Formaldehyde dehydrogenase and method for preparing formaldehyde using same

Abstract

This invention relates to a novel formaldehyde dehydrogenase expressed by a formaldehyde dehydrogenase gene and having independent reduction activity for formic acid, a method of preparing the formaldehyde dehydrogenase from a strain transformed with a recombinant expression vector including the gene, and a method of producing formaldehyde from formic acid through a reduction reaction of the formaldehyde dehydrogenase.

Claims

1. A method of producing formaldehyde from a substrate through a reduction reaction with formaldehyde dehydrogenase having reduction activity and comprising an amino acid sequence of SEQ ID NO:2 by treating the substrate with the formaldehyde dehydrogenase using NADH as a coenzyme, wherein the substrate is formic acid.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the vector map of vector pET28a in which a fragment having a formaldehyde dehydrogenase gene, selected from chromosomes of Burkholderia multivorans (KTCT 2970), is cloned into a vector used for E. coli;

(2) FIG. 2 shows a process of manufacturing an expression vector including the formaldehyde dehydrogenase gene derived from Burkholderia multivorans;

(3) FIG. 3 shows the SDS-PAGE gel of formaldehyde dehydrogenase derived from Burkholderia multivorans, 1 indicating the size marker, 2 indicating the water-soluble protein expressed using a strain transformed with an expression vector, 3 indicating the insoluble protein of formaldehyde dehydrogenase, and 4 indicating the water-soluble protein of formaldehyde dehydrogenase;

(4) FIG. 4 shows the optimal pH for producing formaldehyde through a reduction reaction; and

(5) FIG. 5 is a graph showing the kinetic parameters of formaldehyde dehydrogenase.

MODE FOR INVENTION

(6) A better understanding of the present invention may be obtained via the following examples, which are set forth to illustrate, but are not to be construed as limiting the present invention.

EXAMPLE 1: Cloning of Novel Formaldehyde Dehydrogenase Gene from Burkholderia multivorans

(7) In the case of genes having similar functions, individual base sequences and sizes are known to be similar to some extent. Thus, the gene of formaldehyde dehydrogenase of Burkholderia multivorans is estimated to have a size of about 1.2 kb, and all genes of formaldehyde dehydrogenase of Burkholderia multivorans were cloned based on already known formaldehyde dehydrogenase base sequences of other strains.

(8) An E. coli pET28a vector was used in the cloning process. An LB medium having a typical composition was used for culturing E. coli, and culturing of Burkholderia multivorans was performed on malt extract peptone agar. The plate media of E. coli, were an LB agar plate and an agar plate composed of 3 to 5% of sugar, 0.3 to 0.5% of a beef extract, 0.9 to 1.1% of Bacto peptone, and 1.3 to 1.7% of agar. If necessary, 50 g/ml of ampicillin was added. Burkholderia multivorans was inoculated into a 250 ml Erlenmeyer flask containing 50 ml of a culture medium and cultured at 37 C. and 200 rpm for 5 days, and E. coli was cultured at 37 C. and 200 rpm for 16 hr.

(9) Most DNA was identified on agarose gel (TAE buffer, 0.5%) using an electrophoresis method, and the DNA band was purified on the gel using a QiaXII gel extraction device (QIAGEN, USA), and DNA ligation was used T4 DNA ligase (NEB). Also, RNA extraction of Burkholderia multivorans was performed using a Qiagen plant total RNA kit (QIAGEN), and the reverse transcriptase for the synthesis of cDNA was Oligo-dT RT-mix (Intron).

(10) In order to clone the formaldehyde dehydrogenase gene, the Burkholderia multivorans chromosome was separated. To partially amplify the Burkholderia multivorans formaldehyde dehydrogenase gene, nonspecific primers (degenerated primers), BinFalDH_5-SP1 atttgyggcagcgatcwrcatatgkwysrc (SEQ ID NO:3) and BmFaIDH_3-SP1-Attggcrthccgggnytgtaygtgmcc (SEQ ID NO:4) were manufactured, based on the already known formaldehyde dehydrogenase base sequences of other strains, and were used so that a portion of the formaldehyde dehydrogenase gene 780 bp long was amplified in the Burkholderia multivorans chromosome using PCR.

(11) Using Sac1, Not1, Xho1 and Sal1 as restriction enzymes having no restriction sites in the base sequence of the amplified portion as above, genomic DNA of Burkholderia multivorans was completely cleaved. Furthermore, a radiolabeled probe was manufactured using the DNA fragment obtained by PCR, and was used to search for a DNA fragment having a gene of interest through southern hybridization. A desired gene was searched for using a fragment cut with Sac1 of about 2.7 kb and a fragment cut with Sal1 of about 5.3 kb. A DNA fragment of about 2.7 kb, separated after cleavage of the Burkholderia multivorans chromosome with Sac1, and a DNA fragment of about 5.3 kb, cut with Sal1, were cloned into WC and called pUC-faldh (FIG. 1).

