WO2024077702A1 - 一种低温高活力碱性蛋白酶突变体及其应用 - Google Patents

一种低温高活力碱性蛋白酶突变体及其应用 Download PDF

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WO2024077702A1
WO2024077702A1 PCT/CN2022/131434 CN2022131434W WO2024077702A1 WO 2024077702 A1 WO2024077702 A1 WO 2024077702A1 CN 2022131434 W CN2022131434 W CN 2022131434W WO 2024077702 A1 WO2024077702 A1 WO 2024077702A1
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mutant
alkaline protease
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amino acid
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路福平
刘逸寒
李玉
马向阳
刘夫锋
张会图
王洪彬
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天津科技大学
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Definitions

  • the invention belongs to the technical field of bioengineering, and specifically relates to alkaline protease mutants with improved enzyme activity obtained by iterative saturation mutation through overlapping PCR technology, and the preparation and application thereof.
  • Proteases are widely found in animals, plants and microorganisms. Microorganisms have become an important source of biological enzymes because of their fast growth rate, simple growth conditions, special metabolic processes and wide distribution. Its application mainly revolves around its function of hydrolyzing protein peptide bonds. In production and life, there are several main needs: to convert complex macromolecular protein structures into simple small molecule peptide chains or amino acids, so that they are easy to absorb or wash away, such as in the fields of food, detergents, feed, etc.; to partially destroy the protein structure to separate the material components, which is very effective in the processing of protein-rich materials such as leather and silk; to promote the degradation of environmental pollutants and be used in the field of environmental protection. Proteases can catalyze both hydrolysis reactions and their reverse reactions, and have high activity and specificity, which is very suitable for the production needs of the pharmaceutical industry for certain specific molecules.
  • proteases can be divided into alkaline proteases, acidic proteases and neutral proteases.
  • the optimal pH of alkaline proteases is 8.0-11.0. Most of them are derived from microorganisms, especially alkaline proteases produced by industrial microorganisms, which have more obvious advantages in hydrolysis ability and alkaline resistance. Compared with alkaline proteases from animals and plants, microbial alkaline proteases can be secreted outside the cell, and have the characteristics of relatively simple downstream technical processing, low price, wide source, easy cultivation of bacteria, and easy industrial mass production. Therefore, alkaline protease research has become a hot spot in protease research.
  • alkaline proteases are mainly used in the detergent industry because of their high stability and activity under alkaline conditions. Their use in detergent formulations accounts for 89% of their total sales. Due to its wide application, alkaline proteases have attracted the attention of more and more researchers, and people are committed to exploring new alkaline proteases with unique properties and higher activity.
  • Serine alkaline proteases are one of the most important enzymes in industrial detergents, accounting for about 35% of the total sales of microbial enzymes.
  • the first commercial detergent containing bacterial proteases was produced by Gebrüder schnyder in 1959.
  • liquid detergents on the market have become more popular with consumers than solid detergents.
  • Adding alkaline proteases to detergents can maintain the original color of clothes, improve the product's decontamination ability, reduce the amount of surfactant added, and save water, energy, and protect the environment. According to survey results, the washing temperature in my country is usually concentrated between 10-20°C, while most of the proteases added to commercially available detergents are medium-temperature alkaline proteases, with an optimum temperature of 40-60°C.
  • the essence of directed protein evolution is to construct a molecular diversity library and screen mutants with improved traits from the library.
  • library construction it can be divided into four strategies: random evolution, reorganization technology, semi-rational evolution and rational evolution.
  • the general idea is to start from a target gene or a family of related family genes, create a molecular diversity library by mutating or recombining the coding gene; screen the library to obtain genes that can encode improved traits as a template for the next round of evolution; complete the evolution that takes thousands of years in nature in a short time, so as to obtain proteins with improved functions or new functions.
  • the design and modification of enzyme molecules is the result of the complementary development and penetration of genetic engineering, protein engineering and computer technology.
  • Overlap PCR is a technique that uses primers with complementary ends to form overlapping chains in PCR products, thereby extending the overlapping chains in the subsequent amplification reaction to overlap and splice the amplified fragments from different sources. It is reasonable and practical to use this technology to perform site-directed mutagenesis on gene sequences and thus achieve directed modification of proteins.
  • the Bacillus expression system has the following advantages: 1. It can efficiently secrete various proteins; 2. Many Bacillus have been used in the fermentation industry for a long time, are non-pathogenic, and do not produce any endotoxins; 3. The genetic background of Bacillus microorganisms has been very clear, and they grow rapidly and have no special requirements for nutrients; 4. The codon preference is not obvious; 5. The fermentation process is simple. Bacillus is an aerobic bacteria and does not require anaerobic fermentation equipment. After the fermentation is completed, the fermentation liquid and bacterial cells can be simply separated to enter the separation, purification and recovery stage of the target protein; 6. It has stress resistance and can produce a variety of heat-resistant enzyme preparations.
  • the alkaline protease gene from Bacillus Clausii is molecularly modified by overlapping PCR, and a Bacillus subtilis expression system is used for high-throughput screening to obtain an alkaline protease mutant gene with improved enzyme activity at low temperature.
  • the purpose of the present invention is to provide a mutant of alkaline protease with high activity at low temperature.
  • the alkaline protease gene (apr) from Bacillus clausii was combined with the shuttle vector pLY-3 to construct a recombinant expression vector pLY-3-apr, which was expressed in Bacillus subtilis WB600; the key hotspot area was determined by homology modeling of alkaline protease (ALK) and analysis of its docking results with substrate AAPF (Suc-Ala-Ala-Pro-Phe-pNA); the gene apr was iteratively saturated mutated using overlapping PCR technology, and the mutants were high-throughput screened using AAPF to select mutants with high activity at low temperature.
  • ALK alkaline protease
  • the alkaline protease gene apr from Bacillus clausii was saturated mutagenesis, and the Bacillus subtilis expression system was used to screen out the high-activity ALK mutants G95P, G95P/A96D, G95P/A96D/S99W, G95P/A96D/S99W/S101T, G95P/A96D/S99W/S101T/P127S, G95P/A96D/S99W/S101T/P127S/S126T and their encoding genes aprm1, aprm2, aprm3, aprm4, aprm5, and aprm6 at 10°C. Efficient expression of each mutant was achieved in Bacillus amyloliquefaciens, and ALK mutants with improved enzyme activity were obtained through fermentation, extraction and other techniques.
  • One of the technical solutions provided by the present invention is an alkaline protease mutant, wherein the mutant is obtained by causing at least one of the following mutations of the mature peptide, such as G95P, A96D, S99W, S101T, P127S, S126T, etc., on the basis of the wild-type alkaline protease zymogen region shown in SEQ ID NO.1;
  • alkaline protease mutant is a G95P mutant having an amino acid sequence shown in SEQ ID NO.3;
  • the encoding gene aprm1 of the G95P mutant has a nucleotide sequence shown in SEQ ID NO.4;
  • alkaline protease mutant is a G95P/A96D mutant having an amino acid sequence shown in SEQ ID NO.5;
  • the encoding gene aprm2 of the G95P/A96D mutant has a nucleotide sequence shown in SEQ ID NO.6;
  • alkaline protease mutant is a G95P/A96D/S99W mutant having an amino acid sequence shown in SEQ ID NO.7;
  • the encoding gene aprm3 of the G95P/A96D/S99W mutant has the nucleotide sequence shown in SEQ ID NO.8;
  • alkaline protease mutant is a G95P/A96D/S99W/S101T mutant having an amino acid sequence shown in SEQ ID NO.9;
  • the encoding gene aprm4 of the G95P/A96D/S99W/S101T mutant has the nucleotide sequence shown in SEQ ID NO.10;
  • alkaline protease mutant is a G95P/A96D/S99W/S101T/P127S mutant having an amino acid sequence shown in SEQ ID NO.11;
  • the encoding gene aprm5 of the G95P/A96D/S99W/S101T/P127S mutant has the nucleotide sequence shown in SEQ ID NO.12;
  • alkaline protease mutant is a G95P/A96D/S99W/S101T/P127S/S126T mutant having an amino acid sequence shown in SEQ ID NO.13;
  • the encoding gene aprm6 of the G95P/A96D/S99W/S101T/P127S/S126T mutant has the nucleotide sequence shown in SEQ ID NO.14.
