CN117737044A - Neutral protease with high activity and tolerance, and encoding gene and application thereof - Google Patents
Neutral protease with high activity and tolerance, and encoding gene and application thereof Download PDFInfo
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- CN117737044A CN117737044A CN202311791578.9A CN202311791578A CN117737044A CN 117737044 A CN117737044 A CN 117737044A CN 202311791578 A CN202311791578 A CN 202311791578A CN 117737044 A CN117737044 A CN 117737044A
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Abstract
The invention relates to the field of genetic engineering, in particular to neutral protease, and a coding gene and application thereof. The full length of the gene HtpX capable of encoding neutral protease with high activity and tolerance is 873bp, the nucleotide sequence of the gene HtpX encoding the neutral protease is 290 amino acids shown in SEQ No. 1. The neutral protease prepared by fermenting the recombinant strain constructed by the invention has higher enzyme activity and better temperature and pH tolerance.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to neutral protease with high activity and tolerance, and a coding gene and application thereof.
Background
The enzyme preparation is used as a green and environment-friendly catalyst, and can meet the requirements of regulations and requirements of customers on clean technology and a clean sustainable production method in industrial production, thereby reducing the influence on the environment. However, most of the natural enzyme preparations are sensitive to temperature and pH, are easily inactivated by the influence of external environment, and cannot meet the industrial production requirements. Therefore, screening for enzyme resources with high activity and tolerance becomes a key in the industrial popularization of enzymes.
Insects are the largest number, variety and distribution of the largest biological groups on the earth, and are huge biological resources that have not been fully developed and utilized. The insect intestinal tissues contain abundant microbial resources, and can be used as beneficial microbial resources for development and utilization. An alkalophilic strain producing alkaline protease, which is alkaline halobacillus sp PAIA262 isolated from the intestinal tract of periplaneta americana and is occluded in China center for culture collection of microorganisms at 3 and 23 in 2023, and the preservation number is GDMCC No.63124, and the application thereof are disclosed in the patent document with publication number of CN 117070394A. The alkaline protease in the patent document can be applied to the fields of detergent production, leather tanning processing, food processing, medical industry, protein waste treatment and the like.
Disclosure of Invention
The invention provides a neutral protease with high activity and tolerance, and a gene and application of the neutral protease, which aim to solve the technical problems that a natural protease preparation is sensitive to temperature and pH, is easy to be inactivated due to the influence of external environment and cannot meet the industrial production requirement.
The technical scheme adopted by the invention is as follows:
the nucleotide sequence of the gene HtpX capable of encoding neutral protease with high activity and tolerance is shown as SEQ ID No. 1.
The method comprises the steps of extracting the whole genome of the Gryllotalpa intestinal tract from the Gryllotalpa intestinal tract, sending the Gryllotalpa intestinal tract whole genome TO Hangzhou Linchuan biotechnology Co Ltd for metagenome analysis, and predicting and obtaining a protease gene HtpX containing a complete sequence through KEGG functional annotation of a metagenome result, wherein the number of the protease gene HtpX in a KNGG database is BG04_5331, and the CDS number is TO3825. Designing a pair of primers containing recognition sites of restriction enzymes BamHI and SmaI according to HtpX protease gene sequence obtained by screening intestinal microbial metagenome of Gryllotalpa, and designing the nucleotide sequence 5' of forward primerCGGGAT CCTGCTAAAACGAATTTCACTGTTT-3 '(underlined as BamHI cleavage site), the nucleotide sequence 5' of the reverse primer, shown in SEQ No.3TCCCCCGGGTTATAGCGAATGCAAGCGC-3' (underlined as SmaI cleavage site), as shown in SEQ No.4, PCR amplification is carried out by taking extracted whole genome DNA of the mole cricket intestinal microorganism as a template, thus obtaining a gene HtpX capable of encoding neutral protease with high activity and tolerance.
The total length of the gene HtpX capable of encoding neutral protease with high activity and tolerance is 873bp, the nucleotide sequence of the gene HtpX encoding neutral protease is 290 amino acids shown in SEQ No. 1.
