CN114958805B - Feruloyl esterase and mutant N.9-98 thereof and application - Google Patents
Feruloyl esterase and mutant N.9-98 thereof and application Download PDFInfo
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Abstract
The invention discloses a ferulic acid esterase, a mutant N.9-98 thereof and application. The ferulic acid esterase has an amino acid sequence shown as SEQ ID No.3, has stable property, the reaction pH is 6-8, and the reaction temperature is 37-54 ℃. On the basis of the amino acid sequence of the ferulic acid esterase, the invention respectively obtains the ferulic acid esterase mutant N.9-98 by increasing the number of disulfide bonds in the ferulic acid esterase, changing the side chain group of a carbohydrate binding module of the ferulic acid esterase and changing the charge of a catalytic structural domain. The feruloyl esterase and the mutant obtained by the invention have stable properties and are beneficial to the industrial application of the feruloyl esterase.
Description
Technical Field
The invention belongs to the field of enzyme engineering, and particularly relates to ferulic acid esterase, a mutant N.9-98 thereof and application of the ferulic acid esterase.
Background
The feruloyl esterase is an enzyme which can hydrolyze ester bonds in ferulic acid methyl ester, oligosaccharide ferulic acid ester and polysaccharide ferulic acid ester and further free ferulic acid, is a subclass of carboxylic ester hydrolase and is also an extracellular enzyme. At present, ferulic acid esterase is generally used in the food industry to break the crosslinking of ferulic acid and polysaccharides in cell wall materials such as bran and straws, efficiently degrade the polysaccharides and obtain trans-ferulic acid. Feruloyl esterase can be used in feed industry and paper industry to improve digestibility of fiber feed and help to remove lignin. Therefore, the feruloyl esterase has wide application prospect.
Feruloyl esterase in nature is widely present in plants and microorganisms, and is mainly derived from microorganisms, fungi, bacteria and yeasts can secrete feruloyl esterase. To date, researchers have isolated and identified over 80 ferulic acid esterases from microorganisms, of which the major origin is fungal. However, the ferulic acid esterase secreted by the wild strain has relatively low activity and takes a long time in the enzyme production process, so that a novel ferulic acid esterase with higher enzyme activity needs to be obtained through abundant gene resources.
Disclosure of Invention
The invention aims to provide ferulic acid esterase, a mutant N.9-98 thereof and application. The feruloyl esterase has stable property, and the enzyme activity of the mutant obtained by site-directed mutagenesis is obviously improved.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
the invention provides feruloyl esterase which has an amino acid sequence shown as SEQ ID No. 3.
Further, the reaction pH of the ferulic acid esterase is 6-8, and the reaction temperature is 37-54 ℃; zn 2+ 、Fe 2+ 、Fe 3+ Can obviously inhibit the activity of ferulic acid esterase.
Further, the optimum reaction pH of the ferulic acid esterase is 7, and the optimum reaction temperature is 45 ℃.
The invention also provides a feruloyl esterase mutant N.1-300, the amino acid sequence of which is shown as SEQ ID No.13, and the feruloyl esterase mutant is obtained by changing the amino acid at the 300 th site of the feruloyl esterase with the amino acid sequence of SEQ ID No.1 from glycine to cysteine.
The invention also provides the coding gene of the ferulic acid esterase mutant N.1-300, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 14.
The invention also provides a feruloyl esterase mutant N.7-16, the amino acid sequence of which is shown as SEQ ID No.15, and the feruloyl esterase mutant is obtained by changing the 16 th amino acid of the feruloyl esterase with the amino acid sequence of SEQ ID No.2 from lysine to arginine.
The invention also provides the coding gene of the ferulic acid esterase mutant N.7-16, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 16.
The invention also provides a feruloyl esterase mutant N.9-98, the amino acid sequence of which is shown as SEQ ID No.17, and the feruloyl esterase mutant is obtained by changing the 98 th amino acid of the feruloyl esterase with the amino acid sequence of SEQ ID No.3 from lysine to glutamic acid.
The invention also provides the coding gene of the ferulic acid esterase mutant N.9-98, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 18.
Further, the specific enzyme activity of the ferulic acid esterase mutant N.1-300, the ferulic acid esterase mutant N.7-16 or the ferulic acid esterase mutant N.9-98 is obviously higher than that of the ferulic acid esterase.
The invention also provides application of the ferulic acid esterase, or the ferulic acid esterase mutant N.1-300, or the ferulic acid esterase mutant N.7-16, or the ferulic acid esterase mutant N.9-98 in preparation of a preparation for degrading methyl ferulate.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention obtains 3 novel ferulic acid esterase N.1, N.7, N.9 which are different from the existing ferulic acid esterase by a gene technology, and obtains 3 ferulic acid esterase mutants N.1-300, N.7-16 and N.9-98 with obviously improved specific enzyme activities by site-specific mutagenesis on the basis of amino acid sequences of the ferulic acid esterase, the specific enzyme activities of the ferulic acid esterase mutants are respectively 9.91%, 11.47% and 14.86% higher than that of the original ferulic acid esterase, and simultaneously proves that the increase of the number of disulfide bonds in the ferulic acid esterase, the change of side chain groups of Carbohydrate Binding Modules (CBM) of the ferulic acid esterase and the change of structure domain charges can influence the enzyme activity of the ferulic acid esterase, improve the enzyme activity of the ferulic acid esterase and provide a basis and a mutation mode for improving the enzyme activity of the ferulic acid esterase and obtaining the ferulic acid esterase with better activity. The feruloyl esterase and the mutant obtained by the invention have stable properties, are more favorable for the decomposition of methyl ferulate and the preparation of ferulic acid, expand the preparation way and the acquisition mode of the feruloyl esterase and are favorable for the application of the feruloyl esterase in industrial production.
Drawings
FIG. 1 shows the result of PCR amplification of ferulic acid esterase gene; wherein lanes 1, 4 and 6 are PCR amplified ferulic acid esterase genes N.1, N.7 and N.9 respectively.
FIG. 2 shows the result of double digestion of plasmid pHBM905BDM; wherein 1 is a control plasmid pHBM905BDM; lane 2 is a double restriction of plasmid pHBM905 BDM.
FIG. 3 shows the successful expression of feruloyl esterase N.1, N.7, N.9 in the culture supernatant; wherein lanes 1, 2 and 3 are feruloyl esterase N.1, N.7 and N.9, respectively.
FIG. 4 shows the results of detecting the activity of feruloyl esterase by using a plate, wherein 1, 2 and 3 are transparent rings generated by hydrolyzing a substrate by using feruloyl esterase N.1, N.7 and N.9 respectively.
FIG. 5 is a graph showing the results of purification of feruloyl esterase; wherein lanes 1, 2 and 3 are feruloyl esterase N.1, N.7 and N.9, respectively.
FIG. 6 shows the results of pH adaptation of the reactions of feruloyl esterases N.1, N.7, N.9.
FIG. 7 shows the results of pH stability of feruloyl esterases N.1, N.7, N.9.
FIG. 8 shows the results of suitable temperatures for the reactions of feruloyl esterases N.1, N.7, N.9.
FIG. 9 shows the results of temperature stability of feruloyl esterases N.1, N.7, N.9.
FIG. 10 shows the results of the effect of metal ions on the activity of feruloyl esterase N.1, N.7, N.9.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific examples. Experimental procedures without specific conditions noted in the following examples, generally according to conventional conditions, or according to conditions recommended by the manufacturer; materials of which specific sources are not indicated are all commercially available products.