  • the second technical solution provided by the present invention is a recombinant plasmid or recombinant strain containing the above mutant encoding gene;
  • the expression vector used is pLY-3, and the host is Escherichia coli or Bacillus amyloliquefaciens;
  • the host cell is Escherichia coli WB600, or the host cell is Bacillus amyloliquefaciens CGMCC No.11218;
  • the recombinant strain is obtained by connecting the mutant encoding gene to the expression vector pLY-3 and expressing it in the host Bacillus amyloliquefaciens CGMCC No.11218.
  • the third technical solution provided by the present invention is the application of the above-mentioned recombinant plasmid or recombinant strain, especially in the production of alkaline protease.
  • the fourth technical solution provided by the present invention is the application of the alkaline protease mutant described in the first technical solution, especially in the fields of detergents, leather making, food, feed, etc.; more particularly in the field of detergents, specifically the application of adding the alkaline protease to a detergent used at low temperature, and further, the low temperature is 20°C or below, especially 10-20°C.
  • the mutated ALK encoding gene was transferred into Bacillus subtilis WB600 by constructing a recombinant plasmid, and the protease activity was determined using the AAPF method.
  • ALK mutants with improved enzyme activity at 10°C compared with the wild-type were obtained through screening, and ALK mutant encoding genes aprm1, aprm2, aprm3, aprm4, aprm5, and aprm6 were obtained through sequencing.
  • the plasmids pLY-3-aprm1, pLY-3-aprm2, pLY-3-aprm3, pLY-3-aprm4, pLY-3-aprm5, and pLY-3-aprm6 containing the ALK mutant encoding genes with improved enzyme activity were preserved.
  • the high-activity mutants screened were fermented and cultured, and the ALK protein was purified and then the enzyme activity was rescreened. The specific enzyme activity of each mutant was calculated.
  • a recombinant strain of Bacillus amyloliquefaciens containing an alkaline protease encoding gene and a process for preparing an alkaline protease with improved enzyme activity using the recombinant strain of Bacillus amyloliquefaciens comprising the following steps:
  • ALK mutant encoding genes aprm1, aprm2, aprm3, aprm4, aprm5, and aprm6 were connected with the Bacillus amyloliquefaciens expression plasmid pLY-3 to obtain new recombinant plasmids pLY-3-aprm1, pLY-3-aprm2, pLY-3-aprm3, pLY-3-aprm4, pLY-3-aprm5, and pLY-3-aprm6;
  • the recombinant plasmid was transferred into Bacillus amyloliquefaciens CGMCC No.11218, and the recombinant strain was obtained through kanamycin (Kan) resistance screening and enzyme digestion verification. The recombinant strain was then cultured and fermented to obtain alkaline protease.
  • the generally accepted IUPAC nomenclature for amino acid residues is used, either as a single letter or three letter code.
  • the generally accepted IUPAC nomenclature for DNA nucleic acid sequences is used.
  • the mutated amino acid in the ALK mutant is represented by "amino acid replaced by the original amino acid position".
  • Gly95Pro means that the amino acid at position 95 is replaced by Pro from Gly of wild-type ALK, and the position number corresponds to the amino acid sequence number of the wild-type ALK mature peptide in SEQ ID NO.19.
  • lowercase italic apr represents the coding gene of wild-type alkaline protease ALK
  • lowercase italic aprm1 represents the coding gene of mutant G95P
  • lowercase italic aprm2, aprm3, aprm4, aprm5, and aprm6 represent the coding genes of mutants G95P/A96D, G95P/A96D/S99W, G95P/A96D/S99W/S101T, G95P/A96D/S99W/S101T/P127S, and G95P/A96D/S99W/S101T/P127S/S126T, respectively, and the specific information is as shown in the following table.
  • the present invention utilizes iterative saturation mutagenesis technology to mutate the ALK wild type to obtain mutants G95P, G95P/A96D, G95P/A96D/S99W, G95P/A96D/S99W/S101T, G95P/A96D/S99W/S101T/P127S, G95P/A96D/S99W/S101T/P127S/S126T, whose enzyme activities at 10°C are improved compared with those of the wild type, and the maximum values of fermentation enzyme activities in the Bacillus amyloliquefaciens expression system are 118.48 U/mL, 142.61 U/ml, 146.88 U/ml, 166.22 U/ml, 207.25 U/ml, and 231.95 U/ml, respectively.
  • the present invention uses the Bacillus amyloliquefaciens expression system to achieve efficient expression and preparation of ALK mutants with improved enzyme activity.
  • Figure 1 is the electrophoresis of PCR amplification of wild-type alkaline protease zymogen gene
  • M is DNA Marker
  • 1 is alkaline protease zymogen gene apr
  • Figure 2 is a diagram showing the restriction enzyme cutting verification of pLY-3-apr plasmid
  • M is DNA Marker
  • 1 is the double restriction enzyme digestion image of pLY-3-apr by BamHI and SmaI;
  • LB medium g/L: yeast extract 5.0, trypsin 10.0, NaCl 10.0, and the rest is water.
  • Solid medium was supplemented with 2% agar.
  • Fermentation medium 64% corn flour, 40% soybean meal, 2.7% amylase, 4% Na 2 HPO 4 , 0.3% KH 2 PO 4 , and the rest water; keep at 90°C for 30 min and sterilize at 121°C for 20 min.
  • the zymogen region sequence of the wild-type alkaline protease ALK is shown in SEQ ID NO.1:
  • OEGA Bacterial DNA Kit
  • the amplification primers for the alkaline protease gene apr of the present invention are as follows:
  • Upstream primer P1 (SEQ ID NO.15):
  • Downstream primer P2 (SEQ ID NO.16):
  • P1 and P2 were used as upstream and downstream primers, and the genome of Bacillus clausii alkaline protease was used as template for amplification.
  • the amplification reaction system is:
  • Upstream primer P1 2.0 ⁇ L Downstream primer P2 2.0 ⁇ L DNA template 2.0 ⁇ L Primer Star Max Enzyme 25 ⁇ L ddH2O 19 ⁇ L
  • the amplification procedure was as follows: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 54°C for 20s, extension at 72°C for 7s, and reaction for 30 cycles; extension at 72°C for 10min.
  • the PCR amplification product was subjected to 0.8% agarose gel electrophoresis to obtain a band of 1062bp ( Figure 1).
  • the PCR product was recovered using a small amount of DNA recovery kit to obtain the wild-type alkaline protease zymogen region gene apr of the present invention (SEQ ID NO.2).
  • the apr and pLY-3 plasmid were double-digested with restriction endonucleases BamHI and SmaI, respectively.
  • the apr recovered from the gel was connected to the vector pLY-3 to obtain the recombinant plasmid pLY-3-apr.
  • the enzyme digestion verification was shown in Figure 2, and it was transformed into Escherichia coli JM109 and Bacillus subtilis WB600.
  • P1 and 95-R were used as upstream and downstream primers
  • P2 and 95-F were used as upstream and downstream primers.
  • Plasmid pLY-3-apr was used as template to perform PCR1 reaction to obtain upstream fragments and downstream fragments, respectively.
  • the reaction system for upstream fragment amplification is:
  • the reaction system for downstream fragment amplification is:
  • the amplification program was as follows: pre-denaturation at 98°C for 30 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 54°C for 20 s, and extension at 72°C for 7 s; and extension at 72°C for 10 min.
  • the amplification program was as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 54°C for 20 s, extension at 72°C for 7 s, 5 cycles; and extension at 72°C for 10 min.