According to another aspect of the present invention, there is also provided a neutral protease having high activity and tolerance, the amino acid sequence of which is shown in SEQ No. 2.
According to another aspect of the invention, there is provided a recombinant vector comprising a PCR product expression vector pHT43, double enzyme digestion and purification, wherein the PCR product expression vector pHT43 is subjected to ligation overnight under the action of T4 ligase at a temperature of 16 ℃, transformed into E.coli DH5 alpha competent cells, the competent cells are coated into LB solid plate medium containing chloramphenicol, cultured overnight at 37 ℃, monoclonal is selected, positive clones are verified by bacterial liquid PCR, and then bacterial species are preserved and sequenced. And (3) selecting bacterial liquid PCR to verify positive clones and correct sequencing results, inoculating the clones to LB liquid medium containing amikacin, carrying out shake culture at 37 ℃ for overnight, extracting plasmids to obtain recombinant plasmids pHT43-HtpX, electrically transforming the recombinant plasmids to bacillus subtilis WB800N competent cells, diluting and coating the recombinant plasmids on LB plates containing chloramphenicol antibiotics, and screening positive transformants.
According to another aspect of the present invention, there is provided a recombinant strain which is preserved in China Center for Type Culture Collection (CCTCC) at the time of 24 days of 11.2023, and after detection by the preserving agency at the time of 1 day of 12.2023, the detection result is survival, and the classification name is bacillus subtilis WB800N/pHT43-HtpX (Bacillus subtilis), and the preservation number is CCTCC No: m20232325, the address of the preservation institution is located at the university of Wuhan, china, and the preservation institution is called CCTCC for short.
Recombinant strain-Bacillus subtilis WB800N/pHT43-HtpX (Bacillus subtilis) with preservation number of CCTCC M20232325, including the recombinant expression vector-recombinant plasmid pHT43-HtpX.
Recombinant strain-preservation number is CCTCC No: bacillus subtilis WB800N/pHT43-HtpX (Bacillus subtilis) of M20232325 is inoculated in a proportion of 1% -2% into 50 ml fermentation medium containing chloramphenicol at a final concentration of 10 μg/ml, and cultured at 37℃and a rotational speed of 180 rpm to OD 600 And (3) adding 1-4 mmol/L isopropyl-beta-D-thiogalactoside into the mixture, and culturing the mixture for 24-36 hours at 37 ℃ and a rotating speed of 150-220 r/min to induce the production of neutral protease, wherein the activity of the prepared neutral protease is 321-998 units/ml.
According to another aspect of the present invention, there is also provided a recombinant strain having a preservation number of cctcc no: the construction method of the bacillus subtilis WB800N/pHT43-HtpX (Bacillus subtilis) of M20232325 comprises the following steps:
6A, providing a gene HtpX capable of encoding a neutral protease having high activity and tolerance, an expression vector and an expression strain;
6B, amplifying a neutral protease gene HtpX capable of encoding the neutral protease gene with high activity and tolerance, and connecting an amplified product with an expression vector pHT43 to obtain a recombinant expression vector pHT43-HtpX;
6C, integrating the recombinant expression vector pHT43-HtpX into an expression strain, namely bacillus subtilis WB800N competent cells, so as to obtain a recombinant strain, wherein the preservation number of the recombinant strain is CCTCC No: bacillus subtilis WB800N/pHT43-HtpX (Bacillus subtilis) of M20232325.
In the step 6B, the whole genome DNA of the extracted mole cricket intestinal microorganisms is used as a template, and the amplified primers comprise a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ No.3, and the nucleotide sequence of the reverse primer is shown as SEQ No. 4.
According to another aspect of the present invention, there is also provided a method for preparing neutral protease having high activity and tolerance, the method comprising the steps of:
(1) Culturing the recombinant strain with the preservation number of CCTCC No: bacillus subtilis WB800N/pHT43-HtpX (Bacillus subtilis) of M20232325, induces the expression of the gene HtpX encoding neutral protease;
(2) Separating and purifying the expressed neutral protease.
