CN106906198B - Cellulase for improving temperature resistance - Google Patents
Cellulase for improving temperature resistance Download PDFInfo
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- 108010059892 Cellulase Proteins 0.000 title claims abstract description 90
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Classifications
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- 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)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2445—Beta-glucosidase (3.2.1.21)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
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Abstract
The invention relates to a cellulase for improving temperature tolerance, wherein the amino acid sequence is a sequence of adding Cysteine (Cysteine) to the N end of the sequence number 2, and adding Glycine (Glycine) and Cysteine (Cysteine) or Proline (Proline) and Cysteine (Cysteine) to the C end.
Description
Technical Field
The invention relates to cellulase, in particular to cellulase for improving temperature resistance.
Background
Cellulose is one of the main components of plant cell walls and is also the source of the main biomass energy (bioglass) in the earth, so many enzyme proteins capable of effectively decomposing cellulose are widely used in different industries nowadays. Cellulose is a long-chain polysaccharide formed by bonding glucose with beta-1, 4-glycosidic bonds (beta-1, 4-glycosidic bonds), and the polysaccharides are organized together to form a compact crystalline cellulose so as to resist the decomposition of the outside. However, many herbivores and microorganisms in the biological world need to be able to use the polysaccharide cellulose in plant cell walls as a source of survival energy by breaking down the polysaccharide cellulose into glucose monosaccharides that can be absorbed by the body. The catalytic mechanism of cellulase is mainly hydrolysis of beta-1, 4-glycosidic bond connecting two monosaccharides by acid-base reaction, and further decomposition of polysaccharide cellulose. Cellulases can be divided into three basic groups, namely, endoglucanase (endo-glucanase), exoglucanase (cellobiohydrolase), and glucosidase (β -glucanase). Endoglucanases are capable of randomly cleaving long-chain cellulose into many small segments of oligosaccharides; the exoglucanase can be decomposed from the reducing end or the non-reducing end of the long-chain cellulose, and the main product is cellobiose; as for the glucosidase, cellobiose is decomposed into glucose which is a monosaccharide.
The cellulase has wide industrial application, whether in food, feed or textile industries, and even can be applied to the most concerned biomass energy sources at present. Aiming at different industrial applications, the cellulase also needs to meet different applicable conditions and ranges. For example: the feed industry is adapted to the more acidic and temperature tolerant enzyme proteins, whereas the textile industry is biased towards alkaline cellulases. Therefore, it is also an object of the present invention to find out enzyme proteins that are more industrially desirable, and which are more suitable for industrial purposes. In many related studies, existing enzyme proteins are modified in order to obtain better enzymes, in addition to being screened in nature. At present, there are two major modification strategies, one of which is random mutation or random arrangement of enzyme genes, and then screening enzyme proteins more conforming to their action conditions under specific action conditions. The strategy has the advantages that the structure or the action mechanism of the enzyme does not need to be deeply researched, and better enzyme protein can be randomly found out directly under specific conditions; however, the disadvantage is that it requires a lot of manpower and time to perform a lot of screening or a lot of screening methods are well suited. Another modification strategy is to search the structure and mechanism of action of the enzyme to find out the amino acids that are critical to the activity or property of the enzyme, and to mutate and test these specific amino acids, thereby obtaining a more functional modified enzyme protein. This advantage is achieved without the time and labor required to perform numerous mutagenesis and screening steps, but the protein structure and mechanism of action of the enzyme need to be known in advance to find out the specific amino acids with the potential for modification.
Different industrial processes require the matching and participation of enzyme proteins in accordance with their different environments of action. Even though cellulases have been widely used industrially for a long time, many industrial enzymes are selected from mesophilic bacteria such as Trichoderma reesei (Trichoderma reesei), and thus are inferior in heat resistance. On the other hand, heat-resistant cellulases can be effectively used in industries requiring high-temperature environments, including beer production, biomass energy industry, and the like. The protein with heat resistance has higher protein stability relatively, so the protein can exist stably in a high-temperature environment and even has better function. No matter in the working environment or in the post-treatment process, the enzyme protein itself is not damaged by high temperature. In addition, the activity of enzyme proteins is also a major focus of improving industrial enzymes, and higher enzyme activity represents a decrease in cost and an increase in profit.
