Joint Appointment Research Fellow
President (2004-), National Chung-Hsing University
Ph.D., Biochemistry and Molecular Biology, University of Arkansas, USA (1977)
1. SENESCENCE ASSOCIATED GENES OF PAPAYA FRUIT RIPENING AND BROCCOLI YELLOWING.
Broccoli (Brassica oleracea) florets senesce and turn yellow rapidly after harvest. Subtractive hybridization technique was used to clone over 300 cDNAs with enhanced expression during floret senescence. These include genes involved in cell wall degradation, chlorophyll degradation, carbohydrate metabolism, protein and amino acid metabolism, nucleic acid metabolism, lipid metabolism, signal transduction, environmental and oxidative stess, and many other unknown functional genes. Some of these senescence associated genes have been cloned, expressed and characterized, including broccoli ACC oxidase, cysteine protease, caffeoyl CoA 3-o-methyltransferase, chlorophyllase, ethylene receptors (ETR1 and ERS), CTR1, metallothionein, bifuctional nuclease and antifungal protein. A mutated broccoli boers-1gene was transferred into broccoli and petunia, both transgenic plants showed delayed senescence.
Similar subtractive hybridization method was also used to clone 426 cDNAs which are upregulated during papaya fruit ripening. These include genes involved in in cell wall degradation, carbohydrate metabolism, protein and amino acid metabolism, nucleic acid metabolism, lipid metabolism, signal transduction, environmental and oxidative stress, light sensing, and many other unknown functional genes. The unknown functional cDNAs including 59 genes similar to Arabidopsis unknown proteins, 6 genes similar to Zea may proteins, and 272 genes without similarity to the genes deposited in data bank. Several papaya genes have been cloned, expressed and characterized. These 56 57 Annual Report 2005 include 2 ACC oxidases, 3 ACC synthases, a antifungal protein and two tubby-like proteins. Sense and antisense ACO gene have been successfully transferred into papaya and the transgenic papaya fruit has increased storage life for about 3 days. [Biochem. Biophys. Res. Commun. 1996, 225: 697-700; J. Agric. Food Chem. 1997, 45: 526-530; Plant Physiol. 1998, 117: 717; Plant Physiol. 1998, 117: 1126; Plant Physiol. 1998, 116: 1193; Plant Physiol. 2000, 122: 1457; Plant Physiol. 2000, 122: 1457; Mol. Breeding. 2002, 9: 211; Plant Sci 2003, 164: 531-540; J. Agric. Food Chem. 2003, 51: 2569-2575; Plant Physiol. Biochem. 2004, 42:663-670].
2. FUNCTIONAL GENOMICS OF ARABIDOPSIS TULP PROTEINS WHICH ARE NOVEL TYPE OF F-BOX PROTEINS.
We have identified and characterized eleven TULP genes (AtTULP1-11) in Arabidopsis. Sequence comparisons of the deduced proteins showed that AtTULPs show a well-conserved tubby domain in the Cterminal. Unlike the highly diverse Nterminal sequence of animal TULPs, a conserved F-box (51-57 residues) containing domain, is present in all AtTULP members except AtTULP8. RT-PCR analysis indicates that all these genes are expressed (except AtTULP4) and there are both distinct and overlapping expression patterns among family members. Expression analysis by microarray showed that AtTULP genes (AtTULP2, AtTULP7, AtTULP9, and AtTULP10) are involved in phytohormone signaling including auxin, cytokinin, ethylene and ABA. Environmental stresses and light signals also impose influences on the expression of AtTULP genes. Bioinformatic analysis predicts that AtTULPs are novel type of F-box protein, and we hypothesized that AtTULPs may also be involved in ubiquitinmediated proteolysis. The results suggest that AtTULPs are putative F-box protein containing bipartite transcription factors, which regulate transcription of particular genes through ubiquitin-mediated proteolysis of transcription activators or repressors. We have experimentally proved that one TULP member mediate ABA signaling and auother member control plant size. The patent is pending. [Plant Physiol. 2004, 134:1586-1597]
3. PRODUCTION OF HIGH-MALTOSE SYRUP AND HIGH-PROTEIN FOOD FROM CROPS