According to another aspect of the present invention, there is also provided a neutral protease having high activity and tolerance, a gene HtpX capable of encoding the neutral protease having high activity and tolerance, a recombinant vector pHT43-HtpX containing the gene HtpX, the use of a recombinant strain containing the recombinant vector, bacillus subtilis WB800N/pHT43-HtpX (Bacillus subtilis) having a preservation number of CCTCCNo: M20232325, for degrading cattle hide and/or sheep skin solid waste, wherein the neutral protease has a reaction temperature of 45℃to 55℃and a reaction pH=6.0 to 8.0 in degrading cattle hide and/or sheep skin solid waste. The recovery rate of the sheep skin collagen is 10.22-18.83% when the sheep skin solid waste is degraded, and the recovery rate of the cow hide collagen is 30.12-50.85% when the sheep skin solid waste is degraded.
The invention has the substantial characteristics and the remarkable technical progress that:
the invention fully digs the non-culturable enzyme resource in the intestinal tract of the mole cricket, constructs the gene HtpX for encoding neutral protease with high activity and tolerance, the total length of the gene HtpX is 873bp, and the recombinant strain constructed by the gene has the preservation number of CCTCC No: the neutral protease prepared by fermenting the bacillus subtilis WB800N/pHT43-HtpX (Bacillus subtilis) of M20232325 has the enzyme activity of 321-998 units/ml and higher enzyme activity. The composition has better tolerance in buffer solutions with pH value of 3.0-10.0, the relative enzyme activity is over 50 percent, and the composition has no enzyme activity damage when being stored for 3 days at the temperature of 37 ℃ with the pH value of 6.0-8.0. The optimal reaction temperature is 45-55 ℃, no enzyme activity loss exists in 24 hours at 30-40 ℃, and the relative enzyme activity is kept above 50% in 24 hours at 45 ℃, so that the temperature and pH tolerance of neutral protease are better improved.
The strain of the invention is preserved in China Center for Type Culture Collection (CCTCC) at the time of 11/24 of 2023, and after detection by a preserving organization at the time of 12/1 of 2023, the detection result is survival, and the classification name is bacillus subtilis WB800N/pHT43-HtpX (Bacillus subtilis), and the preservation number is CCTCC No: m20232325, the address of the preservation institution is located at the university of Wuhan, china, and the preservation institution is called CCTCC for short.
In addition to the objects, features and advantages described above, the present invention has other objects, features and advantages. The invention will be described in further detail with reference to the accompanying drawings.
Drawings
The drawings of the invention are as follows:
FIG. 1 is a schematic diagram showing gel electrophoresis of a gene HtpX capable of encoding neutral protease having high activity and tolerance prepared in accordance with the present invention.
Since the DNA of different sizes are separated under the action of current, the length of the PCR product can be roughly seen by the means and Marker (control), the total length of the neutral protease gene HtpX is 873bp, and if the length of the band is roughly within the range, the successful amplification from the insect intestinal tract is preliminarily judged according to FIG. 1 to obtain the neutral protease gene HtpX.
FIG. 2 is a recombinant strain prepared by the invention, the preservation number of which is CCTCC No: colony PCR verification effect graph of M20232325 bacillus subtilis WB800N/pHT43-HtpX (Bacillus subtilis).
After the gene HtpX capable of encoding neutral protease is successfully connected with the expression vector pHT43, the forward and reverse primers designed by the invention can be used for PCR reaction, if the connection is successful, the band can be obtained by PCR, and if the connection is unsuccessful, the band is not obtained. The length of the PCR band successfully connected with that of the neutral protease gene HtpX is 873bp, and compared with the control used in the invention, the length of the PCR band is 750-1000 bp, and the band shown in FIG. 2 is in the range, so that the successful connection of the neutral protease gene HtpX with the expression vector pHT43 is illustrated according to FIG. 2.
FIG. 3 is a plasmid map of the expression vector pHT43 in the present invention.
FIG. 4 is a schematic diagram showing construction of recombinant plasmid pHT43-HtpX according to the present invention.
FIG. 5 is a schematic of gel electrophoresis of neutral protease prepared according to the present invention.