Preparing a culture medium:
1. BMGY solid medium (per 100 mL): yeast extract 1.0g, tryptone 2.0g, containing (NH) 4 ) 2 SO 4 1.34g of YNB, 1g of glycerol, 100mM of potassium phosphate buffer pH =6.0, sterilized at 115 ℃ for 20min;
2. BMMY liquid medium (per 100 mL): yeast extract 1.0g, tryptone 2.0g, containing (NH) 4 ) 2 SO 4 YNB 1.34g,100mM potassium phosphate buffer pH = -6.0, sterilized at 115 ℃ for 20min;
3. MD solid medium (per 100 mL): glucose 2.0g, containing (NH) 4 ) 2 SO 4 1.34g of YNB, 1.6g of agar and 20min of sterilization at 115 ℃;
4. YPD liquid medium (per 100 mL): 1.0g of yeast extract, 2.0g of tryptone and 2.0g of glucose, and sterilizing at 115 ℃ for 20min;
5. YPD solid Medium (per 100 mL): yeast extract 1.0g, tryptone 2.0g, glucose 2.0g, agar 1.6g,115 ℃ sterilization for 20min.
Example 1: preparation and purification of feruloyl esterase
1. Preparation of feruloyl esterase
1. PCR amplification reaction
Converting amino acid sequences of ferulic acid esterase N.1, ferulic acid esterase N.7 and ferulic acid esterase N.9 (shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 respectively) from Shanghai biological Limited company into gene sequences, carrying out codon optimization on the gene sequences according to the preference of expression strain Pichia codon, and finally obtaining ferulic acid esterase N.1 gene, ferulic acid esterase N.7 gene and ferulic acid esterase N.9 gene with the lengths of 1065bp, 1203bp and 864bp respectively, wherein the nucleotide sequences are shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 respectively. 3 pairs of amplification primers shown in Table 1 were designed based on the nucleotide sequence of the feruloyl esterase gene, and PCR amplification was performed using the templates thereof.
TABLE 1 Feruloyl esterase Gene amplification PCR primers
The PCR reaction system is shown in Table 2, and the PCR procedure is shown in Table 3.
TABLE 2 PCR System
TABLE 3 PCR procedure
After the reaction, the sample was stored temporarily in a refrigerator at 4 ℃.
The target gene amplified by PCR was detected by 0.8% agarose electrophoresis, and it was confirmed that the amplified fragment was identical in size to the target gene as shown in FIG. 1.
2. Plasmid pHBM905BDM double digestion
The plasmid pHBM905BDM double enzyme digestion system is shown in Table 4:
TABLE 4 pHBM905BDM double enzyme digestion System
After the reaction at 37 ℃ for 5h, verifying whether the band is completely digested by agarose gel electrophoresis, terminating the reaction at 80 ℃ for 15min, recovering the target fragment from the gel, and storing in a refrigerator at-20 ℃.
The restriction enzymes Not I and Rsr II cut the vector plasmid pHBM905BDM to obtain vector fragments as shown in FIG. 2, and the plasmid is completely cut compared with the control.
3. Recovering the DNA fragment of interest
Separating target fragment by agarose gel electrophoresis, staining the agarose gel with nucleic acid dye for 1h, cutting target band under a blue light instrument, recovering the target band with Omega gel recovery kit, storing the recovered DNA sample at-20 deg.C.
4. Ligation of vector fragments
The system for vector fragment ligation is shown in Table 5:
TABLE 5 ligation reaction System
E.coli DH5 α was transformed after 5min on ice.
5. Linearization of recombinant pHBM905BDM expression plasmid
The constructed pHBM905BDM expression plasmid Sal I digestion linearization system with the inserted target sequence is shown in Table 6:
TABLE 6 Sal I cleavage System
Reacting at 37 ℃ for 3h and at 80 ℃ for 10min, recovering the sample gel and storing in a refrigerator at-20 ℃.
6. Preparation of Pichia pastoris GS115 competent cell
(1) Plate scribing: in a clean bench, marking a Pichia pastoris GS115 strain on a YPD plate by using an inoculating loop in three zones, sealing a culture dish by using a sealing film, and inversely placing the culture dish in a constant temperature incubator at 28 ℃ for culturing for 48 hours;
(2) Inoculation: selecting single colony in 20mL YPD medium, culturing bacterial liquid OD at 28 deg.C and 220r/min 600 ≈3；
(3) Transferring: the bacterial solution was transferred to 100mL YPD medium to obtain starting OD 600 Culturing at 28 deg.C and 220r/min of 0.3;
(4) And (3) centrifugal bacterium collection: as bacterial liquid OD 600 When = 1.5-2.0, 3000r/min, 5min of centrifugation;
(5)ddH 2 and O, washing bacteria: removing supernatant in an ultraclean workbench, and gently resuspending cells for 2 times with about 30mL of precooled ultrapure water, 3000r/min, and centrifuging for 5min;
(6) SB bacterial washing: resuspending the cells with 8mL of SB solution, shaking at 220r/min for 30min at 30 ℃;
(7) 1M Sorbitol wash: centrifuging at 3000r/min for 5min, removing supernatant, resuspending cells with 15mL of precooled 1M sorbitol for 3 times, and then resuspending cells with 1mL of precooled 1M sorbitol;
(8) Subpackage competence: 80 mu L of competence of each tube is packed in a freezing tube, and the competence cells are firstly put into a refrigerator with the temperature of 20 ℃ below zero for slowly freezing for a plurality of hours and then put into the refrigerator with the temperature of 80 ℃ below zero for storage.
7. Transformation of Pichia pastoris GS115 competent cells
(1) Preparing an electric revolving cup: cleaning a yeast electric rotating cup, placing the cleaned yeast electric rotating cup in deionized water, performing ultrasonic treatment for 15min by using 75% ethanol after the deionized water is poured off, performing ultrasonic treatment for 15min by using absolute ethanol, performing ultrasonic treatment for 15min, and placing the electric rotating cup in an oven at 55 ℃;
(2) Mixing 80 mu L of prepared Pichia pastoris GS115 competent cells with about 1 mu g of linearly recovered expression plasmids, and transferring the mixture into an electric rotating cup precooled on ice for standing for 5min;
(3) Electric conversion: the electric rotor was wiped dry and put into an electric rotor (Biorad) for electric shock (electric shock parameters 1500V, 25. Mu.F, 200. Omega., 2 mm); immediately adding 200 mu L of MD and 200 mu L of 1M sorbitol after electrotransfer, gently mixing uniformly, transferring to a 1.5ml Ep tube, and incubating for 1-2 h at 28 ℃ and 220 r/min;
(4) Plate coating culture: centrifuging the incubated mixture at 3000r/min for 2min, removing supernatant, sucking and mixing the left bacterial solution with 100 μ l, coating MD plate, and culturing the plate in 28 deg.C incubator for 48-72h.
8. PCR screening of recombinant Pichia pastoris
(1) Single colonies on MD plates were picked up in 1.5ml EP tubes, 50. Mu.l deionized water was added, and 1/4 volume of deionized water glass beads (0.5 mm diameter) were added.
(2) Placing on a beat machine, shaking vigorously for 30s, placing on ice for 10s, shaking for 10 times, 12000r/min, and centrifuging for 10min.
(3) 1 μ l of the supernatant was used for PCR to verify positive clones. The PCR conditions are shown in Table 7 and the PCR procedure is shown in Table 3.
TABLE 7 PCR System
9. 24-pore plate screening recombinant pichia pastoris
(1) The yeast single colony which is positive clone verified by PCR is inoculated into a 24-hole plate containing 5ml BMGY in each hole, the same single colony is marked consistently, and the yeast single colony is cultured for 36 to 48 hours at the temperature of 28 ℃ and at the speed of 220 r/min;
(2) Removing supernatant from the centrifugal bacterial liquid, adding 2ml BMMY into each hole, culturing at 28 ℃ at 220r/min, and adding 20 mu L of methanol every 12h to induce yeast to express target protein;
(3) After 96h of induction, the supernatant of the bacterial liquid is taken and is subjected to polyacrylamide gel electrophoresis (SDS-PAGE) to screen strains capable of expressing the target protein.