We have invented an enzymatic method for simultaneous production of high-maltose syrup and high-protein food from crops and the patent was licenced to a company. Recently we have produced a transgenic "sweet rice" which expressed high amount of thermostable amylopullulanse. The rice grain starch was completely degraded into syrups at 80¢XC for 4 h without exogenous addition of amylases, therefore it can be used for improving the rice nutrition and obtaining syrups for industrial uses such as wine and vinegar production. This has been patented. [US Patent 6,737,563, 2004]
BIOCATALYSIS
Enzymes are very efficient biocatalysts that not only regulate metabolism of organisms but also can be used in industries for biotransformation. Our major interests are structure, function and application of enzymes with a major focus on lipases/esterases. Lipases/esterases are broad specificity enzymes which catalyze the hydrolysis, esterification, and interesterification of various substrates, including glycerides, esters, thioesters. Although the catalytic triads Ser/Asp(Glu)/His are well conserved, the overall amino acid sequence homologies are quite low and the substrate specificities vary greatly due to the structure differences among this group of enzymes. We are interested in the systematic studies of the structure and function of this group of enzymes by site-directed mutagenesis, NMR, X-ray diffraction, computer modeling and DNA shuffling. These would lay the foundation for the rational design of a novel biocatalyst for biotechnological applications.
Figure 1. Schematic diagrams of the overall architecture of TAP. (a) Stereoview of the
Ca trace of the TAP structure with every tenth residue numbered. (b) Ribbon diagram presents the secondary elements of
TAP, in rainbow colors. Five b strands are numbered sequentially from b1 to b5. Seven a helices are labeled aA to aG.
The pink and cyan balls indicate the Nterminal and C-terminal, respectively. The break region between aB and b2 is the
disordered residue Gln32. The sulfate ion is shown as a stick.
1. We have invented new methods for the synthesis of carbohydrate esters, kojic acid esters, fatty acid esters, propylene glycol monoesters by lipasecatalyzed reactions. Three patents were awarded. [J. Am. Oil Chem. Soc. 2004, 81: 533-536]
2. We have discovered a novel family of lipases / esterases which have diverse functions and share a new GDSLS motif. 3D structures of E. coli. thioesterase I (TAP, Figure 1) and Vibrio mimicus arylesterase have been solved and revealed flexible active site structures which account for the multiple functions of these enzymes. [Prog. Lipid Res. 2004, 43(6):528-544]
3. Candida rugosa lipases are widely used in bioindustries. We discovered they contain five isoforms with high homology but quite different specificities. The expressions of isoforms are differentially regulated by culture conditions. We have overcome the codon usage problem and succesfully expressed active enzymes in E. coli and Pichia pastoris. The different substrate specificities were demonstrated to be greatly affected by lid structure. The patent is pending and licensed to a company.[Lipids 2004, 39(6): 513-526]
Shaw, J. F., Chang, R. C., Yen, Y. T., Wang, F. F., and Wang, Y. J. 1994. Nucleotide Sequence of a novel arylesterase gene from Vibrio mimicus and characterization of the enzyme expressed in E. coli, Biochem. J. 298: 675-680.
Lin, C. T., Lin, M. T., Chen, Y. T., and Shaw, J. F. 1995. Subunit interaction enhances enzyme activity and stability of cytosolic Cu-Zn-superoxide dismutase. Plant Mol. Biol. 28: 303-311.
Shaw, J. F., Chou, Y. S., Chang, R. C., and Yang, S. F. 1996. Characterization of the ferrous ion binding sites of apple 1-aminocyclopropane-1-carboxylate oxidase by site-directed mutagenesis. Biochem. Biophys. Res. Commun. 225: 697-700.
Lee, K. C., Tang, S. J., Sun, K. H., and Shaw, J. F. 1999. Analysis of the gene family encoding lipases in Candida rugosa by competitive reverse transcription-PCR. Appl. Environ. Microb. 65: 3888-3895.
