TWI325442B - Pseudomonas putida glutathione-independent formaldehyde dehydrogenase and the gene and amino acid sequence thereof - Google Patents

Description

〇〇 442 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention is a modified enzyme, particularly for a glutathione-independent citrate Dehydrogenase. [Prior Art] % NADH is a highly reducing compound in biological cells, mainly used as a coenzyme for enzymes in living cells, which supplies hydrogen necessary for the enzyme reaction: atom, and then oxidizes to NAD. In general, oxidoreductases require NAD(H) as a coenzyme' to drive the reaction. In vivo, the concentration ratio of NADH to NAD is fixed, maintaining the necessary physiological activity in the living body. Because NADH has high reduction activity, it is not easy to purify and extract from cells. Currently, commercially available NADH is made by electroporation cell extraction to produce '4 Berg' mussels (2,200 yuan/g), so it cannot be applied to redox fermentation. Prime process. Furfural dehydrogenase is an enzyme that can oxidize furfural to citric acid. It can be divided into two types: glutathione-dependent and glutathione-independent. Among them, furfural dehydrogenase, which is associated with sputum, is present in most organisms, including Escherichia coli cells. This type of furfural dehydrogenase converts formaldehyde to citric acid using Chuanqi and its NAD as a coenzyme. Since it is necessary to use aminosulfur to participate in the enzyme reaction, 'industrial use increases operating costs and costs.' Therefore, it is not suitable for large-scale applications. Non-glutamate-sulfur-related A 1334542 aldehyde dehydrogenase was found in 1979 in Pseudomonas syriae bCRC 13897 (Susumu Ogushi, M. Ando, D. Tsuru, ^formaldehyde dehydrogenase from Pseudomonas putida: a Zinc metalloenzyme", J. biochem" 96, 1587-1591, 1984.). The advantage of non-branched aminosulfur-related formaldehyde dehydrogenase is that it does not have to pass through the surface aminosulfur as a coenzyme to directly convert NAD and furfural to formic acid and high-priced NADH. Because NADH has high reductibility, it can be widely used in industrial and residential applications. Therefore, non-brome aminosulfur-related formaldehyde dehydrogenase has high market application value. However, due to the low specific activity (17U/mg) and low matrix specificity of the non-faceted aminosulfur-related formaldehyde dehydrogenase in the wild strain, the application to the industrial process is unfavorable. The viable application of furfural dehydrogenase cannot be commercialized and produced. Therefore, many people have invested in improving the production of formaldehyde dehydrogenase. Japanese Patent Publication No.

剌6303981 towel will not be 'transformed the gene of formate dehydrogenase into E. coli and ferment this large intestine rod S to obtain a large amount of dehydrogenase:: This method has not solved its low activity Question ° and Yasuyc

The specific activity of the hydrogenase activity of Fujitsu et al. published in 2GG4, 'σ fruit is 31.5 U/mg (37 °c pH=9 Q)', but the specific activity of the enzyme is still low, and it is not suitable. Industrial applications for mass production. : :: The invention through the mutation of the furfural dehydrogenase gene sequence to change the sequence of the amine soil &amp; The cost of yeast use. Enzyme activity and specificity can increase the application of formaldehyde dehydrogenation 1325442 to industrial and people's livelihood. SUMMARY OF THE INVENTION Based on the above background, the main object of the present invention is to provide a gene sequence of Pseudomonas putida non-glutamate-based sulfur-related furfural dehydrogenase, wherein (i) the 522th position is mutated to thoracic pyrimidine. 711 positions were mutated to thoracic pyrimidine, 762 positions were mutated to pleuropyrimidine, 818 positions were mutated to thoracpool, 876 positions were mutated to thoracpool, and 903 positions were mutated to • guano 嘌呤, or (ii The 522th position was mutated to thoracic pyrimidine, 826 positions were mutated to guanosene, 876 positions were mutated to pleuropyrimidine, and 903 positions were mutated to guano, or (iii) 522th position was mutated to chest line. Pyrimidine, 774 positions were mutated to pleuropyrimidine, 777 positions were mutated to pleuropyrimidine, 876 positions were mutated to thymidine, 903 positions were mutated to guanosine, and 1126 positions were mutated to thymidine, and The specific activity of non-rough amine-sulfur-related furfural dehydrogenase of Pseudomonas loves is higher than that of wild strain. The present invention further provides an amino acid sequence of Pseudomonas putida non-glutamate sulfur-related furfural dehydrogenase, wherein the 273th position is mutated to valine, the 275th position is mutated to glutamic acid or The 375th position was mutated to cysteine, and the specific activity of the non-glutamate sulphur-related furfural dehydrogenase of P. syphilis was higher than that of the wild strain. The present invention also provides a non-glutamate-sulfur-related furfural dehydrogenase of Pseudomonas putida, wherein the amino acid sequence is mutated to a valeric acid at the 273th position, and the 275th position is mutated to a face acid or a The 375 positions were mutated to cysteamine 1325442 acid, and its specific activity was higher than that of wild plants. The present invention further provides a cell comprising the aforementioned gene sequence of Pseudomonas putida non- face amine sulfur-related furfural dehydrogenase. This cell includes bacteria or yeast. The present invention further provides a vector comprising the aforementioned gene sequence of Pseudomonas putida non- face amine sulfur-related furfural dehydrogenase. The present invention further provides a cell comprising the above vector. This cell includes bacteria or yeast. The above and other objects, features and advantages of the present invention will become more <RTIgt; The genetic sequence, amino acid sequence and furfural dehydrogenase of the non-glutamate-sulfur-related furfural dehydrogenase of Pseudomonas putida. One embodiment of the present invention is a gene sequence of Pseudomonas putida non-glutamate-based sulfur-related furfural dehydrogenase, wherein 522, 711, 762, 774, 777, 818, 826, 876, 903 and / or 1126 positions produce mutations. In some embodiments, the 522th position of (1) of the above sequence is mutated to thymidine, 711 position mutations to thymidine, 762 positions to thymidine, 818 positions to thymidine, 876 positions Mutation to thoracic pyrimidine and 903 position mutations to guanosin, or (ii) mutation at position 522 to thymidine, 826 position mutation to guanosene, 876 1325442 positions to thymidine and 903 Position mutation to guano sputum, or (iii) mutation at position 522 to thymidine, 774 position mutation to thymidine, 777 position mutation to thoracic, 876 position mutation to thoracic, 903 The position was mutated to guanosin and 1126 positions were mutated to thoracic. The specific activity of the non-glutamate-sulfur-related furfural dehydrogenase of P. syringae was higher than that of the wild-type strain, and its specific activity was about 30-150 U/mg, while the specific activity of the wild-type strain was 17 U/mg. The above Pseudomonas putida includes Pseudomonas putida BCRC13897. In one embodiment, the specific activity is about 100-150 U/mg. In a preferred embodiment, the 522th position is mutated to pleuropyrimidine, 774 positions are mutated to pleuropyrimidine, 777 positions are mutated to pleuropyrimidine, 876 positions are mutated to pleuropyrimidine, and 903 positions are mutated to The guano and 1126 positions were mutated to thoracic pyrimidine. Another embodiment of the present invention is an amino acid sequence of Pseudomonas putida non-glutamate sulfur-related formaldehyde dehydrogenase wherein a mutation occurs at positions 273, 275 and/or 375. In some embodiments, the 273th position is mutated to proline, the 275th position is mutated to glutamic acid or the 375th position is mutated to cysteine. The specific activity of the non-glutamate-sulfur-related formaldehyde dehydrogenase of Pseudomonas putida is higher than that of the wild plant, and its specific activity is about 30-150 U/mg, while the specific activity of the wild-type strain is 17 U/mg. The above Pseudomonas putida includes Pseudomonas putida BCRC13897. In one embodiment, the specific activity of the above non-glutamate-based sulfur-related furfural dehydrogenase is approximately 100-150 U/mg. In a preferred embodiment, the 375th position is mutated to cysteine.

