拮抗性臘狀芽孢桿菌菌株28-9幾丁質水解酵素系統研究

黃健瑞; Huang, Chien-Jui

DC Field	Value	Language
dc.contributor	陳昭瑩	en
dc.contributor	臺灣大學：植物病理與微生物學研究所	zh_TW
dc.contributor.author	黃健瑞	zh
dc.contributor.author	Huang, Chien-Jui	en
dc.creator	黃健瑞	zh
dc.creator	Huang, Chien-Jui	en
dc.date	2006	en
dc.date.accessioned	2007-11-27T08:16:31Z	-
dc.date.accessioned	2018-06-29T07:05:47Z	-
dc.date.available	2007-11-27T08:16:31Z	-
dc.date.available	2018-06-29T07:05:47Z	-
dc.date.issued	2006	-
dc.identifier	en-US	en
dc.identifier.uri	http://ntur.lib.ntu.edu.tw//handle/246246/58041	-
dc.description.abstract	利用幾丁質培養基自百合根圈土壤分離出一幾丁質分解性臘狀芽胞桿菌菌株28-9，經對峙培養、切離葉等防治試驗得知此菌株具有防治百合灰黴病的潛力。為了解幾丁質水解能力與拮抗百合灰黴病菌的關係，分別從蛋白質與基因組方向進行，得到兩個幾丁質水解酵素基因，命名為chiCH及chiCW。其演譯之蛋白質除訊息序列外，ChiCH具有一催化功能區，ChiCW則具一催化功能區，一fibronectin type III相似功能區及一幾丁質結合功能區。分別自大腸桿菌表現及純化出ChiCH和ChiCW兩蛋白質，經抑菌試驗分析得知ChiCH僅輕微地抑制百合灰黴病菌分生孢子萌芽，而ChiCW可有效地抑制百合灰黴病菌分生孢子萌芽，推測ChiCW是菌株28-9拮抗百合灰黴病菌之重要因子。此外，本研究亦發展出利用融合glutathione-S-transferase的重組幾丁質水解酵素可在大腸桿菌中大量表現幾丁質水解酵素，經動力學、水解產物及基質結合力分析可知ChiCW為內切型而ChiCH為外切型幾丁質水解酵素。最後，本研究對ChiCW的C端區域在酵素功能上的影響進行分析，相對於ChiCW，在高溫及高酸鹼值的條件下， ChiCWΔFC對醣酐幾丁質有較高之活性，動力學分析結果則顯示ChiCW較ChiCWΔFC容易水解寡分子及聚分子基質，推測菌株28-9將ChiCW修飾成ChiCWΔFC可以提升自身在不同環境下分解幾丁質的效率，效地將大分子基質水解成更小的寡分子基質。	zh_TW
dc.description.abstract	Bacillus cereus 28-9 is a chitinolytic bacterium isolated from rhizosphere of lilies. This bacterium showed antagonistic effect against Botrytis elliptica, the pathogen of lily leaf blight, by detached leaf assays. To investigate the involvement of the chitinolytic activity in antagonism, two chitinase-encoding genes, chiCH and chiCW, have been cloned. ChiCH consists of a signal peptide followed by a catalytic domain. ChiCW consists of a signal peptide, a catalytic domain, a fibronectin type III-like domain, and a chitin-binding domain. ChiCH and ChiCW are slightly and effectively inhibitory to conidial germination of B. elliptica, respectively, indicating that ChiCW may contribute to the antagonistic activity of B. cereus 28-9 against B. elliptica. In addition, an ideal method for high-level expression of chitinases in E. coli as glutathione-S-transferase fusion proteins was established in this study. According to the results of kinetics, hydrolysis products, and binding activities, ChiCW is an endo-chitinase and ChiCH is an exo-chitinase. Finally, the functions of the C-terminal region on ChiCW activity were investigated. Compared with ChiCW, ChiCWΔFC exhibited higher activity at high temperature and pH although both enzymes had the same optimal temperature and pH for enzyme activities. The kinetic properties of ChiCW and ChiCWΔFC indicate that ChiCW hydrolyzes oligomeric and polymeric substrates more efficiently than ChiCWΔFC. Based on the results, we suggest that B. cereus 28-9 proteolytically modifying ChiCW to ChiCWΔFC could improve efficiency of chitin degradation in different environments.	en
dc.description.tableofcontents	中文摘要............................................................................................................................. 1 Abstract ............................................................................................................................... 2 Chapter 1: Introduction...................................................................................................... 3 1.1 Chitinases ................................................................................................................ 3 1.2 Endo- and exo-chitinases.......................................................................................... 4 1.3 Catalytic domains of chitinases ................................................................................ 4 1.4 Noncatalytic domains found in chitinases................................................................. 7 1.5 Chitinases in the control of phytopathogenic fungi ................................................. 10 1.6 Biocontrol of gray mold disease of lilies................................................................. 15 1.7 The goal of this study............................................................................................. 16 Chapter 2: Gene cloning and biochemical characterization of ....................................... 18 chitinase CH from Bacillus cereus 28-9............................................................................ 18 Abstract ....................................................................................................................... 18 Introduction ................................................................................................................. 19 Materials and Methods................................................................................................. 20 Identification of a bacterial strain................................................................................. 20 Chitinase activity measurements.................................................................................. 