According to literature studies, disulfide bonds contribute to stabilization of protein structure and improvement of heat resistance. Trichoderma reesei has many kinds of cellulases, among which Cel5A cellulase belonging to GH family 5, the Protein structure (ID 3QR3) of which was published in 2011, has four disulfide bond positions inside the structure at C16-C22, C92-C99, C232-C2683 and C273-C323, and thus has a higher dissolution temperature (Tm value), and further, the structure of Cel5A exhibits the structure type of α/β TIM barrel (Toni MLee, Mary F Farrow, France H Arnold, and Stephen L Mayo (2011) Protein Structure report, nov 27; 20(11): 5-40. 19310). In 2004, Simon R.Andrews et al found that Cellvibrio japonica xylanase protein CjXyn10A and CmXyn10B of Cellvibrio mixtus, which have a disulfide bond at the N-and C-termini, further stabilized the structure and improved the temperature resistance, and that CjXyn10A and CmXyn10B are also of alpha/beta TIMbarl type (Andrews S.R., Taylor E.J., Pell G.VincentF., Ducros V.M., Davies G.J., Lakey J.H., and Gilbert H.J., 2004 J.biol.Chem.Dem.24; 279(52 54369-79): 2004).
Therefore, the present invention intends to improve the temperature tolerance of cellulase by modifying genes to increase disulfide bond linkage of cellulase, thereby effectively increasing the industrial value of cellulase in industrial application.
Disclosure of Invention
The invention aims to modify the existing cellulase, and increase the disulfide bond bonding of the cellulase by utilizing structural analysis and point mutation technology so as to effectively improve the temperature resistance of the cellulase and further increase the industrial application value of the cellulase.
In order to achieve the above object, one of the broader embodiments of the present invention provides a cellulase having an amino acid sequence in which Cysteine (Cysteine) is added to the N-terminus of SEQ ID No. 2, and Glycine (Glycine) and Cysteine (Cysteine) or Proline (Proline) and Cysteine (Cysteine) are added to the C-terminus. Wherein the gene encoding the sequence number 2 is a gene isolated from Trichoderma reesei (Trichoderma reesei) and optimized.
In one embodiment, the amino acid sequence of the cellulase is the amino acid sequence of seq id No. 4.
In one embodiment, the amino acid sequence of the cellulase is the amino acid sequence of seq id No. 6.
In another broad aspect, the present invention provides a nucleic acid molecule encoding the cellulase and a recombinant plasmid comprising the nucleic acid molecule.
Drawings
FIG. 1 shows the nucleotide sequence and amino acid sequence of WT cellulase.
FIG. 2 shows the mutant primer sequence of primer one.
FIG. 3 shows the mutant primer sequence of primer two.
FIG. 4 shows the mutant primer sequence of primer three.
FIG. 5 shows the nucleotide sequence and amino acid sequence of the modified CGC cellulase.
Fig. 6 shows the nucleotide sequence as well as the amino acid sequence of the engineered CPC cellulase.
FIG. 7 shows the temperature tolerance analysis of two muteins, WT cellulase and CGC cellulase and CPC cellulase.
FIG. 8 shows SDS-PAGE electrophoretic analysis to assess disulfide bonding.
Detailed Description
Some exemplary embodiments that embody features and advantages of the invention will be described in detail in the description that follows. It is to be understood that the invention is capable of other embodiments and that various changes may be made therein without departing from the scope of the invention, and that the description and drawings are to be taken as illustrative and not restrictive in nature.
The cellulase adopted by the invention is a gene separated from a Trichoderma reesei (Trichoderma reesei) strain, and the N-terminal 91 amino acid sequence is optimized and removed to improve the protein expression capability of the cellulase. The gene is not mutated, so it is called wild-type cellulase (hereinafter referred to as WT cellulase). The WT cellulase is ligated to pPICZ alpha A vector at both ends by EcoRI and NotI restriction enzyme sites, and subjected to sequencing and protein expression. FIG. 1 shows the nucleotide sequence and amino acid sequence of WT cellulase, wherein the WT cellulase gene comprises 984 bases (including a stop codon, the nucleotide sequence is designated by SEQ ID NO: 1) and 327 amino acids (the amino acid sequence is designated by SEQ ID NO: 2).
