Tu, Jenn (杜 鎮)
- Applied and evolutionary microbiology, Prokaryotic and eukaryotic mobile elements, Protein engineering
Research Interests: Applied and evolutionary microbiology; Enzymology; Protein engineering.
Isolation of polysaccharide-degrading bacteria and enzyme engineering
(1) Molecular cloning and engineering of Paenibacillus xylanase
Bioresource technology for depollution and biomass reuse is one of our major interests. For this purpose, a multiple glycosyl hydrolase-producing strain, Paenibacillus campinasensis BL11 (accession number: DQ232773),was isolated from black liquor of brownstock at washing stage of kraft pulping process. The xylanase (endo-1, 4-β-xylanase, gene name: xylX, accession number: DQ241676) of strain BL11 has been cloned and characterized. The gene encoding this enzyme composed of 1,131 nucleotides and encodes a protein of 41 kDa. The N-terminal of the enzyme contains a deduced signal peptide of 39 amino acids. Optimum temperature and pH for crude BL11 xylanase activity were 60°C and pH 7, respectively. Its activity was 2392 IU/ mg under optimal condition. At pH 11, the activity was still as high as 517 IU/mg. To fit industrial demands, we created a mutagenesis library of xylX using error-prone PCR and isolated mutants of xylX showing improved thermostability. From the library two activity-improved mutants, an amino acid substitution mutant (T44A) and a downstream deletion mutant, were obtained. The deletion mutant lost most part of its carbohydrate-binding domain (Fig. 1). Between 67.5oC and 70 oC, the substitution mutant showed two times increased activity than the wildtype enzyme whereas the deletion mutant had three times increased activity than the wild-type enzyme. Besides, we have got a thermostability-decreased mutant (T44M). The mutant carries a different amino acid substitution at same site of the activity-improved one (T44A mutant), but its thermostability decreased distinctly. Based on this result, we suggest that the Thr-44 is a critical amino acid affecting the thermostability of xylanase X.。
(2)Molecular cloning and engineering of Bacillus glucanase
In another work a thermophilic bacteria strain, Bacillus subtilis WL-A12, was isolated from a hot spring near downriver of Nan-shi, Wulai, Taipei. Strain WL-A12 exhibited multiple extracellular polysaccharide hydrolyzing enzymes. The thermostable 1,3-1,4-β-glucanase (lichenase) of WL-A12 has been cloned. The cloned DNA fragment encodes a polypeptide of 27 kDa. The signal peptide of the enzyme locates in the N-terminal of the enzyme and contains 28 amino acids. Its activity under the optimal condition (pH 6, 50oC) was 17.72 IU/mg. The enzyme is now subjected for mutagenesis to create mutants for improved thermostability and low end-product (glucose) inhibition.
Selected publication list
- Ko,C.H., Tsai,C.H., Tu,J., Tang,S.H., and Liu,C.C. (2011). Expression and thermostability of Paenibacillus campinasensis BL11 pectate lyase and its applications in bast fibre processing. Ann. Appl. Biol. (in press).
- Ko,C.H., Tsai,C.H., Tu,J., Lee,H.Y., Ku,L.T., Kuo,P.A., and Lai,Y.K. (2010). Molecular cloning and characterization of a novel thermostable xylanase from Paenibacillus campinasensis BL11. Process Biochemistry. 45:1638-1644.
- Ko, C.H., Tsai, C.H., Lin, P.H., Chang, K.C., Tu, J., Wang, Y.N., and Yang, C.Y. Characterization and pulp refining activity of a Paenibacillus campinasensis cellulase expressed in Escherichia coli. BIORESOURCE TECHNOLOGY. 101(20), 7882-7888,2010-06.
- Ko, C.H., Tsai C.H., Tu, J., Lee, H.Y., Ku, L.T., Kuo, P. A., and Lai, Y.K.. Molecular cloning and characterization of a novel thermostable xylanase from Paenibacillus campinasensis BL11. PROCESS BIOCHEMISTRY. 45, 1638-1644, 2010-08.
- Ko, C.H., Lin, Z.P., Tu, J., Tsai, C.H., Liu, C.C., Chen, H.T., and Wang, T.P. Xylanase production by Paenibacillus campinasensis BL11 and its pretreatment of hardwood kraft pulp bleaching. INTERNATIONAL BIODETERIORATION & BIODEGRADATION. 64(1), 13-19, 2010-01.
- Huang, C.Y., Hsieh, S.P., Kuo, P.A., Jane, W.N., Tu, J., Wang, Y.N., Ko, C.H. Impact of disinfectant and nutrient concentration on growth and biofilm formation for a Pseudomonas strain and the mixed cultures from a fine papermachine system.INTERNATIONAL BIODETERIORATION & BIODEGRADATION. 63(8), 998-1007, 2009-12.
- Y. C. Charng, K. T. Li, H. K.Tai, N.S. Li, and Tu, J., An inducible transposon system to terminate the function of a selectable marker in transgenic plants. MOLECULAR BREEDING. 21(3), 359-368, 2008.
- C. H. Ko, W. L. Chen, C. H.Tsai, W. N. Jane, C. C. Liu, and Tu, J., Paenibacillus campinasensis BL11: a wood material-utilizing bacterial strain isolated from black liquor. BIORESOURCE TECHNOLOGY. 98(14), 2727-2733, 2007.
- Charng YC, Wu G, Hsieh CS, Chuan HN, Huang JY, Yeh LC, Shieh YH, Tu, J., The inducible transposon system for rice functional genomics. . Botanical Studies. 48(1),1-11, 2007-01.
- Charng, Y. C., Li, H., Ping., Li, K. T., Hseu, T. H., and Tu, J. ,Fusion of the transposase with a classical nuclear localization signal to increase the transposition efficiency of Ac transposon. Bot. Bull. Academia Sinica 45:267-274,2004.
- Tsai, P.-J., Tu, J., and Chen, T.-H. Cloning of a Ca2+/calmodulin-dependent protein kinase gene from the filamentous fungus Arthrobotrys dactyloides. FEMS Microbiol. Lett. 212: 7-13,2002.
- Yang, Y. C., Yang, M. K., Kuo, T. T., and Tu, J.,Structural and functional characterization of the lexA gene of Xanthomonas campestris pathovar citri. Mol. Gen. Genet. 265: 316-326,2001.
- Charng, Y. C., Pfitzner, A. J. P., Pfitzner, U. M., Charng-Chang, K. F., Chen, C. M., Tu, J., and Kuo, T. T. Construction of an inducible transposon, INAc, to develop a gene tagging system in higher plants. Mol. Breed. 6: 353-367,2000.
- Liu, C. C., Huhne, R., Tu, J., Lorbach, E., and Droge, P. The resolvase encoded by Xanthomonas campestris transposable element ISXc5 constitutes a new subfamily closely related to DNA invertases. Genes Cells 3: 221-233,1998.
- Charng, Y. C., Ma, C., Tu, J., and Kuo, T. T. A 200-bp constructed inducible PR-1a promoter fusion to the Ac transposase gene drives higher transposition of a Ds element than the native PR-1a promoter fusion drives. Plant Sci. 130: 73-86,1997.
- Cheng, C. M., Tu, J., Yang, P. C., and Kuo, T. T. Rifampicin: an inhibitor of Xp12-specific protein phosphorylation in Xanthomonas campestris pv. oryzae. FEMS Microbiol. Lett. 143: 141-149,1996.