CN117947012A - Algin lyase mutant with high activity and high thermal stability and application thereof - Google Patents
Algin lyase mutant with high activity and high thermal stability and application thereof Download PDFInfo
- Publication number
- CN117947012A CN117947012A CN202410207807.6A CN202410207807A CN117947012A CN 117947012 A CN117947012 A CN 117947012A CN 202410207807 A CN202410207807 A CN 202410207807A CN 117947012 A CN117947012 A CN 117947012A
- Authority
- CN
- China
- Prior art keywords
- alya
- algin lyase
- algin
- mutant
- lyase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 235000010443 alginic acid Nutrition 0.000 title claims abstract description 89
- 229920000615 alginic acid Polymers 0.000 title claims abstract description 89
- 108090000856 Lyases Proteins 0.000 title claims abstract description 81
- 102000004317 Lyases Human genes 0.000 title claims abstract description 76
- 230000000694 effects Effects 0.000 title abstract description 33
- 108090000623 proteins and genes Proteins 0.000 claims description 25
- 101710190981 50S ribosomal protein L6 Proteins 0.000 claims description 21
- 102000004169 proteins and genes Human genes 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 15
- 238000000855 fermentation Methods 0.000 claims description 14
- 230000004151 fermentation Effects 0.000 claims description 14
- 241000194108 Bacillus licheniformis Species 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 8
- 230000001131 transforming effect Effects 0.000 claims description 8
- 239000013604 expression vector Substances 0.000 claims description 6
- 229940072056 alginate Drugs 0.000 claims description 5
- -1 alginate oligosaccharides Chemical class 0.000 claims description 5
- 244000005700 microbiome Species 0.000 claims description 5
- 229920001542 oligosaccharide Polymers 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 abstract description 33
- 108090000790 Enzymes Proteins 0.000 abstract description 33
- 150000001413 amino acids Chemical group 0.000 abstract description 22
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 238000010438 heat treatment Methods 0.000 abstract description 2
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 2
- 239000013612 plasmid Substances 0.000 description 30
- 239000013598 vector Substances 0.000 description 27
- 230000035772 mutation Effects 0.000 description 23
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 20
- 238000012258 culturing Methods 0.000 description 20
- 239000001888 Peptone Substances 0.000 description 15
- 108010080698 Peptones Proteins 0.000 description 15
- 239000007788 liquid Substances 0.000 description 15
- 235000019319 peptone Nutrition 0.000 description 15
- 108010076504 Protein Sorting Signals Proteins 0.000 description 11
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 10
- 240000008042 Zea mays Species 0.000 description 10
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 10
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 10
- 235000005822 corn Nutrition 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 239000000843 powder Substances 0.000 description 10
- 238000012216 screening Methods 0.000 description 10
- 239000011780 sodium chloride Substances 0.000 description 10
- 238000003259 recombinant expression Methods 0.000 description 9
- 125000003275 alpha amino acid group Chemical group 0.000 description 6
- 238000003776 cleavage reaction Methods 0.000 description 6
- 230000007017 scission Effects 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 239000004382 Amylase Substances 0.000 description 5
- 102000013142 Amylases Human genes 0.000 description 5
- 108010065511 Amylases Proteins 0.000 description 5
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 244000068988 Glycine max Species 0.000 description 5
- 235000010469 Glycine max Nutrition 0.000 description 5
- 102000003960 Ligases Human genes 0.000 description 5
- 108090000364 Ligases Proteins 0.000 description 5
- 239000004098 Tetracycline Substances 0.000 description 5
- 238000000246 agarose gel electrophoresis Methods 0.000 description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 5
- 235000011130 ammonium sulphate Nutrition 0.000 description 5
- 235000019418 amylase Nutrition 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 210000000988 bone and bone Anatomy 0.000 description 5
- 239000001110 calcium chloride Substances 0.000 description 5
- 229910001628 calcium chloride Inorganic materials 0.000 description 5
- 238000007865 diluting Methods 0.000 description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 5
- 238000004520 electroporation Methods 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 238000002744 homologous recombination Methods 0.000 description 5
- 230000006801 homologous recombination Effects 0.000 description 5
- 239000002054 inoculum Substances 0.000 description 5
- 230000001404 mediated effect Effects 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 229960002180 tetracycline Drugs 0.000 description 5
- 229930101283 tetracycline Natural products 0.000 description 5
- 235000019364 tetracycline Nutrition 0.000 description 5
- 150000003522 tetracyclines Chemical class 0.000 description 5
- 238000012795 verification Methods 0.000 description 5
- 241000589565 Flavobacterium Species 0.000 description 3
- 108010004131 poly(beta-D-mannuronate) lyase Proteins 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 102200157180 rs7997 Human genes 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241001474374 Blennius Species 0.000 description 1
- 201000004569 Blindness Diseases 0.000 description 1
- 102000010911 Enzyme Precursors Human genes 0.000 description 1
- 108010062466 Enzyme Precursors Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108091058545 Secretory proteins Proteins 0.000 description 1
- 102000040739 Secretory proteins Human genes 0.000 description 1
- 238000007068 beta-elimination reaction Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 108091022901 polysaccharide lyase Proteins 0.000 description 1
- 102000020244 polysaccharide lyase Human genes 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 244000000000 soil microbiome Species 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Abstract
The invention belongs to the technical field of enzyme genetic engineering, and discloses a high-activity high-thermal-stability algin lyase mutant and application thereof. The invention aims at wild type algin lyase, and the amino acids at positions 76, 120, 155 and 263 are respectively replaced by S, T, L, E, so that the enzyme activity of the obtained algin lyase mutant is improved by 87.41 percent, the enzyme activity is 75.54 percent compared with the rest enzyme activity after heat treatment of 30min at 60 ℃, the enzyme activity is obviously superior to that of the wild type algin lyase, and a theoretical basis is provided for the large-scale production and application of the algin lyase.