10 1325442 The above-mentioned modified non-glutamate sulfur-related formaldehyde dehydrogenase-derived furfural and acetaldehyde reaction rate ratio (enzyme specificity) is higher than that of wild plants, in one embodiment, furfural The ratio of the reaction rate with acetaldehyde is about 200 to 300, and the ratio of the reaction rate of furfural to acetaldehyde of the wild strain enzyme is 4.53. In still another embodiment, the ratio of the rate of reaction of the modified enzyme furfural to acetaldehyde is about 44-66 times that of the wild strain. The difference between the modified enzyme and the lowest reaction of furfural and acetaldehyde is more than 1〇3. A further embodiment of the present invention is a non-rough amine-sulfur-related furfural dehydrogenase of Pseudomonas putida, wherein the amino acid sequence is mutated to valeric acid at position 273 and mutated to bran at position 275. The amino acid or the 375th position is mutated to cysteine and its specific activity is higher than that of the wild strain. Its specific activity is about 30-150 U/mg, while the specific activity of wild strain is 17 U/mg. The above Pseudomonas putida includes Pseudomonas putida BCRC13897. In one embodiment, the specific activity of the above non-glutamate-based sulfur-related formaldehyde dehydrogenase is approximately 100-150 U/mg. In a preferred embodiment, the 375th position of the amino acid sequence of the enzyme is mutated to cysteine. In one embodiment, the ratio of the reaction rate of furfural to acetaldehyde (enzyme specificity) of the enzyme is higher than that of the wild strain. The ratio of the reaction rate of furfural to acetaldehyde is about 200 to 300, and the ratio of the reaction rate of formaldehyde to acetaldehyde of wild strain enzyme is 4.53. In still another embodiment, the ratio of the rate of reaction of the modified enzyme furfural to acetaldehyde is about 44-66 times that of the wild strain. In a preferred embodiment, the modified enzyme has a minimum reactivity of more than 103 for furfural and acetaldehyde, respectively. 11 1325442 - In the implementation form, the 'gene sequence of the present invention can be placed in a vector or a cell' for the purpose of mass production of the modified dehydrogenase" carrier can include pTrc His A, pTrc His B, pTrc His C, pET 41 a, pET 17b, pET 16b, and the cells may be Escherichia coli and yeast. In other embodiments, the aforementioned vector is placed in a cell, and the cells include Escherichia coli and yeast. This modified formaldehyde dehydrogenase special chemical and chemical production process, formaldehyde removal, sterol detection and biofuel cells. In addition, the use of enzyme gene expression technology for enzyme mass production can reduce the cost of enzyme production. The modified furfural dehydrogenase can be used in the novel NADH regeneration system and reacted with the phthalic acid dehydrogenase to form a multi-enzyme NADH regeneration reaction. On the other hand, furfural dehydrogenation can be applied to biosensors and biofuel cells. [Examples] A method for obtaining the gene sequence of Pseudomonas putida non-glutamate-based sulfur-related formaldehyde de-enzyme of the present invention is described below. Preparation of plastid DNA containing Pseudomonas aeruginosa formaldehyde dehydrogenase gene First, Pseudomonas putida BCRC13897 strain was provided, which was placed in NA or C83 medium and cultured at 26 ° C for 44 hours. Next, the chromosomal DNA of Pseudomonas putida BCRC13897 strain was extracted as a template DNA amplified by the A. dehydrogenase machine gene. 1325442 Designed a primer for the furfural dehydrogenase gene to perform a polymerase chain reaction (PCR). The furfural dehydrogenase gene fragment of Pseudomonas syriae BCRC13897 is 12〇〇 bp.