Further, the structure was analyzed by PyMOL software, and it was found that the space distance between the N-terminus and the C-terminus of WT cellulase was aboutGreater than the distance at which disulfide bonds are formed. Therefore, the invention tries to add Cysteine (Cysteine) to the two ends of the N end and the C end respectively, and adds Glycine (Glycine) with smaller molecules or Proline (Proline) which enables a long chain to generate angle deflection in front of the Cysteine at the C end to reduce the space distance between the N end and the C end to generate disulfide bond connection, thereby stabilizing the N end and the C end of the protein and further improving the temperature resistance of the protein. In other words, the present invention performs two kinds of mutation transformation, wherein the first mutation transformation is to add Cysteine to the N-terminal of the WT cellulase and to add Glycine and Cysteine to the C-terminal, and the second mutation transformation is to add Cysteine to the N-terminal of the WT cellulase and to add Proline and Cysteine to the C-terminal. The modified protein comprises 330 amino acids, Cysteine at the N terminal is positioned at the 1 st position of an amino acid sequence, Glycine or Proline at the C terminal is positioned at the 329 th position of the amino acid sequence, Cysteine at the C terminal is positioned at the 330 th position of the amino acid sequence, so that the first mutation modification of the invention is represented by C1G329C330For short, CGC cellulase, and the second mutant modification is represented by C1P329C330, for short CPC cellulase.
The method for modifying cellulase and the modified cellulase obtained by the method of the invention are described in detail below.
The invention uses the point mutation technology to carry out mutation reconstruction. Firstly, after adding Cysteine to the N-terminal of cellulase by using a first primer (shown in figure 2), adding Glycine and Cysteine to the C-terminal of cellulase by using a second primer (shown in figure 3) to obtain a modified gene of C1G329C330(CGC), and mutating the 329 th amino acid Glycine point of the modified gene of C1G329C330 into Proline by using a third primer (shown in figure 4) to obtain the modified gene of C1P329C330 (CPC). FIG. 5 shows the nucleotide sequence and amino acid sequence of the modified CGC cellulase, wherein the CGC cellulase gene comprises 993 bases (containing a stop codon, the nucleotide sequence is shown in SEQ ID NO: 3) and 330 amino acids (the amino acid sequence is shown in SEQ ID NO: 4). Fig. 6 shows the nucleotide sequence and amino acid sequence of the CPC cellulase after modification, wherein, the CPC cellulase gene comprises 993 bases (containing a stop codon, the nucleotide sequence is marked with sequence number 5) and 330 amino acids (the amino acid sequence is marked with sequence number 6).
After linearization of the two mutant-modified DNA plasmids with the Pme I restriction enzyme, the plasmids were transferred by electroporation into the yeast Pichia pastoris X33, and the transformed bacterial suspension was applied to YPD plates containing 100. mu.g/ml zeocin antibiotic and incubated in an incubator at 30 ℃ for two days. Then selecting single colony to 5ml YPD to culture at 30 ℃, and then inoculating to 50ml BMGY to culture at 30 ℃ for one day; the cells were then replaced with 20ml BMMY for four days of induced protein expression. Sampling every 24 hours, adding 0.5% methanol, centrifuging the bacterial liquid at 3500rpm, collecting supernatant, and performing protein amount measurement and cellulase activity measurement.
The cellulase activity test method comprises mixing 0.2ml of 1% Carboxymethyl cellulose (CMC, pH 4.8,0.05M sodium citrate) with 0.2ml of cellulase protein solution (dilution buffer 0.05M sodium citrate; pH 4.8) with appropriate concentration, and mixingThen reacted at 50 ℃ for 15 minutes, followed by addition of 1.2ml of 1% DNS and boiling in 100 ℃ boiling water for 5 minutes to stop the reaction and color development, followed by cooling in cold water for 10 minutes at OD540The absorbance was measured by wavelength and converted to units of enzyme activity (unit). Wherein the standard curve of enzyme activity is established between 0-0.35. mu.g/ml of glucose standard solution and 1unit is defined as the amount of enzyme protein required to release 1. mu. mole of product per minute.
The temperature test is that cellulase protein solution (dilution buffer solution is 0.05M sodium citrate; pH 4.8) with proper concentration is placed at different temperatures for processing for 2 minutes, the cellulase protein solution is taken out and placed at 4 ℃ for cooling for 10 minutes, then placed at room temperature for warming for 10 minutes, then enzyme activity detection at 50 ℃ is carried out, and the relative residual activity percentage after heat treatment is respectively calculated by taking an enzyme protein sample without heat treatment as 100 percent control.
