CN115851679A - Low-temperature high-activity alkaline protease mutant and application thereof - Google Patents
Low-temperature high-activity alkaline protease mutant and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological engineering, and particularly relates to an alkaline protease enzyme mutant with improved enzyme activity, which is obtained by iterative saturation mutation through an overlapped PCR technology, and preparation and application thereof. The invention utilizes the overlapping PCR technology to carry out iterative saturation mutation on the alkaline protease gene apr from the Bacillus clausii, screens out the mutant with high activity at low temperature, and carries out high-efficiency expression and preparation in the Bacillus amyloliquefaciens, thereby solving the problem of low activity of the alkaline protease in the low-temperature washing environment.
Description
The technical field is as follows:
the invention belongs to the technical field of biological engineering, and particularly relates to an alkaline protease enzyme mutant with improved enzyme activity, which is obtained by iterative saturation mutation through an overlapped PCR technology, and preparation and application thereof.
Technical background:
the protease is widely existed in animals, plants and microorganisms, and the microorganisms are important sources of the biological enzyme due to the characteristics of high growth speed, simple growth condition, special metabolic process, wide distribution and the like. Its application mainly goes around its function of hydrolyzing protein peptide bonds, and in production and life, there are several major requirements: the complex macromolecular protein structure is changed into simple micromolecular peptide chain or amino acid, so that the complex macromolecular protein structure is easy to absorb or wash away, and the complex macromolecular protein structure can be used in the fields of food, detergent, feed and the like; the protein structure is partially destroyed, so that the separation of the substance components is realized, which is very effective when materials containing abundant protein such as leather, silk and the like are processed; promoting the degradation of environmental pollutants and being used in the field of environmental protection. The protease can catalyze the hydrolysis reaction and the reverse reaction, has high activity and specificity, and is very suitable for the production requirement of the pharmaceutical industry on certain specific molecules.
Proteases are classified into alkaline proteases, acid proteases and neutral proteases according to their optimum pH. The most suitable pH of the alkaline protease is 8.0-11.0, the alkaline protease is mostly derived from microorganisms, particularly the alkaline protease produced by industrial microorganisms has more obvious advantages of hydrolysis capability and alkali resistance, compared with the alkaline protease derived from animals and plants, the alkaline protease of microorganisms can be secreted out of cells, and the alkaline protease has the characteristics of relatively simple downstream technical treatment, low price, wide sources, easy culture of bacteria, easy realization of industrial mass production and the like, so the alkaline protease research becomes a hotspot of the protease research. However, the low enzyme activity is still one of the most important limiting factors in the industrial production of alkaline proteases, and therefore, the development of high-activity alkaline proteases is of great importance in the industrial production thereof. The pH of the detergent is generally 9.0-11.0, and the alkaline protease is mainly applied to the detergent industry due to higher stability and activity under alkaline conditions, and the usage of the alkaline protease in the detergent formula accounts for 89% of the total sales amount. Alkaline proteases have been receiving attention from more and more researchers due to their wide use, and efforts have been made to develop novel alkaline proteases having unique properties and higher activities.
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 detergents containing bacterial proteases were produced by Gebr ü der schnyder in 1959. In recent years, liquid detergents on the market have become more popular with consumers than solid detergents. The alkaline protease is added into the detergent, so that the original color of clothes can be kept, the dirt-removing capacity of the product is improved, the addition amount of the surfactant is reduced, and the detergent also has the effects of water saving, energy saving and environmental protection. According to the investigation result, the washing temperature in China is usually concentrated between 10 ℃ and 20 ℃, while the protease added in the current commercial detergent is mostly medium-temperature alkaline protease, and the optimal temperature is 40 ℃ to 60 ℃. Natural low temperature alkaline proteases are poorly stable at temperatures above 20 ℃ and have low yields on a large scale, such that such enzymes often do not meet the demands of the washing industry for low temperature alkaline proteases. Based on the background, the ALK mutant with high activity at low temperature is screened by carrying out molecular modification on the ALK derived from the Bacillus clausii so as to meet the requirements of the current washing industry.
The essence of directed evolution of proteins is to construct a molecular diversity library and screen mutants with improved properties from the library, and the method can be divided into four strategies of random evolution, shuffling technology, semi-rational evolution and rational evolution according to different library construction principles, wherein the general idea is that a target gene or a family of related family genes is started, and the molecular diversity library is created by mutating or recombining coding genes; screening the library to obtain a gene capable of coding the improved character as a template for the next round of evolution; the evolution in nature is required to be completed for thousands of years in a short time, thereby obtaining proteins with improved or completely new functions. The design and modification of enzyme molecule is based on the complementary development and infiltration of gene engineering, protein engineering and computer technology, and it marks that human may modify enzyme molecule according to his will and need, even to design new enzyme molecule which does not exist originally in nature. Under the condition that the artificial modification of enzyme molecules is not mature, a large number of enzyme molecules are successfully modified by a site-directed mutagenesis technology, and industrial enzyme with higher activity and better stability than natural enzyme is obtained. The Overlap PCR technique (Overlap PCR) is a technique in which primers having complementary ends are used to form overlapping strands of PCR products, so that amplified fragments from different sources are overlapped and spliced together through extension of the overlapping strands in a subsequent amplification reaction. The technology is used for carrying out site-directed mutagenesis on the gene sequence, thereby realizing the directional modification of the protein, and being reasonable and practical.
