activity and stability of the present invention is characterized in that the alkylsulfatase mutant is K633H, K633N and T556H;

[0013] Amino acid sequence of K633H:

[0014] ; Among them, L, P, F, I, Q, and E are not shown in the sequence listing;

[0015] MKNASTATHGSRSSVKWKSTSAASVSGSWAATAKATDATKAANDAKDDKTSDAHKGAAKGKGNMWDSKYGKGAADTTNSWRSNSGVTDGYVRNYDSNMTVGKDGTDSTAKAADYYKHRKKVVAVYTHSHVDHYGGVRGVVDADVKAGKVKYAGHAVANVMAGTAMSRRASYMYGNDAKGGAGGTTTSAGTVTTDKTGTHVDGTYMYAGSAAMYYKKKANAADSTHTHNTYSRGAKRDAWSKYNAKWGDDVVMYAMHHWVWGNKVRSRDMYRYNDTRANKGYTMTAVKKKATKSNRGYYGSNHNVKATYVYGWGNATWADKAKRYVMMGGADAVKKAKYYDKGDRWVAVVNHVVANNAAKNMADAGYASGWRNYTGARNGVTDTASDTVKAMDDDAMRKGDVADKHTNDTDKKYTMVNGVNHTGMAKNADATVTTRTNNVMHTTKDASSGDKGDKGKMSYMDNDWNVT

[0016] Amino acid sequence of K633N:

[0017] MKLNALSTATHGSRSSPVKLWKFSTSFLLAASIIVSGQSWAAETAKPATDATKAANDALLKELPFDDKTSFDLAHKGFIAPLPAEPIKGEKGNMIWDPSKYGFIKEGEAAPDTTNPSLWRQSQLINISGLFEVTDGIYQVRNYDLSNMTIVEGKDGITIFDPLISQETAKAALDLYYKHRPKKPVVAVIYTHSHVDHYGGVRGVVDEADVKAGKVKIYAPLGFLEHAVAENVMAGTAMSRRASYMYGNLLPPDAKGQLGAGLGTTTSAGTVTLIPPTDIIKETGETHVIDGLTYEFMYAPGSEAPAEMLYYIKEKKALNAAEDSTHTLHNTYSLRGAKIRDPLAWSKYLNEALKLWGDDVQVMYAMHHWPVWGNKEVREQLSLQRDMYRYINDETLRLANKGYTMTEIAEQVKLPKKIATKFSNRGYYGSLNHNVKATYVLYLGWFIGNPATLWELPPADKAKRYVEMMGGADAVLKKAKEYYDKGDFRWVAEVVNHVVFAEPNNQAAKNMQADALEQLGYQAESGPWRNFYLTGAQELRNGVQQLPTPDTASPDTVKAMDLDLFFDFLAMRLKGPDVADKHITLNLDFTDLKQKYTLEMVNGVLNHTEGMQAKNADATVTLTRETLNNVMLNQTTLKDAESSGDIKIEGDKGKLEELMSYMDNFDFWFNIVTP; wherein L, P, F, I, Q, E are not shown in the sequence listing;

[0018] MKNASTATHGSRSSVKWKSTSAASVSGSWAATAKATDATKAANDAKDDKTSDAHKGAAKGKGNMWDSKYGKGAADTTNSWRSNSGVTDGYVRNYDSNMTVGKDGTDSTAKAADYYKHRKKVVAVYTHSHVDHYGGVRGVVDADVKAGKVKYAGHAVANVMAGTAMSRRASYMYGNDAKGGAGGTTTSAGTVTTDKTGTHVDGTYMYAGSAAMYYKKKANAADSTHTHNTYSRGAKRDAWSKYNAKWGDDVVMYAMHHWVWGNKVRSRDMYRYNDTRANKGYTMTAVKKKATKSNRGYYGSNHNVKATYVYGWGNATWADKAKRYVMMGGADAVKKAKYYDKGDRWVAVVNHVVANNAAKNMADAGYASGWRNYTGARNGVTDTASDTVKAMDDDAMRKGDVADKHTNDTDKKYTMVNGVNHTGMAKNADATVTTRTNNVMNTTKDASSGDKGDKGKMSYMDNDWNVT;