  • PCR 3 amplification program was as follows: 98°C pre-denaturation for 30 s; 98°C denaturation for 10 s, 54°C annealing for 20 s, 72°C extension for 10 s, 5 cycles; 72°C extension for 10 min.
  • the PCR amplification product was subjected to 0.8% agarose gel electrophoresis, and the PCR product was recovered using a small amount of DNA recovery kit to obtain the alkaline protease site-directed mutant gene aprm G95.
  • Short peptide substrate The AAPF method uses Suc-Ala-Ala-Pro-Phe-pNA as a substrate, in which the two amino acid residues Ala-Ala form a hydrophobic group.
  • the Pro amino acid has steric hindrance, which makes it difficult for its C-terminal peptide bond to be cut. Therefore, alkaline protease is more inclined to hydrolyze the peptide bond connecting Phe-pNA and release pNA (p-nitroaniline). Free pNA can be detected at OD410.
  • the absorbance of the solution is measured at OD410 using a spectrophotometer.
  • the enzyme activity is proportional to the absorbance, and the enzyme activity of the alkaline protease can be calculated.
  • the amount of enzyme that hydrolyzes AAPF to produce 1 ⁇ mol pNA in 1mL of enzyme solution for 1min is defined as one enzyme activity unit U.
  • mutant strain plasmid pLY-3-aprm G95 was sent to GENEWISE for sequencing, and it was determined that the mutant was the gene aprm1 of alkaline protease in which the amino acid Gly at position 95 was mutated to Pro.
  • the 96th amino acid Ala of aprm1 was subjected to NNK saturation mutation according to the above embodiment. After preliminary screening, a mutant with the highest enzyme activity at 10°C and higher than G95P was obtained. The plasmid of the mutant strain was sent to GeneWeichi for sequencing, and it was determined that the mutant was the gene aprm2 of alkaline proteinase in which the 96th amino acid Ala was mutated to Asp.
  • the 97th amino acid Ser of aprm2 was subjected to NNK saturation mutation according to the above example, and an effective mutant with higher alkaline protease activity than G95P/A96D was obtained after preliminary screening.
  • the 98th amino acid Gly of aprm2 was subjected to NNK saturation mutation according to the above example, and an effective mutant with higher alkaline protease activity than G95P/A96D was obtained after preliminary screening.
  • the 99th amino acid Ser of aprm2 was subjected to NNK saturation mutation according to the above embodiment. After preliminary screening, a mutant with the highest enzyme activity at 10°C and higher than G95P/A96D was obtained. The plasmid of the mutant strain was sent to GeneWeichi for sequencing, and it was determined that the mutant was the gene aprm3 of alkaline protease in which the 99th amino acid Ser was mutated to Trp.
  • the 100th amino acid Gly of aprm3 was subjected to NNK saturation mutation according to the above example, and an effective mutant with higher alkaline protease activity than G95P/A96D/S99W was obtained after preliminary screening.
  • the 101st amino acid Ser of aprm3 was subjected to NNK saturation mutation according to the above embodiment. After preliminary screening, a mutant with the highest enzyme activity at 10°C and higher than G95P/A96D/S99W was obtained. The plasmid of the mutant strain was sent to GeneWeichi for sequencing, and it was determined that the mutant was the gene aprm4 of alkaline protease in which the 99th amino acid Ser was mutated to Thr.
  • amino acid Val at position 102 of aprm4 was subjected to NNK saturation mutation according to the above example, and an effective mutant with higher alkaline protease activity than G95P/A96D/S99W/S101T was obtained after primary screening.
  • the 127th amino acid Pro of aprm4 was subjected to NNK saturation mutation according to the above embodiment. After preliminary screening, a mutant with the highest enzyme activity at 10°C and higher than G95P/A96D/S99W/S101T was obtained. The plasmid of the mutant strain was sent to GENEWIZ for sequencing, and it was determined that the mutant was the gene aprm5 of alkaline protease in which the 127th amino acid Pro was mutated to Ser.
  • the 126th amino acid Ser of aprm5 was subjected to NNK saturation mutation according to the above embodiment. After preliminary screening, a mutant with the highest enzyme activity at 10°C and higher than G95P/A96D/S99W/S101T/P127S was obtained. The plasmid of the mutant strain was sent to GENEWIZ for sequencing, and it was determined that the mutant was the gene aprm6 of alkaline protease in which the 126th amino acid Ser was mutated to Thr.
  • the 125th amino acid Gly of aprm6 was subjected to NNK saturation mutation according to the above example, and an effective mutant with higher alkaline protease activity than G95P/A96D/S99W/S101T/P127S/S126T was obtained after preliminary screening.
  • the 124th amino acid Leu of aprm6 was subjected to NNK saturation mutation according to the above example, and an effective mutant with higher alkaline protease activity than G95P/A96D/S99W/S101T/P127S/S126T was obtained after preliminary screening.
  • the 123rd amino acid Ser of aprm6 was subjected to NNK saturation mutation according to the above example, and after preliminary screening, an effective mutant with higher alkaline protease activity than G95P/A96D/S99W/S101T/P127S/S126T was obtained.
  • the high-activity alkaline protease mutant recombinant strain WB600/pLY-3-aprmx (x is 1, 2, 3, 4, 5, 6, the same below) and the wild-type recombinant strain WB600/pLY-3-apr obtained in Example 2 were inoculated into 5 mL of LB liquid culture medium (containing kanamycin, 50 ⁇ g/mL), cultured overnight at 37°C, 220 r/min, and transferred to 50 mL of fresh LB culture medium (containing kanamycin, 50 ⁇ g/mL) according to a 2% inoculum amount, and continued to be cultured at 37°C, 220 r/min for 48 h.
  • LB liquid culture medium containing kanamycin, 50 ⁇ g/mL
  • fresh LB culture medium containing kanamycin, 50 ⁇ g/mL
  • the fermentation broth was centrifuged to obtain the supernatant, and the impurities were first removed by salting out with ammonium sulfate at 25% saturation, and then the saturation was increased to 65% to precipitate the target protein.
  • the salt was removed by dialysis, and the active components obtained after salting out and desalting were dissolved in 0.02mol/L Tris-HCl (pH 7.0) buffer, loaded onto a cellulose ion exchange chromatography column, and then the unadsorbed proteins were first eluted with the same buffer, and then gradient eluted with 0.02mol/L Tris-HCl (pH 7.0) buffer containing different concentrations of NaCl (0-1mol/L) to collect the target protein.
  • the active components obtained by ion exchange were first equilibrated with 0.02mol/L Tris-HCl (pH 7.0) buffer containing 0.15mol/L NaCl, loaded onto a sephadex g25 gel chromatography column, and then eluted with the same buffer at a rate of 0.5mL/min to obtain a purified enzyme solution for enzyme activity determination.
  • the enzyme activity determination method is the same as in Example 2.
  • the protein concentration was determined by the BCA protein concentration assay kit according to its instructions;
  • Alkaline protease specific enzyme activity ratio of enzyme activity (U/ml) to protein concentration (mg/ml).
  • the wild-type ALK encoding gene apr and mutant encoding genes aprm1, aprm2, aprm3, aprm4, aprm5, and aprm6 were connected with the Bacillus amyloliquefaciens expression plasmid pLY-3 to obtain new recombinant plasmids pLY-3-apr, pLY-3-aprm1, pLY-3-aprm2, pLY-3-aprm3, pLY-3-aprm4, pLY-3-aprm5, and pLY-3-aprm6;
  • the recombinant plasmid was transferred into Bacillus amyloliquefaciens CGMCC No.11218, and after kanamycin (Kan) resistance screening and enzyme digestion verification, the wild-type recombinant strain CGMCC No.11218/pLY-3-apr and the mutant recombinant strain CGMCC No.11218/pLY-3-aprmx were obtained.