20 Purification of chitinase CH from the culture supernatnat of B. cereus 28-9................. 20 SDS-PAGE and zymogram analysis ............................................................................ 21 Peptide N-terminal sequencing .................................................................................... 21 Cloning of ChiCH-encoding gene................................................................................ 22 Expression and purification of ChiCH from E. coli cells.............................................. 23 Biochemical characterization of ChiCH....................................................................... 23 Antifungal assay of ChiCH.......................................................................................... 24 Nucleotide sequence accession number ....................................................................... 24 Results and Discussion................................................................................................. 25 Purification and N-terminal sequence of ChiCH.......................................................... 25 ChiCH-encoding gene ................................................................................................. 25 Analysis and comparison of the amino acid sequence of ChiCH with other chitinases . 26 Biochemical properties of ChiCH................................................................................ 28 Inhibition by ChiCH on conidial germination of B. elliptica ........................................ 28 Figure 1. SDS-PAGE and zymogram analysis. ........................................................... 30 Figure 2. Southern blot analysis of the genomic DNA of B. cereus 28-9. .................... 31 ii Figure 3. Nucleotide and the deduced amino acid sequences of chiCH........................ 32 Figure 4. Sequence comparison of three chitinases from B. cereus strains................... 33 Figure 5. Effects of pH on the chitinase activity and stability of ChiCH...................... 34 Figure 6. Effects of temperature on the chitinase activity and stability of ChiCH. ....... 35 Chapter 3: Identification of an antifungal chitinase from a potential biocontrol agent, Bacillus cereus 28-9 ........................................................................................................... 36 Abstract ....................................................................................................................... 36 Introduction ................................................................................................................. 37 Bacterial strains and culture conditions........................................................................ 38 Preparation of colloidal chitin...................................................................................... 38 Expression and purification of chitinase ChiCW from recombinant E. coli cells .......... 38 Southern blot analysis.................................................................................................. 39 SDS-PAGE and zymogram analysis ............................................................................ 40 Chitinase activity measurements.................................................................................. 40 Antifungal assay of ChiCW......................................................................................... 41 Nucleotide sequence accession number ....................................................................... 41 Results ......................................................................................................................... 42 Sequence anlysis of ChiCW-encoding gene................................................................. 42 Expression and purification of ChiCW from recombinant E. coli................................. 42 Analysis and comparison of the amino acid sequence of ChiCW with other chitinases 43 Inhibition by ChiCW on conidial germination of B. elliptica ....................................... 44 Discussion ................................................................................................................... 46 Table 1. Purification of ChiCW from periplasmic fraction of E. coli DH5α(pNTU55) 48 Table 2. Effect of ChiCW on conidial germination of B. ellipticaa .............................. 49 Figure. 1. Nucleotide sequence of the chiCW gene of B. cereus 28-9 and the deduced amino acid sequence of the gene product. .................................................................... 51 Figure 2. Southern blot analysis of the genomic DNA of B. cereus 28-9. .................... 52 Figure 3. SDS-PAGE and in-gel activity assay............................................................ 53 Figure 4. Alignment of the peptide sequences of the catalytic domains of chitinases from different Bacillus spp. ......................................................................................... 