[0019] Amino acid sequence of T556:

[0020] MKLNALSTATHGSRSSPVKLWKFSTSFLLAASIIVSGQSWAAETAKPATDATKAANDAL

[0021] LKELPFDDKTSFDLAHKGFIAPLPAEPIKGEKGNMIWDPSKYGFIKEGEAAPDTTNPSLWRQ

[0022] SQLINISGLFEVTDGIYQVRNYDLSNMTIVEGKDGITIFDPLISQETAKAALDLYYKHRPKKP

[0023] VVAVIYTHSHVDHYGGVRGVVDEADVKAGKVKIYAPLGFLEHAVAENVMAGTAMSRRAS

[0024] YMYGNLLPPDAKGQLGAGLGTTTSAGTVTLIPPTDIIKETGETHVIDGLTYEFMYAPGSEAP

[0025] AEMLYYIKEKKALNAAEDSTHTLHNTYSLRGAKIRDPLAWSKYLNEALKLWGDDVQVMY

[0026] AMHHWPVWGNKEVREQLSLQRDMYRYINDETLRLANKGYTMTEIAEQVKLPKKIATKFS

[0027] NRGYYGSLNHNVKATYVLYLGWFIGNPATLWELPPADKAKRYVEMMGGADAVLKKAKEY

[0028] YDKGDFRWVAEVVNHVVFAEPNNQAAKNMQADALEQLGYQAESGPWRNFYLTGAQELR

[0029] NGVQQLPTPDTASPDHVKAMDLDLFFDFLAMRLKGPDVADKHITLNLDFTDLKQKYTLEM

[0030] VNGVLNHTEGMQAKNADATVTLTRETLNNVMLKQTTLKDAESSGDIKIEGDKGKLEELMSYMDNFDFWFNIVTP; wherein L, P, F, I, Q, E are not shown in the sequence listing;

[0031] MKNASTATHGSRSSVKWKSTSAASVSGSWAATAKATDATKAANDAKDDKTSDAHKGAAKGKGNMWDSKYGKGAADTTNSWRSNSGVTDGYVRNYDSNMTVGKDGTDSTAKAADYYKH RKKVVAVYTHSHVDHYGGVRGVVDADVKAGKVKYAGHAVANVMAGTAMSRRASYMYGNDAKGGAGGTTTSAGTVTTDKTGTHVDGTYMYAGSAAMYYKKKANAADSTHTHNTYSRGA KRDAWSKYNAKWGDDVVMYAMHHWVWGNKVRSRDMYRYNDTRANKGYTMTAVKKKATKSNRGYYGSNHNVKATYVYGWGNATWADKAKRYVMMGGADAVKKAKYYDKGDRWVAVVNH VVANNAAKNMADAGYASGWRNYTGARNGVTDTASDHVKAMDDDAMRKGDVADKHTNDTDKKYTMVNGVNHTGMAKNADATVTTRTNNVMKTTKDASSGDKGDKGKMSYMDNDWNVT.

[0032] Preferably, the optimum temperature of the three mutants T556H, K633N and K633H is 70°C, which is consistent with the wild type; at the same time, under the optimum temperature conditions, the enzyme activities of T556H, K633N and K633H are greater than those of the wild type; the half-life of the T556H, K633N and K633H mutants is longer than that of the wild type.

[0033] The invention discloses an application of the alkylsulfatase mutants T556H, K633N and K633H with improved enzyme activity and stability in the degradation of sodium dodecyl sulfate (SDS).

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention obtains SdsAP alkylsulfatase mutants with improved activity and stability by designing mutations in one or more different amino acid sites, wherein the activity of the T556H mutant is increased to 1.7 times compared with the wild type, while the optimum temperature, optimum pH and half-life (t 1 / 2 ), also increased to varying degrees, among which t 1 / 2 The improvement is quite significant, 2.8 times that of the wild type. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1The results of polyacrylamide gel electrophoresis analysis of purified SdsAp wild-type and mutant proteins; in the figure: (A) M: protein standard marker, 1-12: WT D550E D550H T551A A552C T556F K633N S239F A268CP547AV271D F568L; (B) M: protein standard marker, 13-24: F568T M631L M631F N664Q K633QK633SK633H K338L V271L T556C T556H N664T; (C) M: protein standard marker, 25-31: F667TE90PA254P T283P D358P A579P E609P.