The bacillus expression system has the following advantages: 1. can secrete various proteins with high efficiency; 2. the use of many bacilli in the fermentation industry has a long history, is not pathogenic, and does not produce any endotoxin; 3. the research on the background of the genetics of the microorganism of the genus bacillus is clear, the growth is rapid, and no special requirements on nutrient substances are required; 4. codon preference is not obvious; 5. the fermentation process is simple, the bacillus belongs to aerobic bacteria, anaerobic fermentation equipment is not needed, and after the fermentation is finished, the fermentation liquor and the bacterial thalli can be simply separated and enter the separation, purification and recovery stages of target protein; 6. has stress resistance, and can be used for producing various thermostable enzyme preparations.
Therefore, in the invention, the alkaline protease gene derived from the Bacillus clausii is subjected to molecular modification by overlapping PCR, and a Bacillus subtilis expression system is used for high-throughput screening to obtain the alkaline protease mutant gene with improved enzyme activity at low temperature.
The invention content is as follows:
based on the problem of low temperature of washing environment, in order to obtain alkaline protease with high activity at low temperature, the prior properties of the alkaline protease need to be further improved, and the invention aims to provide a mutant of the alkaline protease with high activity at low temperature. Constructing a recombinant expression vector pLY-3-apr by using an alkaline protease gene (apr) derived from Bacillus clausii (Bacillus clausii) and a shuttle vector pLY-3, and expressing in Bacillus subtilis WB 600; determining a key hot spot area of the alkaline protease (ALK) by homologous modeling of the ALK and analysis of a docking result of the ALK and a substrate AAPF; iterative saturation mutation is carried out on apr by using an overlapping PCR technology, and AAPF is used for carrying out high-throughput screening on mutants to select the mutants with high activity at low temperature.
The technical route for achieving the purpose of the invention is summarized as follows:
carrying out saturation mutation on an alkaline protease gene apr from Bacillus clausii (Bacillus clausii), screening by using a Bacillus subtilis expression system to obtain high-activity ALK mutants G95P, G P/A96D, G P/A96D/S99W, G P/A96D/S99W/S101T, G P/A96D/S99W/S101T/P127S, G P95/A96D/S99W/S101T/S127S 126T at 10 ℃, and encoding genes aprm1, aprm2, aprm3, aprm4, aprm5 and aprm6, realizing high-efficiency expression of the mutants in the Bacillus amyloliquefaciens, and obtaining the ALK with improved enzyme activity by using technologies such as fermentation and extraction.
One of the technical schemes provided by the invention is an alkaline protease mutant which is obtained by carrying out at least one of mutations such as G95P, A96D, S W, S T, P S, S T and the like on the basis of a wild type alkaline protease zymogen region shown in SEQ ID NO. 1;
further, the alkaline protease mutant is a G95P mutant and has an amino acid sequence shown in SEQ ID NO. 3;
furthermore, the encoding gene aprm1 of the G95P mutant has a nucleotide sequence shown in SEQ ID NO. 4;
further, the alkaline protease mutant is a G95P/A96D mutant and has an amino acid sequence shown in SEQ ID NO. 5;
furthermore, the encoding gene aprm2 of the G95P/A96D mutant has a nucleotide sequence shown in SEQ ID NO. 6;
further, the alkaline protease mutant is a G95P/A96D/S99W mutant and has an amino acid sequence shown in SEQ ID NO. 7;
furthermore, the coding gene aprm3 of the G95P/A96D/S99W mutant has a nucleotide sequence shown in SEQ ID NO. 8;
further, the alkaline protease mutant is a G95P/A96D/S99W/S101T mutant and has an amino acid sequence shown in SEQ ID NO. 9;
furthermore, the coding gene aprm4 of the G95P/A96D/S99W/S101T mutant has a nucleotide sequence shown in SEQ ID NO. 10;
further, the alkaline protease mutant is a G95P/A96D/S99W/S101T/P127S mutant, and has an amino acid sequence shown in SEQ ID NO. 11;
furthermore, the gene aprm5 of the G95P/A96D/S99W/S101T/P127S mutant has a nucleotide sequence shown in SEQ ID NO. 12;
further, the alkaline protease mutant is a G95P/A96D/S99W/S101T/P127S/S126T mutant and has an amino acid sequence shown in SEQ ID NO. 13;
furthermore, the coding gene aprm6 of the G95P/A96D/S99W/S101T/P127S/S126T mutant has a nucleotide sequence shown in SEQ ID NO. 14.
The second technical scheme provided by the invention is a recombinant plasmid or a recombinant strain containing the mutant coding gene;
further, the adopted expression vector is pLY-3, and the host is escherichia coli or bacillus amyloliquefaciens;
furthermore, the host cell is Escherichia coli WB600, or the host cell is Bacillus amyloliquefaciens CGMCC No.11218;
preferably, the recombinant strain is obtained by connecting the mutant coding gene with an expression vector pLY-3 and then expressing the mutant coding gene in a host bacillus amyloliquefaciens CGMCC No.11218.
The third technical scheme provided by the invention is the application of the recombinant plasmid or the recombinant strain, in particular to the application in the production of alkaline protease.
