CN114807091A - Thermomyces lanuginosus lipase with improved heat resistance and application thereof - Google Patents
Thermomyces lanuginosus lipase with improved heat resistance and application thereof Download PDFInfo
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- CN114807091A CN114807091A CN202210390426.7A CN202210390426A CN114807091A CN 114807091 A CN114807091 A CN 114807091A CN 202210390426 A CN202210390426 A CN 202210390426A CN 114807091 A CN114807091 A CN 114807091A
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- China
- Prior art keywords
- ala
- gly
- lipase
- thermomyces lanuginosus
- thr
- 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.)
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- 101000984201 Thermomyces lanuginosus Lipase Proteins 0.000 title claims abstract description 30
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 13
- 241000235058 Komagataella pastoris Species 0.000 claims abstract description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 4
- 241000894006 Bacteria Species 0.000 claims description 8
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 108090001060 Lipase Proteins 0.000 abstract description 33
- 102000004882 Lipase Human genes 0.000 abstract description 31
- 239000004367 Lipase Substances 0.000 abstract description 30
- 235000019421 lipase Nutrition 0.000 abstract description 30
- 102000004190 Enzymes Human genes 0.000 abstract description 13
- 108090000790 Enzymes Proteins 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 10
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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Abstract
The invention discloses a thermomyces lanuginosus lipase with improved heat resistance and application thereof, wherein the amino acid sequence of the thermomyces lanuginosus lipase is shown as SEQIDNO.2. The thermomyces lanuginosus lipase with improved heat resistance provided by the invention carries out site-directed mutation on the thermomyces lanuginosus lipase gene, and expresses the thermomyces lanuginosus lipase gene in a eukaryotic exogenous protein expression system pichia pastoris so as to improve the heat resistance of the thermomyces lanuginosus lipase. The experiment proves that the relative enzyme activity of the mutant R209H and lipase at high temperature is higher than that before mutation, R209H tolerates 90min at 50 ℃, the enzyme activity is 60.34 percent, while the wild type lipase is 53.68 percent, tolerates 90min at 70 ℃, the residual activity is 49.32 percent, and the wild type lipase is 44.75 percent.
Description
Technical Field
The invention relates to Thermomyces lanuginosus lipase, and in particular relates to Thermomyces lanuginosus lipase with improved heat resistance and application thereof.
Background
Lipase (EC 3.1.1.3) is triacylglycerol acyl hydrolase, and Lipase not only can catalyze natural substrate grease to hydrolyze to generate fatty acid, glycerol and monoglyceride or diester, but also can catalyze esterification, ester exchange, alcoholysis, acidolysis and other reactions. At present, lipase is widely applied to a plurality of fields of feed industry, food processing, detergents, textiles, paper making and the like. The lipase has wide sources and mainly exists in animals, plants and microorganisms, wherein the lipase from the microorganisms has various types, short period for industrially producing the lipase, low cost and wide temperature and pH action range, so the lipase from the microorganisms is an important source of the lipase for industrial use.
Nowadays, people pay attention to health problems of foods, and people are fattened due to the fact that the foods are high in fat content, so that a plurality of health problems are caused. Lipases can be used to catalyze the production of structured lipids, which are modified from natural fats or fatty acids and have lipids that can meet nutritional needs for consumption or use in medicine.
The lipase can also be used as a biocatalyst for transesterification, and can catalyze the transesterification of triglyceride and short-chain alcohol to generate long-chain fatty acid monoalkyl ester, namely biodiesel.
Moreover, lipases can catalyze the hydrolysis of fats (triglycerides) in animal cortex to glycerol and free fatty acids, which can remove fats from various leathers. Degreasing is the most important process in the leather-making process, the quality of leather is directly influenced by the degreasing degree, and lipase plays an important role in the leather-making process.
In addition, in the paper industry, resin in the paper making process is an important factor influencing the paper quality, the main components of the resin are triglyceride glycerol and wax, and lipase is used in the paper industry to remove the resin in the paper pulp and improve the paper quality.
Thermomyces lanuginosus is a fungus that is widely present in nature and has a high upper limit on growth temperature. It produces lipases which have good thermostability compared to lipases of other microbial origin, but which still do not meet the ever more stringent industrial requirements.
Therefore, increasing the thermostability of Thermomyces lanuginosus lipase would allow the Thermomyces lanuginosus lipase to be used more widely in industry.
Disclosure of Invention
The invention aims to provide Thermomyces lanuginosus lipase with improved heat resistance and application thereof, solves the problem of heat resistance of Thermomyces lanuginosus, and can improve the heat resistance of Thermomyces lanuginosus.
In order to achieve the above object, the present invention provides a Thermomyces lanuginosus lipase with improved thermostability, the amino acid sequence of the Thermomyces lanuginosus lipase is shown in SEQ ID NO. 2.