Description
Technical Field
The invention belongs to the technical field of enzyme genetic engineering, and particularly relates to a high-activity high-thermal-stability algin lyase mutant and application thereof.
Background
Algin lyase is a polysaccharide lyase, which can specifically cleave the marine polysaccharide algin by a beta-elimination mechanism to obtain alginate oligosaccharides (Alginate oligosaccharides, AOS). Along with the deep research of algin lyase, the development and the utilization of the algin lyase are more extensive, the algin lyase not only can efficiently degrade algin, but also can be used in the fields of fermentation production of ethanol, seaweed waste treatment, medical treatment and the like, and is an indispensable tool enzyme in the development and the utilization of ocean resources. Algin lyase is mainly derived from marine bacteria, soil bacteria, fungi and other microorganisms. A plurality of strains for producing algin lyase are screened from the nature, the enzyme yield is low, the enzyme activity is low, and the application of the enzyme is limited. Therefore, the algin lyase with excellent performance is further excavated, the catalytic performance of the algin lyase is improved, and the algin lyase is an important way for expanding the application of the algin lyase.
In order to make the discovered algin lyase better applied to industrial production and meet the requirements of high catalytic efficiency and high heat resistance required by industrial production, more and more researches are being conducted on molecular modification of the algin lyase, such as site-directed mutagenesis, site-directed saturation mutagenesis, assembly and truncation of multi-domain enzyme, and rational mutagenesis under computer guidance, etc., which stabilize proteins by introducing favorable biophysical properties at proper positions of protein structures, and are reasonably designed to overcome their own defects. The rational mutation under the guidance of a computer avoids blindness of residue mutation, has the advantages of simplicity, high efficiency, wide applicability and the like, and can be used for precise directional design and optimization of biological macromolecules such as proteins and the like.
Disclosure of Invention
The invention aims to provide an algin lyase mutant with high activity and high thermal stability, wherein the amino acid sequence of the algin lyase is shown as SEQ ID NO. 17.
The invention also aims to provide the application of the alginate lyase mutant with high activity and high thermal stability in the preparation of the alginate oligosaccharides.
In order to achieve the above purpose, the invention adopts the following technical scheme:
Algin lyase alyA derived from Flavobacterium (encoded protein is shown as SEQ ID NO. 1), the applicant replaces original signal peptide with signal peptide SPsacC (encoded protein is shown as SEQ ID NO. 2) which is highly secreted in Bacillus licheniformis to obtain algin lyase with replaced signal peptide, and further, amino acids 76, 120, 155 and 263 of the algin lyase are replaced with S, T, L, E respectively to obtain algin lyase four mutant T76S-K120T-Y155L-Q263E (shown as SEQ ID NO. 17).
The protection scope of the invention also comprises:
A gene encoding a protein represented by SEQ ID NO. 17;
an expression vector containing the above gene;
expressing a recombinant microorganism comprising the protein shown in SEQ ID NO. 17;
The application of the substances in preparing algin lyase;
The application of the substances in preparing brown alginate oligosaccharides.
The preparation method of the algin lyase four mutant comprises the following steps: inserting the gene encoding the protein shown in SEQ ID NO.17 into an expression vector, and transforming into bacillus licheniformis BL10 for fermentation expression.
