Progress 11/15/02 to 11/14/05
Outputs
There are six specific objectives in this project:1) purification of secondary alcohol dehydrogenase (2-ADH) to homogeneity from M. luteus WIUJH-20; 2) based on available amino acid sequence to design and synthesize degenerated oligonucleotides; 3) use degenerated oligonucleotides as primer to prepare nucleotide acid probes by polymerase Chain Reaction; 4) construct a partial DNA bank from M. luteus WIUJH-20; 5) clone and characterize 2-ADH gene; and 6) localize 2-ADH in M. luteus WIUJH-20. We have accomplished the specific aims of 1, 2, and 4, and a portion of the specific aim 3. With regard to the specific aim 1, the enzyme, 2-ADH, has been purified to homogeneity, and is a homotetramer. The native molecular weight of this enzyme is about 160 KDa; the isoelectric point of this enzyme is about 4.8. This enzyme is NAD+ dependent and the optimal pH is about 9. With regard to the specific aim 2, the 10 amino acid residues of its N-terminus have been sequenced by the
Protein Facility at University of Illinois at Urbana-Champaign. With regard to the specific aim 3, based on the available N-terminal amino acids residues, the degenerated oligonucleotides probes were synthesized by Integrated DNA Technologies, Inc. We did not do in vitro synthesis of a specific probe by PCR because we had difficulty to obtain enough internal peptide fragments for N-terminal sequencing. We are now able to obtain internal peptide for N-terminal sequencing. With regard to the specific aims 4 and 5, the degenerated oligonucleotide probes hybridized to the DNA fragments of 0.5-0.7, 1.0-1.6, and 2.0-3.0 kilo base pairs, respectively, in a Southern genomic blot. The corresponding DNA fragment were then isolated from an agarose gel and purified. The purified DNA fragments were then ligated with pBluescript SK (-) vector that has been subjecting to Bam HI digestion to construct a partial DNA bank from M. luteus WIUJH-20. The ligation product was used to transform JM109
competent cells, and positive transformants were selected in the presence of X-gal and IPTG. Four positive transformants were obtained for chimera plasmid isolation and DNA sequencing. None of the transformants contained the sequence corresponding to the degenerated oligonucleotide probes. We are now able to obtain enough internal peptide fragment for N-terminal sequencing, which will allow us to synthesize a part 2-ADH by PCR to facilitate cloning of 2-ADH in the future.
Impacts
The long-term objective of this project is strain improvement of Nocardial cholesterolicum NRRL 5767 for production of 10-hydroxyl fatty acid derivatives from soapstock (a by-product of edible oil refinery), which contains about 10-15% oleic and linoleic acids. Currently, soapstock is sold to chicken industry in low cash value as an ingredient of feed. 10-hydroxyl fatty acid derivatives are potential for industrial applications. Purification of 2-ADH and study of its gene will facilitate our long-term goal to develop an economic feasible process to convert oleic and linoleic acid in soapstock to value-added compounds for industrial usage. Eventually, corn and soybean farmers will benefit from this research.
Publications
- Weber, S.A., Holton, N.W., Parks, J. K., Huang, J.-K., Wen, L. 2005. Purification And CNBr Cleavage of Secondary Alcohol Dehydrogenase From Micrococcus Luteus WIUJ-H20. Central States Universities Incorporated (CSUI) Conference, November 4-5.