The invention determines that the mutation site is that the 209 th arginine is changed into histidine by analyzing and simulating the protein sequence of the Thermomyces lanuginosus lipase. A new lipase gene is obtained by a site-directed mutagenesis method by taking a Thermomyces lanuginosus lipase gene (SEQ ID NO.3, the amino acid sequence of the Thermomyces lanuginosus lipase is shown as SEQ ID NO. 1) as a template, the mutated gene and a pPIC9K vector are combined to construct a recombinant plasmid, the recombinant plasmid is transferred into a corresponding host bacterium GS115 for heterologous expression, a transformant is selected for fermentation, and the heat resistance of the mutated site is improved.
Another purpose of the invention is to provide the encoding gene of the Thermomyces lanuginosus lipase with improved heat resistance, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 4.
Another object of the present invention is to provide a vector containing the encoding gene.
Preferably, the vector is selected from pPIC9K, pPIC9, pPICZaA \ B \ C, pPICZA \ B \ C or PGAPZaA \ B \ C.
The invention also aims to provide an engineering bacterium containing the carrier.
Preferably, the bacterium is Pichia pastoris.
The thermolysin gossypii with improved heat resistance and the application thereof,
solves the heat resistance problem of the thermomyces lanuginosus and has the following advantages:
the thermomyces lanuginosus lipase with improved heat resistance provided by the invention carries out site-directed mutation on the thermomyces lanuginosus lipase gene, and expresses the thermomyces lanuginosus lipase gene in a eukaryotic exogenous protein expression system pichia pastoris so as to improve the heat resistance of the thermomyces lanuginosus lipase. The experiment proves that the relative enzyme activity of the mutant R209H and lipase at high temperature is higher than that before mutation, R209H tolerates 90min at 50 ℃, the enzyme activity is 60.34 percent, while the wild type lipase is 53.68 percent, tolerates 90min at 70 ℃, the residual activity is 49.32 percent, and the wild type lipase is 44.75 percent. The Thermomyces lanuginosus lipase with improved heat resistance can be applied to the catalytic production of structured lipids, as a biocatalyst for transesterification, degreasing and paper industry.
Drawings
FIG. 1 is a graph showing the optimum temperature profile of Experimental example 2 of the present invention.
FIG. 2 is a graph showing the tolerance curve at 50 ℃ in Experimental example 3 of the present invention.
FIG. 3 is a graph showing the tolerance of Experimental example 3 of the present invention at 70 ℃.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The experimental materials used in the following experiments are as follows:
1. expression of host bacteria: pichia pastoris GS115, available from Invitrogen corporation (USA);
2. carrier: pPIC9K, available from Invitrogen corporation (USA);
3. cloning host bacteria: DMT competent cells, purchased from chromotago;
4. the main reagents are as follows: site-directed Mutagenesis kit Fast Mutagenesis System (Beijing Quanji corporation); plasmid Extraction Kit Plasmid Mini Kit I and Gel Extraction Kit (Omega company, USA); GoldView nucleic acid dye (Bekkaike, Beijing); restriction endonucleases EcoR I, Not I (TaKaRa Co.); p-nitrophenol palmitate pNPP (Sigma);
5. an experimental instrument: ETC811PCR instrument (Touchen, Beijing); PowerB nucleic acid electrophoresis apparatus (beijing kaiyuan jingi); universal Hood II gel imager (Bio-Red company); mini P-4 protein gel electrophoresis apparatus (Beijing Kaiyun Xinrui Co.); SpectraMax Plus384 enzyme-labeled analyzer (Molecular Devices, Inc.); legend micro21 high speed centrifuge (Thermo corporation, USA); HWS12 constant temperature water bath (shanghai henkoke); TS-211B constant temperature speed regulation shaking table (Shanghai Tian Xia company); MK-20 metal baths (TOMOS Co.).
6. Main culture Medium
LB liquid medium: 2g of tryptone, 1g of yeast extract and 2g of sodium chloride, and diluting the mixture to a constant volume of 200mL by using double distilled water;
ampicillin-containing LB solid medium: 2g tryptone, 1g yeast extract, 2g sodium chloride and 2% (w/v) agar, adding 1 per mill of ampicillin which is sterilized by filtration through a 0.22 mu m filter membrane at about 45 ℃ after sterilizing by using double distilled water until the volume is 200mL and the temperature is 121 ℃ for 30min, and uniformly mixing for use.
Experimental example 1 preparation of lipase mutant
(1) The TLL-pPIC9K (pPIC 9K plasmid with the target sequence SEQ ID NO.3 inserted) plasmid was extracted, and site-directed mutagenesis amplification was performed using the TLL-pPIC9K recombinant plasmid as a template and the following upstream primers using a site-directed mutagenesis kit.