Reference numeral 1 in fig. 5 is before induction; reference numeral 2 is after induction; protein analysis by polyacrylamide gel electrophoresis revealed that 2 had a distinct recombinant band at 37-52 kDa, and 1 did not show that this band was produced by expression after induction, indicating that the obtained gene encoding neutral protease HtpX with high activity and tolerance was successfully expressed.
FIG. 6 is a schematic representation of a neutral protease prepared according to the invention spotted on a 2% milk plate.
FIG. 6 shows that 2. Mu.L of neutral protease prepared by the invention is spotted on a 2% milk plate, and the generation of hydrolysis circles can be observed after 20 minutes, which indicates that the recombinant strain in the invention, the preservation number is CCTCCNo: bacillus subtilis WB800N/pHT43-HtpX (Bacillus subtilis) of M20232325 has the ability to produce neutral proteases.
FIG. 7 is a schematic diagram showing the enzyme activities of neutral proteases prepared according to the present invention at different temperatures.
FIG. 7 shows that the neutral protease prepared by the invention has the highest enzyme activity at 45-55 ℃, so that the optimal reaction temperature of the neutral protease is 45-55 ℃.
FIG. 8 is a schematic view showing the effect of the neutral protease prepared by the invention on heat stability at 30-60 ℃.
FIG. 8 shows that the neutral protease has no enzyme activity loss within 24 hours at 30-40℃and the relative enzyme activity at 45℃for 24 hours is maintained at 50% or more, and that the enzyme activity loss of the neutral protease is rapid at 50℃and above.
FIG. 9 is a schematic of the pH optimum reaction and pH tolerance effects of neutral proteases prepared according to the present invention.
Fig. 9 illustrates that the neutral protease prepared by the invention has better tolerance in buffer solution with pH=3.0-10.0, the relative enzyme activity is over 50%, and the neutral protease has no enzyme activity damage when stored for 3 days at 37 ℃ at the pH=6.0-8.0.
FIG. 10 is a schematic diagram showing gel electrophoresis of collagen obtained by degrading cowhide solid waste and sheep skin solid waste with neutral protease prepared in the present invention.
In fig. 10, reference numeral 1 denotes collagen extracted by treating cowhide; the mark 2 is collagen extracted by sheep skin treatment. Collagen hydrolyzed by neutral protease of cow leather and sheep skin is composed of 4 chains, namely 2 different alpha peptide chains alpha 1 and alpha 2,1 alpha peptide chain dimer beta peptide chain and 1 alpha peptide chain trimer gamma peptide chain. Typical type I collagen is a trimer consisting of 2 α1 and 1 α2 chains, whereas collagen extracted from the waste cow leather and sheep leather in fig. 8 meets the peptide chain characteristics of typical type I collagen.
Detailed Description
The embodiments of the present invention will be further described with reference to the accompanying drawings, which are to be construed as illustrative, but not limitative of the present invention, and all equivalent technical means as defined in the claims are replaced by the following description without departing from the scope of the present invention.
Examples
The Gryllotalpa is collected from Hebei quyang farmland.
Coli strain DH 5. Alpha. Competent cells were purchased from Hebei three lion biotechnology Co., ltd, and Bacillus subtilis WB800N competent cells were purchased from Hua-Viea biotechnology Co., ltd.
Expression vector pHT43 was purchased from Va.
Primer synthesis and gene sequencing were performed by the biological engineering Co., ltd.
Sequencing of the bowel metagenome of Gryllotalpa was performed by Hangzhou Union Biotechnology Co.
The intestinal genome of the Gryllotalpa is extracted by a radix et rhizoma Rhei fecal genome extraction kit.
Plasmid extraction, gel purification, restriction enzymes BamHI and SmaI, T4 ligase, and PCR kit were all purchased from New England Biotechnology (Beijing) Inc.
Recombinant strain-bacillus subtilis WB800N/pHT43-HtpX (Bacillus subtilis) CCTCC No: the culture medium of M20232325 is LB culture medium and fermentation culture medium, and the fermentation culture medium comprises the following raw materials: 2-6 g/l of sodium chloride, 5-10 g/l of yeast extract, 10-16 g/l of peptone, 10-20 g/l of skimmed milk powder, 10-20 g/l of maltose and sterilizing at 115 ℃ for 20 minutes.