Compared with the prior art, the invention has the following advantages:
The wild algin lyase has poor thermal stability, especially when the temperature exceeds 60 ℃, enzyme molecules are rapidly deactivated, which is unfavorable for production under a high-temperature environment process, and is difficult to produce on a large scale. According to the invention, molecular transformation is carried out on the algin lyase, and through predicting the evolution site of the algin lyase, single site amino acid is replaced by amino acid which promotes the algin lyase to have a more stable structure, so that the enzyme activity of the algin lyase is improved to a certain extent. The mutant strain constructed by the invention effectively promotes the high-efficiency expression of the algin lyase and obtains a high-temperature-resistant algin lyase mutant, thereby providing a theoretical basis for the large-scale production and application of the algin lyase.
Detailed Description
The technical content of the present invention is further described below with reference to examples, but the present invention is not limited to these examples, and the scope of the present invention is not limited to the following examples, and the technical solutions of the present invention are all conventional in the art unless specifically described otherwise; the reagents or materials, unless otherwise specified, are commercially available. The high-activity high-thermal stability algin lyase mutant can be obtained by adopting a direct synthesis mode or by constructing engineering strains of the high-activity high-thermal stability algin lyase mutant and fermenting.
Example 1:
Expression of the amino acid mutant at position 76 of the original sequence of algin lyase:
The algin lyase alyA (the coded protein is shown as SEQ ID NO. 1) from flavobacterium is used as a template, the original signal peptide is replaced by a signal peptide SPsacC (the coded protein is shown as SEQ ID NO. 2) suitable for efficient secretion of protein in bacillus licheniformis, and the 76 th amino acid of the original amino acid sequence is changed from T to S, Q or Y to obtain mutants T76S (SEQ ID NO. 3), T76Q (SEQ ID NO. 4) and T76Y (SEQ ID NO. 5) of 3 algin lyase.
In the present invention, the above three mutants were obtained in the following manner:
An algin lyase gene alyA which is mediated by a PPrbs + ykza promoter and is terminated by an amylase terminator TamyL is inserted between XbaI and EcoRI enzyme cleavage sites of a vector pHY300PLK, the sequence of the gene alyA plus the promoter and the terminator is shown as SEQ ID NO.18, the obtained vector is pHY-alyA, and a mutant skeleton is amplified by using a plasmid pHY-alyA as a template and using a designed mutation primer.
The primers for the mutation vector pHY-alyA-T76S are as follows:
T76S-KF：GGAGTAACCACGGCTAATAGTCATTATTCTCGTTCCGAG
T76S-KR：CTCGGAACGAGAATAATGACTATTAGCCGTGGTTACTCC
the primers for the mutation vector pHY-alyA-T76Q are as follows:
T76Q-KF：GGAGTAACCACGGCTAATCAACATTATTCTCGTTCCGAG
T76Q-KR：CTCGGAACGAGAATAATGTTGATTAGCCGTGGTTACTCC
the primers for the mutation vector pHY-alyA-T76Y are as follows:
T76Y-KF：GGAGTAACCACGGCTAATTACCATTATTCTCGTTCCGAG
T76Y-KR：CTCGGAACGAGAATAATGGTAATTAGCCGTGGTTACTCC
And (3) carrying out agarose gel electrophoresis on the PCR product, respectively recovering correct strips through a gel recovery kit, splicing two ends of the linear plasmid by using homologous recombination ligase, synchronously transforming the linear plasmid into escherichia coli, and screening positive strains through a resistance plate and PCR verification. Plasmid was then extracted and tested, and the correctly sequenced plasmids (pHY-alyA-T76S, pHY-alyA-T76Q and pHY-alyA-T76Y) and template plasmid (pHY-alyA) were transformed into Bacillus licheniformis BL10 (CN 104630123A, CCTCC NO: M2013400) by electroporation, respectively, and the algin lyase mutant recombinant expression strain BL10/alyA-T76S, BL/alyA-T76Q, BL10/alyA-T76Y and the control strain BL10/alyA were obtained by resistance plate and PCR screening.
Inoculating the recombinant expression strain and the control strain on a tetracycline resistance flat plate, culturing for 12 hours at 37 ℃, picking single colony, inoculating in 5mL of LB culture medium, culturing for 12 hours at 37 ℃ at 230rpm/min, inoculating in 1mL of bacterial liquid to 50mL of LB seed liquid, culturing for about 12 hours at 37 ℃ at 230rpm/min until OD 600 reaches 4.0-4.5, inoculating in 30mL of corn steep liquor culture medium at 3% of inoculum size, and culturing for 48 hours at 37 ℃ at 230 rpm/min. Taking 2mL of fermentation liquor, centrifuging at 12000rpm/min for 10min, taking supernatant, diluting to a proper multiple, and measuring enzyme activity by adopting a DNS method.