The fourth technical scheme provided by the invention is the application of the alkaline protease mutant in the first technical scheme, in particular to the application in the fields of detergents, leathers, foods, feeds and the like; more particularly in the field of detergents, in particular in detergents used at low temperatures, further wherein said low temperature is 20 ℃ and below, in particular 10-20 ℃, to which said alkaline protease is added.
The experimental scheme of the invention is as follows:
1. obtaining an ALK mutant coding gene, comprising the following steps:
(1) The wild ALK coding gene apr shown in SEQ ID NO.2 is used as a starting gene to construct an expression vector pLY-3-apr, and iterative saturation mutation is carried out.
(2) And (3) transferring the mutated ALK coding gene into the bacillus subtilis WB600 after constructing a recombinant plasmid, and determining the activity of the protease by using an AAPF method.
(3) ALK mutants with improved activity relative to wild-type enzymes at 10 ℃ are obtained by screening, ALK mutant coding genes aprm1, aprm2, aprm3, aprm4, aprm5 and aprm6 are obtained by sequencing, and plasmids pLY-3-aprm1, pLY-3-aprm2, pLY-3-aprm3, pLY-3-aprm4, pLY-3-aprm5 and pLY-3-aprm6 containing ALK mutant coding genes with improved enzyme activity are stored.
And (3) carrying out fermentation culture on the screened high-activity mutant, purifying to obtain ALK protein, and then re-screening the enzyme activity. And the specific enzyme activity of each mutant was calculated.
2. The recombinant bacillus amyloliquefaciens strain containing the coding gene of the alkaline protease and the process for preparing the alkaline protease with improved enzyme activity by using the recombinant bacillus amyloliquefaciens strain comprise the following steps:
(1) Connecting the ALK mutant coding genes aprm1, aprm2, aprm3, aprm4, aprm5 and aprm6 with a 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;
(2) Transferring the recombinant plasmid into bacillus amyloliquefaciens CGMCC No.11218, screening resistance of kanamycin (Kan), performing enzyme digestion verification to obtain a recombinant strain, and then culturing and fermenting the recombinant strain to obtain the alkaline protease.
The following definitions are used in the present invention:
1. nomenclature for amino acid and DNA nucleic acid sequences
The accepted IUPAC nomenclature for amino acid residues is used, in single or three letter code form. DNA nucleic acid sequences employ the accepted IUPAC nomenclature.
2. Identification of alkaline protease mutants
The "amino acid substituted at the original amino acid position" is used to indicate the amino acid mutated in the ALK mutant. Such as Gly95Pro, which means the amino acid at position 95 is replaced by Gly from wild type ALK to Pro, and the numbering of the position corresponds to the numbering of the amino acid sequence of the wild type ALK mature peptide in SEQ ID NO. 19.
In the present invention, lower italic apr represents the coding gene of wild-type alkaline protease ALK, lower italic aprm1 represents the coding gene of mutant G95P, lower italic aprm2, aprm3, aprm4, aprm5 and aprm6 represent the coding genes of mutant G95P/A96D, G P/A96D/S99W, G P/A96D/S99W/S101 3532 zxft 3595P/A96D/S99W/S101T/P127S, G P/A96D/S99W/S101T/P127S 126T, respectively, and the specific information is as shown in the following table.
Has the advantages that:
1. the invention utilizes iterative saturation mutation technology to mutate ALK wild type, obtains mutant G95P, G P/A96D, G P/A96D/S99W, G P/A96D/S99W/S101T, G P/A96D/S99W/S101T/P127S, G P/A96D/S99W/S101T/P127S/S126T with enzyme activity improved relative to the wild type at 10 ℃, and the highest values of the fermentation enzyme activity in a bacillus amyloliquefaciens expression system are 118.48U/mL, 142.61U/mL, 146.88U/mL, 166.22U/mL, 207.25U/mL and 231.95U/mL respectively.
2. The high-efficiency expression and preparation of the ALK mutant with improved enzyme activity are realized by using a Bacillus amyloliquefaciens expression system.
Description of the drawings:
FIG. 1 is a PCR amplification electrophoresis chart of wild type alkaline protease zymogen gene
Wherein: m is DNA Marker,1 is alkaline protease zymogen gene apr;
FIG. 2 shows the restriction enzyme digestion verification of pLY-3-apr plasmid
Wherein: m is DNA Marker,1 is pLY-3-apr, and the map is cut by BamHI and SmaI;
the specific implementation mode is as follows:
the technical content of the present invention is further illustrated by the following examples, but the present invention is not limited to these examples, and the following examples should not be construed as limiting the scope of the present invention.
The culture medium used in the examples of the present invention was as follows:
LB medium (g/L): 5.0 parts of yeast extract, 10.0 parts of tryptone, 10.0 parts of NaCl and the balance of water.
Solid media was supplemented with 2% agar.
Fermentation medium (g/L): corn flour 64, bean cake powder 40, 2.7 amylase, 4Na 2 HPO 4 ，0.3KH 2 PO 4 The balance of water; keeping the temperature at 90 ℃ for 30min, and then sterilizing at 121 ℃ for 20min.
In the invention, the zymogen region sequence of the wild type alkaline protease ALK is shown in SEQ ID NO. 1: AEEAKEKYLIGFNEQEAVSEFVEQVEANDEVAILSEEEEVEIELLHEFETIPVLSVELSPEDVDALELDPAISYIEEDAEVTTMAQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR.