[0037] Figure 2 The standard curve of protein concentration.

[0038] Figure 3 This is the standard curve of SDS substance amount.

[0039] Figure 4 Comparison of the relative enzyme activities of wild-type and mutant SdsAp; In the figure: 1: WT; 2: D550H; 3: T551A; 4: A552C; 5: T556F; 6: K633N; 7: D550E; 8: K633S; 9: P547A; 10: K633H; 11: T556H; 12: F568T; 13: F568L; 14: M631F; 15: N664Q; 16: K633Q; 17: V271D; 18: M631L; 19: S239F; 20: T556C; 21: K338L; 22: A268C; 23: N664T; 24: V271L; 25: F667T

[0040] Figure 5 The optimal pH, optimal reaction temperature, and half-life (t 1 / 2 ) comparison; in the figure: (A) is the optimal pH; (B) is the optimal reaction temperature; (C) is the half-life (t 1 / 2 ). DETAILED DESCRIPTION

[0041] The specific implementation modes of the present invention are further described below in conjunction with the accompanying drawings, but the description of the embodiments does not impose any limitation on the protection scope of the present invention.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0043] Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels; the parts not described in the present invention are prior art.

[0044] This example provides an alkylsulfatase mutant with improved enzyme activity and stability, and methods for constructing its vector, expressing and purifying it, and characterizing its enzymatic properties.

[0045] 1. Vector construction and expression purification

[0046] The wild-type alkylsulfatase SdsAp gene (GenBank: HQ189533.1) (Long, M., L. Ruan, F. Li, Z. Yu, and X. Xu, Heterologous expression and characterization of a recombinantthermostable alkylsulfatase (sdsAP). Extremophiles, 2011. 15 (2): p. 293-301) contains 2025 bp of bases, encoding 674 amino acids, and a 41-amino acid signal peptide at the N-terminus. The gene fragment encoding the signal peptide was truncated to 123 bp, and restriction enzyme sites (BamH I, EcoR I), designed the corresponding primers SdsAp-BamHI-F / SdsAp-EcoRI-R (see Table 2), used pET-his-SdsAp (Long, M., L.Ruan, F.Li, Z.Yu, and X.Xu, Heterologous expression and characterization of a recombinant thermostable alkylsulfatase (sdsAP).Extremophiles, 2011.15(2): p.293-301) donated by Xu Xun Laboratory of the Third Institute of Oceanography, State Oceanic Administration as a template, used 2×Es Taq Master Max DNA polymerase (purchased from Beijing Biolab Technology Co., Ltd.) to amplify the target gene (wild-type sulfatase SdsAp), and inserted it into the expression vector pET28a(+) (purchased from Novagen) to construct the recombinant expression vector of pET28a-SdsAp. The recombinant expression vector of pET28a-SdsAp was transformed into Escherichia coli DH5α competent cells (purchased from GenScript Biotech Co., Ltd.), and positive transformants were screened and sequenced to verify that the pET28a-SdsAp expression vector was successfully constructed.

[0047] In order to improve the catalytic activity and stability of SdsAp, we used homologous complex structure alignment, molecular docking analysis of catalytic activity pockets, and HotSpot Wizard 3.0 online server structure prediction based on catalytic activity pockets and substrate binding channels. At the same time, we combined Consurf to perform sequence conservation analysis to remove highly conservative mutation sites, and then eliminated unreasonable mutation sites based on structural analysis to further narrow the screening range. Finally, a small electronic library containing 24 active mutants was constructed (Table 1). Primer Premier 5.0 computational software was used to design site-directed mutagenesis primers (see Table 2). The introduced codons were selected according to the codon preference of Escherichia coli. QuickChangeTM site-directed mutagenesis kit (purchased from Stratagene, La Jolla, CA, USA) was used to achieve site-directed mutagenesis. The expression vector pET28a-SdsAp was used as a template. The amplification conditions were: 95℃ pre-denaturation for 5min, 98℃ denaturation for 15s, 68℃ annealing for 30s, 72℃ extension for 8min, 26 cycles, and 72℃ extension for 12min. The PCR product was digested with Dpn I for 4 hours. After the template was digested, the product was purified using a gel recovery kit and transferred into Escherichia coli DH5α competent cells for culture. Positive transformants were screened using kanamycin-resistant plates, plasmids were extracted, and the mutation site was determined by first-generation sequencing analysis. The plasmid containing the mutation site was transferred into Escherichia coli BL21 (DE3) competent cells (purchased from GenScript Biotech Co., Ltd.) to obtain 24 SdsAp mutant expression strains.