  • the mutant recombinant strain of Bacillus amyloliquefaciens CGMCC No.11218/pLY-3-aprmx and the wild-type recombinant strain CGMCC No.11218/pLY-3-apr were inoculated into 5 mL of fermentation medium (containing kanamycin, 50 ⁇ g/mL), cultured overnight at 37°C, 220 r/min, and transferred to 50 mL of fresh fermentation medium (containing kanamycin, 50 ⁇ g/mL) according to 2% inoculation amount, and continued to be cultured at 37°C, 220 r/min for 48 hours.
  • the activity of alkaline protease obtained by fermentation of Bacillus amyloliquefaciens was determined by the short peptide substrate method in Example 2 (the fermentation broth was centrifuged and the supernatant was taken to determine the enzyme activity).
  • the fermentation broth enzyme activity of alkaline protease in Bacillus amyloliquefaciens was 27.13 U/ml
  • the enzyme activity of the ALK mutant G95P was 118.48 U/ml
  • the enzyme activities of the ALK mutants G95P/A96D, G95P/A96D/S99W, G95P/A96D/S99W/S101T, G95P/A96D/S99W/S101T/P127S, and G95P/A96D/S99W/S101T/P127S/S126T were 142.61 U/ml, 146.88/ml, 166.22 U/ml, 207.25 U/ml, and 23
  • the supernatant obtained by centrifugation of the fermentation broth was first salted out with 25% saturation ammonium sulfate to remove impurities, and then the saturation was increased to 65% to precipitate the target protein.
  • the salt was removed by dialysis, and the active components obtained after salting out and desalting were dissolved in 0.02mol/L Tris-HCl (pH 7.0) buffer, loaded onto a cellulose ion exchange chromatography column, and then the unadsorbed protein was first eluted with the same buffer, and then gradient eluted with 0.02mol/L Tris-HCl (pH 7.0) buffer containing different concentrations of NaCl (0-1mol/L) to collect the target protein.
  • the active components obtained by ion exchange were first balanced with 0.02mol/L Tris-HCl (pH 7.0) buffer containing 0.15mol/L NaCl, loaded onto a sephadex g25 gel chromatography column, and eluted with the same buffer at a rate of 0.5mL/min to obtain a purified enzyme solution, which was freeze-dried to obtain pure alkaline protease enzyme powder.
  • the prepared alkaline protease mutant enzyme powder can be applied to the fields of detergent, leather making, food, feed, etc.
  • the pure enzyme solution of the G95P/A96D/S99W/S101T/P127S/S126T mutant prepared in Example 4 was applied to the washing of protein-stained cloth.
  • the whiteness value of the front and back sides of the protein-stained cloth JB-02 cloth
  • the protein-stained cloth was washed with 500 mL of cloth washing liquid at 10° C. for 20 minutes, and then the cloth was dried.
  • the whiteness value of the cloth after drying was measured by a whiteness meter, which was 31.06.
  • V/V Dirty cloth washing liquid 0.2% standard laundry detergent, 0.1% enzyme solution, 500mL water.
  • Standard laundry detergent (code: SLD, refer to GB/T13174-2021): 8% alkylbenzene sulfonic acid (by active matter), 4% polyethoxylated fatty alcohol (average EO addition number is 9), 2% sodium ethoxylated alkyl sulfate (2EO, by active matter), 0.5% triethanolamine, 0.6% trisodium citrate dihydrate, 0.1% preservative, and the balance is water.
  • Laboratory preparation method Add various ingredients to a certain amount of water in turn, stir and dissolve (heat if necessary), and adjust the pH value of the solution to 8.5-9.0 with sodium hydroxide, and make up the water to 100%.

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Abstract