Example 1
Construction of Gene HtpX clone and expression vector encoding neutral protease having high Activity and tolerance according to claim 1
Extracting the whole intestinal genome of the Gryllotalpa from the intestinal tract of the Gryllotalpa by adopting a root fecal genome extraction kit, sending the whole intestinal genome of the Gryllotalpa TO Hangzhou Linchuan biotechnology Co-Ltd for metagenome analysis, and predicting the result of the metagenome through KEGG functional annotation TO obtain the protease gene HtpX containing the complete sequence, wherein the number of the protease gene HtpX in the KNGG database is BG045331, and the CDS number is TO3825. Designing restriction enzyme based on neutral protease gene sequence obtained by screening intestinal microorganism metagenome of GryllotalpaA pair of primers of BamHI and SmaI recognition sites, nucleotide sequence 5' of forward primerCGGGATCCTGCTAAAACGAATTTCACTGTTT-3 '(underlined as BamHI cleavage site), the nucleotide sequence 5' of the reverse primer, shown in SEQ No.3TCCCCC GGGTTATAGCGAATGCAAGCGC-3' (underlined as SmaI cleavage site), as shown in SEQ No.4, PCR amplification is carried out by taking extracted whole genome DNA of the mole cricket intestinal microorganism as a template, so as to obtain a gene HtpX capable of encoding neutral protease with high activity and tolerance, as shown in FIG. 1, and a PCR reaction system is shown in the specification: primeSTAR Max DNA Polymerase 25. Mu.l each of the forward primer and the reverse primer (10 mM) was 2.0. Mu.l each, 2.0. Mu.l of the DNA template, and the double distilled water was made up to 50.0. Mu.l. PCR reaction conditions: 98 ℃ for one minute; 55 ℃, sixty seconds; 72 ℃, one minute, 30 cycles; 4 ℃ for fifty minutes.
The PCR product expression vector pHT43 is subjected to double enzyme digestion and purification, is connected overnight under the action of T4 ligase at the temperature of 16 ℃, is transformed into competent cells of escherichia coli DH5 alpha, is coated in LB solid plate medium containing chloramphenicol, is cultured overnight at 37 ℃, is picked up and is subjected to positive cloning by bacterial liquid PCR verification, and the strain is preserved and sequenced, and the plasmid map of the expression vector pHT43 is shown in figure 3. Selecting bacterial liquid PCR to verify positive clones with correct sequencing results, inoculating the clones to LB liquid medium containing ampicillin, shaking and culturing overnight at 37 ℃, extracting plasmids to obtain recombinant plasmids pHT43-HtpX, and particularly as shown in fig. 4, electrically converting the plasmids into bacillus subtilis WB800N competent cells, diluting and coating the bacillus subtilis WB800N/pHT43-HtpX (Bacillus subtilis) on LB flat plates containing chloramphenicol antibiotics to screen positive transformants, thus obtaining recombinant strains, namely bacillus subtilis WB800N/pHT43-HtpX (Bacillus subtilis), which are preserved in China center for type culture collection on 24 11 months of 2023, and after detection by a preserving mechanism on 1 day of 12 months of 2023, the detection results are survival, and the classification name is bacillus subtilis WB800N/pHT43-HtpX (Bacillus sub tilis), and the preservation number is CCTCC No: m20232325, the address of the preservation institution is located at the university of Wuhan, china, and the preservation institution is called CCTCC for short.