The primers designed were: 5' GAA TTC AGG CCG CGC TGA AGG TCT TGT GCG 3, and 5' GAA TTC ATG TCT GGT AAT CGT GGT GTC GTT3'. PCR is then carried out to amplify the desired formate dehydrogenase gene fragment. Put the dehydrogenase gene fragment into pTrc His B (kanamycin and ampicillin (ampiciiHn)), while the wild strain of Pseudomonas putida BCRC13897 is the furfural dehydrogenase gene sequence For the sequence identification number: 1; the amino acid sequence is the sequence identification number: 2. The prepared plastids were transformed into E. coli BL-21 Blue competent cells (〇0!1^616 plus (^11) (purchased from 81 plus & § §6). Confirmed that the transformed competent cells have been After having the target plastid, a large number of competent cells are cultured, and the plastid DNA is extracted for preservation. Preparation of the mitochondrial enzyme-modified strain The morphological DNA is used to perform any point mutation using GeneM〇rph II Random mutagenesis kit (from stratagene). The PCR conditions of PCR are shown in Table 。. After that, the pCR product is electrophoresed, and the target gene fragment is excised from the ginseng for purification. After the target gene is inserted into the plastid, it is then subjected to E. coli BL21 competent cells. Transplantation reaction. After overnight culture in 2YT S] medium, select the modified strain. 1325442 Table 1. PCR operating conditions Temperature time 94 〇C 2 min 94 〇C 30 sec Primer Tm-5°C 30 sec 72〇C 1 min/kb 72 °C 10 min Screening of modified strains for rapid screening of modified strains includes the following steps: (1) 100 μΐ of 2ΥΤ medium containing ampomycin is placed in the culture well of a 96-well shallow culture dish. (2) using modified toothpicks to modify bacteria The plants were transferred from solid medium to 96-well shallow culture plates, and the first culture well of each row was the control group (wild strain); (3) After overnight culture, 10 μΐ of the bacteria from the 96-well shallow culture plate was taken. The solution was added to a 96-well deep culture dish containing 0.5 ml of 2 ΥΤ (containing ampomycin) per well. The concentration of the broth in the well was 0.5, and 5 μM of 100 mM IPTG was added; (4) After 16 hours of culture, take out ΙΟΟμΙ of the bacterial solution to a 96-well shallow culture dish, centrifuge at 5000 rpm for 10 minutes, then pour out the supernatant; (5) add 20 μΐ lysozyme buffer solution (lysozyme buffer) to the 96-well shallow culture plate after centrifugation, after shaking evenly Place at room temperature for 1 hour; (6) Remove in a -80 °C freezer for 45 minutes, remove it, wait until the cells are completely warmed, then put in -80 °0 refrigerator for 45 minutes; (7) from the refrigerator The 96-well shallow culture plate to be tested was taken out, and after the cells were warmed up, 180 μl of the active 1325442 test solution was added to the eight-claw micropipette, and the position of the strain with the blue change faster than the control group was recorded; (8) 100- After 200 strains, repeat steps (1) to (7) until the candidate strain Decrease to 10 strains; (9) Incubate the candidate strain with a 250 ml flask, add 0.5 ml of 100 mM IPTG when the amount of bacteria is 0.5, and centrifuge the culture solution at 7000 rpm for 10 minutes after 6 hours of culture; (10) After the supernatant, add lysozyme buffer (add volume: 0D value * 0.5 mg cells * X ml culture solution / 50 mg = Y ml buffer solution), and react at room temperature for 1 hour; (11) Break with ultrasound The cells were stopped for 15 seconds in 15 seconds and repeated 10 times. Centrifuge at 12000 i*pm for 4 min at 4 °C, take the supernatant as a crude enzyme solution; (12) Add the enzyme activity test solution to test the activity and protein concentration of the crude enzyme solution, and select the one with the highest specific activity for the next generation. Maternal strains, and analysis of furfural dehydrogenase DNA and protein structure mutations; (13) Repeated strain modification and strain screening experiments until the specific activity of furfural dehydrogenase reached 50 U / mg 〇 enzyme activity test solution The composition is shown in Table 2, and the results of the modified strain are shown in Table 3.

15 1325442 Table 2. Composition of enzyme activity test solution

Sample μΐ/well stock (stock)/disk (ml) Crude enzyme solution 20 Enzyme activity test 60 mM 168.7 Liquid composition Na2C03-NaHC03 buffer (pH 8.8) 16.87 lOOmMHCHO 3 0.3 100 mM NAD 3 0.3 13.4mMNBT 2.3 0.23 ImMPMS 3 0.3 Total volume 200 18 ml Table 3, strains modified with furfural dehydrogenase