10. Polyacrylamide gel (SDS-PAGE) electrophoresis detection of protein expression
(1) The split gum at 12% and the concentrated gum at 5% were prepared with the components shown in tables 8 and 9:
TABLE 8 fraction of separating gel
Table 9% concentrated gum component
(2) Boiling samples: and (3) adding 20 mu L of 5 Xprotein loading into 80 mu L of supernatant, boiling at 100 ℃ for 10min, and centrifuging at 12000r/min for 1min after the sample is cooled to room temperature.
(3) Glue running: 20 mu L of centrifuged sample is added into the upper layer gel hole, and the electrophoresis condition is 80V for gel concentration and 120V for gel separation.
(4) Dyeing: and (3) when the target protein is electrophoresed to the bottom of the separation gel, ending the electrophoresis, and dyeing for 4 hours or overnight at room temperature by using Coomassie brilliant blue dyeing solution.
(5) And (3) decoloring: and (3) washing the dyed glue with clear water, placing the glue into Coomassie brilliant blue decoloration solution, decoloring at room temperature until strips appear, and analyzing the result.
As a result, as shown in FIG. 3, the culture supernatant successfully expressed the target protein.
11. Shake flask expression of feruloyl esterase in Pichia pastoris
(1) Inoculation: inoculating the recombinant positive single colony to 100mL BMGY liquid medium, culturing at 28 deg.C and 220r/min to OD 600 ＝8～12。
(2) Transferring: the culture was collected by centrifugation, the supernatant discarded and resuspended in 30mL BMMY medium.
(3) Induction: the incubation was continued for 120h by adding 30. Mu.L of methanol to the culture solution every 12 h.
(4) Collecting a supernatant: centrifuging the induced bacterial liquid at 10000r/min for 20min, and temporarily storing the supernatant in a 4 ℃ refrigerator or concentrating the supernatant by using an ultrafiltration tube and then placing the concentrated supernatant in a-80 ℃ ultra-low temperature refrigerator.
2. Feruloyl esterase protein concentration determination and purification
1. Protein concentration determination
Protein concentration standard curves (BSA protein diluted in PBS) and protein concentration of interest were plotted according to the Bradford protein concentration assay kit instructions.
2. Protein purification
(1) An appropriate amount of Ni-NTA beads were loaded onto a small column (60ml, 26.2X 134 mM) and washed 3 times with PBS and 3 times with 10mM imidazole to activate the Ni beads.
(2) The crude enzyme solution was mixed with Ni beads at 4 ℃ for 60min using a silent mixer.
(3) After the target protein is combined with the Ni beads, the supernatant flowing out through the sieve plate is collected. Washing Ni beads, washing 3 times with PBS; the hybrid protein was eluted 3 times with 10mM and 20mM imidazole; eluting the target protein 3 times by using 200mM imidazole; the Ni beads were washed with 1M imidazole.
(4) SDS-PAGE electrophoresis detection analyzes the purity of the target band, and the enzymology property is determined by using feruloyl esterase without hybrid protein.
The purification result is shown in FIG. 4, SDS-PAGE shows that the purified protein is cleaner and can be used for enzyme activity property determination.
Example 2: determination of feruloyl esterase enzymatic Properties
1. Definition of Feruloyl esterase Activity
The amount of enzyme required for the hydrolysis of ferulic acid methyl ester by ferulic acid esterase per minute to produce 1. Mu. Mol of ferulic acid is defined as 1 enzyme activity unit, expressed as U, under certain conditions of temperature and pH. The specific enzyme activity of the enzyme refers to the number of enzyme activity units per mg of the feruloyl esterase under specific conditions.
2. Principle of enzyme activity determination
The ferulic acid esterase hydrolyzes ferulic acid methyl ester to generate ferulic acid under the conditions of certain temperature and pH, and the reduction of the ferulic acid methyl ester obtained by enzymolysis is calculated by measuring the change of absorbance of the ferulic acid methyl ester in a system before and after reaction at 0D=350nm, so as to obtain the activity of the ferulic acid esterase.
3. Flat plate screening of feruloyl esterase
Adding 1g of substrate ferulic acid methyl ester and 1.5g of agar powder into 100ml of deionized water, heating and dissolving the mixture by a microwave oven to obtain a uniform solution, pouring the heated solution into a culture dish, and cooling the culture dish at room temperature. And (3) dropping 5 mul of fermentation supernatant on the cooled solid plate, culturing overnight at 37 ℃, detecting whether an enzymolysis loop exists in the fermentation supernatant or not, and judging whether the enzyme activity exists in the fermentation supernatant.
The results are shown in FIG. 4, and hydrolysis circles appear on the plates, which indicates that the ferulic acid esterase N.1, N.7, N.9 can decompose the ferulic acid methyl ester in the plates, i.e. the ferulic acid esterase N.1, N.7, N.9 expressed in Pichia pastoris has activity.
4. Drawing of methyl ferulate concentration standard curve
(1) Preparing 100mM of methyl ferulate mother liquor by using methanol as a solvent, wherein the mother liquor is diluted into standard liquor with different concentrations by using corresponding pH solutions at each integral point of pH because the light absorption values of methyl ferulate solutions with the same concentration are different under different pH values: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0mM; pH 3.0-7.0 (0.1M citrate phosphate), pH 7.0-9.0 (0.1M Tris-HCl), pH 9.0-11.0 (0.1M Glycine-NaOH).
(2) Adding 100 mu L of standard solution into a 96-well plate, and measuring the absorbance of the standard solution by using an enzyme-linked immunosorbent assay (ELISA) instrument at the wavelength of 350 nm;
(3) Drawing a standard curve by taking the concentration of the ferulic acid methyl ester as an abscissa and the absorbance as an ordinate to obtain a linear regression equation and a regression coefficient (R) 2 ) If the content is more than 0.999, the product can be used.
5. Effect of pH on Feruloyl esterase enzyme Activity
(1) Optimum pH of feruloyl esterase
Each hole of the enzyme label plate is used as a reaction container, a reaction system of 100 mul is adopted, and 10mM methyl ferulate is used as a substrate. The reaction temperature is 37 deg.C, the reaction time is 1h under the wavelength of 350nm, the light absorption values of pH at 4, 5, 6, 7, 8, 9 and 10, pH 3.0-7.0 (0.1M citrate phosphate), pH 7.0-9.0 (0.1M Tris-HCl) and pH 9.0-11.0 (0.1M Glycine-NaOH) are measured every 3 min. The highest enzyme activity is taken as 100%, and the relative enzyme activities under different pH values are calculated to obtain the optimum pH value of the ferulic acid esterase reaction.
(2) pH stability of Feruloyl esterase
Selecting the optimum pH value and the two pH points of the ferulic acid esterase as incubation buffer solutions, taking ferulic acid methyl ester as a reaction substrate, incubating the equivalent enzyme at 37 ℃, taking the enzyme activity before incubation as 100%, and sampling at intervals to determine the residual enzyme activity.
The results of pH optima of feruloyl esterase reactions are shown in FIG. 6, and the optimum pH of each of the three feruloyl esterases was 7. The pH stability results of the ferulic acid esterase are shown in fig. 7, the three ferulic acid esterases have better pH stability, the best pH stability is ferulic acid esterase N.7, and the residual enzyme activity is about 90% after incubation for 8h at pH =6, 7 and 8 respectively.
6. Effect of temperature on Feruloyl esterase enzyme Activity
(1) Optimum temperature of feruloyl esterase
Reacting at 18 deg.C, 28 deg.C, 37 deg.C, 45 deg.C, 53 deg.C and 62 deg.C with 10mM methyl ferulate as substrate for 30min, boiling to terminate the reaction, centrifuging, collecting 100 μ l of supernatant, and determining OD 350 Absorbance values of (A) and (B). The highest enzyme activity is taken as 100%, and the relative enzyme activities at different temperatures are calculated to obtain the optimal temperature of the ferulic acid esterase reaction.