54 Figure 5. Alignment of the peptide sequences of the fibronectin type III-like domains of chitinases from different Bacillus spp. ......................................................................... 55 Figure 6. Alignment of the peptide sequences of the chitin- and cellulose-binding iii domains of chitinases from different Bacillus spp. ....................................................... 56 Figure 7. Effect of ChiCW on conidial germination of B. elliptica. ............................. 57 Chapter 4: High-level expression and characterization of two chitinases, ChiCH and ChiCW, of Bacillus cereus 28-9 in Escherichia coli.......................................................... 58 Abstract ....................................................................................................................... 58 Introduction ................................................................................................................. 59 Materials and Methods................................................................................................. 61 Bacterial strains, plasmid, and media........................................................................... 61 Construction of expression vectors .............................................................................. 61 Bacterial expression of recombinant chitinases ............................................................ 61 Purification of recombinant chitinases ......................................................................... 62 PreScission protease cleavage of GST-chitinases......................................................... 63 SDS-PAGE and chitinolytic zymography assay........................................................... 63 Chitinase activity measurements and protein concentration determination ................... 64 Kinetic properties characterization............................................................................... 64 Thin-layer chromatography ......................................................................................... 65 Substrate-binding assay ............................................................................................... 65 Affinity electrophoresis ............................................................................................... 66 Results ......................................................................................................................... 67 Vector Construction and expression of GST-chitinase fusion proteins in E. coli .......... 67 Purification of recombinant chitinases ......................................................................... 67 Kinetics of chitinases................................................................................................... 68 Hydrolysis products of chitinases ................................................................................ 69 Binding activity of chitinases to polysaccharides ......................................................... 69 Discussion ................................................................................................................... 71 Table 1. Primers used.................................................................................................. 77 Table 2. Purification of GST-chitinase fusion proteins ................................................ 78 Table 3. Kinetics of chitinases and GST-chitinase fusion proteins............................... 79 Figure 1. Schematic diagrams of constructed plasmids, pGW59 (A) and pGH60 (B), for overexpression of ChiCW and ChiCH. ........................................................................ 80 Figure 2. Expression of GST-ChiCH fusion protein in E. coli cells. ............................ 81 Figure 3. Purification of GST-ChiCH and recombinant ChiCH after cleavage by PreScission protease. ................................................................................................... 82 iv Figure 4. Purification of GST-ChiCW and recombinant ChiCW after cleavage by PreScission protease. ................................................................................................... 83 Figure 5. Thin- layer chromatography of hydrolytic products from various Nacetylchitooligosaccharides. ........................................................................................ 84 Figure 6. Domain structures of ChiCH and ChiCW from B. cereus 28-9..................... 85 Figure 7. Affinity electrophoresis of chitinases in the presence (+) and absence (-) of glycol chitin (A), CM-cellulose (B), and laminarin (C). ............................................... 86 Chapter 5: Functions of the C-terminal region of chitinase ChiCW from Bacillus cereus 28-9 in substrate-binding and hydrolysis of chitin........................................................... 