LB medium: peptone 10g/L, sodium chloride 10g/L, yeast powder 5g/L
Algin lyase fermentation medium: 10g/L of soybean peptone, 10g/L of bone peptone, 10g/L of corn steep liquor centrifugate, 15g/L of yeast powder, 3g/L of dipotassium hydrogen phosphate, 6g/L of ammonium sulfate, 20g/L of glucose, 10g/L of sodium chloride and 0.66g/L of calcium chloride, regulating pH to 7.0 and 30mL of liquid per bottle
The enzyme activity of the algin lyase is detected by adopting a DNS method, and the experimental result is as follows:
from the table, the 76 th amino acid is changed from T to S, Q or Y, and the enzyme activity of the algin lyase is improved by at least 45.39%.
Example 2:
expression of the amino acid mutant at position 120 of the initial sequence of algin cleavage zymogen:
The algin lyase alyA from flavobacterium is used as a template, the original signal peptide is replaced by a signal peptide SPsacC (SEQ ID NO. 2) suitable for efficient secretory protein in bacillus licheniformis, and the 120 th amino acid of the original amino acid sequence is changed from K to T, A or I to obtain mutants K120T (SEQ ID NO. 6), K120A (SEQ ID NO. 7) and K120I (SEQ ID NO. 8) of the 3 algin lyase. The specific preparation method of the invention is as follows:
An algin lyase gene alyA which is mediated by a PPrbs + ykza promoter and terminated by an amylase terminator TamyL is inserted between XbaI and EcoRI cleavage sites of a vector pHY300PLK, the obtained vector is pHY-alyA, and a mutant skeleton is amplified by using a plasmid pHY-alyA as a template and using a designed mutation primer.
The primers for the mutation vector pHY-alyA-K120T are as follows:
K120T-KF：TCAAAAGAGCCGGATGGCACCTACAGCCGCGTCATTATC
K120T-KR：GATAATGACGCGGCTGTAGGTGCCATCCGGCTCTTTTGA
The primers for the mutation vector pHY-alyA-K120A were as follows:
K120A-KF：TCAAAAGAGCCGGATGGCGCATACAGCCGCGTCATTATC
K120A-KR：GATAATGACGCGGCTGTATGCGCCATCCGGCTCTTTTGA
the primers for the mutation vector pHY-alyA-K120I were as follows:
K120I-KF：TCAAAAGAGCCGGATGGCATTTACAGCCGCGTCATTATC
K120I-KR：GATAATGACGCGGCTGTAAATGCCATCCGGCTCTTTTGA
And (3) carrying out agarose gel electrophoresis on the PCR product, respectively recovering correct strips through a gel recovery kit, splicing two ends of the linear plasmid by using homologous recombination ligase, synchronously transforming the linear plasmid into escherichia coli, and screening positive strains through a resistance plate and PCR verification. The plasmids are extracted and tested, plasmids (pHY-alyA-K120T, pHY-alyA-K120A and pHY-alyA-K120I) and template plasmids (pHY-alyA) which are sequenced correctly are respectively transformed into bacillus licheniformis BL10 (CN 104630123A, CCTCC NO: M2013400) by electroporation, and the algin lyase mutant recombinant expression strain BL10/alyA-K120T, BL/alyA-K120A, BL10/alyA-K120I and the control strain BL10/alyA are obtained by resistance plate and PCR screening.
Inoculating the recombinant expression strain and the control strain on a tetracycline resistance flat plate, culturing for 12 hours at 37 ℃, picking single colony, inoculating in 5mL of LB culture medium, culturing for 12 hours at 37 ℃ at 230rpm/min, inoculating in 1mL of bacterial liquid to 50mL of LB seed liquid, culturing for about 12 hours at 37 ℃ at 230rpm/min until OD 600 reaches 4.0-4.5, inoculating in 30mL of corn steep liquor culture medium at 3% of inoculum size, and culturing for 48 hours at 37 ℃ at 230 rpm/min. Taking 2mL of fermentation liquor, centrifuging at 12000rpm/min for 10min, taking supernatant, diluting to a proper multiple, and measuring enzyme activity by adopting a DNS method.