(2) Temperature stability of feruloyl esterase
Taking ferulic acid methyl ester as a reaction substrate, incubating equivalent enzyme at an optimal temperature and two temperatures around the optimal temperature, taking the enzyme activity before incubation as 100%, and sampling at intervals to determine the residual enzyme activity.
The results of optimum temperature for the ferulic acid esterase reaction are shown in FIG. 8, where three ferulic acid esterases were active in the range of 18 to 63 ℃ and the optimum reaction temperatures were all 45 ℃. The results of the temperature stability of the feruloyl esterases are shown in fig. 9, the higher the temperature is, the lower the enzyme activities of the three feruloyl esterases are; the best temperature stability is ferulic acid esterase N.7, and the residual enzyme activity is up to more than 90% after incubation for 12 hours at 37 ℃; and secondly, the temperature stability is better, the ferulic acid esterase N.1, and the residual enzyme activity is more than 60 percent after incubation for 9 hours at 45 ℃.
7. Effect of Metal ions on enzyme Activity of Feruloyl esterase
Respectively adding metal ions (Ca) with a final concentration of 5mmol/L into an enzymatic reaction system 2+ 、Na + 、K + 、Zn 2+ 、Mg 2+ 、Mn 2+ 、Cu 2+ 、Fe 2+ 、Fe 3+ 、Co 3+ ) Incubating with feruloyl esterase for 10min, adding ferulic acid methyl ester, pH =7 (0.1M Tris-HCl), reacting at 37 deg.C for 30min, boiling to terminate reaction, centrifuging at 12000r/min, and centrifuging for 5min to determine absorbance at OD 350. The reaction system without metal ions was used as a control.
As shown in FIG. 10, different metal ions all have certain influence on the activity of feruloyl esterase, and metal ions having obvious inhibition effect on feruloyl esterase N1, N.7, N.9 have Zn 2+ 、Fe 2+ 、Fe 3+ (P<0.05 The inhibition degree of the three metal ions is different; ca 2+ Can obviously improve the activity (P) of the feruloyl esterase N.1 and N.7<0.05)。
8. Specific enzyme activity of feruloyl esterase
Preparing 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0mM of ferulic acid methyl ester respectively, adding equivalent amount of purified ferulic acid esterase, reacting for 1h at the optimum temperature and pH, measuring the absorbance at the wavelength of 350nm every 2min, performing double reciprocal mapping and calculating the obtained K m And V max 。
Measuring the reaction rate of the recombinant enzyme catalyzing the ferulic acid methyl ester with different concentrations under the conditions of the optimal pH and temperature, and obtaining the corresponding V according to a Linewear-Burk mapping max And K m Value, calculated to obtainK cat 、K cat /K m The value is obtained. The ferulic acid esterase kinetic parameters are shown in table 10.
V max Represents the maximum value of the enzymatic reaction rate reached with the increase of the substrate concentration, provided that the enzyme is present in a certain amount; k m Affinity between the enzyme and the substrate, K m The larger the affinity of the enzyme for the substrate; k cat Indicating the ability of the enzyme to catalyze a particular substrate, K cat The larger the catalyst, the stronger the catalytic ability; k cat /K m The catalytic efficiency of the enzyme on the substrate is reflected, the value reflects the affinity and the catalytic capacity of the enzyme on the substrate at the same time, K cat /K m The larger the catalyst the higher the efficiency. As can be seen from Table 10, V of feruloyl esterase N.1 max The maximum amount of 96.26U mg -1 Although feruloyl esterase N.7 has the highest affinity for methyl ferulate, the high catalytic ability of feruloyl esterase n.1 to methyl ferulate maximizes the catalytic efficiency of the final feruloyl esterase n.1.
TABLE 10 enzymatic kinetic parameters of feruloyl esterases
Example 3: acquisition of Feruloyl esterase mutants
The nucleotide sequences of the ferulic acid esterase N.1 gene, the ferulic acid esterase N.7 gene and the ferulic acid esterase N.9 gene obtained in example 1 are respectively used as templates, the catalytic domain and the substrate binding domain of the ferulic acid esterase are analyzed in a comparison manner, and site-directed mutation is carried out according to the amino acid sequences of the catalytic domain and the substrate binding domain.
Increase the number of disulfide bonds of feruloyl esterase N.1 (nucleotide sequence SEQ ID No. 4) to obtain feruloyl esterase N.1-300, feruloyl esterase N.1-300 is a mutant containing G300C single-point mutation (the amino acid sequence is shown as SEQ ID No.13, the coding nucleotide sequence is shown as SEQ ID No.14, the 300 th amino acid is changed from glycine to cysteine, and the corresponding DNA sequence is changed from GGT to TGT). Changing a side chain group of feruloyl esterase N.7 (nucleotide sequence SEQ ID No. 5) Carbohydrate Binding Module (CBM), researching the influence of the region change on enzyme activity to obtain feruloyl esterase N.7-16, feruloyl esterase N.7-16 is a mutant containing K16R single point mutation (the amino acid sequence is shown as SEQ ID No.15, the coding nucleotide sequence is shown as SEQ ID No.16, the 16 th amino acid is changed from lysine to arginine, and the corresponding DNA sequence is changed from AAG to AGA). Basic amino acids in a catalytic domain of the ferulic acid esterase N.9 (nucleotide sequence SEQ ID No. 6) are mutated into acidic amino acids, the influence of the charge change of the catalytic domain on enzyme activity is researched to obtain the ferulic acid esterase N.9-98, the ferulic acid esterase N.9-98 is a mutant containing K98E single-point mutation (the amino acid sequence is shown as SEQ ID No.17, the coding nucleotide sequence is shown as SEQ ID No.18, the 98 th amino acid is changed from lysine into glutamic acid, and the corresponding DNA sequence is changed from AAG into GAG).
The ferulic acid esterase mutant is obtained by converting pichia pastoris according to the method in the embodiment 1 to express the pichia pastoris, and then determining the specific enzyme activities of ferulic acid esterase N.1, N.7 and N.9 and the specific enzyme activity of the ferulic acid esterase mutant before mutation by using a substrate ferulic acid methyl ester, wherein the specific enzyme activities of the ferulic acid esterase mutant are improved in different ranges as shown in table 11, wherein the specific enzyme activities of the ferulic acid esterase mutant N.1-300 are improved by 9.91% compared with the specific enzyme activity of N.1, the specific enzyme activities of the ferulic acid esterase mutant N.7-16 are improved by 11.47% compared with N.7, and the specific enzyme activities of the ferulic acid esterase mutant N.9-98 are improved by 14.86% compared with N.9.