[0048] The wild-type SdsAp and its 24 mutant expression strains were cultured in the same liquid culture medium, and the culture environment and induction time were controlled to be the same: a single clone colony was picked and placed in 20 ml of liquid Luria-Bertani (LB) medium containing 0.005 wt% kanamycin, cultured at 37°C for 12 to 15 hours, 500 μl of bacterial solution was aspirated, 500 μl of 30 wt% glycerol was added as seed solution, the mass ratio was (1:1), and stored in a -80°C refrigerator. 200 μl of seed solution was aspirated and added to 20 ml of liquid LB containing 0.005 wt% kanamycin, cultured at 37°C for 4 to 5 hours; then the culture was expanded to 1 L LB, cultured at 37°C for 2 to 3 hours, and the OD of the bacterial solution was measured. 600When the pH reaches 0.6-0.8, cool to 16°C and add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.4 mM to induce for 6 hours; adjust the centrifuge speed to 4000 rpm, discard the supernatant after centrifugation for 16 minutes, and collect the bacterial precipitate; resuspend the cell precipitate with pre-cooled cell lysis buffer, set the high-pressure cell disruptor to 4°C temperature and 720 MPa pressure to disrupt the cells for 3 minutes, centrifuge at 4°C and 12000 rpm for 50 minutes, and the supernatant collected is the crude enzyme solution.

[0049] The N-terminus of the pET28a-SdsAp expression vector contains 6 His-tags that bind nickel ions, which can be purified by nickel ion affinity chromatography. Therefore, all crude enzyme samples are added to an agarose affinity chromatography column containing nickel ions (purchased from Invitrogen), and the speed of each drop of the flow-through liquid is adjusted to 6 to 8 seconds. After the crude enzyme solution is finished flowing through the column, a 10mM imidazole eluent is prepared and added to the nickel ion agarose affinity chromatography column. The speed of each drop of the flow-through liquid is adjusted to 2 to 3 seconds. During the flow process, the protein content of the eluted flow-through liquid is continuously tested using the Coomassie Brilliant Blue G250 working solution until the Coomassie Brilliant Blue G250 working solution does not change color. The target protein is eluted according to the 10mM, 20mM, 40mM, and 60mM imidazole concentration gradient, and samples are taken respectively. Finally, 300mM imidazole eluent was prepared and added to the nickel ion agarose affinity chromatography column. All the flow-through was collected in a 50ml centrifuge tube until the Coomassie Brilliant Blue G250 working solution did not change color. The wild-type and mutant proteins collected by nickel ion metal affinity chromatography were analyzed by SDS-PAGE electrophoresis (eg Figure 1 The purified target protein has uniform bands and reaches electrophoresis-level purity, which is consistent with the expected SdsAp molecular weight result.

[0050] Table 2 Primers for SdsAp active mutants

[0051]

[0052]

[0053] 2. Determination of basic enzymatic properties of SdsAp and its mutants

[0054] (1) Coomassie Brilliant Blue method for protein content determination

[0055] Store detergent-compatible Coomassie Brilliant Blue reagent in a refrigerator at 4°C away from light, and shake well before use. Dilute 2mg / ml bovine serum albumin (BSA) in gradients from 25μg / ml to 2000μg / ml, and 0 to 50μg / ml. Pipette 500μl of BSA working solution of different concentrations into 1500μl of Coomassie Brilliant Blue staining solution, shake evenly, let it stand at room temperature for 10 minutes, pipette 200μl of the mixture into a 96-well plate, place the 96-well plate in a preheated and balanced microplate reader, set the wavelength to 595, measure its absorbance value, and draw a standard curve (see Figure 2 The protein concentration was determined by comparing the color reaction with that of the prepared bovine serum albumin (BSA) standard using the Pierce Coomassie Protein Assay Kit (purchased from Thermo Fisher Scientific (China) Co., Ltd.).