In the present invention, the mature peptide sequence of the wild-type alkaline protease ALK is shown in SEQ ID NO. 19: AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR.
The invention will be further illustrated by the following specific examples.
EXAMPLE 1 acquisition of wild-type alkaline protease Gene
1. The Kit (OMEGA: bacterial DNA Kit) is used for extracting the genome DNA of Bacillus clausii (Bacillus clausii), and the extraction steps are as follows:
(1) The strain was inoculated on an LB solid plate using an inoculating loop and incubated overnight at 37 ℃.
(2) A single colony was picked from a plate on which the cells were cultured, inoculated into a liquid tube medium, and cultured overnight at 37 ℃ under shaking at 220 r/min.
(3) 3mL-5mL of the bacterial solution is put into a sterilized EP tube, centrifuged at 12000r/min for 2min, and the supernatant is discarded.
(4) Add 200. Mu.L sterile water to the EP tube to resuspend the thallus, add 50. Mu.L lysozyme, mix well by aspiration, and keep the temperature at 37 ℃ for 20min.
(5) Adding 100 μ L BTL buffer and 20 μ L proteinase K into EP tube, mixing by vortex oscillation, keeping temperature at 55 deg.C for 40min, and mixing by oscillation every 20min.
(6) Add 5. Mu.L RNase, reverse and mix several times, and leave at room temperature for 10min.
(7) Centrifuge at 12000r/min for 2min to remove the undigested fraction, transfer the supernatant to a fresh EP tube, add 220. Mu.L BDL buffer, and water bath at 65 ℃ for 15min.
(8) Adding 220 mu L of absolute ethyl alcohol, blowing, sucking and mixing evenly.
(9) Transferring the liquid in the EP tube into a recovery column, standing for 1min, centrifuging for 1min at 12000r/min, pouring the filtrate into the recovery column again, repeating twice, and pouring off the waste liquid.
(10) Adding 500. Mu.L HBC buffer, centrifuging at 12000r/min for 1min, and discarding the filtrate.
(11) Add 700. Mu.L of DNA wash buffer, let stand for 1min, centrifuge at 12000r/min for 1min, discard the filtrate.
(12) Adding 500. Mu.L of DNA wash buffer, standing for 1min, centrifuging at 12000r/min for 1min, and discarding the filtrate.
(13) The column was emptied at 12000r/min for 2min, the waste tube discarded and the recovery column placed in a new EP tube.
(14) Drying in 55 deg.C metal bath for 10min.
(15) Adding 50 μ L of 55 deg.C sterile water, standing at room temperature for 5min, centrifuging at 12000r/min for 2min, and removing the column to obtain genome in EP tube.
2. The extracted genome of the Bacillus clausii is used as a template, a pair of primers are designed on the upstream and downstream of an ORF frame, restriction enzyme cutting sites BamHI and SmaI are respectively introduced, and the amplification primers of the alkaline protease gene apr are as follows:
upstream primer P1 (SEQ ID NO. 15):
5’-CGCGGATCCGCTGAAGAAGCAAAAGA-3’
downstream primer P2 (SEQ ID NO. 16):
5’-TCCCCCGGGTTAGCGTGTTGCCGCTTCT-3’
and (3) amplifying by taking the P1 and the P2 as upstream and downstream primers and taking the bacillus clausii alkaline protease genome as a template.
The reaction system for amplification is as follows:
the amplification procedure was: pre-denaturation at 98 ℃ for 30s; denaturation at 98 ℃ for 10s, annealing at 54 ℃ for 20s, and extension at 72 ℃ for 7s, and reacting for 30 cycles; extension at 72 ℃ for 10min. The PCR amplification product was subjected to 0.8% agarose gel electrophoresis to obtain a 1062bp band (FIG. 1), the PCR product was recovered using a small amount of DNA recovery kit to obtain the wild-type alkaline protease pro-region gene apr of the present invention (SEQ ID NO. 2), the apr and pLY-3 plasmids were double digested with restriction enzymes BamHI and SmaI, respectively, the recovered apr after gel cutting was ligated to the vector pLY-3 to obtain the recombinant plasmid pLY-3-apr, which was verified by digestion as shown in FIG. 2 and transformed into E.coli JM109 and Bacillus subtilis WB 600.
Example 2 construction of alkaline protease mutant library screening of high-Activity alkaline protease mutants
1. Carrying out saturation mutation based on an overlapping PCR technology to construct novel alkaline protease, and designing mutation primers as follows:
mutation upstream primer 95-F (SEQ ID NO. 17):
5’-GTTAAAGTATTANNKGCGAGCGGTTCA-3’
mutation downstream primer 95-R (SEQ ID NO. 18):
5’-TGAACCGCTCGCMNNTAATACTTTAAC-3’
in the overlapping PCR first-step reaction system, P1 and 95-R are used as an upstream primer and a downstream primer, and P2 and 95-F are used as an upstream primer and a downstream primer, respectively. And carrying out PCR1 reaction by taking the plasmid pLY-3-apr as a template to respectively obtain an upstream fragment and a downstream fragment.