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Progress 02/01/04 to 02/01/05
Outputs
RESULTS FROM NRI SUPPORT CSREES award number: Seed Grant 2003-35504-12870 The amount of support: $74,973 plus $17,284 matching funds from Western Illinois Univ. The period of support: 11-15-02 to 11-14-05 (includes approved one year extension) Title of the project: Molecular cloning of a secondary alcohol dehydrogenase from A Micrococcus luteus WIUJH 20. Summary of the results: The objective of the project is to purify, characterize, and clone a microbial secondary alcohol dehydrogenase. In the previous report (11-15-02 to 02-01-04) we reported the purification and characterization of the secondary alcohol dehydrogenase; used the available N-terminus amino acid sequence to synthesize degenerate oligonucleotide probes to hybridize the genomic blot (Micrococcus luteus WIUJH 20 DNA/Bam HI digestion), and identified the candidate DNA fragments for partial gemonic DNA library construction. In this report, we report that: (1).The degenerate oligonucleotide probes hybridized
to fragments of 0.5-0.7,1.0-1.6, and 2.0-3.0 Kb, respectively. (2).The corresponding fragmenrts were isolated from agarose gel and purified. (3).The purified DNA fragments were ligated with pBluescript SK(-) vector that has been cut with Bam HI restriction enzyme and transformed to JM 109 host cells. The positive transformants were selected in the presence of X-gal and IPTG. (4). Four positive candidate clones were selected for individual chimera plasmid isolation and subjected to DNA sequencing. Unfortunately, none of the clones contained the sequence that corresponded to the degenerate oligonucleotide probes. We pursued another approach to digest the purified secondary alcohol dehydrogenase with trypsin and purified the peptide fragments with the state-of-the-arts HPLC. Several fragments were purified, and will be sent to the Protein Facility at University of Illinois at Urbana-Champaign for N-terminal amino acid sequence of the fragments. Eventually, the available amino acid
sequences will be used for synthesis of oligonucleotide probes for polymerase Chain Reaction (PCR) to amplify the secondary alcohol dehydrogenase. This new approach should give better chance of success in identifying the gene codes for secondary alcohol dehydrogenase. Our project has been delaying due to the unexpected results. We had requested a one year extension (which will be ended in 11-14-05) to accomplish our proposed work. Our request has been approved. Professional presentation (* graduate student) James T. Lamer*, Jenq Kuen Huang, and Lisa Wen (2004) "Secondary alcohol dehydrogenase from micrococcus luteus WIUJH20." 15th Annual Illinois Student Research Conference, Northeastern Illinois University, Chicago, Illinois. March 13. J. T. Lamer*, J. K. Huang, and L. Wen (2004) "Characteristics of secondary alcohol dehydrogenase from micrococcus luteus WIUJH20." 96th Annual Meeting, Illinois State Academy of Science, April.
Impacts
The purification of this enzyme will allow us to proceed the proposed work for the second year to study the gene responsible for the synthesis of this enzyme, and to introduce this gene into Nocardial cholesterolicum NRRL 5767.
Publications
- Lamer, J. T., Huang, J.-K., and Wen L. 2004. Characteristics of secondary alcohol dehydrogenase from micrococcus luteus WIUJH20. 96th Annual Meeting, Illinois State Academy of Science, April.
- Lamer, J. T., Huang, J.-K., and Wen, L. 2004. Secondary alcohol dehydrogenase from micrococcus luteus WIUJH20. 15th Annual Illinois Student Research Conference, Northeastern Illinois University, Chicago, Illinois. March 13.
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Progress 10/01/02 to 09/30/03
Outputs
The short-term objective of this project is to isolate and purify a secondary alcohol dehydrogenase from a Micrococcus luteus (WIUJH20). The long-term objective of this project is to do the microbial strain improvement of Nocardial cholesterolicun NRRL 5767 (NC NRRL 5767) by molecular cloning approach. We proposed to purify the enzyme (secondary alcohol dehydrogenase) and do physical and chemical property studies of this enzyme in the first year. We have accomplished most of our proposed work for this period. The results are presented as follows. The activity of this enzyme was found in two fractions (the pellet and the floated proteins) when the 40-75% ammonium sulfate fractionation was subjected to centrifugation at 20,000 x g for 60 min. The protein patterns of both fractions are very similar by SDS-PAGE. The floated protein fraction, presumably the lipid associated secondary alcohol dehydrogenase, was further purified by a hydrophorbic and ion-exchange
chromatographic column, respectively, to near homogeneity. The molecular weight of the native enzyme is about 160KD and the molecular weight of the denatured enzyme is about 40kD. The optimal pH of this enzyme is about 9; the pI of this enzyme is about 4.8. The enzyme is NAD+ dependent. Its N-terminal amino acid sequence (10 residues) has been obtained. The deduced oligonucleotides have been used to study the gene responsible for the synthesis of this enzyme (secondary alcohol dehydrogenase).
Impacts
The purification of this enzyme will allow us to proceed the proposed work for the second year to study the gene responsible for the synthesis of this enzyme, and to introduce this gene into Nocardial cholesterolicum NRRL 5767.
Publications
- R. Oduor, J.-K. Huang, V.C. Sershon, L.Wen, and K.C. Keudell. "Isolation and partial purification of secondary alcohol dehydrogenase from Bacillus sphaericus 5d4 (NRRL B-14865)." The 14th Annual Illinois Student Research Conference at Western Illinois University, April 4 and
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