87 Abstract ....................................................................................................................... 87 Introduction ................................................................................................................. 88 Materials and Methods................................................................................................. 89 Bacterial strains, plasmid, and media........................................................................... 89 Construction of expression vectors .............................................................................. 89 Bacterial expression of truncated chitinases................................................................. 89 Purification of ChiCWΔFC.......................................................................................... 90 SDS-PAGE and chitinolytic zymography assay........................................................... 90 Chitinase activity measurement and protein concentration determination..................... 91 Optimal condition for enzyme activity......................................................................... 91 Substrate specificity and binding assay........................................................................ 91 Kinetic properties characterization............................................................................... 92 Results ......................................................................................................................... 93 Expression of ChiCW derivatives ................................................................................ 93 Purification of ChiCWΔFC.......................................................................................... 93 Optimal conditions for enzyme activity ....................................................................... 93 Substrate specificity and binding activity..................................................................... 94 Kinetics of ChiCW and ChiCWΔFC............................................................................ 95 Discussion ................................................................................................................... 96 Table 1. Primers used in this study............................................................................ 100 Table 2. Purification of ChiCWΔFC ......................................................................... 101 Table 3. Substrate specificity of ChiCW and ChiCWΔFC......................................... 102 Table 4. Kinetics of ChiCW and ChiCWΔFC............................................................ 103 Figure 1. Schematic diagram of the domain structures of ChiCW and two derivatives. v ................................................................................................................................. 104 Figure 2. Elution profile of ChiCWΔFC purification by anion exchange chromatography......................................................................................................... 105 Figure 3. SDS-PAGE and zymography assay of ChiCW and ChiCWΔFC................. 106 Figure 4. Effect of temperature on the chitinase activity of ChiCW and ChiCWΔFC. 107 Figure 5. Effect of pH on the chitinase activity of ChiCW and ChiCWΔFC. ............. 108 Figure 6. Insoluble substrate binding ability of ChiCW and ChiCWΔFC. ................. 109 Future aspect................................................................................................................... 110	zh_TW
dc.format.extent	4503485 bytes	-
dc.format.mimetype	application/pdf	-
dc.language	en-US	en
dc.language.iso	en_US	-
dc.subject	幾丁質水解酵素	en
dc.subject	臘狀芽胞桿菌	en
dc.subject	百合灰黴病菌	en
dc.subject	蛋白質大量表現	en
dc.subject	Chitinase	en
dc.subject	Bacillus cereus	en
dc.subject	Botrytis elliptica	en
dc.subject	Protein overexpression	en
dc.title	拮抗性臘狀芽孢桿菌菌株28-9幾丁質水解酵素系統研究	zh
dc.title	Chitinase system of antagonistic Bacillus cereus 28-9	en
dc.type	other	-
dc.identifier.uri.fulltext	http://ntur.lib.ntu.edu.tw/bitstream/246246/58041/1/ntu-95-F89633001-1.pdf	-
dc.relation.reference	[1] Adams, P. B. 1990. The potential of mycoparasites for biological control of plant diseases. Annu. Rev. Phytopathol. 28: 59-72. [2] Armand, S., Tomita, H., Heyraud, A., Gey, C., Watanabe, T., and Henrissat, B. 1994. Stereochemical course of the hydrolysis reaction catalyzed by chitinase A1 and D from Bacillus circulans WL-12. FEBS Lett. 343: 177-180. [3] Arora, N., Ahmad, T., Rajagopal, R., and Bhatnagar, R. K. 2003 A constitutively expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1. Biochem. Biophy. Res. Commun. 307: 620-625. [4] Aunpad, R. and Panbangred, W. 2003. Cloning and characterization of the constitutively expressed chitinase C gene from a marine bacterium, Salinivibrio costicola strain 5SM-1. J. Biosci. Bioeng. 