LB medium: 10g/L of peptone, 10g/L of sodium chloride and 5g/L of yeast powder;
Algin lyase fermentation medium: 10g/L of soybean peptone, 10g/L of bone peptone, 10g/L of corn steep liquor centrifugate, 15g/L of yeast powder, 3g/L of dipotassium hydrogen phosphate, 6g/L of ammonium sulfate, 20g/L of glucose, 10g/L of sodium chloride and 0.66g/L of calcium chloride, and regulating the pH to 7.0 and the liquid volume per bottle to 30mL.
The enzyme activity of the algin lyase is detected by adopting a DNS method, and the experimental result is as follows:
From the table, the 120 th amino acid is changed from K to T, A or one of I, and the enzyme activity of algin lyase is improved by at least 47.51%.
Example 3:
expression of the amino acid mutant at position 155 of the original sequence of alginate lyase:
The algin lyase alyA from bacillus subtilis is used as a template, the original signal peptide is replaced by a signal peptide SPsacC (SEQ ID NO. 2) suitable for efficient secretion protein in bacillus licheniformis, the 155 th amino acid of the original amino acid sequence is changed from Y to one of L, W or V, and 3 algin lyase mutants Y155L (SEQ ID NO. 9), Y155W (SEQ ID NO. 10) and Y155V (SEQ ID NO. 11) are obtained. The specific preparation method of the invention is as follows:
An algin lyase gene alyA which is mediated by a PPrbs + ykza promoter and terminated by an amylase terminator TamyL is inserted between XbaI and EcoRI cleavage sites of a vector pHY300PLK, the obtained vector is pHY-alyA, and a mutant skeleton is amplified by using a plasmid pHY-alyA as a template and using a designed mutation primer.
The primers for the mutation vector pHY-alyA-Y155L were as follows:
Y155L-KF：CCGCCTATTTTGAAAGTCCTTTGGGATAAAGGAAAGATT
Y155L-KR：AATCTTTCCTTTATCCCAAAGGACTTTCAAAATAGGCGG
the primers for the mutation vector pHY-alyA-Y155W were as follows:
Y155W-KF：CCGCCTATTTTGAAAGTCTGGTGGGATAAAGGAAAGATT
Y155W-KR：AATCTTTCCTTTATCCCACCAGACTTTCAAAATAGGCGG
The primers for the mutation vector pHY-alyA-Y155V were as follows:
Y155V-KF：CCGCCTATTTTGAAAGTCGTTTGGGATAAAGGAAAGATT
Y155V-KR：AATCTTTCCTTTATCCCAAACGACTTTCAAAATAGGCGG
And (3) carrying out agarose gel electrophoresis on the PCR product, respectively recovering correct strips through a gel recovery kit, splicing two ends of the linear plasmid by using homologous recombination ligase, synchronously transforming the linear plasmid into escherichia coli, and screening positive strains through a resistance plate and PCR verification. Then extracting plasmid for measurement, respectively converting the plasmids (pHY-alyA-Y155L, pHY-alyA-Y155W and pHY-alyA-Y155V) and template plasmid (pHY-alyA) with correct sequence into bacillus licheniformis BL10 (CN 104630123A, CCTCC NO: M2013400) by electroporation method, and obtaining algin lyase mutant recombinant expression strain BL10/alyA-Y155L, BL/alyA-Y155W, BL10/alyA-Y155V and control strain BL10/alyA by resistance plate and PCR screening.
Inoculating the recombinant expression strain and the control strain on a tetracycline resistance flat plate, culturing for 12 hours at 37 ℃, picking single colony, inoculating in 5mL of LB culture medium, culturing for 12 hours at 37 ℃ at 230rpm/min, inoculating in 1mL of bacterial liquid to 50mL of LB seed liquid, culturing for about 12 hours at 37 ℃ at 230rpm/min until OD 600 reaches 4.0-4.5, inoculating in 30mL of corn steep liquor culture medium at 3% of inoculum size, and culturing for 48 hours at 37 ℃ at 230 rpm/min. Taking 2mL of fermentation liquor, centrifuging at 12000rpm/min for 10min, taking supernatant, diluting to a proper multiple, and measuring enzyme activity by adopting a DNS method.
LB medium: peptone 10g/L, sodium chloride 10g/L, yeast powder 5g/L
Algin lyase fermentation medium: 10g/L of soybean peptone, 10g/L of bone peptone, 10g/L of corn steep liquor centrifugate, 15g/L of yeast powder, 3g/L of dipotassium hydrogen phosphate, 6g/L of ammonium sulfate, 20g/L of glucose, 10g/L of sodium chloride and 0.66g/L of calcium chloride, and regulating the pH to 7.0 and the liquid volume per bottle to 30mL. The enzyme activity of the algin lyase is detected by adopting a DNS method, and the experimental result is as follows:
From the table, the enzyme activity of the algin lyase is improved by at least 47.27% by changing the 155 th amino acid from Y to one of L, W or V.