The PCR reaction system is as follows:
the nucleotide sequence of Forward-R209H is as follows (SEQ ID NO. 5):
CCTAGACTCCCTCCACACGAGTTCGGTTA；
the nucleotide sequence of Reverse-R209H is as follows (SEQ ID NO. 6):
TGTGGAGGGAGTCTAGGGACAATATCATTGG。
the PCR reaction parameters are as follows: pre-denaturation at 94 ℃ for 5 min; then denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 1.5min, and heat preservation at 72 ℃ for 10min after 30 cycles.
(2) Transformation of mutant plasmids
After amplification, DMT competent cells were thawed on ice for 5min, 5. mu.L of the digested product (after PCR amplification, the product after degradation of the methylated plasmid template by DMT) was added to 50. mu.L of DMT competent cells and mixed well, ice-washed for about 30min, then heat-shocked in a 42 ℃ molecular water bath for 45 sec, ice-washed for 5min, 500. mu.L of LB medium was added to a clean bench, incubated for 1h in a shaker at 180rpm and 37 ℃, then centrifuged for 5min at 8000rmp of a centrifuge, and finally 200. mu.L of the bacterial solution was spread on a kan + resistant LB dish and cultured overnight in a 37 ℃ incubator.
(3) Screening and identification
Randomly picking a single colony on the plate in the step (2) in the next morning for positive clone verification, sequencing and comparing positive bacteria, comparing a sequencing result with a template sequence (a non-mutated Thermomyces lanuginosus lipase gene sequence), and determining whether mutation is successful.
(4) Electric converter
Inoculating the strain with successful sequencing into a liquid culture medium of LB for culturing, extracting plasmids in the morning next day, performing linearization treatment by using restriction nuclease, adding absolute ethyl alcohol and sodium acetate for settling overnight after linearization treatment, and transferring the recombinant vector into pichia pastoris by electric shock in the next day to obtain a pichia pastoris recombinant strain transformant.
And (3) fermenting the recombinant strain transformant to obtain fermentation liquor, and measuring the lipase activity.
Experimental example 2 measurement of optimum temperature of Lipase
According to the determination method of lipase activity, under the condition of optimum pH, reaction systems (Tris-HCL buffer solution, p-nitrophenylpalmitate substrate, enzyme solution, SDS stop solution and sodium carbonate developing solution) are placed at different temperatures of 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃ and 80 ℃ for reaction.
As shown in FIG. 1, the optimum temperatures for the enzymatic reactions of lipases are 45 ℃ for all Thermomyces lanuginosus lipase R209H, and the mutants are improved by 5 ℃ compared to the optimum temperature for lipases.
Experimental example 3 temperature tolerance assay for Lipase
The enzyme solution was diluted to the corresponding fold (10 fold) and then placed at different temperatures: tolerance is carried out for 1min, 5min, 10min, 15min, 20min, 25min, 30min, 45min, 60min, 75min and 90min at 50 ℃; and tolerance is carried out at 70 deg.C for 1min, 5min, 10min, 15min, 20min, 25min, 30min, 35min, 45min, 60min, 75min, 90 min; then according to the lipase activity determination method, the reaction is carried out at the optimum pH value and the temperature of 37 ℃.
The temperature tolerance of the lipase at high temperature is shown in fig. 2 and fig. 3, the relative enzyme activity is continuously reduced along with the increase of temperature, the relative enzyme activity is gradually reduced along with the increase of time, R209H is tolerant for 90min at 50 ℃, the enzyme activity is 60.34 percent, the wild-type lipase is 53.68 percent, the residual half time of the relative enzyme activity after mutation at 70 ℃ is approximately tolerant for 90min, and the tolerance before mutation is 75min, so that the half-life is reached.
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.