通过重叠PCR技术进行迭代饱和突变获得酶活力提高的碱性蛋白酶酶突变体及其制备与应用。利用重叠PCR技术对克劳氏芽抱杆菌来源的碱性蛋白酶基因apr进行迭代饱和突变，筛选出低温下高活力的突变体，并在解淀粉芽抱杆菌中进行高效表达和制备，解决了低温洗涤环境下碱性蛋白酶活性较低的问题。

Description

一种低温高活力碱性蛋白酶突变体及其应用 技术领域：
本发明属于生物工程技术领域，具体涉及通过重叠PCR技术进行迭代饱和突变获得酶活力提高的碱性蛋白酶酶突变体及其制备与应用。
技术背景：
蛋白酶广泛存在于动物、植物和微生物中，由于微生物具有生长速度快，生长条件简单、代谢过程特殊和分布广等特点，使之成为生物酶的重要来源。其应用主要围绕其水解蛋白质肽键的功能展开，生产生活中，有几个主要的需求：使复杂的大分子蛋白质结构变成简单的小分子肽链或者氨基酸，从而变得易于吸收或洗去，比如食品、洗涤剂、饲料等领域；部分破坏蛋白质结构，使物质组分之间实现分离，这在皮革、丝绸等含蛋白质丰富的材料加工时十分有效；促进环境污染物降解，用于环保领域。蛋白酶既能催化水解反应，也能催化其逆反应，而且具备高度的活性和专一性，非常适合医药工业对某些特定分子的生产需求。
按照最适pH值的不同，蛋白酶可分为碱性蛋白酶、酸性蛋白酶和中性蛋白酶。碱性蛋白酶的最适pH为8.0-11.0，其多来源于微生物，特别是工业微生物产生的碱性蛋白酶其水解能力与耐碱性具有更加明显的优势，与动植物来源的碱性蛋白酶相比，微生物碱性蛋白酶可分泌到细胞外，具有下游技术处理相对简单，价格低廉、来源广、菌易培养、同时易于实现工业化大批量生产等特点，因此，碱性蛋白酶研究已成为蛋白酶研究的热点。然而，酶活力不高仍是当前碱性蛋白酶工业化生产的一个最主要的限制因素，因此，开发高活力碱性蛋白酶对其实现工业化生产具有重要意义。洗涤剂的pH一般在9.0-11.0，碱性蛋白酶由于其在碱性条件下具有较高的稳定性和活性，被主要应用于洗涤剂行业，它们在洗涤剂配方中的使用占其总销售额的89％。碱性蛋白酶由于其广泛的应用，受到了越来越多研究者的关注，人们致力于挖掘具有独特性质和更高活性的新型碱性蛋白酶。
丝氨酸碱性蛋白酶是工业洗涤剂中最重要的酶类之一，约占微生物酶总销售额的35％。第一个含有细菌蛋白酶的商业洗涤剂是由Gebrüder schnyder在1959年生产的。近年来，市场上的液体洗涤剂比固体洗涤剂更受到消费者的欢迎。在洗涤剂中加入碱性蛋白酶能保持衣物原有的颜色，提高产品的去污能力，降低表面活性剂的添加量，还能有节水、节能和环保的作用。根据调查结果显示，我国 的洗衣温度通常集中在10-20℃之间，而目前市售的洗涤剂中添加的蛋白酶大多为中温碱性蛋白酶，其最适温度为40-60℃。天然的低温碱性蛋白酶在高于20℃的温度下稳定性差且大规模生产时产量低，使得这类酶通常不能满足洗涤行业对低温碱性蛋白酶的需求。基于以上背景，通过对克劳氏芽孢杆菌来源的碱性蛋白酶(ALK)进行分子改造，筛选低温下高活力的ALK突变体，以满足目前洗涤行业的需求。
蛋白质定向进化的本质是构建分子多样性文库以及从文库中筛选到性状有改进的突变体，根据文库构建原理的不同，可分为随机进化、改组技术、半理性进化和理性进化四种策略，其大致思路均为由某一靶基因或一族相关的家族基因起始，通过对编码基因进行突变或重组，创建分子多样性文库；筛选文库获得能够编码改进性状的基因，作为下一轮进化的模板；在短时间内完成自然界中需要成千上万年的进化，从而获得具有改进功能或全新功能的蛋白质。对酶分子的设计与改造方面，是基于基因工程、蛋白质工程和计算机技术互补发展和渗透的结果，它标志着人类可以按照自己的意愿和需要改造酶分子，甚至设计出自然界中原来并不存在的全新的酶分子。酶分子人为改造还不成熟的情况下，通过定点突变技术改造成功了大量的酶分子，获得了比天然酶活力更高、稳定性更好的工业用酶。重叠PCR技术(Overlap PCR)，是采用具有互补末端的引物，使PCR产物形成了重叠链，从而在随后的扩增反应中通过重叠链的延伸，将不同来源的扩增片段重叠拼接起来的技术。运用这项技术对基因序列进行定点突变，从而实现对蛋白质的定向改造，是合理且实用的。
芽孢杆菌表达***具有以下优点：1、能够高效的分泌各种蛋白质；2、许多芽孢杆菌在发酵工业上的使用已有相当长的历史，无致病性，不产生任何内毒素；3、芽孢杆菌属微生物遗传学背景研究的十分清楚，并且生长迅速，对营养物质无特殊要求等优点；4、密码子偏爱性不明显；5、发酵过程简单，芽孢杆菌属于好氧菌，无需厌氧发酵设备，发酵结束后，简单的分离发酵液和细菌菌体可进入目的蛋白的分离、纯化回收阶段；6、具有抗逆性，可以生产多种耐热性酶制剂。
因此，本发明中，通过重叠PCR对来源于克劳氏芽孢杆菌的碱性蛋白酶基因进行分子改造，利用枯草芽孢杆菌表达***进行高通量筛选，得到低温下酶活力提高的碱性蛋白酶突变体基因。
发明内容：
基于洗涤环境低温的问题，为了获得低温下高活力的碱性蛋白酶，需对其现有性质作进一步的提升，本发明的目的在于提供一种低温下高活力碱性蛋白酶的 突变体。将克劳氏芽孢杆菌(Bacillus clausii)来源的碱性蛋白酶基因(apr)同穿梭载体pLY-3构建重组表达载体pLY-3-apr，在枯草芽孢杆菌WB600中进行表达；通过对碱性蛋白酶(ALK)的同源建模及其与底物AAPF(Suc-Ala-Ala-Pro-Phe-pNA)的对接结果分析，确定其关键热点区域；利用重叠PCR技术对基因apr进行迭代饱和突变，并利用AAPF对突变体进行高通量筛选，选出低温下高活力的突变体。
实现本发明目的的技术路线概述如下：
对来自克劳氏芽孢杆菌(Bacillus clausii)的碱性蛋白酶基因apr进行饱和突变，利用枯草芽孢杆菌表达***筛选得到10℃下高活力ALK突变体G95P、G95P/A96D、G95P/A96D/S99W、G95P/A96D/S99W/S101T、G95P/A96D/S99W/S101T/P127S、G95P/A96D/S99W/S101T/P127S/S126T，及其编码基因aprm1、aprm2、aprm3、aprm4、aprm5、aprm6，并实现了各突变体在解淀粉芽孢杆菌中的高效表达，通过发酵、提取等技术获得酶活力提高的ALK突变体。
本发明提供的技术方案之一，是一种碱性蛋白酶突变体，所述突变体是在SEQ ID NO.1所示野生型碱性蛋白酶酶原区基础上发生成熟肽的G95P、A96D、S99W、S101T、P127S、S126T等突变中的至少一种获得的；
进一步地，所述碱性蛋白酶突变体为G95P突变体，具有SEQ ID NO.3所示的氨基酸序列；
更进一步地，所述G95P突变体的编码基因aprm1具有SEQ ID NO.4所示的核苷酸序列；
进一步地，所述碱性蛋白酶突变体为G95P/A96D突变体，具有SEQ ID NO.5所示的氨基酸序列；
更进一步地，所述G95P/A96D突变体的编码基因aprm2具有SEQ ID NO.6所示的核苷酸序列；
进一步地，所述碱性蛋白酶突变体为G95P/A96D/S99W突变体，具有SEQ ID NO.7所示的氨基酸序列；
更进一步地，所述G95P/A96D/S99W突变体的编码基因aprm3，具有SEQ ID NO.8所示的核苷酸序列；
进一步地，所述碱性蛋白酶突变体为G95P/A96D/S99W/S101T突变体，具有SEQ ID NO.9所示的氨基酸序列；
更进一步地，所述G95P/A96D/S99W/S101T突变体的编码基因aprm4，具有SEQ ID NO.10所示的核苷酸序列；
进一步地，所述碱性蛋白酶突变体为G95P/A96D/S99W/S101T/P127S突变体，具有SEQ ID NO.11所示的氨基酸序列；
更进一步地，所述G95P/A96D/S99W/S101T/P127S突变体的编码基因aprm5，具有SEQ ID NO.12所示的核苷酸序列；
进一步地，所述碱性蛋白酶突变体为G95P/A96D/S99W/S101T/P127S/S126T突变体，具有SEQ ID NO.13所示的氨基酸序列；
更进一步地，所述G95P/A96D/S99W/S101T/P127S/S126T突变体的编码基因aprm6，具有SEQ ID NO.14所示的核苷酸序列。
本发明提供的技术方案之二，是包含上述突变体编码基因的重组质粒或重组菌株；
进一步地，采用的表达载体为pLY-3，宿主为大肠杆菌或解淀粉芽孢杆菌；
更进一步地，宿主细胞为大肠杆菌WB600，或者宿主细胞为解淀粉芽孢杆菌CGMCC No.11218；