Example 4:
expression of amino acid mutant at 263 rd position of original sequence of alginate lyase:
The algin lyase alyA from bacillus subtilis is used as a template, the original signal peptide is replaced by a signal peptide SPsacC (SEQ ID NO. 2) suitable for efficient secretion protein in bacillus licheniformis, and the 263 rd amino acid of the original amino acid sequence is changed from Q to E, D or C to obtain mutants Q263E (SEQ ID NO. 12), Q263D (SEQ ID NO. 13) and Q263C (SEQ ID NO. 14) of 3 algin lyase. The specific preparation method of the invention is as follows:
An algin lyase gene alyA which is mediated by a PPrbs + ykza promoter and terminated by an amylase terminator TamyL is inserted between XbaI and EcoRI cleavage sites of a vector pHY300PLK, the obtained vector is pHY-alyA, and a mutant skeleton is amplified by using a plasmid pHY-alyA as a template and using a designed mutation primer.
The primers for the mutation vector pHY-alyA-Q263E were as follows:
Q263E-KF：GCAAAGGTAAAAATCTATTCTCTTGAAGTTACTCATTAA
Q263E-KR：TTAATGAGTAACTTCAAGAGAATAGATTTTTACCTTTGC
the primers for the mutation vector pHY-alyA-Q263D were as follows:
Q263D-KF：GCAAAGGTAAAAATCTATTCTCTTGACGTTACTCATTAA
Q263D-KR：TTAATGAGTAACGTCAAGAGAATAGATTTTTACCTTTGC
the primers for the mutation vector pHY-alyA-Q263C were as follows:
Q263C-KF：GCAAAGGTAAAAATCTATTCTCTTTGCGTTACTCATTAA
Q263C-KR：TTAATGAGTAACGCAAAGAGAATAGATTTTTACCTTTGC
And (3) carrying out agarose gel electrophoresis on the PCR product, respectively recovering correct strips through a gel recovery kit, splicing two ends of the linear plasmid by using homologous recombination ligase, synchronously transforming the linear plasmid into escherichia coli, and screening positive strains through a resistance plate and PCR verification. The plasmids were then extracted and tested, and the plasmids with correct sequencing (pHY-alyA-Q263E, pHY-alyA-Q263D and pHY-alyA-Q263C) and the template plasmid (pHY-alyA) were respectively transformed into Bacillus licheniformis BL10 (CN 104630123A, CCTCC NO: M2013400) by electroporation, and the algin lyase mutant recombinant expression strains BL10/alyA-Q263E, BL/alyA-Q263D, BL10/alyA-Q263C and the control strain BL10/alyA were obtained by resistance plate and PCR screening.
Inoculating the recombinant expression strain and the control strain on a tetracycline resistance flat plate, culturing for 12 hours at 37 ℃, picking single colony, inoculating in 5mL of LB culture medium, culturing for 12 hours at 37 ℃ at 230rpm/min, inoculating in 1mL of bacterial liquid to 50mL of LB seed liquid, culturing for about 12 hours at 37 ℃ at 230rpm/min until OD 600 reaches 4.0-4.5, inoculating in 30mL of corn steep liquor culture medium at 3% of inoculum size, and culturing for 48 hours at 37 ℃ at 230 rpm/min. Taking 2mL of fermentation liquor, centrifuging at 12000rpm/min for 10min, taking supernatant, diluting to a proper multiple, and measuring enzyme activity by adopting a DNS method.
LB medium: peptone 10g/L, sodium chloride 10g/L, yeast powder 5g/L
Algin lyase fermentation medium: 10g/L of soybean peptone, 10g/L of bone peptone, 10g/L of corn steep liquor centrifugate, 15g/L of yeast powder, 3g/L of dipotassium hydrogen phosphate, 6g/L of ammonium sulfate, 20g/L of glucose, 10g/L of sodium chloride and 0.66g/L of calcium chloride, and regulating the pH to 7.0 and the liquid volume per bottle to 30mL. The enzyme activity of the algin lyase is detected by adopting a DNS method, and the experimental result is as follows:
from the table, the 263 th amino acid is changed from Q to E, D or any one of C, and the enzyme activity of the algin lyase is improved by at least 42.28%.