<110> university of Yunnan Master
<120> Thermomyces lanuginosus lipase with improved heat resistance and application thereof
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Ala Thr Pro Leu Thr Ser Pro Gly Ala Ser Gly Val Gly Ala Val Thr
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Gly Pro Leu Ala Leu Ala Ala Thr Ala Leu Leu Ile Val Leu Ser Pro
65 70 75 80
Ala Gly Ser Ala Ser Ile Gly Ala Thr Ile Gly Ala Leu Ala Pro Ala
85 90 95
Leu Leu Gly Ile Ala Ala Ile Cys Ser Gly Cys Ala Gly His Ala Gly
100 105 110
Pro Thr Ser Ser Thr Ala Ser Val Ala Ala Thr Leu Ala Gly Leu Val
115 120 125
Gly Ala Ala Val Ala Gly His Pro Ala Thr Ala Val Val Pro Thr Gly
130 135 140
His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Ala Leu Ala
145 150 155 160
Gly Ala Gly Thr Ala Ile Ala Val Pro Ser Thr Gly Ala Pro Ala Val
165 170 175
Gly Ala Ala Ala Pro Ala Gly Pro Leu Thr Val Gly Thr Gly Gly Thr
180 185 190
Leu Thr Ala Ile Thr His Thr Ala Ala Ile Val Pro Ala Leu Pro Pro
195 200 205
Ala Gly Pro Gly Thr Ser His Ser Ser Pro Gly Thr Thr Ile Leu Ser
210 215 220
Gly Thr Leu Val Pro Val Thr Ala Ala Ala Ile Val Leu Ile Gly Gly
225 230 235 240
Ile Ala Ala Thr Gly Gly Ala Ala Gly Pro Ala Ile Pro Ala Ile Pro
245 250 255
Ala His Leu Thr Thr Pro Gly Leu Ile Gly Thr Cys Leu
260 265
<210> 2
<211> 269
<212> PRT
<213> Artificial sequence (Lipase mutant)
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Gly Val Ser Gly Ala Leu Pro Ala Gly Pro Ala Leu Pro Ala Gly Thr
1 5 10 15
Ser Ala Ala Ala Thr Cys Gly Leu Ala Ala Ala Ala Pro Ala Gly Thr
20 25 30
Ala Ile Thr Cys Thr Gly Ala Ala Cys Pro Gly Val Gly Leu Ala Ala
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Ala Thr Pro Leu Thr Ser Pro Gly Ala Ser Gly Val Gly Ala Val Thr
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Gly Pro Leu Ala Leu Ala Ala Thr Ala Leu Leu Ile Val Leu Ser Pro
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Ala Gly Ser Ala Ser Ile Gly Ala Thr Ile Gly Ala Leu Ala Pro Ala
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100 105 110
Pro Thr Ser Ser Thr Ala Ser Val Ala Ala Thr Leu Ala Gly Leu Val
115 120 125
Gly Ala Ala Val Ala Gly His Pro Ala Thr Ala Val Val Pro Thr Gly
130 135 140
His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Ala Leu Ala
145 150 155 160
Gly Ala Gly Thr Ala Ile Ala Val Pro Ser Thr Gly Ala Pro Ala Val
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gccgatacgt taagacagaa ggtggaggat gctgtgagag agcatccaga ctatagagtg 420
gtgtttaccg gacatagctt gggtggtgca ttggcaactg ttgccggagc agacctgaga 480
ggaaatggtt atgatatcga cgtgttttca tatggagccc ctagagtcgg aaacagagct 540
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<213> Artificial sequence (Coding gene)
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tgcccagagg tagagaaggc cgatgcaacg tttctctact cgtttgaaga ctctggagtg 180
ggagatgtca ccggattcct tgctctcgac aacacgaaca aattgatcgt cctctctttc 240
agaggatcta gatccattga gaactggatc ggaaatctta acttcgactt gaaagagatc 300
aatgacattt gctccggata aggaggacat gacggtttca cttcgtcctg gagatctgta 360
gccgatacgt taagacagaa ggtggaggat gctgtgagag agcatccaga ctatagagtg 420
gtgtttaccg gacatagctt gggtggtgca ttggcaactg ttgccggagc agacctgaga 480
ggaaatggtt atgatatcga cgtgttttca tatggagccc ctagagtcgg aaacagagct 540
tttgcagagt tcctgaccgt acagaccgga ggaacactct acagaattac ccacaccaat 600
gatattgtcc ctagactccc tccacacgag ttcggttaca gacattctag cccagagtac 660
tggatcaaat ctggaaccct tgtcccagtc accagaaacg atatcgtgaa gattgaagga 720
atcgatgcca ccggaggaaa caaccagcct aacattcctg atatccctgc ccacctatgg 780
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Claims (6)
1. Thermomyces lanuginosus lipase with improved heat resistance is characterized in that the amino acid sequence of the Thermomyces lanuginosus lipase is shown as SEQ ID No. 2.
2. The gene encoding Thermomyces lanuginosus lipase with improved thermostability according to claim 1, wherein the nucleotide sequence of the encoding gene is represented by SEQ ID No. 4.
3. A vector comprising the gene according to claim 2.
4. The vector of claim 3, wherein the vector is selected from the group consisting of pPIC9K, pPIC9, pPICZaA \ B \ C, pPICZA \ B \ C and PGAPZaA \ B \ C.
5. An engineered bacterium comprising the vector of claim 3 or 4.
6. The engineering bacterium of claim 5, wherein Pichia pastoris is selected for use.
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| CN115927250A (en) * | 2022-08-26 | 2023-04-07 | 云南师范大学 | Thermomyces lanuginosus lipase mutant with 256-site mutation and application thereof |
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| WO2009158343A1 (en) * | 2008-06-23 | 2009-12-30 | Teva Pharmaceutical Industries, Ltd. | Stereoselective enzymatic synthesis of (s) or (r)-iso-butyl-glutaric ester |
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