Liu, K. J., Nag, A., and Shaw, J. F. 2001. Lipase-catalyzed synthesis of diethanolamide in organic solvent. J. Agric. Food. Chem. 49: 5761-5764.
Shaw, J. F., Chen, H. H., Kuo, C. I., and Huang, L. C. 2002. Delaying petunia senescence by transgenic anisense ERS. Molecular Breeding. 9: 211-216.
Lee, G. C., Lee, L. C., and Shaw, J. F. 2002. Multiple mutagenesis of nonuneversal serine codons of Candida rugosa LIP2 gene and the biochemical characterization of the purified LIP2 lipase over expressed in Pichia pastoris. Biochem. J. 366: 603-611.
Yang C. Y., Chu, F. H., Wang, Y. T., Chen, Y. T., Yang, S. F., and J. F. Shaw, 2003. Novel broccoli 1-Aminocyclopropane-1-carboxylate oxidase gene (Bo-ACO3) associated with the late stage of postharvest floret senescence. J. Agric. Food Chem. 51: 2569-2575.
Tyukhtenko, S. I., Shaw, J. F., Liaw, Y. C., and Huang, T. H. 2003. Sequential structural changes of E. coli thioesterase/protease-1 in the serial formation of Michaelis and tetrahedral complexes with diethyl p-nitrophenyl phosphate. Biochemistry 42: 8289-8297.
Lo, Y. C., Lin, S. E., Shaw, J. F., and Liaw, Y. C. 2003. Crystal structure of Escherichia coli thioesterase 1/protease 1/ lysophospholipase L1. J. Mol. Biol. 330: 539-551.
Lai, C. P., Lee, C. L., Chen, P. H., Wu, S. H., Yang, C. H., and Shaw, J. F. 2004. Molecular analysis of the Arabidopsis Tubby-like protein gene family. Plant Physiol. 134: 1586-1597.
Akoh, C. C., Lee, G. C., and Shaw, J. F. 2004. Protein Engineering and Applications of Candida rugosa Lipase Isoforms. Lipids 39: 513-526.
Akoh, C. C., Lee, G. C., and Shaw, J. F. 2004. GDSL Family of Serine Esterases/Lipases. Progress in Lipid Research 43: 528-544.
Chen, L. F. O., Huang, J. Y., Wang, Y. H., Chen, Y. T., and Shaw, J. F. 2004. Ethylene insensitive and postharvest yellowing retardation in mutant ethylene response sensor (boers) gene transformed broccoli (Brassica olercea var. italica) Mol. Breed. 14: 199-213.
Lee, G. C., Shieh, C. J., and Shaw, J. F. 2004. Protein Engineering of recombinant Candida rugosa Lipases. In: Hou, C. T. (Ed.). Handbook of Industrial Biocatalysis. Marcel Dekker Inc. New York.
Chang, S. W., Shieh, C. J., Lee, G. C., and Shaw, J. F. 2005. Multiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 expressed in Pichia pastoris. Appl. Microbiol. Biotechnol. 67: 215-224.
Lo, Y. C., Lin, S. C., Shaw, J. F., and Liaw, Y. C. 2005. Substrate specificities of Escherichia coli thioesterase I/protease I/lysophospholipase L1 are governed by its switch loop movement. Biochemistry 44: 1971-1979.
Chang, S. W., Shaw, J. F., Yang, K. H., Shih, I. L., Hsieh, C. H., and Shieh, C. J. 2005. Optimal lipasecatalyzed formation of hexyl laurate. Green Chem. 7: 547-551.
Chiang, C. M., F. S. Yeh, L. F. Huang, T. H. Tseng, M. C. Chung, C. S. Wang, H.S. Lur, J. F. Shaw and S. M. Yu (Dr. Shaw and Dr. Yu's group contributed equally 2005) Expression of a bi-functional and thermostable amylopullulanase in transgenic rice seeds leads to autohydrolysis and altered composition of starch. Mol. Breed. 15: 125-143.