The reaction system for amplifying the upstream fragment is as follows:
| P1 | 2μL |
| 95-R | 2μL |
| wild type alkaline protease gene | 2μL |
| Primer Star Max enzyme | 25μL |
| ddH 2 O | 19μL |
The reaction system for downstream fragment amplification is as follows:
the amplification procedure was: pre-denaturation at 98 ℃ for 30min; denaturation at 98 ℃ for 10s, annealing at 54 ℃ for 20s, and extension at 72 ℃ for 7s for 30 cycles; extension at 72 ℃ for 10min.
2. After cutting the gel and recovering the upstream and downstream fragments, carrying out PCR 2, wherein the reaction system is as follows:
| upstream segment | 2.0μL |
| Downstream fragment | 2.0μL |
| Primer Star Max enzyme | 25μL |
| ddH 2 O | 21μL |
The amplification procedure was: pre-denaturation at 98 ℃ for 30s; denaturation at 98 ℃ for 10s, annealing at 54 ℃ for 20s, and extension at 72 ℃ for 7s, and reacting for 5 cycles; extension at 72 ℃ for 10min.
3. After the PCR 2 is finished, 2 μ L of each of the primers P1 and P2 is added into the system, and the PCR 3 amplification program is carried out as follows: pre-denaturation at 98 ℃ for 30s; denaturation at 98 ℃ for 10s, annealing at 54 ℃ for 20s, and extension at 72 ℃ for 10s, and reacting for 5 cycles; extension for 10min at 72 ℃. And (3) carrying out 0.8% agarose gel electrophoresis on the PCR amplification product, and recovering the PCR product by using a small-amount DNA recovery kit to obtain the alkaline protease site-directed mutant gene aprm G95.
4. The alkaline protease site-directed mutant gene aprm G95 is connected with an expression vector pLY-3 and then transformed into JM109, the plasmid is extracted to obtain a recombinant plasmid pLY-3-aprm G95, and the recombinant plasmid pLY-3-aprm G95 is transformed into Bacillus subtilis WB600 to obtain a recombinant strain WB600/pLY-3-aprm G95. The transformants of the subtilisation were activated on a new Kan plate streaked with partitions, cultured for 12 hours at 37 ℃ in an inverted manner, and then the mutant strains were selected by using a high-throughput screening system, as follows:
(1) Liquid LB medium containing Kan resistance was dispensed under sterile conditions into sterile 48-well plates, 700. Mu.L per well.
(2) Single colonies of the mutants were picked and plated onto 48-deep well plates (labeled) with four blanks (i.e., WB 600/pLY-3-apr) left in each plate and incubated overnight at 37 ℃ with shaking at 600 r/min.
(3) Under aseptic conditions, liquid LB medium containing Kan resistance was dispensed into sterile 24-depth well plates, 1mL per well, 20. Mu.L of the suspension was aspirated and added to each well as a label, and shaking culture was performed at 37 ℃ and 600r/min for 48 hours.
(4) After the completion of the culture, the 24-depth well plate was removed, and the bacterial concentration of the bacterial suspension in each well was measured at OD 600.
(5) And (3) putting the 24-deep-hole plate into a hole plate centrifuge, centrifuging for 30min at a speed of 5000r/min, and taking the supernatant as enzyme liquid for enzyme activity determination.
5. Determination of alkaline protease activity by short peptide substrate
Short peptide substrate: the AAPF method uses Suc-Ala-Ala-Pro-Phe-pNA as a substrate, wherein Ala-Ala two amino acid residues form a hydrophobic group, pro amino acid has steric hindrance, and a peptide bond at the C terminal of the Pro amino acid is not easy to be cut, so that alkaline protease is more prone to hydrolyzing a peptide bond connected between Phe-pNA, pNA (p-nitroaniline) is released, free pNA can be detected at OD410, a spectrophotometer is used for measuring the light absorption value of a solution at OD410, and the enzyme activity is in direct proportion to the light absorption, namely the enzyme activity of the alkaline protease can be calculated.
The determination method comprises the following steps: 20. Mu.L of 4mM AAPF solution was pipetted into a 96-well plate, 80. Mu.L of boric acid buffer (pH = 10.5) was added thereto, and the mixture was mixed by pipetting and incubated at 10 ℃ for 2min. Adding 100 mu L of diluted enzyme solution into the sample group, adding 100 mu L of boric acid buffer solution into the control group, and uniformly mixing by blowing and sucking. After reacting for 10min at 10 ℃, the absorbance at 410nm is measured by using a microplate reader. Subtracting the OD value of the control group from the OD value measured by the experimental group to obtain delta OD, and substituting the delta OD into the following formula to calculate the corresponding enzyme activity:
the amount of enzyme that hydrolyzes AAPF at 10 ℃ under pH =10.5 for 1min with 1mL of enzyme solution to yield 1 μmol of pNA is defined as one enzyme activity unit U.
Through preliminary screening, obtaining a mutant with highest enzyme activity at 10 ℃ and higher than that of a wild type, putting the mutant strain plasmid pLY-3-aprm G95 out of Jin Weizhi company for sequencing, and determining that the mutant is the gene aprm1 of alkaline protease with 95-amino acid Gly mutated into Pro.