Modification number 2-5 8B9 4-1 5-1 6-23 7-2 8-5 Parent strain source WT Μ 2-5 2-12 8B9 5-1 4-1 6-23 Number of crude sieve strains in the first stage 2997 1674 2924 2352 3554 4536 6384 4032 Number of crude strains in the second stage 217 70 91 ~100 280 336 399 201 Number of strains in the third stage 15 13 12 38 26 26 16 Specific activity (U/mg) 4828 50.88 50.88 32.59 40 25.71 15.70 120.9 Gene sequence changes c522t' c876t, c903g c522t, c876t, c903g c522t, g71U, g762t, a818t, c876t, c903g c522t&gt; a826g' c876t, c903g c522t, c774t, c876t, c903g c522t, c774t, c777t, c876t, c903g C522t, c774t' c777t, c876t, c903g' gll26t amino acid sequence change one - E273V K275E — — — G375C 16 1325442 It can be seen from Table 3 that the selected strains have varying numbers of gene sequences. There are 8B9, 4-1 and 8-5 strains with a change in amino acid and higher specific activity. The gene mutation of 8B9 was changed to the 522th position to thymidine, 711 position mutation to thymidine, 762 position mutation to thoracic acid, 818 position mutation to thoracic acid, and 876 position mutation to thoracic acid. And 903 positions were mutated to guano (SEQ ID NO: 3); amino acid sequence changes to 273th position mutation to proline (sequence number: 4). The 4-1 gene changes to the 522th position mutation to thoracic pyrimidine, 826 position mutation to guanosene, 876 position mutation to thoracic pyrolysis, and 903 position mutation to bird feces (sequence identification number: 5) The amino acid sequence was changed to the 275th position mutation to glutamic acid (SEQ ID NO: 6). The gene mutation of 8-5 was changed to the 522th position to thymidine, 774 to thymidine, 777 to thymidine, 876 to thymidine, and 903 to The guano and 1126 positions were mutated to thoracic acid (SEQ ID NO: 7); the amino acid sequence was changed to the 375th position mutation to cysteine (sequence number: 8). On the other hand, the mutation of the 8-5 amino acid sequence of the mutant having the preferred activity of the present invention was changed to a glycine acid at position 375 and mutated to cysteine. Although this amino acid is a member of the inactive center, it is located in an α-helix (α-helix) on the NAD binding region. Glycine is an essential amino acid having the smallest steric structure of polarity, but since hydrogen atoms in the molecule cannot form hydrogen bonds, they do not cause steric structural obstruction. The cysteine has a sulfhydryl group, and 1325442 is changed from the original glycine to cysteine to cause a change in the conformation of the wild-type dehydrogenase. This disulfide bond occurs in the α-helix. The inner side, which affects the space in which the active center of the enzyme reacts with the matrix, may increase the diffusion effect of the matrix and the enzyme, and accelerate the rate of binding or detachment of the matrix to the enzyme. Kinetic parameter analysis of furfural dehydrogenase The enzyme kinetic parameters were analyzed for the formaldehyde dehydrogenase of the comparative example (commercially available) and the inventive example (8_5). The experimental method of enzyme kinetics was as follows: Α. For NAD test Km and Vmax: (1) The concentration of furfural in the immobilized enzyme activity test solution is 7 mM; (2) Adjusting the NAD concentration in the enzyme activity test solution to be 0.12 mM to 12 mM; (3) adding the purified furfural dehydrogenase to Ιμΐ (4) Record the value of OD34〇nm every other minute for 5 minutes in a row; (5) Calculate the slope of each sample by linear regression to obtain the reaction rate V; (5) to 1 The /NAD concentration is plotted against the 1/reaction rate. The slope is Klt^Vmax and the φ intercept is 1/Vmax. (6) Km and Vmax are obtained from the slope and intercept. B. Test Km and Vmax for furfural. (1) In the immobilized enzyme activity test solution, the concentration of SiAD is 1〇 times Km(NAD); (2) The concentration of formaldehyde in the test enzyme activity test solution is 〇12mM to 12mM; (3) Adding ΐμΐ purified citrate Hydrogenase to the test solution; (4) Record the value of 〇D34〇nm every other minute for five minutes; (5) Calculate the slope of each sample by linear regression to obtain the reaction rate V; 6) Plot the 1/曱 disk concentration versus 1/reaction rate, and find the slope as Km/Vmax with an intercept of 1/Vmax. (7) Find Km 1325442 and Vmax by slope and intercept. The steps A to B are repeated until the difference between Vmax (NAD) and Vmax (furfural) is less than 5%. It is the kinetic parameter of furfural dehydrogenase. The results of the comparative examples are shown in Tables 4 and 5, panels 1 and b (fixed brewing concentration (2 mM), 1/reaction rate versus 1/NAD inversion graph and reaction rate versus NAD concentration at different NAD concentrations) and 2a and b (fixed NAD concentration (4 mM), 1/reaction rate vs. furfural concentration map and reaction rate vs. furfural concentration plot for different furfural concentrations). From the above results, it was deduced that the comparative example had a Km (NAD) of 0.384 mM and a Km (furfural) of 368.368 mM. Table 4, fixed aldehyde concentration (2 mM) of the comparative example, change of NAD concentration to measure Km (NAD) NAD concentration (mM) 0.120 0.600 1.200 3.600 4.000 6.000 9.000 12.000 Reaction time 1 minute NADH concentration OD340 0.035 0.085 0.107 0.123 0.131 0.126 0.091 0.103 Reaction time 2 minutes NADH concentration OD340 0.071 0.168 0.227 0.244 0.266 0.256 0.198 0.204 Reaction time 3 minutes NADH concentration OD^ 0.101 0.249 0.332 0.372 0.380 0.377 0.304 0.319 Reaction time 4 minutes NADH concentration OD340 0.127 0.316 0.436 0.484 0.511 0.507 0390 0.412 Reaction time 5 minutes NADH concentration OD^ 0.150 0.377 0.520 0.605 0.620 0.615 0.508 0.526 19 1325442 NADH production rate (V) (Δ0D/min) 0.029 0.073 0.104 0.120 0.122 0.123 0.103 0.105 1/NAD concentration 34.965 13.661 9.662 8.306 8.177 8.137 9.747 9.488 1/ Reaction rate 8333 1.667 0.833 0.278 0.250 0.167 0.111 0.083 Table 5, fixed NAD concentration of the comparative example (4 mM), change of furfural concentration to measure Km (曱骏) formaldehyde concentration (mM) 0.02 0.1 0.25 0.5 1 1.5 3 Reaction time 1 Minute NADH concentration 0〇34〇0.173 0.175 0.056 0.205 0.233 0.033 0 .162 Reaction time 2 minutes NADH concentration OD340 0.172 0.190 0.107 0.247 0.313 0.169 0.340 Reaction time 3 minutes NADH concentration ODa^ 0.178 0.214 0.157 0.293 0.395 0.301 0.498 Reaction time 4 minutes NADH concentration OD340 0.185 0.233 0.200 0.334 0.466 0.420 0.636 Reaction time 5 minutes NADH Concentration OD340 0.182 0.244 0.237 0.377 0.532 0.539 0.767 NADH production rate (V) (ΔOD/min) 0.007 0.018 0.046 0.043 0.075 0.126 0.151 1/furfural concentration 153.846 55.249 21.978 23.202 13.316 7.918 6.640 1/reaction rate 50.000 10.000 4.000 2.000 1.000 0.667 0.333