[0056] (2) Standard curve of substrate sodium dodecyl sulfate (SDS)

[0057] 1g SDS (purchased from Sinopharm Chemical Reagent Co., Ltd.) powder was weighed and dissolved in pH 8.0 50mM Tris-HCl solution, and the volume was adjusted to 100ml to prepare a 1wt% SDS solution. Then 1wt% SDS was diluted at different multiples to form a concentration gradient of 0.002wt%, 0.004wt%, 0.006wt%, 0.008wt%, 0.01wt%, 0.012wt%, 0.014wt%, 0.016wt% SDS working solution, and the amount of SDS substance corresponding to this concentration ranged from 0 to 0.05μM. 50 μl of SDS working solution of different concentrations was added to 950 μl of stain-all (purchased from Sangon Biotech (Shanghai) Co., Ltd.) staining working solution, mixed well, and allowed to stand for 2 min at room temperature for color development. 200 μl of the mixed staining solution was then placed in a 96-well plate. The 96-well plate was then placed in a preheated ELISA reader, the wavelength was set to 438 nm, the absorbance was measured, and the SDS standard curve was plotted. The results are shown in Figure 3 , its linearity is good, and the square of the correlation coefficient is greater than 0.99.

[0058] (3) Determination of SdsAp catalytic activity

[0059] The activity of SdsAp was determined using a certain concentration of SDS as a substrate. A 1000μl reaction system contained 880μl pH 8.0 50mM Tris-Hcl, 100μl 0.1wt% SDS, and then 20μl 22μg / ml SdsAp protein was added. The reaction was heated in a 70℃ water bath for 5 minutes. Then 50μl of the reaction solution was mixed with 950μl stain-all stain indicator, and 200μl was added to a 96-well plate. The absorbance value at a wavelength of 438nm was measured. The larger the value, the higher the content of unreacted SDS. The difference in absorbance value corresponding to the reduction in the amount of reactant corresponds to the corresponding change in enzyme activity. By detecting the absorbance value A 438 The change value can be used to calculate the reduction in the substrate SDS, thereby determining the specific activity of the enzyme. Each unit of enzyme activity (U) is expressed as the amount of enzyme protein required to convert 1 μmol of substrate SDS into product per minute under fixed reaction conditions. The formula for calculating protein specific enzyme activity is: U / mg = C 0 ×v×c / (t×m)C 0 represents the initial concentration of the substrate SDS, v represents the reaction volume, c represents the substrate conversion rate, t represents the reaction time, and m represents the amount of enzyme protein involved in the reaction.

[0060] The wild type and 24 mutants of SdsAp were tested for their enzyme activities under the same conditions, and the relative enzyme activities of different mutants and the wild type were compared. Figure 4 As shown, the catalytic activity of most mutants was significantly reduced or even inactivated, among which the relative enzyme activity of the three mutants K633H, K633N and T556H was higher than 100%, the relative activity of mutants S239F, K338L, A268C, V271L and V271D was completely lost, and the activity of other mutants was reduced to 0-60%. Analysis of the spatial structure of SdsAp revealed that the mutants with lost activity were due to the introduction of mutant amino acids that destroyed the interaction between the side chain groups of the original protein amino acid residues and other surrounding amino acids, or destroyed its binding with the substrate.

[0061] Three mutants with improved specific activity, K633H, K633N and T556H, were screened out, with specific activities of 19.8 U.mg -1 、26.4U.mg -1 and 28.1 U.mg -1 , 1.2, 1.6 and 1.7 times of the wild type, respectively. At the same time, the mutants T556H / K633H and T556H / K633N were obtained by superimposing the mutation points with improved activity, but their activity was not improved (see Table 3).