优选地，所述重组菌株是将突变体编码基因与表达载体pLY-3连接后在宿主解淀粉芽孢杆菌CGMCC No.11218中表达所得。
本发明提供的技术方案之三，是上述重组质粒或重组菌株的应用，特别是在生产碱性蛋白酶中的应用。
本发明提供的技术方案之四，是技术方案一所述碱性蛋白酶突变体的应用，特别是在洗涤剂、制革、食品、饲料等领域的应用；更特别地是在洗涤剂领域中的应用，具体是在低温下使用的洗涤剂中添加所述碱性蛋白酶的应用，进一步地，所述低温为20℃及以下，特别是10-20℃。
本发明的实验方案具体如下：
1、ALK突变体编码基因的获得，包括如下步骤：
(1)以SEQ ID NO.2所示的野生型ALK编码基因apr为出发基因，构建表达载体pLY-3-apr，进行迭代饱和突变。
(2)将突变后的ALK编码基因，通过构建重组质粒后转入枯草芽孢杆菌WB600中，利用AAPF法测定蛋白酶活力。
(3)经筛选获得10℃下相对于野生型酶活力提高的ALK突变体，经测序获得ALK突变体编码基因aprm1、aprm2、aprm3、aprm4、aprm5、aprm6，将含有酶活力提高的ALK突变体编码基因的质粒pLY-3-aprm1、pLY-3-aprm2、pLY-3-aprm3、pLY-3-aprm4、pLY-3-aprm5、pLY-3-aprm6保存。
将筛选得到的高活力突变体进行发酵培养，纯化得到ALK蛋白后对酶活进行复筛。并计算了各突变体的比酶活。
2、含有碱性蛋白酶编码基因的解淀粉芽孢杆菌重组菌株及以其制备酶活力提高的碱性蛋白酶的过程，包括如下步骤：
(1)将ALK突变体编码基因aprm1、aprm2、aprm3、aprm4、aprm5、aprm6与解淀粉芽孢杆菌表达质粒pLY-3通过连接得到新的重组质粒pLY-3-aprm1、pLY-3-aprm2、pLY-3-aprm3、pLY-3-aprm4、pLY-3-aprm5、pLY-3-aprm6；
(2)将重组质粒转入解淀粉芽孢杆菌CGMCC No.11218中，经卡纳霉素(Kan)抗性筛选，酶切验证得到重组菌株，之后将重组菌株进行培养发酵，得到碱性蛋白酶。
在本发明中采用如下定义：
1.氨基酸和DNA核酸序列的命名法
使用氨基酸残基的公认IUPAC命名法，用单字母或三字母代码形式。DNA核酸序列采用公认IUPAC命名法。
2.碱性蛋白酶突变体的标识
采用“原始氨基酸位置替换的氨基酸”来表示ALK突变体中突变的氨基酸。如Gly95Pro，表示位置95的氨基酸由野生型ALK的Gly替换成Pro，位置的编号对应于SEQ ID NO.19中野生型ALK成熟肽的氨基酸序列编号。
在本发明中，小写斜体apr表示野生型碱性蛋白酶ALK的编码基因，小写斜体aprm1表示突变体G95P的编码基因，小写斜体aprm2、aprm3、aprm4、aprm5、aprm6分别表示突变体G95P/A96D、G95P/A96D/S99W、G95P/A96D/S99W/S101T、G95P/A96D/S99W/S101T/P127S、G95P/A96D/S99W/S101T/P127S/S126T的编码基因，具体信息如下表。
Figure PCTCN2022131434-appb-000001
有益效果：
1、本发明利用迭代饱和突变技术对ALK野生型进行突变，得到10℃下酶活力相对于野生型提高的突变体G95P、G95P/A96D、G95P/A96D/S99W、G95P/A96D/S99W/S101T、G95P/A96D/S99W/S101T/P127S、G95P/A96D/S99W/S101T/P127S/S126T，在解淀粉芽孢杆菌表达***内发酵酶活最高值分别为118.48U/mL、142.61U/ml、146.88U/ml、166.22U/ml、207.25U/ml、231.95U/ml。
2、本发明使用了解淀粉芽孢杆菌表达***实现酶活力提高的ALK突变体的高效表达和制备。
附图说明：
图1为野生型碱性蛋白酶酶原基因的PCR扩增电泳图
其中：M为DNA Marker，1为碱性蛋白酶酶原基因apr；
图2为pLY-3-apr质粒酶切验证图
其中：M为DNA Marker，1为pLY-3-apr经BamHI和SmaI双酶切图；
具体实施方式：
下面结合实施例对本发明的技术内容做进一步说明，但本发明不只限于这些实施例，不能以下述实施例来限定本发明的保护范围。
本发明实施例所用培养基如下：
LB培养基(g/L)：酵母提取物5.0，胰蛋白胨10.0，NaCl 10.0，其余为水。
固态培养基添加2％琼脂。
发酵培养基(g/L)：玉米粉64，豆饼粉40，加入2.7淀粉酶，4Na 2HPO 4，0.3KH 2PO 4，其余为水；90℃保温30min再121℃灭菌20min。
在本发明中，野生型碱性蛋白酶ALK的酶原区序列如SEQ ID NO.1所示：
Figure PCTCN2022131434-appb-000002
在本发明中，野生型碱性蛋白酶ALK的成熟肽序列如SEQ ID NO.19所示：
Figure PCTCN2022131434-appb-000003
以下将通过具体实施例对本发明作进一步解释说明。
实施例1野生型碱性蛋白酶基因的获得
1、使用试剂盒(OMEGA:Bacterial DNA Kit)提取克劳氏芽孢杆菌(Bacillus clausii)的基因组DNA，提取步骤如下：
(1)用接种环将菌株接种至LB固体平板上，37℃恒温培养过夜。
(2)从培养菌体的平板上挑取单菌落接种到液体试管培养基中，37℃，220r/min振荡培养过夜。
(3)取3mL-5mL菌液置于已灭菌的EP管中，12000r/min离心2min，弃上清。
(4)向EP管中加入200μL无菌水重悬菌体，再加入50μL溶菌酶，吹吸混匀，37℃保温20min。
(5)向EP管中加入100μL BTL buffer和20μL蛋白酶K，旋涡振荡混匀，55℃保温40min，每隔20min振荡混匀。
(6)加入5μL RNA酶，颠倒混匀数次，室温放置10min.
(7)12000r/min离心2min，除去未消化的部分，将上清转移至新的EP管中，加入220μL BDL buffer，65℃水浴15min。
(8)加入220μL无水乙醇，吹吸混匀。
(9)将EP管中的液体转入回收柱静置1min，12000r/min离心1min，将滤液重新倒入回收柱中，重复两次，倒掉废液。
(10)加入500μL HBC buffer，12000r/min离心1min，弃滤液。
(11)加入700μL DNA wash buffer，静置1min，12000r/min离心1min，弃滤液。
(12)加入500μL DNA wash buffer，静置1min，12000r/min离心1min，弃滤液。
(13)12000r/min空离2min，弃废液管，将回收柱放到一个新的EP管中。
(14)置于55℃金属浴烘干10min。
(15)加入50μL 55℃的无菌水，室温静置5min，12000r/min离心2min，弃回收柱，EP管中液体为基因组。
2、以提取的克劳氏芽孢杆菌的基因组为模板，在ORF框上下游设计一对引物，分别引入限制性酶切位点BamHI、SmaI，本发明的碱性蛋白酶基因apr的扩增引物如下：
上游引物P1(SEQ ID NO.15)：
5’-CGCGGATCCGCTGAAGAAGCAAAAGA-3’
下游引物P2(SEQ ID NO.16)：
5’-TCCCCCGGGTTAGCGTGTTGCCGCTTCT-3’
以P1和P2作为上、下游引物，以克劳氏芽孢杆菌碱性蛋白酶基因组为模板进行扩增。
其扩增的反应体系为：
上游引物P1 2.0μL
下游引物P2 2.0μL
DNA模板 2.0μL
Primer Star Max酶 25μL
ddH 2O 19μL
扩增程序为：98℃预变性30s；98℃变性10s，54℃退火20s，72℃延伸7s，反应30个循环；72℃延伸10min。PCR扩增产物经0.8％琼脂糖凝胶电泳，得到1062bp的条带(图1)，用小量DNA回收试剂盒回收PCR产物，得到本发明的野生型碱性蛋白酶酶原区基因apr(SEQ ID NO.2)，apr与pLY-3质粒分别用限制性内切酶BamHI和SmaI进行双酶切，将切胶回收的apr与载体pLY-3载体连接，得到重组质粒pLY-3-apr，酶切验证如图2所示，并将其转化至大肠杆菌JM109及枯草芽孢杆菌WB600中。