Fig. 7 shows the temperature tolerance analysis of the WT cellulase and both CGC cellulase and CPC cellulase muteins, wherein the cellulase activity of the non-heat-treated sample was set to 100%. From the results of FIG. 7, it is clear that the relative residual activities of CPC cellulase, and CGC cellulase were 94%, 70%, and 74%, and 93%, 68%, and 75%, respectively, which were much higher than those of WT cellulase, 66%, 35%, and 43%, respectively, after treatment at 75 ℃, 80 ℃, and 85 ℃ for 2 minutes. In other words, the residual activity of the two mutant proteins of CGC cellulase and CPC cellulase after being treated for 2 minutes at different temperatures is higher than that of the original protein of WT cellulase, so that the temperature resistance is higher, and the industrial application value with greater potential is also shown.
In another aspect, the present invention also provides an assay for assessing disulfide bonds in mutant proteins. The mutant proteins were preliminarily analyzed for disulfide linkage by adding 10mM Dithiothreitol (DTT) to appropriate concentrations of both the CGC cellulase and CPC cellulase mutant proteins and WT cellulase original protein solution, and performing 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
FIG. 8 shows SDS-PAGE electrophoretic analysis for evaluating disulfide bonds by cleaving disulfide bonds in protein structures using DTT and observing the speed of movement of proteins in the electrophoretic gel. From the results of fig. 8, it was found that the molecular weight and size of the CGC cellulase mutein after DTT addition are equivalent to those of the original WT cellulase protein to which DTT is added, while the position of the CGC cellulase mutein without DTT addition is much lower than that of the original WT cellulase protein to which DTT is not added, indicating that the CGC cellulase mutein generates more disulfide bonds to reduce the space of the protein molecules, and the moving speed in the electrophoretic colloid is faster than that of the original protein without disulfide bonds.
In summary, in order to increase the industrial application value of cellulase, the invention utilizes logical mutation design to generate disulfide bond stable protein structures at the N-terminal and the C-terminal to improve the temperature tolerance of cellulase. In the two mutation designs of the invention, the first is to add Cysteine to the N-terminal of the WT cellulase and to add Glycine and Cysteine to the C-terminal of the WT cellulase to obtain CGC cellulase; the second method is to add Cysteine to the N-terminal of the WT cellulase and to add Proline and Cysteine to the C-terminal to obtain the CPC cellulase. According to the temperature resistance test result, compared with the original protein of the WT cellulase, the two mutant proteins of the CGC cellulase and the CPC cellulase have better high temperature resistance, so that the CGC cellulase and the CPC cellulase can be more stable when exposed to temperature impact, the production cost can be reduced, and the industrial value of the cellulase in industrial application can be effectively increased.
While the present invention has been described in detail with respect to the above embodiments, it will be apparent to those skilled in the art that various modifications can be made without departing from the scope of the invention as defined in the appended claims.
Claims (5)
1. The amino acid sequence of the cellulase is the sequence of adding Cysteine (Cysteine) to the N terminal of SEQ ID NO. 2 and Glycine (Glycine) and Cysteine (Cysteine) to the C terminal.
2. The cellulase of claim 1, wherein the gene encoding the SEQ ID NO 2 is a gene isolated from Trichoderma reesei (Trichoderma reesei) and optimized.
3. The cellulase of claim 1, wherein the amino acid sequence of the cellulase is the amino acid sequence of SEQ ID NO. 4.
4. A nucleic acid molecule encoding the cellulase of claim 1.
5. A recombinant plasmid comprising the nucleic acid molecule of claim 4.
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| CN102676476A (en) * | 2011-03-09 | 2012-09-19 | 东莞泛亚太生物科技有限公司 | Dextranase with improved enzyme activity and thermal stability |
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| CN102676476A (en) * | 2011-03-09 | 2012-09-19 | 东莞泛亚太生物科技有限公司 | Dextranase with improved enzyme activity and thermal stability |
| US20140308713A1 (en) * | 2013-04-05 | 2014-10-16 | Toni M. Lee | Endoglucanase having enhanced thermostability and activity |
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