NNK saturation mutation is carried out on 96 th amino acid Ala of the aprm1 according to the embodiment, a mutant with the highest enzyme activity at 10 ℃ and higher than G95P is obtained through primary screening, the mutant strain plasmid is sent to Jin Weizhi company for sequencing, and the mutant is determined to be gene aprm2 of alkaline protease with 96 th amino acid Ala mutated into Asp.
NNK saturation mutation is carried out on amino acid Ser at the 97 th position of the aprm2 according to the embodiment, and an effective mutant with higher basic protease activity than G95P/A96D is obtained through primary screening and unscreened.
NNK saturation mutation is carried out on amino acid Gly at the 98 th position of the aprm2 according to the embodiment, and effective mutants with higher basic protease activity than G95P/A96D are obtained through primary screening and unscreened.
NNK saturation mutation is carried out on the 99 th amino acid Ser of aprm2 according to the embodiment, a mutant with the highest enzyme activity at 10 ℃ and higher than G95P/A96D is obtained through preliminary screening, the mutant strain plasmid is sent to Jin Weizhi company for sequencing, and the mutant is determined to be the gene aprm3 of alkaline protease with the 99 th amino acid Ser mutated into Trp.
NNK saturation mutation is carried out on 100 th amino acid Gly of the aprm3 according to the embodiment, and effective mutants with higher basic protease activity than G95P/A96D/S99W are obtained through primary screening and unscreened.
NNK saturation mutation is carried out on the 101 th amino acid Ser of the aprm3 according to the embodiment, a mutant with the highest enzyme activity at 10 ℃ and higher than G95P/A96D/S99W is obtained through preliminary screening, the mutant strain plasmid is sent to Jin Weizhi company for sequencing, and the mutant is determined to be the gene aprm4 of alkaline protease with the 99 th amino acid Ser mutated into Thr.
NNK saturation mutation is carried out on the 102 th amino acid Val of the aprm4 according to the embodiment, and effective mutants with higher alkaline protease activity than G95P/A96D/S99W/S101T are obtained through primary screening and unscreened.
NNK saturation mutation is carried out on 127 th amino acid Pro of the aprm4 according to the embodiment, a mutant with the highest enzyme activity at 10 ℃ and higher than G95P/A96D/S99W/S101T is obtained through primary screening, the plasmid of the mutant strain is sent to Jin Weizhi company for sequencing, and the mutant is determined to be gene aprm5 of alkaline protease with 127 th amino acid Pro mutated into Ser.
NNK saturation mutation is carried out on the 126 th amino acid Ser of the aprm5 according to the embodiment, a mutant with the highest enzyme activity at 10 ℃ and higher than G95P/A96D/S99W/S101T/P127S is obtained through primary screening, the plasmid of the mutant strain is sent to Jin Weizhi company for sequencing, and the mutant is determined to be the gene aprm6 of alkaline protease with the 126 th amino acid Ser mutated into Thr.
NNK saturation mutation is carried out on amino acid Gly at position 125 of aprm6 according to the embodiment, and effective mutants with higher basic protease activity than G95P/A96D/S99W/S101T/P127S/S126T are obtained through primary screening and unscreened.
NNK saturation mutation is carried out on the 124 th amino acid Leu of the aprm6 according to the embodiment, and an effective mutant with the alkaline protease activity higher than that of G95P/A96D/S99W/S101T/P127S/S126T is obtained through primary screening and unscreened.
NNK saturation mutation is carried out on 123 th amino acid Ser of the aprm6 according to the embodiment, and an effective mutant with higher basic protease activity than G95P/A96D/S99W/S101T/P127S/S126T is obtained through primary screening and unscreened.
Wherein, the partial mutation primers are as follows:
6. and (4) carrying out shake flask fermentation and re-screening on mutant strains which are obtained by primary screening and have the highest enzyme activity at 10 ℃ compared with wild type.
Example 3 evaluation of specific enzyme Activity of high Activity mutants of alkaline protease
The high-activity alkaline protease mutant recombinant strain WB600/pLY-3-aprmx (x is 1, 2, 3, 4, 5, 6, respectively, the same applies hereinafter) obtained in example 2 and the wild-type recombinant strain WB600/pLY-3-apr were inoculated into 5mL of LB liquid medium (containing kanamycin, 50. Mu.g/mL), cultured overnight at 37 ℃ at 220. Mu.g/min, transferred to 50mL of fresh LB medium (containing kanamycin, 50. Mu.g/mL) in an amount of 2%, and cultured at 37 ℃ at 220. Mu.g/min for another 48 hours.
Centrifuging the fermentation liquor to obtain supernatant, separating out foreign proteins by using ammonium sulfate with the saturation of 25%, increasing the saturation to 65%, and precipitating target proteins. After dissolving, dialyzing to remove salt, dissolving the active component obtained after salting-out and desalting by using 0.02mol/L Tris-HCl (pH 7.0) buffer solution, loading the solution to a cellulose ion exchange chromatographic column, eluting unadsorbed protein by using the same buffer solution, performing gradient elution by using 0.02mol/L Tris-HCl (pH 7.0) buffer solution containing NaCl with different concentrations (0-1 mol/L), and collecting the target protein. The active components obtained by ion exchange are balanced by 0.02mol/L Tris-HCl (pH 7.0) buffer solution containing 0.15mol/L NaCl, and the active components are loaded to a sephadex g25 gel chromatographic column and then eluted by the same buffer solution at the speed of 0.5mL/min to obtain purified enzyme solution for enzyme activity determination.