TABLE 11 specific enzyme Activity of feruloyl esterase before and after mutation
The above examples are only intended to illustrate the technical solution of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
<110> Nanjing university of agriculture
<120> ferulic acid esterase, mutant and application thereof
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 355
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Asp Cys Phe Ser Thr Gln Leu Gly Tyr Pro Cys Cys Glu Asn Thr Asn
1 5 10 15
Glu Val Val Ala Val Asp Glu Asn Gly Val Trp Gly Ile Glu Asn Gly
20 25 30
Val Trp Cys Gly Ile Gly His Ser Ile Lys Asn Asp Asp Tyr Glu Ser
35 40 45
Ser Leu Asn Asn Thr Asp Trp Lys Leu Leu Glu Gln Gln Gln Gln Gln
50 55 60
Gln Gln Gln Gln Asn Ile Ile Gln Lys Arg Gln Tyr Gln Gly Asp Tyr
65 70 75 80
Met Ser Lys Leu Arg Val Val Asn Thr Cys Pro Met Glu Ala Arg Phe
85 90 95
Lys Gln Asn Gly Ile Asn Tyr Pro Thr Ala Gln Lys Ile Thr Tyr Phe
100 105 110
Ser Arg Thr Thr Asn Lys Asn Arg Gln Met Asn Ile Ile Leu Pro Val
115 120 125
Gly Tyr Asn Pro Asn Lys Arg Tyr Pro Val Leu Tyr Phe Leu His Gly
130 135 140
Met Leu Gln Tyr Glu Asp Ser Met Leu Glu Glu Asn Ile Gly Thr Ile
145 150 155 160
Ala Ile Pro Thr Tyr Leu Ala Lys Gln Gly Lys Ala Lys Glu Met Ile
165 170 175
Ile Val Leu Pro Asn Val Tyr Ala Pro Pro Pro Gly Lys Glu Ala Pro
180 185 190
Ala Glu Phe Asn Glu Ala His Phe Leu Gly Tyr Asn Asn Phe Ile Asn
195 200 205
Glu Ile Val Asn Asp Ile Met Pro Tyr Met Gln Ser His Tyr Ser Val
210 215 220
Ala Thr Gly Arg Glu Asn Thr Ala Ile Cys Gly Phe Ser Met Gly Gly
225 230 235 240
Arg Thr Ser Ile Tyr Ile Gly Phe Gln Arg Pro Asp Leu Phe Gly Tyr
245 250 255
Val Gly Ala Phe Ser Pro Ala Pro Gly Leu Ile Pro Ala Asp Asp Ser
260 265 270
Asn Gly His His Asn Gly Leu Tyr Thr Val Asn Asn Phe Arg Ser Asn
275 280 285
Ser Pro Ala Pro Ile Val Thr Leu Ile Ser Cys Gly Thr Asn Asp Ser
290 295 300
Ala Val His Gln Phe Pro Lys Glu Tyr His Glu Val Leu Thr Arg Asn
305 310 315 320
Asn Gln Arg His Ile Trp Phe Glu Ile Pro Gly Ala Asp His Asp Ala
325 330 335
Arg Ala Ile Ser Ala Gly Leu Tyr Asn Phe Val Ser Ala Ala Phe Gly
340 345 350
Ala Leu Asn
355
<210> 2
<211> 401
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Glu Cys Trp Ser Glu Glu Tyr Gly Tyr Pro Cys Cys Gln Glu Thr Lys
1 5 10 15
Asp Val Val Lys Thr Asp Glu Ala Gly Ala Trp Gly Ile Glu Asn Gly
20 25 30
Glu Trp Cys Gly Ile Ser Lys Ile Glu Ser Asp Ala Glu Asp Val Ile
35 40 45
Glu Ser Gly Ser Asp Ser Asp Asn Asp Asp Glu Leu Leu Asp Thr Ser
50 55 60
Asp Val Ala Glu Ile Val Glu Pro Thr Glu Ser Thr Val Pro Glu Val
65 70 75 80
Pro Glu Val Pro Gly Ile Pro Glu Asn Pro Phe Gly Glu Ser Pro Phe
85 90 95
Pro Gly Gly Ala Gly Glu Glu Ile Gln Trp Asn Ala Asn Ala Asn Tyr
100 105 110
Thr Pro Ala Glu Ile Pro Asn Thr Ala Val Ser Glu Tyr Met Ser Lys
115 120 125
Leu Val Val Lys Asp Tyr Cys Pro Ala Asp Val Ser Ser Pro Gln Glu
130 135 140
Gly Val Glu Tyr Pro Thr Ala Glu Lys Ile Thr Tyr Tyr Ser Asn Thr
145 150 155 160
Thr Ala Asn Glu Arg Lys Met Asn Val Ile Leu Pro Val Gly Tyr Thr
165 170 175
Glu Ser Lys Lys Tyr Pro Val Leu Tyr Phe Leu His Gly Ile Met Gly
180 185 190
Asp Glu Asp Thr Met Leu Leu Thr Gly Pro Asp Thr Ile Ala Ile Pro
195 200 205
Thr Asn Leu Ile Asn Ser Gly Leu Ala Lys Glu Met Ile Ile Val Leu
210 215 220
Pro Asn Gln Tyr Ala Pro Ala Pro Gly Thr Glu Ile Pro Pro Ala Leu
225 230 235 240
Thr Gln Glu Tyr Phe Asp Gly Tyr Asp Asn Phe Ile Asn Glu Leu Val
245 250 255
Asn Asp Ile Met Pro Tyr Ile Glu Ser Asn Tyr Ser Val Ala Thr Gly
260 265 270
Arg Glu Asn Thr Ala Val Ala Gly Phe Ser Met Gly Gly Arg Asn Ser
275 280 285
Leu Tyr Ile Gly Tyr Lys Arg Ser Asp Leu Phe Gly Tyr Val Gly Ala
290 295 300
Phe Ser Pro Ala Pro Gly Val Val Pro Gly Asp Asp Phe Ser Gly His
305 310 315 320
His Pro Gly Leu Phe Lys Val Glu Ser Glu Phe Arg Thr Asp Tyr Pro
325 330 335
Pro Ile Val Thr Leu Ile Ser Gly Gly Thr Lys Asp Ser Ile Val Gly
340 345 350
Val Phe Pro Lys Ser Tyr His Asp Ile Leu Thr Thr Asn Glu Gln Asp
355 360 365
His Ile Trp Val Glu Val Pro Glu Ala Asp His Asp Gly Thr Ala Leu
370 375 380
Asp Ser Gly Tyr Tyr Asn Phe Ile Gln Thr Ala Phe Gly Ala Leu Asp
385 390 395 400
Asn
<210> 3
<211> 288
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Met Glu Leu Ala Lys Asn Tyr Leu Lys Lys Ile Lys Ile Ile Asn Pro
1 5 10 15
Cys Pro Thr Asn Leu Leu Leu Arg His Ala Gly Val Ser Tyr Gly Asn
20 25 30
Ile Ile Arg Asp Lys Tyr Tyr Ser Lys Thr Ile Asn Asp Ile Lys Pro
35 40 45
Ile Thr Leu Ile Leu Pro Lys Asp Phe Lys Glu Asn Lys Thr Tyr Pro
50 55 60
Val Leu Tyr Leu Leu His Gly Leu Phe Ser Thr Glu Glu Ser Leu Leu
65 70 75 80
Glu Asp Gly Tyr Asn Ala Asp Asn Ile Leu Phe Asn Leu Ile His Glu
85 90 95
Lys Lys Ala Lys Asp Met Ile Leu Ala Leu Pro Asn Gln Tyr Thr Pro
100 105 110
Val Asn Gly Lys Tyr Phe Thr Pro Ala Phe Asp Gln Lys His Tyr Asp
115 120 125
Gly Tyr Asp Asn Phe Ile Asn Asp Leu Val His Asp Ile Met Pro Phe
130 135 140
Met Glu Lys Asn Tyr Pro Ile Ala Lys Gly Arg Glu Asn Thr Ala Ile
145 150 155 160
Ser Gly Phe Ser Met Gly Gly Arg Asn Ser Leu Tyr Ile Gly Tyr Thr
165 170 175
Arg Pro Asp Leu Phe Gly Tyr Val Gly Ala Phe Ser Pro Ala Pro Gly
180 185 190
Val Thr Pro Gly Arg Asp Ile Tyr Asn Glu Leu Lys Gly Leu Phe Lys
195 200 205
Glu Ser Glu Phe Arg Val Lys Asp Glu Lys Leu Thr Pro Lys Val Ser
210 215 220
Leu Ile Cys Gly Gly Thr Asn Asp Phe Ile Val Gly Asn Thr Pro Glu
225 230 235 240
Lys Tyr His Lys Ile Leu Glu Lys Asn Lys Gln Pro His Val Trp Tyr
245 250 255
Pro Ile Pro Gly Ala Asp His Asp Thr Asp Ala Phe Thr Ser Gly Tyr