(12) Colony hybridization was performed using the probe having a size of 780 bp in the pUC-faldh library, and thus a clone having the desired formaldehyde dehydrogenase gene was determined. The base sequence was analyzed using the determined clone, whereby the total 1,197 bp-long gene base sequence of formaldehyde dehydrogenase was found (SEQ ID NO:1), and had a size similar to the formaldehyde dehydrogenase gene as proven in the other strains.

EXAMPLE 2: Preparation of Recombinant Expression Vector and Recombinant Strain

(13) In order to express a large amount of formaldehyde dehydrogenase in E. coli using the gene encoding formaldehyde dehydrogenase of Example 1, the enzyme gene was inserted into BarriFIT and Xho1 sites of the expression vector pET28a (Novagen, USA) and then transformed to E. coli B1-21 (DE3) (NEB, England) (FIG. 2).

EXAMPLE 3: Expression of Recombinant Formaldehyde Dehydrogenase and Isolation

(14) The recombinant strain of Example 2 was inoculated into an LB medium and cultured at 37 C. for 24 hr, and the protein expressed on the SDS-PAGE gel was identified (FIG. 3).

(15) In order to purify the recombinant formaldehyde dehydrogenase expressed using the method of Example 2, the recombinant strain culture solution was centrifuged (8000g, 10 min), and only the cell mass was collected and sonicated to thus lyse the cell wall of E. coli, followed by centrifugation at 20,000g for 20 min to remove the precipitate (cell mass), thus yielding the supernatant. Thereafter, final Ni-NTA His-tag interaction chromatography (Qiagen, Germany) was performed, thereby isolating a recombinant formaldehyde dehydrogenase.

EXAMPLE 4: Optimal pH for Producing Formaldehyde Through Reduction Reaction

(16) The test for the production of formaldehyde using the formaldehyde dehydrogenase of Example 3 was performed under the following conditions. In the method of producing formaldehyde of the present invention using the formaldehyde dehydrogenase, the amount of formaldehyde that was produced was measured depending on changes in pH during the reaction.

(17) Enzyme purification was carried out as in Example 3, and the amount of formaldehyde that was produced was measured using a 100 mM substrate solution at 25 C. in the pH range of 4.0 to 10.0. As shown in FIG. 4, the amount of formaldehyde that was produced was the greatest at pH 7.0. Thus, the optimal pH was determined to be 7.0 in the method of producing formaldehyde of the present invention.

EXAMPLE 5: Metal Ion Effect

(18) In order to evaluate the effect of the metal ion of the purified formaldehyde dehydrogenase on enzymatic activity, this test was performed. Each of MgCl.sub.2, MnCl.sub.2, CoCl.sub.2, ZnCl.sub.2, FeCl.sub.2, CuSO.sub.4, CoCl.sub.2, HgCl.sub.2, BaCl and KCl at final concentrations of 1 mM and 5 mM was added to the enzyme reaction solution, and residual activity of the enzyme was measured. The effects of various metals at concentrations of 1 mM and 5 mM on formaldehyde dehydrogenase activity are shown in Table 1 below. The formaldehyde dehydrogenase of the present invention exhibited 2.7 times as much enzyme activity in the presence of 5 frill Mg.sup.2+ than without it (Table 1).

(19) TABLE-US-00001 TABLE 1 Relative activity (%) Relative activity (%) Metal ions 1 mM 5 mM MnCl.sub.2 62.32 111.6 MgCl.sub.2 147.8 269.6 CaCl.sub.2 50.67 75.96 ZnCl.sub.2 63.99 192.2 CuSO.sub.4 124.3 91.30 CoCl.sub.2 58.39 72.60 BaCl.sub.2 98.55 115.1 KCl 104.0 94.49 FeCl.sub.2 ND ND HgCl.sub.2 ND ND None 15.00 13.00

(20) Table 1 shows the effects of metal ions on the activity of formaldehyde dehydrogenase.

EXAMPLE 6: Kinetic Parameters of Formaldehyde Dehydrogenase

(21) Enzymatic reaction was carried out using formic acid as a substrate at various concentrations (0.125 to 5 mM), after which kinetic parameters thereof were measured through nonlinear regression analysis (FIG. 5). As for the formaldehyde dehydrogenase, the K.sub.m value for NADH was determined to be about 0.19 mM, and the K.sub.m value forformic acid was about 1.7 mM, Vmax being determined to be about 4.7 U mg-protein.sup.1.

EXAMPLE 7: Test for Producing Formaldehyde from Formic Acid

(22) The test for producing formaldehyde using Burkholderia multivorans-derived formaldehyde dehydrogenase was carried out under optimal conditions. A 10 mM substrate was reacted with 20 g of formaldehyde dehydrogenase in the reaction solution for 3 hr under conditions of a pH of 7.0 and a reaction temperature of 30 C., whereby a conversion rate of about 27% resulted. This is the first report on the direct production of formaldehyde from formic acid through hioconversion.