The enzyme activity was measured as in example 2.
The protein concentration is determined by a BCA protein concentration determination kit according to the instruction;
alkaline protease specific activity = ratio of enzyme activity (U/ml) to protein concentration (mg/ml).
The specific enzyme activities of the wild ALK and each mutant at 10 ℃ are finally calculated and obtained as shown in the table below.
| Alkaline protease | Specific activity (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 |
Example 4 expression and preparation of alkaline protease mutants in recombinant strains of Bacillus amyloliquefaciens
Connecting the ALK wild type encoding gene apr and mutant encoding genes aprm1, aprm2, aprm3, aprm4, aprm5 and aprm6 with a 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 is transferred into bacillus amyloliquefaciens CGMCC No.11218, and is subjected to resistance screening by kanamycin (Kan) and enzyme digestion verification to obtain a wild type recombinant strain CGMCC No.11218/pLY-3-apr and a mutant recombinant strain CGMCC No.11218/pLY-3-aprmx.
Respectively inoculating the bacillus amyloliquefaciens mutant recombinant strain CGMCC No.11218/pLY-3-aprmx and the wild type recombinant strain CGMCC No.11218/pLY-3-apr into 5mL of fermentation medium (containing kanamycin and 50 mu g/mL), culturing at 37 ℃ and 220r/min overnight, transferring into 50mL of fresh fermentation medium (containing kanamycin and 50 mu g/mL) according to the inoculum concentration of 2%, and continuously culturing at 37 ℃ and 220r/min for 48h. (fermentation medium (g/L): corn flour 64, bean cake powder 40, 2.7 amylase, na 2 HPO 4 4，KH 2 PO 4 0.3, and the balance of water; keeping the temperature at 90 ℃ for 30min, and then sterilizing at 121 ℃ for 20min. )
The activity of alkaline protease obtained by fermentation of Bacillus amyloliquefaciens was measured by the short peptide substrate method in example 2 (enzyme activity was measured by centrifuging the fermentation broth and collecting the supernatant). Fermentation broth enzyme activity of alkaline protease in bacillus amyloliquefaciens: the enzyme activity of the wild ALK is 27.13U/ml, the enzyme activity of the ALK mutant G95P is 118.48U/ml, and the enzyme activities of the ALK mutant G95P/A96D, G P/A96D/S99W, G P/A96D/S99W/S101T, G P/A96D/S99W/S101T/P127 34 zxft 4234P/A96D/S99W/S101T/P127S 126T are 142.61U/ml, 146.88/ml, 166.22U/ml, 207.25U/ml and 231.95U/ml respectively.
Supernatant obtained by centrifuging fermentation liquor is firstly separated and deproteinized by ammonium sulfate with the saturation of 25 percent, then the saturation is increased to 65 percent, and target protein is precipitated. After dissolution, dialysis is carried out to remove salt, active components obtained after salting-out and desalting are dissolved by 0.02mol/L Tris-HCl (pH 7.0) buffer solution, after the active components are loaded to a cellulose ion exchange chromatographic column, unadsorbed protein is eluted by the same buffer solution, then gradient elution is carried out by 0.02mol/L Tris-HCl (pH 7.0) buffer solution containing NaCl (0-1 mol/L) with different concentrations, and target protein is collected. Balancing the active component obtained by ion exchange with 0.02mol/L Tris-HCl (pH 7.0) buffer solution containing 0.15mol/L NaCl, loading the active component to sephadex g25 gel chromatographic column, eluting the active component with the same buffer solution at the speed of 0.5mL/min to obtain purified enzyme solution, and freeze-drying to obtain the pure enzyme powder of the alkaline protease. The prepared alkaline protease mutant enzyme powder can be applied to the fields of detergents, tanning, foods, feeds and the like.
EXAMPLE 5 use of alkaline protease in washing
The pure enzyme solution of the G95P/A96D/S99W/S101T/P127S/S126T mutant prepared in example 4 is applied to washing of protein-contaminated cloth. Before washing, the whiteness value of the front and back surfaces of the protein dirty cloth (JB-02 dirty cloth) is measured to be 18.73 by using a whiteness instrument, then the protein dirty cloth is washed for 20min at 10 ℃ by using 500mL of dirty cloth washing liquid, then the dirty cloth is dried, and the whiteness value of the dried dirty cloth is measured to be 31.06 by using the whiteness instrument.
Dirty cloth washing solution (V/V): 0.2% of standard laundry detergent, 0.1% of enzyme solution and 500mL of water.
Standard laundry detergent (code: SLD, reference GB/T13174-2021): 8% of alkylbenzene sulfonic acid (calculated by active matter), 4% of polyethoxylated fatty alcohol (average EO addition number is 9), 2% of ethoxylated alkyl sodium sulfate (2 EO, calculated by active matter), 0.5% of triethanolamine, 0.6% of trisodium citrate dihydrate, 0.1% of preservative and the balance of water. The preparation method in the laboratory comprises sequentially adding various components into a certain amount of water, stirring and dissolving (heating if necessary), adjusting the pH value of the solution to 8.5-9.0 with sodium hydroxide, and supplementing water to 100%.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.