96: 529-536. [5] Barboza-Corona, J. E., Nieto-Mazzocco, E., Velazquez-Robledo, R., Salcedo- Hernandez, R., Bautista, M., Jimenez, B., and Ibarra, J. E. 2003. Cloning, sequencing, and expression of the chitinase gene chiA74 from Bacillus thuringiensis. Appl. Environ. Microbiol. 69: 1023-1029. [6] Barnett, H. L. and Binder, F. L. 1973. The fungal host-parasite relationship. Annu. Rev. Phytopathol. 11: 273-292. [7] Benhanou, N. and Chet, I. 1993. Hyphal interactions between Trichodera harzianum and Rhizoctonia solani: ultrastructure and gold cytochemistry of the mycoparasitic process. Phytopathology 83: 1062-1071. [8] Blaak, H., Schnellmann, J., Walter, S., Henrissat, B., and Schrempf, H. 1993. Characteristics of an exochitinase from Streptomyces olivaceoviridis, its corresponding gene, putative protein domains and relationship to other chitinases. Eur. J. Biochem. 214: 659-669. [9] Blaak, H. and Schrempf, H. 1995. Binding and substrate specificities of a Streptomyces olivaceoviridis chitinase in comparison with its proteolytically processed form. Eur. J. Biochem. 229: 132-139. [10] Bork, P. and Doolittle, R. F. 1992. Proposed acquisition of an animal protein domain by bacteria. Proc. Natl. Acad. Sci. USA 89: 8990-8994. [11] Bradford, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. 112 Biochem. 72: 248-254. [12] Brooks, A.V. 1980. Lily diseases and disorders. Universe Books, New York, United States of America. [13] Brun, E., Moriaud, F., Gans, P., Blackledge, M. J., Barras, F., and Marion, D. 1997. Solution structure of the cellulose-binding domain of the endoglucanase Z secreted by Erwinia chrysanthemi. Biochemistry 36: 16074-16086. [14] Brunel, B., Perissol, C., Fernandez, M., Boeufgras, J. M., and Le Petit, J. 1994. Occurrence of Bacillus species on evergreen oak leaves. FEMS Microbiol. Ecol. 14: 331-342. [15] Buxton, E. W., Khalifa, O., and Ward, V. 1965. Effects of soil amendment with chitin on pea wilt caused by Fusarium oxysporum f. sp. pisi. Ann. Appl. Biol. 55: 83-88. [16] Cao, L., Yan, X., Borysenko, C. W., Blair, H. C., Wu, C., and Yu, L. 2005. CHDL: A cadherin-like domain in Proteobacteria and Cyanobacteria. FEMS Microbiol. Lett. 251: 203-209. [17] Carsolio, C. 1997. Role of the endochitinase Ech42 in the mycoparasitism by Trichoderma harzianum. PhD thesis dissertation. CINVESTAV, Irapuato, Mexico, 42- 61. [18] Carsolio, C., Gutierrez, A., Jimenez, B., van Montagu, M., and Herrera-Estrella, A. 1994. Characteriation of ech-42, a Trichoderma harzianum endochitinase gene expressed during mycoparasitism. Proc. Natl. Acad. Sci. USA 91: 10903-10907. [19] Chastagner, G. A., and Riley, K. I. 1990. Occurrence and control of benzimidazole and dicarboximide resistant Botrytis spp. on bulb crops in Western Washington and Oregon. Acta Hort. 266: 437-445. [20] Chernin, L., Ismailov, Z., Haran, S., and Chet, I. 1995. Chitinolytic Enterobacter agglomerans antagonistic to fungal plant pathogens. Appl. Environ. Microbiol. 61: 1720-1726. [21] Chet, I. 1987. Trichoderma – application, mode of action and potential as a biocontrol agent of soilborne plant pathogenic fungi. In: Chet, I. (ed): Innovative approaches to plant disease control. Wiley, New York, 137-160. [22] Chet, I. 1990. Biological control of soil-borne plant pathogens with fungal antagonists in combination with soil treatments. In: Hornby, D. (ed): Biological control of soilborne plant pathogens. C. A. B. International, Oxon, 15-25. [23] Chet, I., Inbar, J., and Hadar, Y. 1997. Fungal antagonists and mycoparasites. In 113 Wichlow, D. T., and Soderstrom (eds): The mycota IV: environmental and microbial relationships. Springer, Heidelberg, 165-184. [24] Chiou, A. L. and Wu, W. S. 2001. Isolation, identification and evaluation of bacterial antagonists against Botrytis elliptica on lily. J. Phytopathol. 149: 319-324. [25] Chiou, A.L. and Wu, W.S. 2003. Formulation of Bacillus amyloliquefaciens B190 for control of lily grey mould (Botrytis elliptica). J. Phytopathol. 151: 13-18. [26] Chong, S., Montello, G. E., Zhang, A., Cantor, E. J., Liao, W., Xu, M., and Benner, J. 1998. Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucleic Acids Res. 26: 5109- 5115. [27] Cohen-Kupiec, R. and Chet, I. 1998. The molecular biology of chitin digestion. Curr. Opin. Biotechnol. 9: 270-277. [28] Cook, R. J. 1993. Making greater use of introduced microorganisms for biological control of plant pathogens. Annu. Rev. Phytopathol. 31: 53-80. [29] Cooper, G. M. 1997. The Cell, a Molecular Approach, American Society for Microbiology, Washington, DC. [30] Coutinho, J. B., Gilkes, N. R., Warren, R. A. J., Kilburn, D. G., and Miller, R. C. Jr. 1992. The binding of Cellulomonas fimi endoglucanase C (CenC) to cellulose and Sephadex is mediated by the N-terminal repeats. Mol. Microbiol. 6: 1243-1252. [31] De la Cruz, J., Hidalgo-Gallega, A., Lora, J. M., Benitez, T., Pintor-Toro, J. A., and Llobell, A. 1992. Isolation and characterization of three chitinases from Trichoderma harzianum. Eur. J. Biochem. 