260 265 270
Tyr Asn Phe Val Thr Ser Ile Phe Asp Ile Leu Asn Lys Lys Lys Asn
275 280 285
<210> 4
<211> 1065
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gattgtttct ctacccaatt gggttaccca tgttgtgaga acactaacga ggttgttgct 60
gttgatgaga acggtgtttg gggtattgag aacggtgttt ggtgtggtat tggtcactct 120
atcaagaacg atgattacga gtcttctttg aacaacactg attggaagtt gttggagcag 180
caacaacaac aacaacagca acaaaacatt attcaaaaga gacaatacca aggtgactac 240
atgtctaagc ttagagttgt taacacttgt ccaatggagg ctcgttttaa gcaaaacggt 300
attaactacc caactgctca aaagattact tacttctcta gaaccaccaa caagaacaga 360
caaatgaaca ttattttgcc agttggttac aaccctaaca agagataccc tgttttgtac 420
ttcttgcacg gtatgttgca atacgaggat tctatgttgg aggagaacat tggtactatt 480
gctattccta cttacttggc taagcaaggt aaagctaagg agatgattat tgttttgcca 540
aacgtctacg ctccaccacc aggaaaggag gctccagctg agttcaacga agctcacttc 600
ttgggttaca acaacttcat caacgagatt gttaacgaca ttatgccata catgcaatct 660
cactactctg ttgctactgg aagagagaac actgctattt gtggtttctc catgggtggt 720
agaacctcta tctacattgg attccaaaga ccagatttgt tcggttacgt tggtgctttc 780
tctcctgctc ctggtttgat tcctgctgat gactctaacg gtcaccacaa cggtttgtac 840
actgttaaca acttcagatc taactctcca gccccaattg ttactttgat ttcttgtggt 900
actaacgatt ctgctgttca ccaattccca aaggaatacc acgaagtttt gactagaaac 960
aaccaaagac acatttggtt cgagattcca ggtgctgatc acgacgctag agctatctct 1020
gctggtttgt acaacttcgt ttctgctgct ttcggtgctt tgaac 1065
<210> 5
<211> 1203
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gagtgttggt ctgaagagta cggttaccca tgttgtcaag aaactaagga tgttgttaag 60
actgatgagg ctggtgcttg gggtattgag aacggtgaat ggtgtggtat ttctaagatt 120
gaatctgatg ctgaggacgt cattgaatcc ggttctgact ctgataacga tgatgaattg 180
ttggatactt cagacgttgc tgaaatcgtt gaaccaactg aatcaactgt tccagaagtt 240
ccagaagttc ctggtattcc agagaaccca ttcggtgaat ctccattccc aggtggtgct 300
ggtgaagaaa ttcaatggaa cgctaacgct aactacactc cagctgaaat tccaaacact 360
gctgtttctg agtacatgtc taagttggtc gttaaggatt actgtccagc tgatgtttct 420
tccccacaag aaggtgttga gtacccaact gctgaaaaga ttacctacta ctcaaacact 480
actgctaacg agagaaagat gaacgttatc ttgccagttg gttacactga gtctaagaag 540
tacccagttt tgtacttctt gcacggtatt atgggtgacg aagatacaat gctgttgact 600
ggtccagaca ctatcgctat tcctactaac ttgattaact ctggtttggc taaggagatg 660
atcattgttt tgccaaacca atacgcccca gctccaggta ccgagatccc acctgccttg 720
actcaagaat acttcgatgg ttatgacaac ttcattaacg aattggtcaa cgacattatg 780
ccatacattg agtctaacta ctcagttgcc actggtagag aaaacactgc tgttgctggt 840
ttttccatgg gtggtagaaa ctctctttac attggttaca agagatccga tctgttcggt 900
tacgtcggtg ctttctcccc agcccctggt gttgttccag gtgacgattt ctctggtcac 960
caccctggtt tgttcaaggt tgaatctgag ttccgtaccg attacccacc aattgttact 1020
ttgatttctg gtggtaccaa ggattctatc gttggtgttt ttccaaagtc ctaccacgac 1080
attttgacta ctaacgaaca agatcacatt tgggttgaag ttccagaagc tgatcatgac 1140
ggtactgctt tggattctgg ttactacaac ttcatccaaa ctgctttcgg tgctttggat 1200
aac 1203
<210> 6
<211> 864
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atggagttgg ctaagaacta cttgaagaag attaagatca ttaacccatg tccaactaac 60
ttgttgttga gacacgctgg tgtttcttac ggtaacatta ttagagacaa gtactactct 120
aagactatta acgatattaa gccaattact ttgattttgc caaaggactt caaggaaaac 180
aagacttacc ctgttttgta cttgttgcac ggtttgttct ccaccgagga atctttgttg 240
gaggacggtt acaacgctga taacattttg ttcaacttga ttcacgaaaa gaaggctaag 300
gatatgattt tggcattgcc aaaccaatac accccagtca acggaaagta cttcacccca 360
gctttcgacc aaaagcacta cgatggttac gataacttca ttaacgattt ggttcatgac 420
attatgcctt tcatggaaaa gaactaccca atcgctaagg gtagagagaa cactgccatt 480
tctggtttct ctatgggtgg tagaaactct ttgtacatcg gatacactag accagacttg 540
ttcggttacg tcggagcttt ctccccagcc ccaggagtca ctccaggtag agatatctac 600
aacgagttga agggtttgtt caaggagtct gagttcagag ttaaggatga gaagttgact 660
ccaaaggttt cattgatttg tggtggaacc aacgatttca tcgttggtaa caccccagaa 720
aagtaccaca agatcttgga aaagaacaag caaccacacg tttggtaccc aattccaggt 780
gctgatcacg atactgatgc tttcacctcc ggttactaca atttcgttac ttctattttc 840
gacattttga acaagaagaa gaac 864
<210> 7
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gaattaagat cccggatgga ttgtttctct acccaattgg gttacc 46
<210> 8
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gcgaattaat tcgcttagtg atggtgatgg tgatggttca aagcaccgaa agcagc 56
<210> 9
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gaattaagat cccggatgga gtgttggtct gaagagtacg g 41
<210> 10
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gcgaattaat tcgcttagtg atggtgatgg tgatggttat ccaaagcacc gaaagcag 58
<210> 11
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gaattaagat cccggatgat ggagttggct aagaactact tgaag 45
<210> 12
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
cgaattaatt cgcttagtga tggtgatggt gatggttctt cttcttgttc aaaatgtcg 59
<210> 13
<211> 355
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Asp Cys Phe Ser Thr Gln Leu Gly Tyr Pro Cys Cys Glu Asn Thr Asn
1 5 10 15
Glu Val Val Ala Val Asp Glu Asn Gly Val Trp Gly Ile Glu Asn Gly
20 25 30
Val Trp Cys Gly Ile Gly His Ser Ile Lys Asn Asp Asp Tyr Glu Ser
35 40 45
Ser Leu Asn Asn Thr Asp Trp Lys Leu Leu Glu Gln Gln Gln Gln Gln
50 55 60
Gln Gln Gln Gln Asn Ile Ile Gln Lys Arg Gln Tyr Gln Gly Asp Tyr
65 70 75 80
Met Ser Lys Leu Arg Val Val Asn Thr Cys Pro Met Glu Ala Arg Phe
85 90 95
Lys Gln Asn Gly Ile Asn Tyr Pro Thr Ala Gln Lys Ile Thr Tyr Phe
100 105 110
Ser Arg Thr Thr Asn Lys Asn Arg Gln Met Asn Ile Ile Leu Pro Val
115 120 125
Gly Tyr Asn Pro Asn Lys Arg Tyr Pro Val Leu Tyr Phe Leu His Gly