Claims (10)
1. An alkaline protease mutant, characterized in that the mutant is obtained by carrying out at least one of G95P, A96D, S W, S T, P127S, S T mutations on the basis of the wild-type alkaline protease shown in SEQ ID NO. 1.
2. The alkaline protease mutant according to claim 1, wherein the alkaline protease mutant is a G95P mutant, a G95P/A96D/S99W/S101T/P127S mutant, or a G95P/A96D/S99W/S101T/P127S/S126T mutant.
3. The alkaline protease mutant according to claim 2, wherein the G95P mutant has an amino acid sequence shown in SEQ ID No. 3; the G95P/A96D mutant has an amino acid sequence shown in SEQ ID NO. 5; the G95P/A96D/S99W mutant has an amino acid sequence shown in SEQ ID NO. 7; the G95P/A96D/S99W/S101T mutant has an amino acid sequence shown in SEQ ID NO. 9; the G95P/A96D/S99W/S101T/P127S mutant has an amino acid sequence shown in SEQ ID NO. 11; the G95P/A96D/S99W/S101T/P127S/S126T mutant has an amino acid sequence shown in SEQ ID NO. 13.
4. The gene encoding the alkaline protease mutant according to claim 2.
5. The coding gene of claim 4, wherein the G95P mutant has the nucleotide sequence shown in SEQ ID NO.4 as the coding gene aprm 1; the encoding gene aprm2 of the G95P/A96D mutant has a nucleotide sequence shown in SEQ ID NO. 6; the coding gene aprm3 of the G95P/A96D/S99W mutant has a nucleotide sequence shown in SEQ ID NO. 8; the encoding gene aprm4 of the G95P/A96D/S99W/S101T mutant has a nucleotide sequence shown in SEQ ID NO. 10; the encoding gene aprm5 of the G95P/A96D/S99W/S101T/P127S mutant has a nucleotide sequence shown in SEQ ID NO. 12; the coding gene aprm6 of the G95P/A96D/S99W/S101T/P127S/S126T mutant has a nucleotide sequence shown in SEQ ID NO. 14.
6. A recombinant plasmid or a recombinant strain comprising the encoding gene of claim 4.
7. The recombinant plasmid or strain of claim 6 wherein the expression vector used is pLY-3 and the host cell is escherichia coli WB600 or the host cell is bacillus amyloliquefaciens CGMCC No.11218.
8. Use of the recombinant plasmid or recombinant strain of claim 6 for the production of alkaline protease.
9. The use of the alkaline protease mutant according to claim 1.
10. Use according to claim 9, in the fields of detergents, tanning, food and feed.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108441489A (en) * | 2018-04-09 | 2018-08-24 | 青岛蔚蓝生物集团有限公司 | A kind of bacillus subtilis of protein production processes and high yield alkali protein |
| CN112458072A (en) * | 2020-12-21 | 2021-03-09 | 山东隆科特酶制剂有限公司 | Alkaline protease mutant and preparation thereof |
| CN112501149A (en) * | 2020-12-21 | 2021-03-16 | 天津科技大学 | Alkaline protease mutant and gene, engineering bacterium, preparation method and application thereof |
| CN112521466A (en) * | 2020-01-22 | 2021-03-19 | 天津科技大学 | Signal peptide mutant for improving secretion of heterologous protein and application thereof |
-
2022
- 2022-10-09 CN CN202211227211.XA patent/CN115851679A/en active Pending
- 2022-11-11 WO PCT/CN2022/131434 patent/WO2024077702A1/en unknown
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108441489A (en) * | 2018-04-09 | 2018-08-24 | 青岛蔚蓝生物集团有限公司 | A kind of bacillus subtilis of protein production processes and high yield alkali protein |
| CN112521466A (en) * | 2020-01-22 | 2021-03-19 | 天津科技大学 | Signal peptide mutant for improving secretion of heterologous protein and application thereof |
| CN112458072A (en) * | 2020-12-21 | 2021-03-09 | 山东隆科特酶制剂有限公司 | Alkaline protease mutant and preparation thereof |
| CN112501149A (en) * | 2020-12-21 | 2021-03-16 | 天津科技大学 | Alkaline protease mutant and gene, engineering bacterium, preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| GENBANK: "Bacillus alcalophilus strain TCCC11004 alkaline protease gene, complete cds，Accession Number：FJ940727.1", NCBI, 26 May 2009 (2009-05-26) * |
| JIALIN LI 等: "Improving the activity and stability of Bacillus clausii alkaline protease using directed evolution and molecular dynamics simulation", ENZYME AND MICROBIAL TECHNOLOGY, vol. 29, no. 13, 24 March 2021 (2021-03-24), pages 2 * |
| 黄磊: "地衣芽胞杆菌碱性蛋白酶嗜碱突变体的特征分析", 食品与发酵工业, vol. 43, no. 6, 31 December 2017 (2017-12-31) * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117417922A (en) * | 2023-12-13 | 2024-01-19 | 北京科为博生物科技有限公司 | High-temperature-resistant alkaline protease and gene and application thereof |
| CN117417922B (en) * | 2023-12-13 | 2024-03-15 | 北京科为博生物科技有限公司 | High-temperature-resistant alkaline protease and gene and application thereof |
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| WO2024077702A1 (en) | 2024-04-18 |
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