The full length of the gene HtpX capable of encoding neutral protease with high activity and tolerance is 873bp, 290 amino acids of the neutral protease are encoded, the nucleotide sequence of the gene HtpX is shown in SEQ No.1, and the gene HtpX is specifically as follows:
CTGCTAAAACGAATTTCACTGTTTATTTTCGTTAATATTTTAGTATTAATTACAATCACAACGATT
ACATCTTTATTAGGTGTTCAAAGCTATATGGGAAGCAGCGGTTACGGCGGCCTGCTGGCATTTAGC
GTAATTGCCGGCTTCAGCGGCGCGATTATTTCTCTTATGATGTCACGCGTGATGGCAAAGTGGATG
ATGGGTGTTCAAGTTATTGATGAACGTTCTCCTCAAGGTGAATATGAGCGATTTGTTTTAGAAGAA
ACGCACCGACTTGCATCTGTTGCAGGATTAAGAAAAATGCCTCAGGTCGGAATTTATCATTCAGCG
GAAGTTAACGCTTTTGCAACAGGACCAAGTAAAAGACGCTCGCTTGTTGCCGTTTCAAGCGGAATG
CTTGAACGAATGGACCGCGATGCGATCAGCGGCGTTATCGCTCACGAAATAGCGCATATTAAAAGC
GGTGATATGGTCACAACTACGCTTCTGCAAGGAGTATTAAATACATTTGTTATTTTCTTCTCTCGT
TTAGTAGCCAAAGCGGTGTCAAACTTTGTGCGAGAAGAGTTTGCGATGGTTGTCTATTTCCTTACA
TCGATTGTATTTGAGATTCTCTTTAGTATCTTAAGCAGCCCAATTATCTTCTGGCATTCAAGAAGA
CGTGAATTCAAAGCCGATGAGCTTGCAGCTAAACTCGGTGGTAAAGAAAAAATGATTTATGCGTTA
GAATCGCTTCGTCATACTACTTCATTAGTAGATGACCGCCAAAAATCAATTGCCGCTTTTAAAATT
AGCGGCAAAGAAAAATTCTCACGCTTATTCTCTACTCACCCGCCGCTTGAAAAACGCA
the amino acid sequence of the neutral protease is shown as SEQ No.2, and is specifically as follows: LLKRISLFIFVNILVLITITTITSLLGVQSYMGSSGYGGLLAFSVIAGFSGAIISLMMSRVMAKWMMGVQVIDERSPQGEYERFVLEETHRLASVAGLRKMPQVGIYHSAEVNAFATGPSKRRSLVAVSSGMLERMDRDAISGVIAHEIAHIKSGDMVTTTLLQGVLNTFVIFFSRLVAKAVSNFVREEFAMVVYFLTSIVFEILFSILSSPIIFWHSRRREFKADELAAKLGGKEKMIYALESLRHTTSLVDDRQKSIAAFKISGKEKFSRLFSTHPPLEKRIERLHSL
Example 2
Culture-preservation number is CCTCC No: bacillus subtilis (Bacillus subtilis) WB800N/pHT43-HtpX of M20232325
The invention is toThe preservation number of the recombinant strain is CCTCC No: bacillus subtilis WB800N/pHT43-HtpX (Bacillus subtilis) of M20232325 is inoculated in a volume percentage of 0.1% into 5 ml LB medium containing chloramphenicol at a final concentration of 10. Mu.g/ml, activated at 37℃at a rotation speed of 180 rpm for 12 hours, inoculated in a volume percentage of 1% into 50 ml fermentation medium containing chloramphenicol at a final concentration of 10. Mu.g/ml, and cultured at 37℃at a rotation speed of 180 rpm to OD 600 And (3) adding 1-4 millimoles/liter isopropyl-beta-D-thiogalactoside into the mixture, culturing the mixture for 24 hours at 37 ℃ and 180 revolutions/minute to induce the production of neutral protease, and centrifuging the mixture for 5 minutes at 12000 revolutions/minute to obtain the neutral protease with high activity and tolerance.
Example 3
Expression and analysis of proteins
Referring to FIG. 5, gel electrophoresis analysis of the neutral protease having high activity and tolerance obtained in example 2 revealed that a significant recombinant band was found at 37-52 kDa by polyacrylamide gel electrophoresis, indicating successful expression of genes obtained from the intestinal microbial metagenome of Gryllotalpa. Referring to FIG. 6, 2. Mu.L of protease solution was spotted on 2% of milk plates, and after 20 minutes, the generation of hydrolysis circles was observed, indicating that the recombinant strain-with a preservation number of CCTCC No: bacillus subtilis WB800N/pHT43-HtpX (Bacillus subtilis) of M20232325 produces neutral proteases with high activity and tolerance.