实施例2构建碱性蛋白酶突变体文库筛选高活力碱性蛋白酶突变体
1、基于重叠PCR技术进行饱和突变，构建新型碱性蛋白酶，设计突变引物如下：
突变上游引物95-F(SEQ ID NO.17)：
5’-GTTAAAGTATTANNKGCGAGCGGTTCA-3’
突变下游引物95-R(SEQ ID NO.18)：
5’-TGAACCGCTCGCMNNTAATACTTTAAC-3’
在重叠PCR第一步反应体系中，分别以P1和95-R作为上、下游引物，以 P2和95-F作为上、下游引物。以质粒pLY-3-apr为模板，进行PCR1反应，分别得到上游片段和下游片段。
上游片段扩增的反应体系为：
P1 2μL
95-R 2μL
野生型碱性蛋白酶基因 2μL
Primer Star Max酶 25μL
ddH 2O 19μL
下游片段扩增的反应体系为：
P2 2μL
95-F 2μL
野生型碱性蛋白酶基因 2μL
Primer Star Max酶 25μL
ddH 2O 19μL
扩增程序为：98℃预变性30min；98℃变性10s，54℃退火20s，72℃延伸7s反应30个循环；72℃延伸10min。
2、切胶回收上、下游片段后进行PCR 2，反应体系为：
上游片段 2.0μL
下游片段 2.0μL
Primer Star Max酶 25μL
ddH 2O 21μL
扩增程序为：98℃预变性30s；98℃变性10s，54℃退火20s，72℃延伸7s，反应5个循环；72℃延伸10min。
3、PCR 2结束后向体系中加入引物P1和P2各2μL，进行PCR 3扩增程序为：98℃预变性30s；98℃变性10s，54℃退火20s，72℃延伸10s，反应5个循环；72℃延伸10min。PCR扩增产物经0.8％琼脂糖凝胶电泳，用小量DNA回收试剂盒回收PCR产物，获得碱性蛋白酶定点突变体基因aprm G95。
4、将碱性蛋白酶定点突变体基因aprm G95与表达载体pLY-3连接后转化至JM109中并提取其质粒即得重组质粒pLY-3-aprm G95，再将重组质粒pLY-3-aprm G95转化至枯草芽孢杆菌WB600中，获得重组菌株WB600/pLY-3-aprm G95。将枯草化转的转化子活化到一个新的分区划线的Kan板上，37℃倒置培养12 小时，然后利用高通量筛选体系对这些突变体菌株进行筛选，操作步骤如下：
(1)在无菌条件下，将含有Kan抗性的液体LB培养基分装到无菌的48孔板中，每个孔700μL。
(2)挑取突变体单菌落接种到48深孔板中(做好标记)，每个孔板中留四个空白对照(即WB600/pLY-3-apr)，37℃，600r/min振荡培养过夜。
(3)在无菌条件下，将含有Kan抗性的液体LB培养基分装到无菌的24深孔板中，每个孔1mL，吸取20μL菌液按标记加到各个孔中，37℃，600r/min振荡培养48h。
(4)培养结束后，取出24深孔板，在OD600下测定每个孔中菌液的菌浓。
(5)将24深孔板放到孔板离心机中，5000r/min，离心30min，上清作为酶液进行酶活测定。
5、短肽底物测定碱性蛋白酶酶活力
短肽底物：AAPF法以Suc-Ala-Ala-Pro-Phe-pNA为底物，其中Ala-Ala两个氨基酸残基会组成一个疏水基团，Pro氨基酸具有空间位阻，导致其C端肽键不容易被切割，因此碱性蛋白酶更倾向于水解Phe-pNA之间连接的肽键，将pNA(对硝基苯胺)释放出来，游离的pNA可以在OD410处被检测到，用分光光度计于OD410处测定溶液的吸光值，酶活力与吸光度成正比，即可计算碱性蛋白酶的酶活力。
测定方法：精准吸取20μL浓度为4mM的AAPF溶液置于96孔板中，加入80μL的硼酸缓冲液(pH＝10.5)，吹吸混匀，10℃保温2min。样品组加入100μL稀释后的酶液，对照组加入100μL的硼酸缓冲液，吹吸混匀。10℃下反应10min后使用酶标仪测定410nm处的吸光值。将实验组测得的OD值减去对照组的OD值得到ΔOD，再将ΔOD代入以下公式算出相应的酶活力：
Figure PCTCN2022131434-appb-000004
在10℃，pH＝10.5的条件下，1mL酶液1min水解AAPF产生1μmol pNA的酶量定义为一个酶活力单位U。
经过初筛，得到在10℃下酶活最高且高于野生型的突变体，将该突变体菌株质粒pLY-3-aprm G95提出送去金唯智公司进行测序，确定突变体为95位氨基酸Gly突变为Pro的碱性蛋白酶的基因aprm1。
对aprm1的第96位氨基酸Ala按上述实施例进行NNK饱和突变，经过初筛，得到在10℃下酶活最高且高于G95P的突变体，将该突变体菌株质粒提出送去金唯智公司进行测序，确定突变体为96位氨基酸Ala突变为Asp的碱性蛋白 酶的基因aprm2。
对aprm2的第97位氨基酸Ser按上述实施例进行NNK饱和突变，经过初筛未筛选获得碱性蛋白酶活力比G95P/A96D高的有效突变体。
对aprm2的第98位氨基酸Gly按上述实施例进行NNK饱和突变，经过初筛未筛选获得碱性蛋白酶活力比G95P/A96D高的有效突变体。
对aprm2的第99位氨基酸Ser按上述实施例进行NNK饱和突变，经过初筛，得到在10℃下酶活最高且高于G95P/A96D的突变体，将该突变体菌株质粒提出送去金唯智公司进行测序，确定突变体为99位氨基酸Ser突变为Trp的碱性蛋白酶的基因aprm3。
对aprm3的第100位氨基酸Gly按上述实施例进行NNK饱和突变，经过初筛未筛选获得碱性蛋白酶活力比G95P/A96D/S99W高的有效突变体。
对aprm3的第101位氨基酸Ser按上述实施例进行NNK饱和突变，经过初筛，得到在10℃下酶活最高且高于G95P/A96D/S99W的突变体，将该突变体菌株质粒提出送去金唯智公司进行测序，确定突变体为99位氨基酸Ser突变为Thr的碱性蛋白酶的基因aprm4。
对aprm4的第102位氨基酸Val按上述实施例进行NNK饱和突变，经过初筛未筛选获得碱性蛋白酶活力比G95P/A96D/S99W/S101T高的有效突变体。
对aprm4的第127位氨基酸Pro按上述实施例进行NNK饱和突变，经过初筛，得到在10℃下酶活最高且高于G95P/A96D/S99W/S101T的突变体，将该突变体菌株质粒提出送去金唯智公司进行测序，确定突变体为127位氨基酸Pro突变为Ser的碱性蛋白酶的基因aprm5。
对aprm5的第126位氨基酸Ser按上述实施例进行NNK饱和突变，经过初筛，得到在10℃下酶活最高且高于G95P/A96D/S99W/S101T/P127S的突变体，将该突变体菌株质粒提出送去金唯智公司进行测序，确定突变体为126位氨基酸Ser突变为Thr的碱性蛋白酶的基因aprm6。
对aprm6的第125位氨基酸Gly按上述实施例进行NNK饱和突变，经过初筛未筛选获得碱性蛋白酶活力比G95P/A96D/S99W/S101T/P127S/S126T高的有效突变体。
对aprm6的第124位氨基酸Leu按上述实施例进行NNK饱和突变，经过初筛未筛选获得碱性蛋白酶活比G95P/A96D/S99W/S101T/P127S/S126T高的有效突变体。
对aprm6的第123位氨基酸Ser按上述实施例进行NNK饱和突变，经过初筛未筛选获得碱性蛋白酶活力比G95P/A96D/S99W/S101T/P127S/S126T高的 有效突变体。
其中，部分突变引物如下：
Figure PCTCN2022131434-appb-000005
6、对初筛得到的相对野生型10℃下酶活最高的突变株进行摇瓶发酵复筛。实施例3碱性蛋白酶高活力突变体比酶活力评估
将上述实施例2所获得的高活力碱性蛋白酶突变体重组菌株 WB600/pLY-3-aprmx(x分别为1、2、3、4、5、6，下同)和野生型重组菌株WB600/pLY-3-apr分别接种于5mL的LB液体培养基(含卡那霉素，50μg/mL)中，37℃，220r/min培养过夜，按照2％接种量转接于50mL新鲜LB培养基(含卡那霉素，50μg/mL)中，继续以37℃，220r/min培养48h。