130 135 140
Met Leu Gln Tyr Glu Asp Ser Met Leu Glu Glu Asn Ile Gly Thr Ile
145 150 155 160
Ala Ile Pro Thr Tyr Leu Ala Lys Gln Gly Lys Ala Lys Glu Met Ile
165 170 175
Ile Val Leu Pro Asn Val Tyr Ala Pro Pro Pro Gly Lys Glu Ala Pro
180 185 190
Ala Glu Phe Asn Glu Ala His Phe Leu Gly Tyr Asn Asn Phe Ile Asn
195 200 205
Glu Ile Val Asn Asp Ile Met Pro Tyr Met Gln Ser His Tyr Ser Val
210 215 220
Ala Thr Gly Arg Glu Asn Thr Ala Ile Cys Gly Phe Ser Met Gly Gly
225 230 235 240
Arg Thr Ser Ile Tyr Ile Gly Phe Gln Arg Pro Asp Leu Phe Gly Tyr
245 250 255
Val Gly Ala Phe Ser Pro Ala Pro Gly Leu Ile Pro Ala Asp Asp Ser
260 265 270
Asn Gly His His Asn Gly Leu Tyr Thr Val Asn Asn Phe Arg Ser Asn
275 280 285
Ser Pro Ala Pro Ile Val Thr Leu Ile Ser Cys Cys Thr Asn Asp Ser
290 295 300
Ala Val His Gln Phe Pro Lys Glu Tyr His Glu Val Leu Thr Arg Asn
305 310 315 320
Asn Gln Arg His Ile Trp Phe Glu Ile Pro Gly Ala Asp His Asp Ala
325 330 335
Arg Ala Ile Ser Ala Gly Leu Tyr Asn Phe Val Ser Ala Ala Phe Gly
340 345 350
Ala Leu Asn
355
<210> 14
<211> 1065
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gattgtttct ctacccaatt gggttaccca tgttgtgaga acactaacga ggttgttgct 60
gttgatgaga acggtgtttg gggtattgag aacggtgttt ggtgtggtat tggtcactct 120
atcaagaacg atgattacga gtcttctttg aacaacactg attggaagtt gttggagcag 180
caacaacaac aacaacagca acaaaacatt attcaaaaga gacaatacca aggtgactac 240
atgtctaagc ttagagttgt taacacttgt ccaatggagg ctcgttttaa gcaaaacggt 300
attaactacc caactgctca aaagattact tacttctcta gaaccaccaa caagaacaga 360
caaatgaaca ttattttgcc agttggttac aaccctaaca agagataccc tgttttgtac 420
ttcttgcacg gtatgttgca atacgaggat tctatgttgg aggagaacat tggtactatt 480
gctattccta cttacttggc taagcaaggt aaagctaagg agatgattat tgttttgcca 540
aacgtctacg ctccaccacc aggaaaggag gctccagctg agttcaacga agctcacttc 600
ttgggttaca acaacttcat caacgagatt gttaacgaca ttatgccata catgcaatct 660
cactactctg ttgctactgg aagagagaac actgctattt gtggtttctc catgggtggt 720
agaacctcta tctacattgg attccaaaga ccagatttgt tcggttacgt tggtgctttc 780
tctcctgctc ctggtttgat tcctgctgat gactctaacg gtcaccacaa cggtttgtac 840
actgttaaca acttcagatc taactctcca gccccaattg ttactttgat ttcttgttgt 900
actaacgatt ctgctgttca ccaattccca aaggaatacc acgaagtttt gactagaaac 960
aaccaaagac acatttggtt cgagattcca ggtgctgatc acgacgctag agctatctct 1020
gctggtttgt acaacttcgt ttctgctgct ttcggtgctt tgaac 1065
<210> 15
<211> 401
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Glu Cys Trp Ser Glu Glu Tyr Gly Tyr Pro Cys Cys Gln Glu Thr Arg
1 5 10 15
Asp Val Val Lys Thr Asp Glu Ala Gly Ala Trp Gly Ile Glu Asn Gly
20 25 30
Glu Trp Cys Gly Ile Ser Lys Ile Glu Ser Asp Ala Glu Asp Val Ile
35 40 45
Glu Ser Gly Ser Asp Ser Asp Asn Asp Asp Glu Leu Leu Asp Thr Ser
50 55 60
Asp Val Ala Glu Ile Val Glu Pro Thr Glu Ser Thr Val Pro Glu Val
65 70 75 80
Pro Glu Val Pro Gly Ile Pro Glu Asn Pro Phe Gly Glu Ser Pro Phe
85 90 95
Pro Gly Gly Ala Gly Glu Glu Ile Gln Trp Asn Ala Asn Ala Asn Tyr
100 105 110
Thr Pro Ala Glu Ile Pro Asn Thr Ala Val Ser Glu Tyr Met Ser Lys
115 120 125
Leu Val Val Lys Asp Tyr Cys Pro Ala Asp Val Ser Ser Pro Gln Glu
130 135 140
Gly Val Glu Tyr Pro Thr Ala Glu Lys Ile Thr Tyr Tyr Ser Asn Thr
145 150 155 160
Thr Ala Asn Glu Arg Lys Met Asn Val Ile Leu Pro Val Gly Tyr Thr
165 170 175
Glu Ser Lys Lys Tyr Pro Val Leu Tyr Phe Leu His Gly Ile Met Gly
180 185 190
Asp Glu Asp Thr Met Leu Leu Thr Gly Pro Asp Thr Ile Ala Ile Pro
195 200 205
Thr Asn Leu Ile Asn Ser Gly Leu Ala Lys Glu Met Ile Ile Val Leu
210 215 220
Pro Asn Gln Tyr Ala Pro Ala Pro Gly Thr Glu Ile Pro Pro Ala Leu
225 230 235 240
Thr Gln Glu Tyr Phe Asp Gly Tyr Asp Asn Phe Ile Asn Glu Leu Val
245 250 255
Asn Asp Ile Met Pro Tyr Ile Glu Ser Asn Tyr Ser Val Ala Thr Gly
260 265 270
Arg Glu Asn Thr Ala Val Ala Gly Phe Ser Met Gly Gly Arg Asn Ser
275 280 285
Leu Tyr Ile Gly Tyr Lys Arg Ser Asp Leu Phe Gly Tyr Val Gly Ala
290 295 300
Phe Ser Pro Ala Pro Gly Val Val Pro Gly Asp Asp Phe Ser Gly His
305 310 315 320
His Pro Gly Leu Phe Lys Val Glu Ser Glu Phe Arg Thr Asp Tyr Pro
325 330 335
Pro Ile Val Thr Leu Ile Ser Gly Gly Thr Lys Asp Ser Ile Val Gly
340 345 350
Val Phe Pro Lys Ser Tyr His Asp Ile Leu Thr Thr Asn Glu Gln Asp
355 360 365
His Ile Trp Val Glu Val Pro Glu Ala Asp His Asp Gly Thr Ala Leu
370 375 380
Asp Ser Gly Tyr Tyr Asn Phe Ile Gln Thr Ala Phe Gly Ala Leu Asp
385 390 395 400
Asn
<210> 16
<211> 1203
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gagtgttggt ctgaagagta cggttaccca tgttgtcaag aaactagaga tgttgttaag 60
actgatgagg ctggtgcttg gggtattgag aacggtgaat ggtgtggtat ttctaagatt 120
gaatctgatg ctgaggacgt cattgaatcc ggttctgact ctgataacga tgatgaattg 180
ttggatactt cagacgttgc tgaaatcgtt gaaccaactg aatcaactgt tccagaagtt 240
ccagaagttc ctggtattcc agagaaccca ttcggtgaat ctccattccc aggtggtgct 300
ggtgaagaaa ttcaatggaa cgctaacgct aactacactc cagctgaaat tccaaacact 360
gctgtttctg agtacatgtc taagttggtc gttaaggatt actgtccagc tgatgtttct 420
tccccacaag aaggtgttga gtacccaact gctgaaaaga ttacctacta ctcaaacact 480
actgctaacg agagaaagat gaacgttatc ttgccagttg gttacactga gtctaagaag 540
tacccagttt tgtacttctt gcacggtatt atgggtgacg aagatacaat gctgttgact 600
ggtccagaca ctatcgctat tcctactaac ttgattaact ctggtttggc taaggagatg 660
atcattgttt tgccaaacca atacgcccca gctccaggta ccgagatccc acctgccttg 720
actcaagaat acttcgatgg ttatgacaac ttcattaacg aattggtcaa cgacattatg 780
ccatacattg agtctaacta ctcagttgcc actggtagag aaaacactgc tgttgctggt 840
ttttccatgg gtggtagaaa ctctctttac attggttaca agagatccga tctgttcggt 900