206: 859-867. [32] Dian, C., Eshaghi, S., Urbig, T., McSweeney, S., Heijbel, A., Salbert, G., and Birse, D. 2002. Strategies for the purification and on-column cleavage of glutathione- Stransferase fusion target proteins. J. Chromatogr. B. 769: 133-144. [33] Doss, R. P., Chastagner, G. A., and Riley, K. L. 1984. Techniques for inoculum production and inoculation of lily leaves with Botrytis elliptica. Plant Dis. 68: 854-856. [34] Drouillard, S., Armand, S., Davies, G., Vorgias, C., and Henrissat, B. 1997. Serratia marcescens chitobiase is a retaining glycosidase utilizing substrate acetamido group participation. Biochem. J. 328: 945-949. [35] Elad, Y. 1996. Mechanisms involved in the biological control of Botrytis cinerea incited diseases. Eur. J. Plant Pathol. 102: 719-732. [36] Elad, Y., Barak, R., Chet, I., and Henis, Y. 1983a. Ultrastructural studies of the 114 interaction between Trichoderma spp. and plant pathogenic fungi. J. Phytopathol. 107: 168-175. [37] Elad, Y., Chet, I., Boyle, P., and Henis, Y. 1983b. Parasitism of Trichoderma spp. on Rhizoctonia solani and Sclerotium rolfsii scanning electron microscopy and fluorescens microscopy. Phytopathology 73: 85-88. [38] Elad, Y., Chet, I., and Henis, Y. 1982. Degradation of plant pathogenic fungi by Trichoderma harzianum. Can. J. Microbiol. 28: 719-725. [39] Emmert, E. A. B. and Handelsman, J. 1999. Biocontrol of plant disease: a (Gram-) positive perspective. FEMS Microbiol. Lett. 171: 1-9. [40] Felse, P. A. and Panda, T. 1999. Regulation and cloning of microbial chitinase genes. Appl. Microbiol. Biotechnol. 51: 141-151. [41] Flach, J., Pilet, P. E., and Jolles, P. 1992. What’s new in chitinase research? Experientia 48: 701-716. [42] Freeman S., Minz O., Kolesnik I., Barbul O., Zveibil A., Maymon M., Nitzani Y., Kirshner B., Rav-David D., Bilu A., Dag A., Shafir S., and Elad Y. 2004. Trichoderma biocontrol of Colletotrichum acutatum and Botrytis cinerea and survival in strawberry. Eur. J. Plant Pathol. 110: 361-370. [43] Fridlender, M., Inbar, J., and Chet, I. 1993. Biological control of soilborne plant pathogens by a β-1, 3-glucanase-producing Pseudomonas cepacia. Soil. Biol. Biochem. 25: 1211-1221. [44] Gay, P. A., Saikumar, K. V., Cleveland, T. E., and Tuzun, S. 1992. Antagonistic effect of chitinolytic bacteria against toxin producing fungi. Phytopathology 82: 1074. [45] Gebler, J., Gilkes, N. R., Claeyssens, M., Wilson, D. B., Beguin, P., Wakarchuk, W. W., Kilburn, D. G., Miller, R. C. Jr., Warren, R. A. J., and Withers, S. G. 1992. Stereoselective hydrolysis catalyzed by related β-1, 4-glucananses and β-1, 4- xylanases. J. Biol. Chem. 267: 12559-12561. [46] Geremia, R., Jacobs, D., Goldman, G. H., van Montagu, M., and Herrera-Estrella, A. 1991. Induction and secretion of hydrolytic enzymes by the biocontrol agent Trichoderma harzianum. In: Bee,ster, A. B. R., Bollen G. J., Gerlagh, M., Ruissen, M. A., Schippers, B., and Tempel, A. (eds): Bioteic interactions and soil-borne disease. Elsevier, Amsterdam, pp.181-186. [47] Gilkes, N. R., Henrissat, B., Kilburn, D. G., Miller, R. C., and Warren, R. A. J. 1991. Domain in microbial β-1, 4-glycanases: sequence conservation, function and enzyme 115 families. Microbiol. Rev. 55: 303-315. [48] Goldstein, M. A., Takagi, M., Hashida, S., Shoseyov, O., Doi, R. H., and Segel, I. H. 1993. Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A. J. Bacteriol. 175:5762-5768. [49] Gould, A. B., Kobayashi, D. Y., and Bergen, M. S. 1996. Identification of bacteria for biological control of Botrytis cinerea on petunia using a petal disk assay. Plant Dis. 80: 1029-1033. [50] Graham, L. S. and Sticklen, M. B. 1994. Plant chitinases. Can. J. Bot. 72: 10571083. [51] Haran, S., Schickler, H., Oppenheim, A., and Chet, I. 1995. New ccomponents of the chitinolytic system of Trichoderma harzianum. Mycol. Res. 99: 441-446. [52] Hart, P. J., Monzingo, A. F., Ready, M. P., Ernst, S. R., and Robertus, J. D. 1993. Crystal structure of an endochitinase from Hordeum vulgare L. seeds. J. Mol. Biol. 229: 189-193. [53] Hart, P. J., Pfluger, H. D., Monzingo, A. F., Hollis, T., and Robertus, J. D. 1995. The refined crystal structure of an endochitinase from Hordeum vulgare L. seeds. J. Mol. Biol. 248: 402-413. [54] Hennig, M., Jansonius, J. N., Terwisscha van Scheltinga, A. C., Dijkstra, B. W., and Schlessier, B. 1995a. Crystal stucture of concavalin B at 1.65 A resolution. An “inactivated” chitinase from seeds of Canavalia ensiformis. J. Mol. Biol. 254: 237-246. [55] Hennig, M., Pfeffer-Hennig, S., Dauter, Z., Wilson, K. S., Schlesier, B., and Nong, V. H. 1995b. Crystal structure of narbonin at 1.8 A resolution. Acta Cryst. D 51: 177-189. [56] Henrissat, B. 1991. A classification of glycosyl hydrolases based on amino acid sequence similarity. Biochem. J. 280: 309-316. [57] Henrissat, B. 1999. Classification of chitinases modules. EXS 87:137-156. [58] Henrissat, B. and Bairoch, A. 1993. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 293: 781-788. [59] Henrissat, B. and Bairoch, A. 1996. Updating the sequence-based classification of glycosyl hydrolases. Biochem. J. 346: 695-696. [60] Henrissat, B., Claeyssens, M., Tomme, P., Lemesle, L., and Mornon, J. P. 1989. Cellulase families revealed by hydrophobic cluster analysis. Gene 81: 83-95. [61] Henrissat, B. and Davies, G. J. 1997. Structural and sequence-based classification of glycosyl hydrolases. Curr. Opin. Struct. Biol. 7: 637-644. [62] Herrera-Estrella, A. and Chet, I. 1999. Chitinases in biological control. EXS 87: 171- 116 184. [63] Hollis, T., Honda, Y., Fukamizo, T., Marcotte, E., Day, P. J., and Robertus, J. D. 1997. Kinetic analysis of barley chitinase. Arch. Biochem. Biophys. 344: 335-342. [64] Hsieh, T. F. and Huang, J. W. 1998. Factors affecting disease development of Botrytis leaf blight of lily caused by Botrytis elliptica. Plant Pathol. Bull. 40: 227-240. [65] Huang, C. J., Wang, T. K., Chung, S. C., and Chen, C. Y. 2004. Identification of an antifungal chitinase from a potential biocontrol agent, Bacillus cereus 28-9. J. Biochem. Mol. Biol. 38: 82-88. [66] Huang, C. J. and Chen, C. Y. 2004. Gene cloning and biochemical characterization of chitinase CH from Bacillus cereus 28-9. Ann. Microbiol. 54: 289-297. [67] Huang, C. J. and Chen, C. Y. 2005. High-level expression and characterization of two chitinases, ChiCH and ChiCW, of Bacillus cereus 28-9 in Escherichia coli. Biochem. Biophy. Res. Commun. 327: 8-17. [68] Imoto, T. and Yogishita, K. 1971. A simple activity measurement of lysozyme. Agric. Biol. Chem. 35: 1154-1156. [69] Iseli, B., Armand, S., Boller, T., Neuhaus, J. M., and Henrissat, B. 1996. Plant chitinases use two different hydrolytic mechanisms. FEBS Lett. 382: 186-188. [70] Jones, J. D. G., Grady, K. L., Suslow, T. V., and Bedbrook, J. R. 1986. Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens. EMBO J. 5: 467-473. [71] Kaomek, M., Mizuno, K., Fujimura, T., Sriyotha, P., and Cairns, J. R. K. 2003. Cloning, expression, and characterization of an antifungal chitinase from Leucaena leucocephala de Wit. Biosci. Biotechnol. Biochem. 67: 667-676. [72] Keim, P., Kalif, A., Schupp, J., Hill, K., Travis, S. E., Richmond, K., Adair, D. M., Hugh-Jones, M., Kuske, C. R., and Jackson, P. 1997. Molecular evolution and diversity in Bacillus anthracis as detected by amplified fragment length polymorphism markers. J. Bacteriol. 179: 818-824. [73] Kim, W. S., Salm, H., and Geider, K. 2004. Expression of bacteriophage φEa1h lysozyme in Escherichia coli and its activity in growth inhibition of Erwinia amylovora. Microbiology 150: 2707-2714. [74] Kobayashi, D. Y., Reedy, R. M., Bick, J. A., and Oudemans, P. V. 2002. Characterization of a chitinase gene from Stenotrophomonas maltophilia strain 34S1 and its involvement in biological control. Appl. Environ. Microbiol. 68: 1047-1054. 117 [75] Kong, H., Shimosaka, M., Ando, Y., Nishiyama, K., Fujii, T., and Miyashita, K. 2001. Species-specific distribution of a modular family 19 chitinase gene in Burkholderia gladioli. FEMS Microbiol. Ecol. 37: 135-141. [76] Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. [77] Limon, M. C., Lora, J. M., Garcia, I., De la Cruz, J., Llobell, A., Benitez, T., and Pintor-Toro, J. A. 1995. Primary structure and expression pattern of the 33-kDa chitinase gene from the mycoparasitic fungus Trichoderma harzianum. Curr. Genet. 28: 478-483. [78] Lin, Y. and Xiong, G. 2004. Molecular cloning and sequence analysis of the chitinase gene from Bacillus thuringiensis serovar alesti. Biotechnol. Lett. 26: 635639. [79] Liu, D. A. and Baker, R. 1980. Mechanism of biological control in soil suppressive to Rhizoctonia solani. Phytopathology 70: 404-412. [80] Logemann, J. and Schell, J. 1993. The impact of biotechnology on plant breeding, or how to combine increases in agricultural productivity with an improved protection of the environment. In: Chet, I. (ed): Biotechnology in plant disease control. Wiley-Liss, New York, pp.1-14. [81] Mabuchi, N. and Araki, Y. 2001. Cloning and sequencing of two genes encoding chitinases A and B from Bacillus cereus CH. Can. J. Microbiol. 47: 895-902. [82] Mabuchi, N., Hashizume, I., and Araki, Y. 2000. Characterization of chitinases excreted by Bacillus cereus CH. Can. J. Microbiol. 46: 370-375. [83] MacDaniels, L.H. and Horst, R.K. 1980. Lily disease problems in North America. Universe Books, New York, United States of America. [84] Makrides, S. C. 1996. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev. 60: 512-538. [85] Manocha, M. S. and Sahai, A. S. 1993. Mechanisms of recognition in necrotrophic and biotrophic mycoparasites. Can. J. Microbiol. 