Example 5:
expression of algin lyase four-site mutant:
The 120 th amino acid of the original amino acid sequence is changed from K to T by using a T76S (SEQ ID NO. 3) template, so as to construct an algin lyase double-site mutant vector T76S-K120T (SEQ ID NO. 15). And then taking T76S-K120T as a template, and changing the 155 th amino acid of the original amino acid from Y to L to obtain an algin lyase three-site mutant T76S-K120T-Y155L (SEQ ID NO. 16). Then taking T76S-K120T-Y155L as a template, and changing the 263 rd amino acid of the original amino acid from Q to E to obtain an algin lyase four-site mutant T76S-K120T-Y155L-Q263E (shown in SEQ ID NO. 17). The specific preparation method of the invention is as follows:
An algin lyase gene alyA which is mediated by a PPrbs + ykza promoter and terminated by an amylase terminator TamyL is inserted between XbaI and EcoRI cleavage sites of a vector pHY300PLK, the obtained vector is pHY-alyA, and a mutant skeleton is amplified by using a plasmid pHY-alyA as a template and using a designed mutation primer.
The primers for the mutation vector pHY-alyA-T76S are as follows:
T76S-KF：GGAGTAACCACGGCTAATAGTCATTATTCTCGTTCCGAG
T76S-KR：CTCGGAACGAGAATAATGACTATTAGCCGTGGTTACTCC
the primers for the mutation vector pHY-alyA-T76S-K120T are as follows:
T76S-K120T-KF：GGAGTAACCACGGCTAATAGTCATTATTCTCGTTCCGAG
T76S-K120T-KR：GATAATGACGCGGCTGTAGGTGCCATCCGGCTCTTTTGA
The primers for the mutation vector pHY-alyA-T76S-K120T-Y155L are as follows:
T76S-K120T-Y155L-KF：GGAGTAACCACGGCTAATAGTCATTATTCTCGTTCCGAG T76S-K120T-Y155L-KR：AATCTTTCCTTTATCCCAAAGGACTTTCAAAATAGGCGG The primers for the mutation vector pHY-alyA-T76S-K120T-Y155L-Q263E were as follows:
T76S-K120T-Y155L-Q263E-KF:GGAGTAACCACGGCTAATAGTCATTATTCTCGTTCC GAG
T76S-K120T-Y155L-Q263E-KR:TTAATGAGTAACTTCAAGAGAATAGATTTTTACCTT TGC
And (3) carrying out agarose gel electrophoresis on the PCR product, respectively recovering correct strips through a gel recovery kit, splicing two ends of the linear plasmid by using homologous recombination ligase, synchronously transforming the linear plasmid into escherichia coli, and screening positive strains through a resistance plate and PCR verification. Then extracting the plasmids for measurement, respectively transforming the plasmids (pHY-alyA-T76S-K120T-Y155L-Q263E) and the template plasmids (pHY-alyA) which are sequenced correctly into Bacillus licheniformis BL10 (CN 104630123A, CCTCC NO: M2013400) by an electroporation method, and obtaining the algin lyase four-site mutant recombinant strain BL10/alyA-T76S-K120T-Y155L-Q263E and the control strain BL10/alyA by a resistance plate and PCR screening. Inoculating the recombinant expression strain and the control strain on a tetracycline resistance flat plate, culturing for 12 hours at 37 ℃, picking single colony, inoculating in 5mL of LB culture medium, culturing for 12 hours at 37 ℃ at 230rpm/min, inoculating in 1mL of bacterial liquid to 50mL of LB seed liquid, culturing for about 12 hours at 37 ℃ at 230rpm/min until OD 600 reaches 4.0-4.5, inoculating in 30mL of corn steep liquor culture medium at 3% of inoculum size, and culturing for 48 hours at 37 ℃ at 230 rpm/min. Taking 2mL of fermentation liquor, centrifuging at 12000rpm/min for 10min, taking supernatant, diluting to a proper multiple, and measuring enzyme activity by adopting a DNS method.
In order to detect the overall thermal stability of the mutant, a proper amount of supernatant is taken out and placed on ice immediately after being treated in a water bath at 60 ℃ for 30min, the untreated and heat-treated fermentation liquor is diluted to corresponding multiples, and the enzyme activity of the algin lyase is detected by adopting a DNS method.
LB medium: 10g/L of peptone, 10g/L of sodium chloride and 5g/L of yeast powder;
Algin lyase fermentation medium: 10g/L of soybean peptone, 10g/L of bone peptone, 10g/L of corn steep liquor centrifugate, 15g/L of yeast powder, 3g/L of dipotassium hydrogen phosphate, 6g/L of ammonium sulfate, 20g/L of glucose, 10g/L of sodium chloride and 0.66g/L of calcium chloride, and regulating the pH to 7.0 and the liquid volume per bottle to 30mL.