The results of the examples are shown in Tables 6, 7 and 3a and b (fixed 曱1325442 aldehyde concentration (7.5 mM), 1/reaction rate vs. l/NAD concentration map and reaction rate versus NAD concentration map at different NAD concentrations) And Figures 4a and b (fixed NAD concentration (7.6 mM), 1/reaction rate versus 1/furfural concentration map and reaction rate versus furfural concentration plot for different formaldehyde concentrations). From the above results, it can be inferred that the Km (NAD) is 1.752 mM, Km (wake up) is 1.71 mM, Vmax is 120.8 U/mg, Kcat is 84/s, and the turnover rate is 30 per minute. Ten thousand times. As in the comparative example, when the NAD concentration exceeds 8 mM, the enzyme activity of the examples is also inhibited, but the tolerance to formaldehyde is relatively high, and the enzyme activity does not change significantly when the concentration of furfural exceeds 16 mM. On the other hand, the Km value of the embodiment is higher than the Km value of the comparative example. The results of the modification of the examples show that 'in the process of upgrading, when the enzyme activity increases, not because of the increased affinity for the matrix, it may be because the structure of the enzyme is changed, so that the enzyme activity is increased, but further experiments are needed. prove. Table 6. Fixed aldehyde concentration (7.5 mM) in the example, varying NAD concentration to measure Km (NAD) NAD concentration (mM) 0.12 0.60 1.20 4.00 6.00 8.00 12.00 15.00 Reaction time 1 minute NADH concentration OD340 0.025 0.101 0.173 0.235 0.254 0.251 0.224 0.202 Reaction time 2 minutes NADH concentration OD340 0.041 0.196 0.319 0.481 0.462 0.496 0.473 0.428 Reaction time 3 minutes 0.048 0.280 0.454 0.728 0.676 0.738 0.695 0.651 1325442 NADH concentration OD340 Reaction time 4 minutes NADH concentration ODw 0.064 0.360 0.584 0.880 0.927 0.878 Reaction time 5 Minute NADH concentration OD340 0.073 0.434 NADH production rate (V) (M) D / min) 0.012 0.083 0.137 0.247 0.209 0.244 0.233 0.225 1 / NAD concentration 84.034 12.048 7.310 4.057 4.780 4.107 4.290 4.442 1 / reaction rate 8.333 1.667 0.833 0.250 0.167 0.125 0.083 0.067 Table 7, fixed NAD concentration (7.6 mM) in the example, changing furfural concentration to measure Km (furfural) formaldehyde concentration (mM) 0.12 0.6 1.2 4 6 8 12 15 Reaction time 1 minute NADH concentration OD34 〇 0.023 0.105 0.124 0.216 0.213 0.259 0.094 0.176 Reaction time 2 minutes NADH Concentration OD340 0.047 0.181 0.301 0.456 0.456 0.501 0.205 0.409 Reaction time 3 minutes NADH concentration OD340 0.069 0.261 0.420 0.691 0.691 0.748 0.312 0.625 Reaction time 4 minutes NADH concentration OD340 0.086 0.337 0.552 0.903 0.903 0.418 Reaction time 5 minutes NADH concentration OD340 0.104 0.405 0.675 0.514 NADH Production rate 0.020 0.076 0.135 0.230 0.231 0.245 0.105 0.225 22 1325442 (V) (AOD/min) 1/furfural concentration 49.751 13.228 7.391 4.355 4.338 4.090 9.497 4.454 1/reaction rate 8.333 1.667 0.833 0.250 0.167 0.125 0.083 0.067 Modified formaldehyde Hydrogenase specificity analysis was carried out using an 8 mM furfural solution and various concentrations of acetaldehyde to carry out the activity analysis of the vine dehydrogenase activity of the example. The results are shown in Fig. 5. The figure shows that when Ludang uses different concentrations of acetaldehyde solution as a substrate, the activity of the enzyme is almost zero (in line). Even after the acetaldehyde concentration was as high as 92 mM, the absorbance of NADH remained at the starting point after five minutes. This result showed that acetaldehyde was not a substrate for the modified formaldehyde dehydrogenase, and the modified enzyme had excellent specificity. The sixth embodiment shows a reaction rate diagram in which different concentrations of acetaldehyde and furfural are used together as a furfural dehydrogenase substrate. It is shown in the figure that when furfural and acetaldehyde coexist in solution, the activity of formaldehyde dehydrogenase is affected by the formaldehyde φ / Chen, and the presence of acetic acid does not increase the reaction rate of the enzyme. When the concentration of acetaldehyde is 8ml, the reaction rate of the enzyme is .UA ODW minutes when the concentration of valproate is 〇mM, and the reaction rate of the enzyme can be increased to O.OSCAOD34^/min when the concentration of valeraldehyde is 〇08mM, which is improved. 8 times. When the respective formaldehyde (8 mM) and acetaldehyde (8 mM) were used as the enzyme substrate, the reaction rate of the enzyme was increased from 0.001 (A〇D34〇/min) to 〇2〇 (a〇d, 4Q/min). The rate increased by more than 200 times, while the response rate of wild strain enzyme to formaldehyde and acetaldehyde was 4.54 times (YasUy0 Fujii, Y〇shiaki Yamasaki, Masahiro Matsumoto, Hiroyuki Nishida, Megumi 23 1325442

Hada, and Katsutoshi Ohkubo, 2004, "The Artifical Evolution of an Enzyme by Random Mutagenesis: The Development of Formaldehyde Dehydrogenase'', Biosci. Biotechnol, Biochem., 68, 8, 1722-1727), from which it is known The enzyme specificity of the inventive examples was about 44 times that of the wild strain.

While the present invention has been described in terms of several preferred embodiments, the invention is not intended to limit the invention, and the scope of the invention is intended to be Those who have the usual knowledge in the field of technology, when they can make any changes and applications,

24 1325442 [Simple description of the diagram] Figure la shows the 1/reaction rate versus 1/NAD concentration plot for the fixed acetal concentration (2 mM) and the different NAD concentrations. Figure lb shows a comparison of the reaction rate versus the NAD concentration for the fixed acetal concentration (2 mM) and the different NAD concentrations. Fig. 2a is a graph showing the 1/reaction rate versus 1/furfural concentration of the fixed NAD concentration (4 mM) and the different furfural concentrations in the comparative example. Figure 2b shows a comparison of the fixed NAD concentration (4 mM) and the reaction rate at different formaldehyde concentrations versus furfural concentration. Figure 3a shows a plot of 1/reaction rate vs. 1/NAD concentration for immobilized furfural concentration (7.5 mM) at different NAD concentrations. Figure 3b shows a plot of reaction rate vs. NAD concentration for immobilized furfural concentrations (7.5 mM) and different NAD concentrations. Figure 4a shows a plot of the fixed NAD concentration (7.6 mM) and the 1/reaction rate versus 1/furfural concentration for different furfural concentrations. Figure 4b shows a graph of the reaction rate versus formaldehyde concentration for the fixed NAD concentration (7.6 mM), different formaldehyde concentrations in the examples. Figure 5 shows the activity analysis of the modified furfural dehydrogenase using 8 mM furfural solution and varying concentrations of acetaldehyde. Fig. 6 is a graph showing the reaction rate of the mixture using different concentrations of acetaldehyde and furfural as a furfural dehydrogenase substrate. [Main component symbol description] None. 25 Sequence Listing [Serial Number] &lt;110> Biotechnology Development Center

&lt;120> Gene sequence of Pseudomonas putida non-glutamate sulfur-related formaldehyde dehydrogenase &lt;160&gt; 8 &lt;210&gt; 1 &lt;211&gt; 1200 &lt;212&gt; DNA &lt;213&gt; Pseudomonas putida &lt;; 400 &gt; 1 atgtctggta atcgtggtgt cgtttatctc ggtgcgggca aagtcgaagt gcagaagatc 60 gactacccga agatgcagga ccctcgcggc aagaagatcg aacacggggt gatcctgaag 120 gtggtctcca ccaacatctg cggctcggac cagcacatgg tgcgtggccg taccaccgcg 180 caggtcggcc tggtgctcgg ccacgagatc accggtgagg tgatcgagaa aggccgtgac 240 gtggaaaacc tgcagatcgg cgacctggta tccgtaccgt tcaacgtggc ctgcggccgc 300 tgccgttcct gcaaggaaat gcacaccggc gtgtgcctga ccgtcaaccc ggcccgtgcc 360 ggcggcgcct acggctatgt cgacatgggc gactggaccg gcggccaggc cgagtacgtg 420 ctcgttcctt acgctgactt caacctgctc aagctgccgg agcgcgacaa ggccatggag 480 aagatccgtg acctgacctg cctctccgac atcctgccca ccggctacca cggcgcggtc 540 accgctggtg tgggcccggg cagcaccgtg tacgttgccg gcgcaggtcc cgtcggcctc 600 gccgccgccg cctccgcccg cctgctgggt gctgccgtgg tcatcgtcgg cgacctcaac 660 cccgcccgcc tggcccacgc caaggcgca g ggcttcgaga ttgccgacct gtcgctggac 720 accccgctgc acgagcagat tgccgcgctg ctgggcgagc cggaagtgga ctgcgccgtc 780 gacgcagtgg gcttcgaagc gcgcggccac ggccatgaag gcgccaagca cgaagctccg 840 gccaccgtgc tcaactcgct gatgcaggtc acccgcgtgg ccggcaagat cggtatcccc 900 ggcctctacg tcaccgaaga tccgggcgcg gtggatgccg ccgccaagat cggcagcctg 960 agcatccgct tcggcctcgg ctgggcgaaa tcccacagct tccacaccgg ccagaccccg 1020 gtgatgaagt acaaccgcgc actcatgcag gcgatcatgt gggaccgcat caacatcgcc 1080 gaagtggtgg gcgtgcaggt catcagcctg Gacgacgcac cgcgtggcta tggcgagttc 1140 gatgccggcg taccgaagaa attcgtcatc gacccgcaca agaccttcag cgcggcctga 1200

&Lt; 210 &gt; 2 &lt; 211> 399 &lt; 212 &gt; PRT &lt; 213 &gt; Pseudomonas putida &lt; 400 &gt; 2 MSGNRGVVYL GAGKVEVQKI DYPKMQDPRG KKIEHGVILK WSTNICGSD QHMVRGRTTA 60 QVGLVLGHEI TGEVIEKGRD VENLQIGDLV SVPEWACGR CRSCKEMHTG VCLTVNPARA 120 GGAYGYVDMG DWTGGQAEYV LVPYADFNLL KLPERDKAME KIRDLTCLSD ILPTGYHGAV 180 TAGVGPGSTV YVAGAGPVGL AAAASARLLG AAWIVGDLN PARLAHAKAQ GFEIADLSLD 240 TPLHEQIAAL LGEPEVDCAV DAVGFEARGH GHEGAKHEAP ATVLNSLMQV TRVAGKIGIP 300 GLYVTEDPGA VDAAAKIGSL SIRFGLGWAK SHSFHTGQTP VMKYNRALMQ AIMWDRINIA 360 EWGVQVISL DDAPRGYGEF DAGVPKKFVI DPHKTFSAA 399 &lt;210> 3 &lt;211&gt; 1200

1325442

&Lt; 212 &gt; DNA &lt; 213 &gt; Pseudomonas putida &lt; 400> 3 atgtctggta atcgtggtgt cgtttatctc ggtgcgggca aagtcgaagt gcagaagatc gactacccga agatgcagga ccctcgcggc aagaagatcg aacacggggt gatcctgaag gtggtctcca ccaacatctg cggctcggac cagcacatgg tgcgtggccg taccaccgcg caggtcggcc tggtgctcgg ccacgagatc accggtgagg tgatcgagaa aggccgtgac gtggaaaacc tgcagatcgg cgacctggta tccgtaccgt tcaacgtggc ctgcggccgc tgccgttcct gcaaggaaat gcacaccggc gtgtgcctga ccgtcaaccc ggcccgtgcc ggcggcgcct acggctatgt cgacatgggc gactggaccg gcggccaggc cgagtacgtg ctcgttcctt acgctgactt caacctgctc aagctgccgg agcgcgacaa ggccatggag aagatccgtg acctgacctg cctctccgac atcctgccca ctggctacca cggcgcggtc accgctggtg tgggcccggg cagcaccgtg tacgttgccg gcgcaggtcc cgtcggcctc gccgccgccg cctccgcccg cctgctgggt gctgccgtgg tcatcgtcgg cgacctcaac cccgcccgcc tggcccacgc caaggcgcag ggcttcgaga ttgccgacct gtcgctggac accccgctgc acgagcagat tgccgcgctg ctgggcgagc cggaagtgga ctgcgccgtc gacgcagtgg gcttcgaagc gcgcggccac ggccatgttg gcgccgagca cgaagctccg Gccaccgtgc tcaac tcgct gatgcaggtc acccgtgtgg ccggcaagat cggtatcccc gggctctacg tcaccgaaga tccgggcgcg gtggatgccg ccgccaagat cggcagcctg agcatccgct tcggcctcgg ctgggcgaaa tcccacagct tccacaccgg ccagaccccg gtgatgaagt acaaccgcgc actcatgcag gcgatcatgt gggaccgcat caacatcgcc gaagtggtgg gcgtgcaggt catcagcctg gacgacgcac cgcgtggcta tggcgagttc gatgccggcg taccgaagaa attcgtcatc gacccgcaca agaccttcag cgcggcctga 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 3 1200

&Lt; 210 &gt; 4 &lt; 211 &gt; 399 &lt; 212 &gt; PRT &lt; 213 &gt; Pseudomonas putida &lt; 400 &gt; 4 MSGNRGWYL GAGKVEVQKI DYPKMQDPRG KKIEHGVILK VVSTNICGSD QHMVRGRTTA 60 QVGLVLGHEI TGEVIEKGRD VENLQIGDLV SVPFNVACGR CRSCKEMHTG VCLTVNPARA 120 GGAYGYVDMG DWTGGQAEYV LVPYADFNLL KLPERDKAME KIRDLTCLSD ILPTGYHGAV 180 TAGVGPGSTV YVAGAGPVGL AAAASARLLG AAWIVGDLN PARLAHAKAQ GFEIADLSLD 240 TPLHEQIAAL LGEPEVDCAV DAVGFEARGH GHEGAEHEAP ATVLNSLMQV TRVAGKIGIP 300 GLYVTEDPGA VDAAAKIGSL SIRFGLGWAK SHSFHTGQTP VMKYNRALMQ AIMWDRINIA 360 EVVGVQVISL DDAPRGYGEF DAGVPKKFVI DPHKTFSAA 399

&Lt; 210 &gt; 5 &lt; 211 &gt; 1200 &lt; 212 &gt; DNA &lt; 213 &gt; Pseudomonas putida &lt; 400 &gt; 5 atgtctggta atcgtggtgt cgtttatctc ggtgcgggca aagtcgaagt gcagaagatc 60 gactacccga agatgcagga ccctcgcggc aagaagatcg aacacggggt gatcctgaag 120 gtggtctcca ccaacatctg cggctcggac cagcacatgg tgcgtggccg taccaccgcg 180 caggtcggcc tggtgctcgg ccacgagatc Accggtgagg tgatcgagaa aggccgtgac 240 gtggaaaacc tgcagatcgg cgacctggta tccgtaccgt tcaacgtggc ctgcggccgc 300 360 360

1325442 tgccgttcct gcaaggaaat gcacaccggc gtgtgcctga ccgtcaaccc ggcccgtgcc ggcggcgcct acggctatgt cgacatgggc gactggaccg gcggccaggc cgagtacgtg ctcgttcctt acgctgactt caacctgctc aagctgccgg agcgcgacaa ggccatggag aagatccgtg acctgacctg cctctccgac atcctgccca ctggctacca cggcgcggtc accgctggtg tgggcccggg cagcaccgtg tacgttgccg gcgcaggtcc cgtcggcctc gccgccgccg cctccgcccg cctgctgggt gctgccgtgg tcatcgtcgg cgacctcaac cccgcccgcc tggcccacgc caaggcgcag ggcttcgaga ttgccgacct gtcgctggac accccgctgc acgagcagat tgccgcgctg ctgggcgagc cggaagtgga ctgcgccgtc gacgcagtgg gcttcgaagc gcgcggccac ggccatgtag gcgccgagca cgaagctccg gccaccgtgc tcaactcgct gatgcaggtc acccgtgtgg ccggcaagat cggtatcccc gggctctacg tcaccgaaga tccgggcgcg gtggatgccg ccgccaagat cggcagcctg agcatccgct tcggcctcgg ctgggcgaaa tcccacagct tccacaccgg ccagaccccg gtgatgaagt acaaccgcgc actcatgcag gcgatcatgt gggaccgcat caacatcgcc gaagtggtgg gcgtgcaggt catcagcctg gacgacgcac cgcgtggcta tggcgagttc gatgccggcg taccgaagaa attcgtcatc gacccgcaca agaccttcag cgcggcctga 42 0 480 540 600 660 *720 780 840 900 960 1020 1080 1140 1200

&lt;210&gt; 6 &lt;211>399 &lt;212&gt; PRT &lt;213&gt; Pseudomonas putida &lt;400&gt; 6 60

MSGNRGWYL GAGKVEVQKI DYPKMQDPRG KKIEHGVILK WSTNICGSD QHMVRGRTTA QVGLVLGHEI TGEVlEKGRD VENLQIGDLV SVPFNVACGR CRSCKEMHTG VCLTVNPARA 5 120 GGAYGYVDMG DWTGGQAEYV LVPYADFNLL KLPERDKAME KIRDLTCLSD ILPTGYHGAV 180 TAGVGPGSTV YVAGAGPVGL AAAASARLLG AAVVIVGDLN PARLAHAKAQ GFEIADLSLD 240 TPLHEQIAAL LGEPEVDCAV DAVGFEARGH GHEGAEHEAP ATVLNSLMQV TRVAGKIGIP 300 GLYVTEDPGA VDAAAKIGSL SIRFGLGWAK SHSFHTGQTP VMKYNRALMQ AIMWDRINIA 360 EWGVQVISL DDAPRGYGEF DAGVPKKFVI DPHKTFSAA 399

&Lt; 210 &gt; 7 &lt; 211 &gt; 1200 &lt; 212 &gt; DNA &lt; 213 &gt; Pseudomonas putida &lt; 400 &gt; 7 atgtctggta atcgtggtgt cgtttatctc ggtgcgggca aagtcgaagt gcagaagatc 60 gactacccga agatgcagga ccctcgcggc aagaagatcg aacacggggt gatcctgaag 120 gtggtctcca ccaacatctg cggctcggac cagcacatgg tgcgtggccg taccaccgcg 180 caggtcggcc tggtgctcgg ccacgagatc accggtgagg tgatcgagaa aggccgtgac 240 gtggaaaacc tgcagatcgg cgacctggta tccgtaccgt tcaacgtggc ctgcggccgc 300 tgccgttcct gcaaggaaat gcacaccggc gtgtgcctga ccgtcaaccc ggcccgtgcc 360 ggcggcgcct acggctatgt cgacatgggc gactggaccg gcggccaggc cgagtacgtg 420 ctcgttcctt acgctgactt caacctgctc aagctgccgg agcgcgacaa ggccatggag 480 aagatccgtg acctgacctg cctctccgac atcctgccca ctggctacca cggcgcggtc 540 accgctggtg tgggcccggg cagcaccgtg tacgttgccg gcgcaggtcc cgtcggcctc 600 gccgccgccg cctccgcccg cctgctgggt gctgccgtgg Tcatcgtcgg cgacctcaac 660 cccgcccgcc tggcccacgc caaggcgcag ggcttcgaga ttgccgacct gtcgctggac 720 780 1325442 accccgctgc acgagcagat gacgcagtgg gcttcgaagc gccaccgtgc tc aactcgct gggctctacg tcaccgaaga agcatccgct tcggcctcgg gtgatgaagt acaaccgcgc gaagtggtgg gcgtgcaggt gatgccggcg taccgaagaa tgccgcgctg ctgggcgagc gcgcggccac ggccatgaag gatgcaggtc acccgtgtgg tccgggcgcg gtggatgccg ctgggcgaaa tcccacagct actcatgcag gcgatcatgt catcagcctg gacgacgcac attcgtcatc gacccgcaca cggaagtgga ctgtgctgtc gcgccaagca cgaagctccg ccggcaagat cggtatcccc ccgccaagat cggcagcctg tccacaccgg ccagaccccg gggaccgcat caacatcgcc cgcgttgcta tggcgagttc agaccttcag cgcggcctga 840 900 960 1020 1080 1140 1200

&lt;210&gt; 8 &lt;211&gt; 399 &lt;212&gt; PRT &lt;213&gt; Pseudomonas putida &lt;400&gt; 8

MSGNRGVVYL GAGKVEVQKI DYPKMQDPRG KKIEHGVILK VVSTNICGSD QHMVRGRTTA QVGLVLGHEI TGEVIEKGRD VENLQIGDLV SVPFNVACGR CRSCKEMHTG VCLTVNPARA GGAYGYVDMG DWTGGQAEYV LVPYADFNLL KLPERDKAME KIRDLTCLSD ILPTGYHGAV TAGVGPGSTV YVAGAGPVGL A / ^ AASARLLG AAWIVGDLN PARLAHAKAQ GFEIADLSLD TPLHEQIAAL LGEPEVDCAV DAVGFEARGH GHEGAKHEAP ATVLNSLMQV TRVAGKIGIP GLYVTEDPGA VDAAAKIGSL SIRFGLGWAK SHSFHTGQTP VMKYNRALMQ AIMWDRINIA EWGVQVISL DDAPRCYGEF DAGVPKKFVI DPHKTFSAA 60 120 180 240 300 360 7 399

Claims (1)

1325442 [Male 1. A genetic sequence of a non-glutamate-based sulfur-related furfural dehydrogenase expressing Pseudomonas putida, which is Pseudomonas putida BCRC13897, And (i) the 522th position is mutated to thoracic pyrimidine, 711 position mutations to thoracic pyrimidine, 762 position mutations to thoracic pyrimidine, 818 position mutations to thoracic pyrimidine, 876 position mutations to thoracic pyrimidine, and 903 positions were mutated to bird droppings; or (ii) The 522th position is mutated to thoracic pyrimidine, 826 positions are mutated to guanosene, 876 positions are mutated to pleuropyrimidine, and 903 positions are mutated to guano; or (iii) the 522th position is mutated to Thymidine, 774 positions were mutated to pleuropyrimidine, 777 positions were mutated to pleuropyrimidine, 876 positions were mutated to pleuropyrimidine, 903 positions were mutated to guanosene, and 1126 positions were mutated to thoracic acid; iS X. Patent application scope: The specific activity of the non-glutamate-based sulfur-related formaldehyde dehydrogenase of Pseudomonas putida is higher than that of wild plants. 2. The isolated and purified gene sequence of Pseudomonas putida non-glutamate sulfur-related formaldehyde dehydrogenase as described in claim 1 of the patent application, wherein the 522th position is mutated to thoracic acid, 774 The positions were mutated to thoracic pyrimidine, 777 positions were mutated to pleuropyrimidine, 876 positions were mutated to pleuropyrimidine, 903 positions were mutated to guanosene, and 1126 positions were mutated to thymidine. 3. A segregation and purification of the amino acid sequence of Pseudomonas syringae non-glutamate thiol 1325442 related to furfural dehydrogenase, wherein Pseudomonas putida is Pseudomonas putida BCRC13897 And the 273th position is mutated to proline, the 275th position is mutated to glutamic acid or the 375th position is mutated to cysteine, and the Pseudomonas putida non-glutamate sulphur-related furfural The specific activity of dehydrogenase-enzyme is higher than that of wild plants. - 4. The amino acid sequence of the non-glutamate-based sulfur-related furfural dehydrogenase of Pseudomonas putida which is isolated and purified as described in claim 3 of the patent application, wherein the 375th position mutation It is cysteine. 5. The isolated and purified amino acid sequence of Pseudomonas putida non- face amine sulfur-related formaldehyde dehydrogenase according to claim 3, wherein the Pseudomonas putida is not bran The ratio of the reaction rate of furfural to acetaldehyde (enzyme specificity) of the amine-sulfur-related furfural dehydrogenase is higher than that of the wild strain. 6. A seed cell comprising the isolated and purified gene sequence of Pseudomonas putida non-glutamate sulfur-related formaldehyde dehydrogenase as described in the scope of the patent application, which causes the cell to exhibit functional love Pseudomonas putida non- face amine sulfur-related furfural dehydrogenase, and the cells include bacteria or yeast. 7. The cell of claim 6, wherein the bacterium is Escherichia coli. 8. A vector comprising the isolated and purified gene sequence of Pseudomonas putida non- face amine sulfur-related A. A cell comprising the vector of claim 8 wherein the cell comprises a bacterium or a yeast. 10. The cell of claim 9, wherein the bacterium is 1325442 E. coli or yeast.