[0062] Table 3 Determination of specific activity of positive mutants and superimposed mutants

[0063]

[0064] (4) Determine the optimal pH, optimal temperature and half-life t of the SdsAp enzymatic reaction system 1 / 2

[0065] Verify whether the introduction of the three mutations with improved activity changes the optimal temperature, optimal pH, and half-life t of alkylsulfatase SdsAp 1 / 2 Etc., and the relative parameters of wild type and mutant were determined respectively. The target protein collected by 300mM imidazole elution buffer was replaced by ultrafiltration concentration tube, and the solution pH gradient was set as 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0 respectively; pH ​​4-6 was adjusted by 50mM sodium acetate-acetate buffer, pH 7-9 was adjusted by 50mMTris-HCl, and pH 10-12 was adjusted by 50mM glycine-sodium hydroxide. SdsAp wild type and mutant diluted with different pH buffers were directly added to the reaction system as catalysts, and other components in the system were dissolved and diluted with buffers of corresponding pH. SDS was used as substrate, and the reaction was carried out in a water bath at 70℃ for 5min. According to the above 3 operations, the catalytic activity of SdsAp wild type and mutant under different pH conditions was determined, and three sets of parallel operations were set to collect data to reduce errors. The protein was replaced by the buffer prepared with the optimal reaction pH of SdsAp, and the reaction was carried out under different temperature gradient conditions, with the temperature set at 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃. The relative catalytic activity of SdsAp and mutants under different temperature gradient conditions was determined. Half-life t 1 / 2 First, the initial relative enzyme activity of the wild type and mutants under certain conditions was determined, with the enzyme activity without heating treatment as 100%, and the relative residual enzyme activity of the wild type and mutants after treatment at 70°C for 5 minutes was determined to screen positive mutants with residual relative enzyme activity greater than that of the wild type; secondly, the wild type and positive mutants were incubated at the optimal temperature, and samples were taken according to different time periods and their residual relative enzyme activity was determined. A time-residual enzyme activity line graph was drawn with different treatment time periods as the horizontal axis and the residual relative enzyme activity as the vertical axis; based on the data in the graph, the half-life t of the SdsAp positive mutant under the optimal pH and optimal temperature conditions was calculated. 1 / 2 .

[0066] The results showed that the optimum temperature of the three mutants was 70°C, which was consistent with the wild type; at the same time, under the optimum temperature conditions, the enzyme activities of T556H, K633N and K633H were greater than those of the wild type, which was consistent with the above results. The optimum pH of the three mutants was partially different from that of the wild type, among which the optimum pH of T556H was 7.0, while the optimum pH of K633N changed to 9.0, and the optimum pH of K633H and the wild type were both 8.0 (see Figure 5 ). The half-life of the wild type under optimal conditions is t 1 / 2 The half-life of mutant K633H was 6 min. 1 / 2 The half-life of mutant K633N was 13.3 min. 1 / 2 The half-life of mutant T556H was 11.5 min. 1 / 2 The half-life of the mutants increased to varying degrees compared with the wild type, with the t 1 / 2 The improvement is quite significant, 2.8 times that of the wild type. Therefore, using the corresponding mutant as a production engineering strain can hydrolyze SDS more effectively and has great potential for bioremediation applications in SDS-contaminated environments.

Claims

1. An alkylsulfatase mutant with improved enzyme activity and stability, characterized in that: The alkylsulfatase mutants are K633H, K633N and T556H; The amino acid sequence of K633H: Amino acid sequence of K633N: ; Amino acid sequence of T556H: .

2. The alkylsulfatase mutant with improved enzyme activity and stability according to claim 1, characterized in that: The optimum temperature of the three mutants, T556H, K633N and K633H, was 70 ℃, which was consistent with the wild type. At the same time, under the optimum temperature conditions, the enzyme activities of T556H, K633N and K633H were greater than those of the wild type. The half-lives of the mutants, T556H, K633N and K633H, were longer than those of the wild type.

3. Use of the alkylsulfatase mutant with improved enzyme activity and stability according to claim 1 or 2 in the degradation of sodium dodecyl sulfate (SDS).

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