发酵液离心取上清，先以25％饱和度的硫酸铵盐析除去杂蛋白，再把饱和度加大到65％，沉淀目的蛋白。溶解后，透析除盐，再将盐析脱盐后得到的活性组分用0.02mol/L Tris-HCl(pH 7.0)缓冲液溶解，上样至纤维素离子交换层析柱后用同样的缓冲液先洗脱未吸附的蛋白，再用含不同浓度NaCl(0～1mol/L)的0.02mol/L Tris-HCl(pH 7.0)缓冲液进行梯度洗脱，收集目的蛋白。离子交换得到的活性组分先用含0.15mol/L NaCl的0.02mol/L Tris-HCl(pH 7.0)缓冲液平衡，上样至sephadex g25凝胶层析柱后用相同的缓冲液以0.5mL/min的速度洗脱，获得纯化的酶液，进行酶活测定。
酶活测定方法如实施例2。
蛋白质浓度由BCA蛋白浓度测定试剂盒测定，按照其说明书进行操作；
碱性蛋白酶比酶活力＝酶活力(U/ml)与蛋白质浓度(mg/ml)的比值。
最终计算得到野生型ALK与各突变体10℃下的比酶活如下表所示。
碱性蛋白酶 比酶活(U/mg)
WT 2.74
G95P 12.09
G95P/A96D 14.12
G95P/A96D/S99W 14.40
G95P/A96D/S99W/S101T 16.79
G95P/A96D/S99W/S101T/P127S 20.52
G95P/A96D/S99W/S101T/P127S/S126T 22.74
实施例4碱性蛋白酶突变体在解淀粉芽孢杆菌重组菌株中的表达及制备
将ALK野生型编码基因apr，以及突变体编码基因aprm1、aprm2、aprm3、aprm4、aprm5、aprm6与解淀粉芽孢杆菌表达质粒pLY-3通过连接得到新的重组质粒pLY-3-apr、pLY-3-aprm1、pLY-3-aprm2、pLY-3-aprm3、pLY-3-aprm4、pLY-3-aprm5、pLY-3-aprm6；
将重组质粒转入解淀粉芽孢杆菌CGMCC No.11218中，经卡纳霉素(Kan)抗性筛选，酶切验证得到野生型重组菌株CGMCC No.11218/pLY-3-apr，和突变体重组菌CGMCC No.11218/pLY-3-aprmx。
分别将解淀粉芽孢杆菌突变体重组菌株CGMCC No.11218/pLY-3-aprmx和野生型重组菌CGMCC No.11218/pLY-3-apr接种于5mL的发酵培养基(含卡那霉素，50μg/mL)中，37℃，220r/min培养过夜，按照2％接种量转接于50mL新鲜发酵培养基(含卡那霉素，50μg/mL)中，继续以37℃，220r/min培养48h。(发酵培养基(g/L)：玉米粉64，豆饼粉40，加入2.7淀粉酶，Na 2HPO 4 4，KH 2PO 4 0.3，其余为水；90℃保温30min再121℃灭菌20min。)
使用实施例2中的短肽底物法测定解淀粉芽孢杆菌发酵所得碱性蛋白酶活力(发酵液离心取上清测定酶活)。在解淀粉芽孢杆菌中碱性蛋白酶的发酵液酶活：ALK野生型酶活为27.13U/ml，ALK突变体G95P的酶活为118.48U/ml，ALK突变体G95P/A96D、G95P/A96D/S99W、G95P/A96D/S99W/S101T、G95P/A96D/S99W/S101T/P127S、G95P/A96D/S99W/S101T/P127S/S126T的酶活分别为142.61U/ml、146.88/ml、166.22U/ml、207.25U/ml、231.95U/ml。
发酵液离心获得的上清，先以25％饱和度的硫酸铵盐析除去杂蛋白，再把饱和度加大到65％，沉淀目的蛋白。溶解后，透析除盐，再将盐析脱盐后得到的活性组分用0.02mol/L Tris-HCl(pH 7.0)缓冲液溶解，上样至纤维素离子交换层析柱后用同样的缓冲液先洗脱未吸附的蛋白，再用含不同浓度NaCl(0～1mol/L)的0.02mol/L Tris-HCl(pH 7.0)缓冲液进行梯度洗脱，收集目的蛋白。离子交换得到的活性组分先用含0.15mol/L NaCl的0.02mol/L Tris-HCl(pH 7.0)缓冲液平衡，上样至sephadex g25凝胶层析柱后用相同的缓冲液以0.5mL/min的速度洗脱，获得纯化的酶液，冷冻干燥后制得碱性蛋白酶纯酶酶粉。所制备的碱性蛋白酶突变体酶粉可应用于洗涤剂、制革、食品、饲料等领域。
实施例5碱性蛋白酶在洗涤方面的应用
将实施例4制备的G95P/A96D/S99W/S101T/P127S/S126T突变体纯酶液应用于蛋白污布的洗涤。洗涤前用白度仪对蛋白污布(JB-02污布)的正反面进行白度值测量为18.73，随后用500mL污布洗涤液在10℃时对蛋白污布洗涤20min，然后将污布烘干，使用白度仪测烘干后污布的白度值为31.06。
污布洗涤液(V/V)：0.2％标准洗衣液，0.1％酶液，500mL水。
标准洗衣液(代号:SLD，参考GB/T13174-2021)：烷基苯磺酸(按活性物计)8％，聚乙氧基化脂肪醇(平均EO加合数为9)4％，乙氧基化烷基硫酸钠(2EO，按活性物计)2％，三乙醇胺0.5％，二水合柠檬酸三钠0.6％，防腐剂0.1％，水余量。试验室配制方法:将各种成分依次加入一定量的水中，同时搅拌溶解(必要时加热)，并用氢氧化钠调节溶液的pH值为8.5～9.0，补足水量至100％即可。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本专利构思的前提下，上述各实施方式还可以做出若干变形、组合和改进，这些都属于本专利的保护范围。因此，本专利的保护范围应以权利要求为准。

Claims (10)

  1. 一种碱性蛋白酶突变体，其特征在于，所述突变体是在SEQ ID NO.1所示野生型碱性蛋白酶的基础上发生G95P、A96D、S99W、S101T、P127S、S126T突变中的至少一种获得的。
  2. 如权利要求1所述的一种碱性蛋白酶突变体，其特征在于，所述碱性蛋白酶突变体为G95P突变体、G95P/A96D突变体、G95P/A96D/S99W突变体、G95P/A96D/S99W/S101T突变体、G95P/A96D/S99W/S101T/P127S突变体、或G95P/A96D/S99W/S101T/P127S/S126T突变体。
  3. 如权利要求2所述的一种碱性蛋白酶突变体，其特征在于，所述G95P突变体，具有SEQ ID NO.3所示的氨基酸序列；所述G95P/A96D突变体，具有SEQ ID NO.5所示的氨基酸序列；所述G95P/A96D/S99W突变体，具有SEQ ID NO.7所示的氨基酸序列；所述G95P/A96D/S99W/S101T突变体，具有SEQ ID NO.9所示的氨基酸序列；所述G95P/A96D/S99W/S101T/P127S突变体，具有SEQ ID NO.11所示的氨基酸序列；所述G95P/A96D/S99W/S101T/P127S/S126T突变体，具有SEQ ID NO.13所示的氨基酸序列。
  4. 权利要求2所述碱性蛋白酶突变体的编码基因。
  5. 如权利要求4所述的编码基因，其特征在于，所述G95P突变体的编码基因aprm1具有SEQ ID NO.4所示的核苷酸序列；所述G95P/A96D突变体的编码基因aprm2具有SEQ ID NO.6所示的核苷酸序列；所述G95P/A96D/S99W突变体的编码基因aprm3，具有SEQ ID NO.8所示的核苷酸序列；所述G95P/A96D/S99W/S101T突变体的编码基因aprm4，具有SEQ ID NO.10所示的核苷酸序列；所述G95P/A96D/S99W/S101T/P127S突变体的编码基因aprm5，具有SEQ ID NO.12所示的核苷酸序列；所述G95P/A96D/S99W/S101T/P127S/S126T突变体的编码基因aprm6，具有SEQ ID NO.14所示的核苷酸序列。
  6. 包含权利要求4所述编码基因的重组质粒或重组菌株。
  7. 如权利要求6所述的重组质粒或重组菌株，其特征在于，采用的表达载体为pLY-3，宿主细胞为大肠杆菌WB600，或者宿主细胞为解淀粉芽孢杆菌CGMCC No.11218。
  8. 权利要求6所述的重组质粒或重组菌株在生产碱性蛋白酶中的应用。
  9. 权利要求1所述碱性蛋白酶突变体的应用。
  10. 如权利要求9所述的应用，其特征在于，是在洗涤剂、制革、食品、饲料领域的应用。
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