The enzyme activity of the algin lyase is detected by adopting a DNS method, and the experimental result is as follows:
From the table, amino acids 76, 120, 155 and 263 are replaced by S, T, L, E respectively, the enzyme activity of the obtained algin lyase mutant is improved by 87.41 percent, the enzyme activity is 75.54 percent relative to the rest enzyme activity after heat treatment for 30 minutes at 60 ℃, and the enzyme activity, the heat stability and the half life of the algin lyase four-site mutant are obviously improved.
Claims (7)
1. And the artificially synthesized algin lyase four mutant is shown as SEQ ID NO. 17.
2. A gene encoding the protein shown in SEQ ID NO. 17.
3. An expression vector comprising the gene of claim 2.
4. A recombinant microorganism expressing a protein comprising the protein shown in SEQ ID No. 17.
5. Use of the mutant according to claim 1, the gene according to claim 2, the expression vector according to claim 3 or the recombinant microorganism according to claim 4 for the preparation of algin lyase.
6. Use of the mutant according to claim 1, the gene according to claim 2, the expression vector according to claim 3 or the recombinant microorganism according to claim 4 for the preparation of brown alginate oligosaccharides.
7. The method for preparing the algin lyase four mutant according to claim 1, comprising: inserting the gene encoding the protein shown in SEQ ID NO.17 into an expression vector, and transforming into bacillus licheniformis BL10 for fermentation expression.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410207807.6A CN117947012A (en) | 2024-02-26 | 2024-02-26 | Algin lyase mutant with high activity and high thermal stability and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410207807.6A CN117947012A (en) | 2024-02-26 | 2024-02-26 | Algin lyase mutant with high activity and high thermal stability and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117947012A true CN117947012A (en) | 2024-04-30 |
Family
ID=90801468
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410207807.6A Pending CN117947012A (en) | 2024-02-26 | 2024-02-26 | Algin lyase mutant with high activity and high thermal stability and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117947012A (en) |
-
2024
- 2024-02-26 CN CN202410207807.6A patent/CN117947012A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102206659B (en) | Method for enhancing activity of endoglucanase on basis of error-prone PCR (Sequential Error-Prone) technology | |
| CN106995811B (en) | A kind of algin catenase, preparation method and application | |
| CN105420154A (en) | Double knockout recombinant rhodococcus as well as construction method and application thereof | |
| CN112725319B (en) | Alginate lyase FaAly7 with polyG substrate specificity and application thereof | |
| CN112553227B (en) | Heat-resistant multifunctional glycoside hydrolase, and encoding gene and application thereof | |
| CN113151270A (en) | Promoter for efficiently expressing alkaline protease and application thereof | |
| CN111269907A (en) | Alginate lyase mutant based on loop region transformation and application thereof | |
| CN102260694A (en) | Acidproof medium-temperature alpha-amylase and preparation method thereof | |
| CN113073074B (en) | Genetically engineered bacterium for efficiently synthesizing riboflavin and application thereof | |
| CN112251428B (en) | Glutamic acid decarboxylase mutant and application thereof in production of gamma-aminobutyric acid | |
| CN100392075C (en) | Glutamine synthetase and its dedicated expression engineered bacteria and uses | |
| CN115948363B (en) | Tn5 transposase mutant and preparation method and application thereof | |
| CN115960875A (en) | Alginate lyase mutant enzyme with improved thermal stability | |
| CN108103049B (en) | Thermophilic L-asparaginase mutant and screening and fermenting method thereof | |
| CN117947012A (en) | Algin lyase mutant with high activity and high thermal stability and application thereof | |
| CN116064616A (en) | Cellulase gene, cellulase, recombinant vector and application | |
| CN115725520A (en) | Preparation method of glutathione synthetase and method for catalytically producing glutathione | |
| CN114395578A (en) | Preparation method and application of recombinant trehalase | |
| CN117925582A (en) | Nattokinase mutant with high activity and high thermal stability and application thereof | |
| CN104087604A (en) | Genetic expression sequence of inulin fructotransferase | |
| CN114752581B (en) | Alpha-galactosidase mutant and application thereof | |
| CN113736807B (en) | Cellobiohydrolase, and coding gene and application thereof | |
| CN110016480A (en) | A kind of bacterium flavine synthesis related gene and its application in the colourless xanthan gum engineering bacteria of building production | |
| CN111621486B (en) | Heat-resistant xylanase XYNB with high enzyme activity at low temperature, mutant gene, application and gene sequence preparation method | |
| CN110004130B (en) | Genetically engineered bacterium for improving thermal gel hydrolysis efficiency and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination |