TW200844228A - Pseudomonas putida glutathione-independent formaldehyde dehydrogenase and the gene and amino acid sequence thereof - Google Patents

Abstract

A gene sequence of Pseudomonas putida glutathione-independent formaldehyde dehydrogenase, wherein position 522 is T, position 711 is T, position 762 is T, position 818 is T, position 876 is T and position 903 is G. The specific activity of the enzyme is higher than that of wild type.

Description

200844228 . IX. INSTRUCTIONS: [Technical Field] The present invention is a modified enzyme, in particular, a glutathione-independent decanoate dehydrogenase . [Previous technique] NADH is a highly reducing compound in biological cells. It is mainly used as a coenzyme for enzymes in living cells. It provides the helium atom necessary for the enzyme reaction and then oxidizes to NAD. In general, the redoxase needs to use 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. Since NADH has a very high reducing activity, it is not easy to purify and extract from cells. Currently, commercially available NADH is produced by electrophoretic cell extraction and is expensive (2,200 yuan/g), so it cannot be applied to the redox enzyme process. Furfural dehydrogenase is an enzyme that can oxidize furfural to citric acid and can be divided into two types: glutathione-dependent and glutathione-independent. Among them, glutamate-related furfural dehydrogenase is present in most organisms, including Escherichia coli cells. This type of formaldehyde dehydrogenase uses glutamic acid and NAD as coenzymes to convert furfural to citric acid. Since it is necessary to use the surface aminosulfur to participate in the enzyme reaction, the industrial operation will increase the operating cost and operate 0296-A22078TWF(N2); 373; daphne 6 200844228 instead of glutamic acid-related a change factor, and thus is not suitable for large-scale applications.

Aldehyde dehydrogenase was discovered in 1979 by Pseudomonas syriae CCRC 13897 (Susumu Ogushi, Μ· Ando, D. Tsuru, "formaldehyde dehydrogenase from Pseudomonas putida: a zinc metalloenzyme", J·biochem· , 96, 1587-1591, 1984.) The advantage of non-glutamate aminosulfur-related furfural dehydrogenase is that it does not have to pass the surface aminosulfur as a coenzyme to directly convert NAD and furfural to tannic acid and high-priced NADH. NADH has high reductibility and can be widely used in industrial and people's livelihood fans. Therefore, non-faceted aminosulfur-related M-dehydrogenase has high market application value. Furfural dehydrogenase has low specific activity (Πυ/mg), high price (5(8) yuan/LJ), and low matrix specificity, which is unfavorable for application in the J1 process, making many deuterium dehydrogenase applications impossible. Commercial development and production. So many people are investing in

The modification of citrate dehydrogenase than that of Fujii et al.

0296-A22078TWF(N2);373;daphne but the specific activity of the enzyme is still the dehydrogenase gene sequence for sudden change, 'to achieve the purpose of enzyme modification, and with matrix specificity' and reduce enzyme 7 200844228 • use cost. The increase in enzyme activity and specificity can extend the application of decanoate dehydrogenase to industrial and residential applications. 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 pleuropyrimidine, 762 positions were mutated to pleuropyrimidine, 818 positions were mutated to pleuropyrimidine, 876 positions were mutated to pleuropyrimidine, and 903 positions were mutated to guanosene, or (ii) The 522th position was mutated to thoracic pyrimidine, 826 positions were mutated to guanosene, 876 positions were mutated to thymidine, and 903 positions were mutated to guanospermium, or (iii) 522th position was 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 pleuropyrimidine, and the love was The specific activity of Pseudomonas putida non-glutamate-based sulfur-related dehydrogenase was higher than that of wild plants. The present invention further provides an amino acid sequence of Pseudomonas putida non-glutamate sulfur-related furfural dehydrogenase, wherein the 272th position is mutated to valine, and the 275th position is mutated to glutamic acid or The 375 positions were mutated to cysteine, and the specific activity of the non-glutamate sulphur-related furfural dehydrogenase 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 at position 272 to valerine, 0296-A22078TWF (N2); 373; daphne 8 200844228 The 275th position was mutated to glutamic acid or the 375th position was mutated to cysteine, and its specific activity was higher than that of wild type strain. The present invention further provides a cell comprising the aforementioned gene sequence of Pseudomonas putida non-glutamate sulfur-related T aldehyde deaminase. This cell includes bacteria or yeast. The present invention further provides a vector comprising the aforementioned gene sequence of Pseudomonas putida non-glutamate 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. An embodiment of the present invention is a genetic sequence of Pseudomonas sinensis 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 black scorpion sputum, or (ii) 522th 0296-A22078TWF (N2); 373; daphne 9 200844228 position mutation to thoracic pyrimidine, 826 position mutation to bird feces, 876 positions were mutated to thoracic pyrimidine and 903 positions were mutated to guanosine, or (iii) 522 positions were mutated to thymidine, 774 positions were mutated to thymidine, and 777 positions were mutated to thymidine. 876 positions were mutated to thoracic pyrimidine, 903 positions were mutated to guanosene, and 1126 positions were mutated to thoracic acid. 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 CCRC13897. In one embodiment, the specific activity is about 100-150 U/mg. In a preferred embodiment, the 522th position is mutated to thoracic pyrimidine, 774 positions are mutated to thymidine, and 777 positions are mutated to chest line. Pyrimidine, 876 positions were mutated to pleuropyrimidine, 903 positions were mutated to guanosin, and 1126 positions were mutated to thymidine. Another embodiment of the present invention is an amino acid sequence of Pseudomonas aeruginosa non-glutamate sulfur-related furfural dehydrogenase in which mutations occur at positions 272, 275 and/or 375. In some embodiments, the 272th 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 above non-glutamate-based sulfur-related furfural dehydrogenase is higher than that of wild plants, and its specific activity is about 30-150 U/mg, while the specific activity of wild-type strain is 17 U/mg. . The above Pseudomonas putida includes Pseudomonas putida CCRC13897. 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 0296-A22078TWF (N2); 373; daphne 10 200844228. The ratio of the rate of reaction of the above-mentioned modified Pseudomonas putida non-viscous W wm non-avoiding sulphur-related transaugmentation enzymes is higher than that of wild plants, in the example - The reaction rate between formaldehyde and acetaldehyde is fast and the ratio of the ratio of the enzyme to the B is 4.53. In another embodiment, the sputum is modified, and the enzyme of the enzyme is reacted with acetaldehyde. The fast-acting value is 66 times that of the wild strain. The difference between the enzymes that have been modified and the lowest response to the combination of the two can reach more than one. Ben 4 Ming and Bei Shi type _ is a species of Pseudomonas sinensis non- face amine sulfur-related dehydrogenase, the amino acid sequence is 272 position mutation to valerine, the 275th position mutation It was mutated to the face acid or the 375th position to cysteine, and its specific activity was higher than that of the wild strain. Its specific activity is about 3〇_15〇 U/mg❿ The specific activity of the wild strain is 17 weeks. The above Pseudomonas putida includes Pseudomonas putida CCR (3) δ97. In the embodiment, the specific activity of the above-mentioned Pseudomonas syphilis sulphur-related sulphur-related dehydrogenase is about 100-150 U/mg. In a preferred embodiment, the amino acid of the enzyme The third position of the sequence was mutated to cysteine. In one embodiment, the ratio of the enzyme plate to the acetic acid reaction rate (Enzyme 14) is higher in the wild strain. A! The ratio of the reaction rate to the ethylene reaction 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 modified enzyme furfural to acetaldehyde is 66 times that of the wild plant. In a preferred embodiment, the difference in the minimum reaction between formaldehyde and acetaldehyde is 〇296-A22078TWF(N2); 373; daphne 200844228 ίο3 or higher. In yet another embodiment, the gene sequence of the present invention can be placed in a vector or a cell so that the application of the modified formaldehyde dehydrogenase to be mass-produced can include pTrc His A, pTrc His Β, pTrc His C, pET41a, 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. And this modified furfural dehydrogenase special chemicals and chemicals production process, formaldehyde removal, sterol detection and biofuel cells. In addition, the use of enzyme gene expression technology for mass production of enzymes can reduce the cost of enzyme production and increase the niche application of enzymes. The modified formaldehyde dehydrogenase can be applied to the novel NADH regeneration system and combined with citrate dehydrogenase to form a multi-enzyme NADH regeneration reaction. On the other hand, formaldehyde dehydrogenation can also be applied to biosensors and biofuel cells. [Examples] A method for obtaining the gene sequence of the non-glutamate-based sulfur-related furfural deactivating enzyme of Pseudomonas putida of the present invention is described below. Preparation of plastid DNA containing Pseudomonas syphilis furfural dehydrogenase gene First, Pseudomonas putida CCRC13897 strain was first placed in NA or C83 medium and cultured at 26 ° C for 44 hours. Next, the chromosomal DNA of Pseudomonas syringae CCRC13897 strain was extracted as a light weight 0296-A22078TWF (N2); 373; daphne 12 200844228 _ dehydrogenase machine gene amplification template DNA. The primer of the furfural dehydrogenase gene was designed to carry out a polymerase chain reaction (PCR). The furfural dehydrogenase gene fragment of Pseudomonas putida CCRC13897 is 1200 bp, and the designed primer is ·· 5, GAA TTC AGG CCG CGC TGA AGG TCT TGT GCG 3' and 5' GAA TTC ATG TCT GGT AAT CGTGGTGTCGTT3, . Thereafter, PCR is carried out to amplify the desired formaldehyde dehydrogenase gene fragment. The furfural dehydrogenase gene fragment was introduced into pTrc His B (kanamycin and ampicillin), while the wild strain of Pseudomonas putida CCRC13897 was obtained from the furfural dehydrogenase gene sequence. For the sequence identification number: 1; the amino acid sequence is the sequence identification number: 2. The prepared plasmid was transformed into E. coli BL-21 Blue competent cell (purchased from Stratagene). After confirming that the transformed cells have the target plastids, the competent cells are cultured in large amounts, and the plastid DNA is taken for storage. Preparation of a furfural dehydrogenase-modified strain PCR was carried out by using the above-described plastid DNA using GeneMorph II Random mutagenesis kit (purchased from Stratagene), and the PCR conditions were as shown in Table 1. The PcR product is then subjected to electrophoresis, and the target gene fragment is excised from the colloid for purification. After the target gene is inserted into the plastid, it is then subjected to a transgenic reaction with E. coli BL21 competent cells. After overnight culture in 2YT solid medium, the modified strain was selected. 0296-A22078TWF(N2);373;daphne 13 200844228 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/lcb 72〇 Screening of C 10 min modified strains for rapid screening and upgrading of 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) Modified by using a sterilized toothpick The strains were transferred from solid medium to 96-well shallow culture plates, in which the first culture well of each row was the control group (wild strain); (3) after overnight culture, 10 μΐ from the 96-well shallow culture plate. The bacterial 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 bacterial solution in the well was 0.5, and 5 μM of 100 mM IPTG was added; (4) After the culture for 16 hours, the broth of ΙΟΟμΙ was taken out to a 96-well shallow culture dish, and centrifuged at 5000 rpm for 10 minutes, and then the supernatant was decanted; (5) Add 20 μM lysozyme buffer to the 96-well shallow culture plate after centrifugation, shake it evenly, and let it stand at room temperature for 1 hour; (6) put it in the -80 °C refrigerator for 45 minutes. Take out, wait until the cells are completely warmed up, then put them into the -80 1 refrigerator for 45 minutes; (7) take out the 96-well shallow culture plate of the activity to be tested from the refrigerator, and return the cells to the temperature and then add them with an eight-claw micropipette. 180μ1 activity 0296-A22078TWF(N2); 373; daphne 14 200844228 test solution, and record the position of the strain with blue change faster than the control group; (8) after selecting 100-200 strains, repeat step (1) ~ ( 7), until the candidate strain is reduced to 10; (9) The candidate strain is cultured in a 250 ml flask, 0.5 ml of 100 mM IPTG is added when the amount of the bacteria is 0.5, and the culture is centrifuged at 7000 rpm for 10 minutes after the culture for six hours. (10) After pouring the supernatant, add lysozyme buffer (add volume: 0D value *0.5 mg cells *X ml culture solution / 50 mg = Y ml buffer solution), react at room temperature for 1 hour; (11) Cellulose with ultrasonic waves for 15 seconds, stop for 45 seconds, repeat 10 times . Centrifuge at 12000 rpm for 15 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 highest activity to be the next generation of the modified mother. Strains, and analysis of citrate dehydrogenase DNA and protein structure mutations; (13) repeated experimental steps of strain modification and strain screening until the specific activity of furfural dehydrogenase reached 50 U / mg. The composition of the enzyme activity test solution is shown in Table 2, and the results of the modified strain are shown in Table 3. 0296-A22078TWF(N2);373;daphne 200844228 Table 2. Composition of enzyme activity test solution μΐ/well storage (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, Furfural decarboxylase-modified strain

Rev. No. 1-4 2-5 8B9 4-1 5-1 6-23 7-2 8-5 Parent source WT 14 2-5 2-12 8B9 5-1 4-1 6-23 First stage rough Number of sieve strains 2997 1674 2924 2352 3554 4536 6384 4032 Second stage coarse screening thief 217 70 91 ~100 280 336 399 201 Number of crude strains in the third stage 15 13 12 38 26 26 16 Specific activity (U/mg) 48.28 50.88 50.88 32.59 40 25.71 15.70 120.9 Gene sequence changes c522t, c876t, c903g c522t, c876t, c903g c522t, g711t, g762t, a818t, c876t, c903g c522t, a826g, c876t, c903g c522t, c774t, c876t, c903g c522t, c774t, c777t, C876t, c903g c522t &gt; c774t, c777t, c876t, c903g, gll26t amino acid sequence changes - E273V K275E one - G375C 0296-A22078TWF (N2): 373; daphne 16 200844228 8B9, 4] and 8_5 three strains. The amino acid changes and the specific activity is higher. There is a single position of 7 and the second is changed to the 522th position and the mutation is chest line. Ding, 711 mouth position is greatly affected by the chest line, 762 positions mutation position mutation to chest line t, 876 position position H8 903 position mutation to bird fecal noise (sequence identification number: heart two = Lian 272 positions were mutated to proline (sequence identification number: 4). The position 4 was set to ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ The mutation was chest line and (4) coffee _峨: 5); the amino acid sequence was mutated to 275 positions to change to face acid (sequence identification number · 6). The genetic changes of 8.5 were changed from the 522th position to the chest line, 4 positions to the peach (4), and the m positions were changed to the chest line and the position was changed to the chest line. The bite, 9 〇 3 positions were mutated to the pterygium and 1126 positions were mutated to the chest line (Sequence Identification No. 7), and the amine group was defined as 帛 375 positions mutated to cysteine Amino acid (sequence recognition 5 tiger · 8 ) 突变 The mutant of the present invention has a better change in the activity of the 8-5 amino acid sequence, and the glycine acid at the 375 position is mutated to cysteine. Although the amino acid is a member of the inactive center, it is located in a helix of the NAD binding region. *Glycine is a must-have female base with the smallest steric structure of polarity. However, since hydrogen atoms in the molecule cannot form hydrogen bonds, they do not cause domains on the three-dimensional structure. Also, cysteine has a thiol group, 0296-A22078TWF(N2); 373; daphne 17 200844228. The change of the original glycine to cysteine causes the wild type furfural to be destructive. Change, this disulfide bond occurs on the inner side of the ce-helix, affecting the space in which the active center of the enzyme reacts with the matrix, may increase the diffusion effect of the matrix enzyme, and accelerate the rate of binding or detachment of the matrix to the enzyme. The kinetic parameter analysis of formaldehyde dehydrogenase The enzyme kinetics of the enzymes in the comparative example (commercially available) and the inventive example (8-5) of the dehydrogenase were analyzed as follows: A·for NAD Test Km and Vmax: (1) The concentration of furfural in the immobilized enzyme activity test solution is 7 mM; (2) Adjust the NAD concentration in the enzyme activity test solution to be 0.12 mM to 12 mM; (3) Add Ιμΐ purified formaldehyde dehydrogenase to (4) Record the value of 〇D34〇nm every other minute' for five minutes in a row; (5) Calculate the slope of each group by linear regression to obtain the reaction rate V; (5) Taking 1/NAD 〉thinity as a plot of 1/reaction rate, the slope of the slope can be determined as Km/Vmax and the intercept is 1/Vmax. (6) Km and Vmax are obtained by slope and intercept. B. Test Km and Vmax for the disk. (1) Km (NAD) in which the concentration of NAD in the immobilized enzyme activity test solution is 1〇; (2) The degree of furfural in the test enzyme activity test solution is 〇 to 12 mM; (3) Addition of Ιμΐ purified formaldehyde dehydrogenation Enzyme to test solution; (4) Record the value of 〇〇34〇_ every minute for five minutes; (5) Calculate the slope of each sample by linear regression, and obtain the reaction rate V; (6) plot 1/furfural concentration versus 1/reaction rate, the slope is Km/Vmax, the intercept is i/Vmax; (7) the slope and intercept are obtained ^] 0296-A22078TWF(N2); 373; daphne 18 200844228 Repeated steps A to b with Vmax 直到 until the difference between Vmax (NAD) and Vmax (furfural) is less than 5%. It is the kinetic parameter of formaldehyde dehydrogenase.

The results of the comparative examples are shown in Tables 4 and 5, panels 1 and b (fixed formaldehyde concentration (2 mM), 1/reaction rate vs. 1/nad concentration map and reaction rate versus NAD concentration at different NAD concentrations) and 2a and Figure b (fixed NAD concentration (4 mM), 1/reaction rate vs. 1 furfural concentration plot and reaction rate versus formaldehyde concentration plot for different formaldehyde concentrations). From the above results, it was found that the Km (NAD) of the comparative example was 0.384 mM, and the Km (NA )) was 368 368 mM. Table 4, fixed aldehyde concentration of the comparative example (2111_, change nad concentration to measure Km(NAD) NAD concentration _) reaction time 1 minute NADH concentration OD34Q ~------ 0.120 0.600 1.200 3.600 4.000 6.000 9.000 12.000 0.035 0.085 0.107 0.123 0.131 0.126 0.091 0.103 Reaction time 2 minutes NADH concentration ODm 〇 0.071 0.168 0.227 0.244 0.266 0.256 0.198 0.204 Reaction time 3 minutes NADH concentration OD34 〇 0.101 0.249 0.332 0.372 0.380 0377 0.304 0.319 Reaction time 4 minutes NADH concentration OD34 〇 0.127 0.316 0.436 0.484 0.511 0.507 0.390 0.412 Reaction time 5 minutes NADH concentration OD34〇0.150 0.377 0.520 0.605 0.620 0.615 0.508 0.526 0296-A22078TWF(N2);373;daphne 19 200844228 NADH production rate (V) (ΔOD/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 8.333 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), varying the concentration of furfural to measure Km (曱Aldehyde concentration (mM) 0.02 0.1 0.25 0.5 1 1.5 3 Reaction time 1 minute NADH Concentration OD34〇0.173 0.175 0.056 0.205 0.233 0.033 0.162 Reaction time 2 minutes NADH Concentration OD34〇0.172 0.190 0.107 0.247 0.313 0.169 0.340 Reaction time 3 minutes NADH Concentration 〇Dmq 0.178 0.214 0.157 0.293 0.395 0.301 0.498 Reaction time 4 minutes NADH Concentration ODm〇0.185 0.233 0.200 0.334 0.466 0.420 0.636 Reaction time 5 minutes NADH concentration OD34 〇 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/formaldehyde 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 0296-A22078TWF (N2); 373; daphne 20 200844228 •- aldehyde concentration (7.5 mM) ), 1/reaction rate versus l/NAD concentration plot and reaction rate vs. NAD concentration plot for different NAD concentrations) and plots 4a and b (fixed NAD concentration (7.6 mM), enthalpy/reaction rate vs. different furfural concentrations) y Furfural concentration map and reaction rate versus furfural concentration). From the above results, it can be inferred that the Km (NAD) is 1.752 niM, I (m (furfural) is 1.71 mM, Vmax is 120·8 U/mg, Kcat is 84/s, and the turnover rate is 3 million times per minute. As in the comparative example, when the NAD concentration exceeds 8 mM, the enzyme activity of the examples is also inhibited by ΐ, ^, but its tolerance to formaldehyde is relatively high, when the concentration of furfural is high. The enzyme activity did not change significantly when it exceeded 16 mM. On the other hand, the Km value of the examples was higher than the Km value of the comparative example. The results of the modification of the examples showed that when the enzyme activity increased during the upgrading process, it was not Because of the increased affinity for the matrix, it may be because the structure of the enzyme is changed, so that the activity of the enzyme is increased, but further experiments are needed. Table 6, the fixed aldehyde concentration (7.5 mM) of the example, the change NAD is rich 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 OD34 〇 0.025 0.101 0.173 0.235 0.254 0.251 0.224 0.202 Reaction time 2 minutes NADH concentration OD34 〇 0.041 0.196 0.319 0.48 1 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 0296-A22078TWF (N2); 373; daphne 21 200844228 NADH concentration OD34 〇 reaction time 4 minutes NADH concentration OD34 〇 0.064 0.360 0.584 0.880 0.927 0.878 reaction time 5 Minute NADH concentration OD34 〇 0.073 0.434 NADH production rate (V) (AOD/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 4780 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, the fixed NAD concentration (7.6 mM) of the examples, the change of furfural concentration to determine the 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 OD34Q 0.047 0.181 0.301 0.456 0.456 0.501 0.205 0.409 Reaction time 3 minutes NADH concentration OD34 〇 0.069 0.261 0.420 0.691 0.691 0.748 0.312 0.625 Reaction time 4 minutes NADH concentration OD34 〇 0.086 0.337 0.552 0.903 0.903 0.418 reaction 5-minute NADH concentration OD34〇0.104 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 0296-A22078TWF(N2); 373;daphne 22 200844228 (V)(M)D/min) 1/Formaldehyde concentration 49.751 1 /reaction rate 8.333 13.228

··355 355 4.338 4.090 9.497 4.454 0.250 0.167 0.125 0.083 0.067 Modified furfural dehydrogenase specificity analysis Using 8 mM furfural solution Yiwu a different degree of elongation of the sample of the furfural dehydrogenase activity analysis , 6 士 &gt; , , ~ fruit as the brother 5 shows. Shown in the figure

When different concentrations of acetaldehyde solution are used as a matrix, the activity of the enzyme is almost zero (in line). Even if the concentration of B-cut, song + &gt; 1讥G黾 is as high as 92 mM, the absorbance of NADH remains at 4 t ± α after five minutes. This result shows that it is not suitable for the modification of formaldehyde dehydrogenation. ^ ^ ^ ^ ^ The matrix of the modified enzyme has excellent specificity. The sixth embodiment shows a reaction rate diagram in which different concentrations of acetic acid and formic acid are used together as a formate dehydrogenase matrix. It is shown in the figure that when tannic acid and ethyl coexist in solution +, the activity of dehydrogenase is affected by the concentration of tannic acid. The presence of B does not increase the reaction rate of the enzyme. When the total concentration of acetaminophen is 8 mM furfural concentration is 〇 mM, the reaction rate of the enzyme is O.OOUAOD^o/min), and when the concentration of furfural is 〇〇8 mM, the reaction rate of the enzyme can be increased to 〇.〇8 ( ΔΟΓ) 34. /min), increased by 8 times. When comparing only furfural (8 mM) and acetaldehyde (8 mM) as the enzyme substrate, the reaction rate of the enzyme was increased from 〇·〇〇1(Δ〇Ι)34()/min) to 0 2〇(a〇). Dw minutes), the reaction rate increased by more than 200 times, while the response rate of wild strain enzyme to furfural and acetaldehyde was 4.54 times (Yasuyo Fujii, Y〇shiaki

Yamasaki, Masahiro Matsumoto, Hiroyuki Nishida, Megumi 0296-A22078TWF (N2); 373; daphne 23 200844228: 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), it is thus known that the enzyme specificity of the examples of the present invention is about 44 times that of the wild strain.

I* * is used in, not in j decoration, it is ^7 dry ^ 0296-A22078TWF (N2); 373; daphne 24 200844228 [Simple description of the figure] The first figure shows the fixed formaldehyde concentration (2mM), different NAD 1/reaction rate versus 1/NAD concentration plot at concentration. 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 plot of the reaction rate versus the furfural concentration for the fixed NAD concentration (4 mM) and the different furfural concentrations in the comparative example. 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/formaldehyde concentration for different furfural concentrations. Figure 4b shows a plot of the reaction rate versus formaldehyde concentration for the fixed NAD concentration (7.6 mM) and different furfural concentrations. Figure 5 shows the activity analysis of the modified furfural deaminase using an 8 mM furfural solution with 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. [Explanation of main component symbols] None 0 0296-A22078TWF(N2);373;daphne 25 200844228 Sequence Listing [Serial Number] &lt;110&gt; Biotechnology Development Center

&lt;12〇>Gene sequence of non-nonylamine sulfur-related furfural dehydrogenase of Pseudomonas putida &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 99c99c9cct 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 caaggcgcag ggcttcg aga ttgccgacct gtcgctggac 720 accccgctgc acgagcagat tgccgcgctg ctgggcgagc cggaagtgga ctgcgccgtc 780 gacgcagtgg gcttcgaagc gcgcggccac ggccatgaag gcgccaagca cgaagctccg 840 0296-A22078TWF (N2); 373; daphne 26 900 200844228 gccaccgtgc tcaactcgct ggcctctacg tcaccgaaga agcatccgct tcggcctcgg gtgatgaagt acaaccgcgc gaagtggtgg gcgtgcaggt gatgccggcg taccgaagaa gatgcaggtc acccgcgtgg tccgggcgcg gtggatgccg ctgggcgaaa tcccacagct actcatgcag Gcgatcatgt catcagcctg gacgacgcac attcgtcatc gacccgcaca ccggcaagat cggtatcccc ccgccaagat cggcagcctg tccacaccgg ccagaccccg gggaccgcat caacatcgcc cgcgtggcta tggcgagttc agaccttcag cgcggcctga 960 1020 1080 1140 1200 /

&lt;210&gt; 2 &lt;211>399 &lt;212&gt; PRT &lt;213&gt; Pseudomonas putida &lt;400&gt; 2

MSGNRGWYL GAGKVEVQKI DYPKMQDPRG KKIEHGVILK WSTNICGSD QHMVRGRTTA QVGLVLGHEI TGEVIEKGRD VENLQIGDLV SVPFNVACGR CRSCKEMHTG VCLTVNPARA / GGAYGYVDMG DWTGGQAEYV LVPYADFNLL KLPERDKAME KIRDLTCLSD ILPTGYHGAV

TAGVGPGSTV YVAGAGPVGL AAAASARLLG AAWIVGDLN PARLAHAKAQ GFEIADLSLD TPLHEQIAAL LGEPEVDCAV DAVGFEARGH GHEGAKHEAP ATVLNSLMQV TRVAGKIGIP GLYVTEDPGA VDAAAKIGSL SIRFGLGWAK SHSFHTGQTP VMKYNRALMQ AIMWDRINIA EWGVQVISL DDAPRGYGEF DAGVPKKFVI DPHKTFSAA &lt; 210 &gt; 3 &lt; 21 1 &gt; 1200 0296-A22078TWF (N2); 373; daphne 60 120 180 240 300 360 399 27 200844228

r &lt; 212 &gt; DNA &lt; 213 &gt; Pseudomonas putida &lt; 400 &gt; 3 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 f

I 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 accccgctgc acgagcagat tgccgcgctg ctgggcgagc cggaagtgga ctgcgccgtc 780 gacgcagtgg gcttcgaagc gcgcggccac ggccatgttg gcgccgagca cgaagctccg 840 gccaccgtgc tcaactcgct gatgcaggtc acccgtgtgg ccggcaagat cggtatcccc 900 gggctctacg 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 Tacc Gaagaa attcgtcatc gacccgcaca agaccttcag cgcggcctga 1200 0296-A22078TWF(N2);373;daphne 28 200844228

&lt;210&gt; 4 &lt;211> 399 &lt;212&gt; PRT &lt;213&gt; Pseudomonas putida &lt;400&gt; 4

MSGNRGWYL GAGKVEVQKI DYPKMQDPRG KKIEHGVILK WSTN work CGSD QHMVRGRTTA QVGLVLGHEI TGEVIEKGRD VENLQIGDLV SVPFNVACGR CRSCKEMHTG VCLTVNPARA GGAYGYVDMG DWTGGQAEYV LVPYADFNLL KLPERDKAME KIRDLTCLSD ILPTGYHGAV TAGVGPGSTV YVAGAGPVGL AAAASARLLG AAWIVGDLN PARLAHAKAQ GFEIADLSLD TPLHEQIAAL LGEPEVDCAV DAVGFEARGH GHEGAEHEAP ATVLNSLMQV TRVAGKIGIP GLYVTEDPGA VDAAAKIGSL SIRFGLGWAK SHSFHTGQTP VMKYNRALMQ AIMWDRINIA EWGVQVISL DDAPRGYGEF DAGVPKKFV work DPHKTFSAA

&Lt; 210 &gt; 5 &lt; 211> 1200 &lt; 212 &gt; DNA &lt; 213 &gt; Pseudomonas putida &lt; 400 &gt; 5 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 0296-A22078TWF (N2); 373; daphne 60 120 180 240 300 360 399 60 120 180 240 29 300 200844228 ggcggcgcct ctcgttcctt aagatccgtg accgctggtg gccgccgccg cccgcccgcc accccgctgc gacgcagtgg gccaccgtgc gggctctacg agcatccgct gtgatgaagt gaagtggtgg gatgccggcg gcaaggaaat acggctatgt acgctgactt acctgacctg tgggcccggg cctccgcccg tggcccacgc acgagcagat gcttcgaagc tcaactcgct tcaccgaaga tcggcctcgg acaaccgcgc gcgtgcaggt taccgaagaa gcacaccggc cgacatgggc caacctgctc cctctccgac cagcaccgtg cctgctgggt caaggcgcag tgccgcgctg gcgcggccac gatgcaggtc tccgggcgcg ctgggcgaaa actcatgcag catcagcctg attcgtcatc gt gtgcctga gactggaccg aagctgccgg atcctgccca tacgttgccg gctgccgtgg ggcttcgaga ctgggcgagc ggccatgtag acccgtgtgg gtggatgccg tcccacagct gcgatcatgt gacgacgcac gacccgcaca ccgtcaaccc gcggccaggc agcgcgacaa ctggctacca gcgcaggtcc tcatcgtcgg ttgccgacct cggaagtgga gcgccgagca ccggcaagat ccgccaagat tccacaccgg gggaccgcat cgcgtggcta agaccttcag ggcccgtgcc cgagtacgtg ggccatggag cggcgcggtc cgtcggcctc cgacctcaac gtcgctggac ctgcgccgtc cgaagctccg cggtatcccc cggcagcctg ccagaccccg caacatcgcc tggcgagttc cgcggcctga 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200

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

MSGNRGWYL · GAGKVEVQKI DYPKMQDPRG KKIEHGVILK WSTNICGSD QHMVRGRTTA QVGLVLGHEI TGEVIEKGRD VENLQIGDLV SVPFNVACGR CRSCKEMHTG VCLTVNPARA 0296-A22078TWF (N2); 373; daphne 30 120 180 200844228 GGAYGYVDMG DWTGGQAEYV LVPYADFNLL KLPERDKAME KIRDLTCLSD ILPTGYHGAV TAGVGPGSTV YVAGAGPVGL AAAASARLLG AAWIVGDLN PARLAHAKAQ GFEIADLSLD TPLHEQIAAL LGEPEVDCAV DAVGFEARGH GHEGAEHEAP ATVLNSLMQV TRVAGKIGIP GLYVTEDPGA VDAAAKIGSL SIRFGLGWAK SHSFHTGQTP VMKYNRALMQ AIMWDRINIA EWGVQVISL DDAPRGYGEF DAGVPKKFVI DPHKTFSAA &lt;210&gt; 7 f ' %

&Lt; 211 &gt; 1200 &lt; 212 &gt; DNA &lt; 213 &gt; Pseudomonas putida &lt; 400 &gt; 7 atgtctggta atcgtggtgt cgtttatctc ggtgcgggca aagtcgaagt gcagaagatc gactacccga agatgcagga ccctcgcggc aagaagatcg aacacggggt gatcctgaag gtggtctcca ccaacatctg cggctcggac cagcacatgg tgcgtggccg taccaccgcg caggtcggcc tggtgctcgg ccacgagatc accggtgagg tgatcgagaa aggccgtgac i - 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 0296-A22078TWF (N2 ); 373; daphne 240 300 360 399 60 120 180 240 300 360 420 480 540 600 660 31 720 780 200844228 accccgctgc acgag cagat gacgcagtgg gcttcgaagc gccaccgtgc tcaactcgct 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>PRT &lt;213&gt; Pseudomonas putida &lt;400&gt; 8

/ MSGNRGWYL GAGKVEVQKI DYPKMQDPRG KKIEHGVILK WSTNICGSD QHMVRGRTTA \

QVGLVLGHEI TGEVIEKGRD VENLQIGDLV SVPFNVACGR CRSCKEMHTG VCLTVNPARA GGAYGYVDMG DWTGGQAEYV LVPYADFNLL KLPERDKAME KIRDLTCLSD ILPTGYHGAV TAGVGPGSTV YVAGAGPVGL AAAASARLLG AAWIVGDLN PARLAHAKAQ GFEIADLSLD TPLHEQIAAL LGEPEVDCAV DAVGFEARGH GHEGAKHEAP ATVLNSLMQV TRVAGKIGIP GLYVTEDPGA VDAAAKIGSL SIRFGLGWAK SHSFHTGQTP VMKYNRALMQ AIMWDRINIA EWGVQVISL DDAPRCYGEF DAGVPKKFVI DPHKTFSAA 0296-A22078TWF (N2); 373; daphne 60 120 180 240 300 360 32 399

Claims (1)

200844228 X. Patent application scope: 1. The gene sequence of a non-glutamate-based sulfur-related furfural dehydrogenase from Pseudomonas putida, in which (i) the 522th position is mutated to thoracic acid and 711 positions are mutated to Thymidine, 762 positions mutated to thoracic pyrimidine, 818 positions were mutated to pleuropyrimidine, 876 positions were mutated to thymidine, and 903 positions were mutated to guanine; or (ii) the 522th position was mutated to Thymidine, 826 positions were mutated to guanosene, 876 positions were mutated to thymidine, and 903 positions were mutated to guanosine; or (iii) 522 positions were mutated to thoracic, and 774 positions were mutated Thymidine, 777 position mutations to thoracic pyrimidine, 876 positions mutation to thoracic pyrimidine, 903 position mutations to guanosene sputum, and 1126 positions mutation to thymidine; and wherein Pseudomonas putida is non- The specific activity of glutamine-sulfur-related furfural dehydrogenase is higher than that of wild plants. 2. The gene sequence of Pseudomonas putida non-glutamate sulfur-related furfural dehydrogenase as described in claim 1, wherein the Pseudomonas putida is Pseudomonas putida CCRC13897. 3. The gene sequence of Pseudomonas putida non-glutamate-sulfur-related furfural dehydrogenase as described in the first paragraph of the patent application, wherein the 522th position is mutated to thoracic acid, and 774 positions are mutated to chest. Linear pyrimidines, 777 positions were mutated to thoracic pyrimidine, 876 positions were mutated to pleuropyrimidine, 903 positions were mutated to guanosin, and 1126 positions were mutated to thoracic acid. 0296-A22078TWF(N2);373;daphne 33 200844228 4. -Pseudomonas aeruginosa non-chamoylthio-related methylamine group, wherein the milk position is mutated to valerine, the 27th position dog For the face acid or the 375th position is mutated to semi-proline, == the specific activity of the non-faceted amine-sulfur-related ADS dehydrogenase is higher than that of the wild strain. 5. The seric acid sequence of Pseudomonas putida, non-face amine, and sulfur-related A (tetra) hydrogenase, as described in claim 4 of claim 4, wherein the fungus is Pseudomonas putida CCRC13897. Cell = as shown in the patent scope, item 4 of the scented singular cell _ off (four) amnesium amino acid sequence, in which the first dog is treated with cysteine. Meimei Furnace It applies for the non-face amine of Pseudomonas sylvesae described in item 4 of the patent scope:: Amino acid sequence of hydrogenase, which::=Correlation--A-enzyme--; ratio (Enzyme specificity) is higher than wild plants.匕(4) 臭假假单钱丝丝相相" _ Hydrogen 1 base &amp; sequence is the 272th position mutated to arginine, 帛 275 dogs to glutamic acid or the 375th position is higher than wild plants. Set a large 4 + (four) acid, and its ratio: · Please ask the patent range of the eighth item of the love stinking single base ^ related formaldehyde dehydrogenase, which is the female bacterium BCRC13897. As described in the second item, the pseudo-A (four)-receptor's amine-order sequence (four) 375 positions protrudes ^96-A22078TWF (N2); 373; daphne 200844228 becomes cysteine. " As described in the patent application The love of odor-based sulfur-related A (10) hydrogenase, its methyl-acetic acid reverse:: the two countries non-Chu-stamin specific - higher than the wild strain. ~ 岐 岐 岐 ( ( 酵 酵 酵 酵 种 种 种 种 酵 种 种 种 种 种 种 种 种 种 种 种 种 酵 种 种 种 种 种 种 种 种 种 种 种 种 种 种 种 种 种 种 种 种 种 种 种 种 种 种 种 种 种The cell of claim 12, wherein the cell is Escherichia coli. Μ,, 囷σσ Μ a carrier, including the Pseudomonas putida non- face amine group described in claim 1 A gene sequence of a sulfur-related furfural dehydrogenase. A cell comprising the vector of claim 14, wherein the cell comprises a bacterium or a yeast. a cell, wherein the bacterium is Escherichia coli or yeast. 〇296-A22078TWF(N2); 373; daphm 35