Example 4
Determination of enzyme Activity
The storage number is CCTCC No: bacillus subtilis WB800N/pHT43-HtpX (Bacillus subtilis) of M20232325 is inoculated in a proportion of 1% -2% into 50 ml fermentation medium containing chloramphenicol at a final concentration of 10 μg/ml, and cultured at 37℃and a rotational speed of 180 rpm to OD 600 0.6 to 0.8 of isopropyl-beta-D-thiogalactoside with the final concentration of 1 to 4 millimoles per liter is added, and the culture is carried out for 24 to 36 hours under the conditions of the temperature of 37 ℃ and the rotating speed of 150 to 220 revolutions per minute to induce neutral proteinaseThe activity of the neutral protease prepared in this example was measured according to the method of appendix B of GB/T23527-2009 protease preparation. Relative enzyme activity calculation formula: w (%) =a i /A 0 X100 (W: relative enzyme Activity; A) i : enzyme activity measured after treatment; a is that 0 : enzyme activity measured at 0 h)
The activity of the prepared neutral protease is 321-998 units/ml.
Example 5
Determination of optimum temperature and temperature stability
According to the method of annex B in GB/T23527-2009 protease preparation, the enzyme activity at 20-60 ℃ is measured, the enzyme activities of neutral proteases at different temperatures are compared, and the optimal reaction temperature is determined. As shown in FIG. 7, the neutral protease has the highest enzyme activity at 45-55deg.C, so that the neutral protease has an optimal reaction temperature of 45-55deg.C.
The enzyme solutions were incubated at 30, 37, 40, 45, 50, 55 and 60℃for 12 hours, and the remaining enzyme activities were measured by spot sampling, and the relative enzyme activities (%) were calculated by using the enzyme activities measured at the same temperature for 0 hour as a control, and the thermostability was analyzed.
The relative enzyme activity is calculated by detecting the enzyme activity of the neutral protease under different temperature conditions, and the result is shown in figure 8, the neutral protease has no enzyme activity loss in 24 hours under the condition of 30-40 ℃, the relative enzyme activity in 24 hours under the condition of 45 ℃ is kept above 50%, and the enzyme activity loss of the neutral protease is faster when the temperature is 50 ℃ and above.
Example 6
Optimum pH and pH stability
The enzyme activities in citrate buffer with pH=3.0 to 5.0, phosphate buffer with pH=6.0 to 8.0, glycine-sodium hydroxide buffer with pH=8.0 to 10.0 were measured at the optimal temperature, and the protease activities under each pH condition were compared to determine the optimal reaction pH. The neutral protease was incubated in buffer solutions at different ph=3.0 to 10.0 at 37 ℃ for 3 days, and relative enzyme activity (%) was calculated and pH stability was analyzed by using the enzyme activity measured at the same pH condition of 0d as a control. The pH optimum reaction and the pH tolerance of the neutral protease are shown in figure 9, the pH optimum reaction of the neutral protease is=7.0, the neutral protease has better tolerance in a buffer solution with the pH value of=3.0-10.0, the relative enzyme activity is over 50 percent, and the neutral protease has no enzyme activity damage when being stored at the temperature of 37 ℃ for 3 days at the pH value of=6.0-8.0.
Example 7
Application of neutral protease in collagen extraction
The sheep skin and the cowhide are soaked for 12 hours for fresh return respectively, the wool fibers are removed, the sheep skin and the cowhide are treated for 12 to 24 hours at the temperature of 4 ℃ by 0.5M sodium hydroxide according to the proportion of 1:5 to 10 (w/v), the enzyme activity of neutral protease is 300 to 1000 units/ml, and the sheep skin and the cowhide treated by sodium hydroxide and the neutral protease are treated for 12 to 24 hours at the temperature of 30 to 40 ℃ according to the proportion of 1:1 to 5 (w/v). The extract was filtered through celite filter paper. And (3) regulating the pH to be about 7.0, precipitating the collagen by using sodium chloride with the final concentration of 2.5M, centrifuging for 7-30 minutes at the temperature of 4 ℃ and the rotating speed of 5000-12000 r/min, and collecting the precipitate to obtain the collagen. The recovery rate was calculated by weighing the collagen after freeze-drying.