tacgtcggtg ctttctcccc agcccctggt gttgttccag gtgacgattt ctctggtcac 960
caccctggtt tgttcaaggt tgaatctgag ttccgtaccg attacccacc aattgttact 1020
ttgatttctg gtggtaccaa ggattctatc gttggtgttt ttccaaagtc ctaccacgac 1080
attttgacta ctaacgaaca agatcacatt tgggttgaag ttccagaagc tgatcatgac 1140
ggtactgctt tggattctgg ttactacaac ttcatccaaa ctgctttcgg tgctttggat 1200
aac 1203
<210> 17
<211> 288
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Met Glu Leu Ala Lys Asn Tyr Leu Lys Lys Ile Lys Ile Ile Asn Pro
1 5 10 15
Cys Pro Thr Asn Leu Leu Leu Arg His Ala Gly Val Ser Tyr Gly Asn
20 25 30
Ile Ile Arg Asp Lys Tyr Tyr Ser Lys Thr Ile Asn Asp Ile Lys Pro
35 40 45
Ile Thr Leu Ile Leu Pro Lys Asp Phe Lys Glu Asn Lys Thr Tyr Pro
50 55 60
Val Leu Tyr Leu Leu His Gly Leu Phe Ser Thr Glu Glu Ser Leu Leu
65 70 75 80
Glu Asp Gly Tyr Asn Ala Asp Asn Ile Leu Phe Asn Leu Ile His Glu
85 90 95
Lys Glu Ala Lys Asp Met Ile Leu Ala Leu Pro Asn Gln Tyr Thr Pro
100 105 110
Val Asn Gly Lys Tyr Phe Thr Pro Ala Phe Asp Gln Lys His Tyr Asp
115 120 125
Gly Tyr Asp Asn Phe Ile Asn Asp Leu Val His Asp Ile Met Pro Phe
130 135 140
Met Glu Lys Asn Tyr Pro Ile Ala Lys Gly Arg Glu Asn Thr Ala Ile
145 150 155 160
Ser Gly Phe Ser Met Gly Gly Arg Asn Ser Leu Tyr Ile Gly Tyr Thr
165 170 175
Arg Pro Asp Leu Phe Gly Tyr Val Gly Ala Phe Ser Pro Ala Pro Gly
180 185 190
Val Thr Pro Gly Arg Asp Ile Tyr Asn Glu Leu Lys Gly Leu Phe Lys
195 200 205
Glu Ser Glu Phe Arg Val Lys Asp Glu Lys Leu Thr Pro Lys Val Ser
210 215 220
Leu Ile Cys Gly Gly Thr Asn Asp Phe Ile Val Gly Asn Thr Pro Glu
225 230 235 240
Lys Tyr His Lys Ile Leu Glu Lys Asn Lys Gln Pro His Val Trp Tyr
245 250 255
Pro Ile Pro Gly Ala Asp His Asp Thr Asp Ala Phe Thr Ser Gly Tyr
260 265 270
Tyr Asn Phe Val Thr Ser Ile Phe Asp Ile Leu Asn Lys Lys Lys Asn
275 280 285
<210> 18
<211> 864
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
atggagttgg ctaagaacta cttgaagaag attaagatca ttaacccatg tccaactaac 60
ttgttgttga gacacgctgg tgtttcttac ggtaacatta ttagagacaa gtactactct 120
aagactatta acgatattaa gccaattact ttgattttgc caaaggactt caaggaaaac 180
aagacttacc ctgttttgta cttgttgcac ggtttgttct ccaccgagga atctttgttg 240
gaggacggtt acaacgctga taacattttg ttcaacttga ttcacgaaaa ggaggctaag 300
gatatgattt tggcattgcc aaaccaatac accccagtca acggaaagta cttcacccca 360
gctttcgacc aaaagcacta cgatggttac gataacttca ttaacgattt ggttcatgac 420
attatgcctt tcatggaaaa gaactaccca atcgctaagg gtagagagaa cactgccatt 480
tctggtttct ctatgggtgg tagaaactct ttgtacatcg gatacactag accagacttg 540
ttcggttacg tcggagcttt ctccccagcc ccaggagtca ctccaggtag agatatctac 600
aacgagttga agggtttgtt caaggagtct gagttcagag ttaaggatga gaagttgact 660
ccaaaggttt cattgatttg tggtggaacc aacgatttca tcgttggtaa caccccagaa 720
aagtaccaca agatcttgga aaagaacaag caaccacacg tttggtaccc aattccaggt 780
gctgatcacg atactgatgc tttcacctcc ggttactaca atttcgttac ttctattttc 840
gacattttga acaagaagaa gaac 864
Claims (4)
1. The ferulic acid esterase mutant N.9-98 is characterized in that the amino acid sequence of the ferulic acid esterase mutant N.9-98 is shown as SEQ ID No.17, and the ferulic acid esterase mutant is obtained by changing amino acid at position 98 of ferulic acid esterase with the amino acid sequence of SEQ ID No.3 from lysine to glutamic acid.
2. The ferulic acid esterase mutant N.9-98 of claim 1, wherein the nucleotide sequence of the coding gene is shown as SEQ ID No. 18.
3. A ferulic acid esterase, which is characterized in that the amino acid sequence of the ferulic acid esterase is shown in SEQ ID No. 3.
4. Use of the ferulic acid esterase mutant N.9-98 of claim 1 or the ferulic acid esterase of claim 3 for preparing a preparation for degrading methyl ferulate.
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| CN102220299A (en) * | 2011-04-22 | 2011-10-19 | 中国科学院成都生物研究所 | Feruloyl esterase A mutant and purpose thereof |
| CN109652392A (en) * | 2019-02-20 | 2019-04-19 | 南京农业大学 | A kind of feruloyl esterase and its preparation method and application |
| CN112980812A (en) * | 2021-03-16 | 2021-06-18 | 南京农业大学 | Feruloyl esterase and mutant and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102220299A (en) * | 2011-04-22 | 2011-10-19 | 中国科学院成都生物研究所 | Feruloyl esterase A mutant and purpose thereof |
| CN109652392A (en) * | 2019-02-20 | 2019-04-19 | 南京农业大学 | A kind of feruloyl esterase and its preparation method and application |
| CN112980812A (en) * | 2021-03-16 | 2021-06-18 | 南京农业大学 | Feruloyl esterase and mutant and application thereof |
| CN114774388A (en) * | 2021-03-16 | 2022-07-22 | 南京农业大学 | Feruloyl esterase and mutant N.7-16 and application thereof |
Non-Patent Citations (4)
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| Jing Ma 等.Characterization of feruloyl esterases from Pecoramyces sp. F1 and the synergistic effect in biomass degradation.《World Journal of Microbiology and Biotechnology》.2022,第1-17页. * |
| Ma,J..Synthetic construct clone FAE_allA07619 feruloyl esterase gene, complete cds.《GenBank Database》.2022,Accession NO:ON942247.1. * |
| 张帅兵 等.黑曲霉阿魏酸酯酶A的克隆、表达及快速酶活检测体系的建立.《应用与环境生物学报》.2009,第276-279页. * |
| 高兆建 等.深绿木霉嗜酸性阿魏酸酯酶酶学性质及生物质转化分析.《食品科学》.2019,第121-128页. * |
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| CN112980812A (en) | 2021-06-18 |
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| CN112980812B (en) | 2022-09-06 |
| CN114774388B (en) | 2023-06-16 |
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