Collagen recovery = dry collagen/dry collagen mass before collagen extraction x 100 dry collagen mass
The recovery rate of the sheepskin collagen is 10.22-18.83% and the recovery rate of the cowhide collagen is 30.12-50.85% according to the calculation of the formula.
The obtained collagen was redissolved in distilled water, and the SDS-PAGESDS-PAGE profile could intuitively determine the subunit composition and type of collagen, and the results are shown in FIG. 10. Collagen hydrolyzed by neutral protease of cow leather and sheep skin is composed of 4 chains, namely 2 different alpha peptide chains alpha 1 and alpha 2,1 alpha peptide chain dimer beta peptide chain and 1 alpha peptide chain trimer gamma peptide chain. Typical type I collagen is a trimer consisting of 2 α1 and 1 α2 chains, whereas the SDS-PAGE diagram of fig. 8 shows that the collagen extracted from the cowhide and sheep skin conforms to the peptide chain characteristics of typical type I collagen.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The neutral protease with high activity and tolerance is characterized in that the amino acid sequence of the neutral protease is shown as SEQ No. 2.
2. A gene HtpX capable of encoding the neutral protease having high activity and tolerance as claimed in claim 1, wherein the nucleotide sequence is shown in SEQ No. 1.
3. A recombinant vector comprising the gene HtpX according to claim 2.
4. A recombinant strain comprising the recombinant vector of claim 3.
5. The recombinant strain according to claim 4, wherein the recombinant strain is bacillus subtilis WB800N/pHT43-HtpX (Bacillus subtilis) with a preservation number of CCTCC No: m20232325.
6. The method for constructing a recombinant strain according to claim 4 or 5, comprising the steps of:
6A, providing a gene HtpX capable of encoding neutral protease having high activity and tolerance, an expression vector pHT43 and an expression strain;
6B, amplifying a neutral protease gene HtpX capable of encoding the neutral protease gene with high activity and tolerance, and connecting an amplified product with an expression vector pHT43 to obtain a recombinant expression vector pHT43-HtpX;
6C, integrating the recombinant expression vector gene into an expression strain to obtain a recombinant strain, wherein the preservation number is CCTCC No: bacillus subtilis WB800N/pHT43-HtpX (Bacillus su btilis) of M20232325;
in the step 6B, the whole genome DNA of the extracted mole cricket intestinal microorganisms is used as a template, and the amplification primers comprise a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ No.3, and the nucleotide sequence of the reverse primer is shown as SEQ No. 4.
7. A method for preparing a neutral protease having high activity and tolerance, characterized in that the method comprises the steps of:
(1) Culturing the recombinant strain of any one of claims 4 to 6, inducing expression of the gene HtpX encoding neutral protease;
(2) Separating and purifying the expressed neutral protease.
8. The method of preparing neutral protease with high activity and tolerance according to claim 7, wherein the step (1) comprises activating the recombinant strain, inoculating to a fermentation medium containing chloramphenicol, and culturing at 37deg.C and 180 rpm to OD 600 0.6 to 0.8, and isopropyl-beta-D-thiogalactoside with the final concentration of 1 to 4 millimoles per liter, and culturing for 24 to 36 hours at the temperature of 37 ℃ and the rotating speed of 150 to 220 revolutions per minute to induce the production of neutral proteinase.
9. Use of the neutral protease with high activity and tolerance according to claim 1, the gene HtpX capable of encoding the neutral protease with high activity and tolerance according to claim 2, the recombinant vector pHT43-HtpX according to claim 3, the recombinant strain according to claim 5 for degrading cow leather and/or sheep leather solid wastes.
10. The use according to claim 9, characterized in that the neutral protease has a reaction temperature of 45-55 ℃ and a pH = 6.0-8.0 in degrading cowhide and/or sheep skin solid waste.
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