39: 269-275. [86] Manoil, C. and Beckwith, J. A genetic approach to analyzing membrane protein topology. Science 233: 1403-1408. [87] Martinez, C., Michaud, M., Belanger, R. R., and Tweddell, R. J. 2002. Identification of soils suppressive against Helminthosporium solani, the causal agent of potato silver scurf. Soil Biol. Biochem. 34: 1861-1868. [88] McCarter, J. D. and Withers, S. G. 1994. Mechanisms of enzymatic glycoside 118 hydrolysis. Curr. Opin. Struct. Biol. 4: 885-892. [89] Migheli, Q., Aloi, C., and Gullino, M. L. 1990. Resistance of Botrytis elliptica to fungicides. Acta Hort. 266: 429-436. [90] Morimoto, K., Karita, S., Kimura, T., Sakka, K., and Ohmiya, K. 1997. Cloning, sequencing, and expression of the gene encoding Clostridium paraputrificum chitinase ChiB and analysis of the functions of novel cadherin-like domains and a chitin-binding domain. J. Bacteriol. 179: 7306-7314. [91] Nilsson, J., Stahl, S., Lundeberg, J., Uhlen, M., and Nygren, P. 1997. Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins. Protein Exp. Purif. 11: 1-6. [92] Ohno, T., Armand, S., Hata, T., Nikaidou, N., Henrissat, B., Mitsutomi, M., and Watanabe, T. 1996. A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT6037. J. Bacteriol. 178: 5065-5070. [93] Ohura, T., Kasuya, K., and Doi, Y. 1999. Cloning and characterization of the polyhydroxybutyrate depolymerase gene of Pseudomonas stutzeri and analysis of the function of substrate-binding domains. J. Bacteriol. 65: 189-197. [94] Okazaki, K., Yamashita, Y., Noda, M., Sueyoshi, N., Kameshita, I., and Hayakawa, S. 2004. Molecular cloning and expression of the gene encoding family 19 chitinase from Streptomyces sp. J-13-3. Biosci. Biotechnol. Biochem. 68: 341-351. [95] Oppenheim, A. B. and Chet, I. 1992. Cloned chitinases in fungal plant-pathogen control strategies. Trends Biotech. 10: 392-394. [96] Orikoshi, H., Nakayama, S., Miyamoto, K., Hanato, C., Yasuda, M., Inamori, Y., and Tsujibo, H. 2005. Roles of four chitinases (chia, chib, chic, and chid) in the chitin degradation system of marine bacterium Alteromonas sp. strain O-7. Appl. Environ. Microbiol. 71: 1811-1815. [97] Perlman, D. and Halvorson, H. O. 1983. A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides. J. Mol. Biol. 167: 391-409. [98] Perrakis, A., Tews, I., Dauter, Z., Oppenheim, A. B., Chet, I., Wilson, K. S., and Vorgias, C. E. 1994. Crystal structure of a bacterial chitinase at 2.3 A resolution. Structure 2: 1169-1180. [99] Peterbauer, C. K., Lorito, M., Hayes, C. K., Harman, G. E., and Kubicek, C. P. 1996. Molecular cloning and expression of the nag1 gene (N-acetyl-β-D-glucosaminidaseencoding gene) from Trichoderma harzianum P1. Curr. Genet. 30: 325-331. 119 [100] Pleban, S., Chernin, L., and Chet, I. 1997. Chitinolytic activity of an endophytic strain of Bacillus cereus. Lett. Appl. Microbiol. 25: 284-288. [101] Raikhel, N. W., Lee, H. I., and Broekaerrt, W. F. 1993. Structure and function of chitin-binding proteins. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44: 591-615. [102] Rao, V., Guan, C., and Van Roey, P. 1995. Crystal structure of endo-β-Nacetylglucosaminidases H at 1.9 A resolution: active-site geometry and substrate recognition. Stucture 3: 449-457. [103] Saito A. and Schrempf, H. 2004. Mutational analysis of the binding affinity and transport activity for N-acetylglucosamine of the novel ABC transporter Ngc in the chitin-degrader Streptomyces olivaceoviridis. Mol Genet Genomics 271: 545-553. [104] Schagger, H. and von Jagow, G. 1987. Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166: 368-379. [105] Schlumbaum, A., Mauch, F. Vogeli, U., and Boller, T. 1986. Plant chitinases are potent inhibitors of fungal growth. Nature 324: 365-367. [106] Schnellmann, J., Zeltins, A., Blaak, H., and Schrempf, H., 1994. The novel lectin- like protein CHB1 is encoded by a chitin-inducible Streptomyces olivaceoviridis gene and binds specifically to crystalline α-chitin of fungi and other organisms. Mol. Microbiol. 13: 807-819. [107] Schrempf, H. 1999. Chitin-binding proteins in streptomycetes. EXS 87: 99-108. [108] Schrempf, H. 2000. Recognition and degradation of chitin by streptomycetes. Antonie Van Leeuwenhoek 79: 285-289. [109] Shapira, R., Ordentlich, A., Chet, I., and Oppenheim, A. B. 1989. Control of plants diseases by chitinases expressed from cloned DNA in Escherichia coli. Phytopathology 79: 1246-1249. [110] Sietsma, J. H., and Wessels, J. G. H. 1979. Evidence for covalent linkages between chitin and β-glucan in a fungal wall. J. Gen. MicrobioI. 114: 99-108. [111] Silo-Suh, L. A., Lethbridge, B. J., Raffel, S. J., He, H., Clardy, J., and Handelsman, J. 1994. Biological activities of two fungistatic antibiotics produced by Bacillus cereus UW85. Appl. Environ. Microbiol. 60: 2023-2030. [112] Simpson, H. and Barras, F. 1999. Functional Analysis of the Carbohydrate-Binding Domains of Erwinia chrysanthemi Cel5 (Endoglucanase Z) and an Escherichia coli Putative Chitinase. Appl. Environ. Microbiol. 181: 4611-4616. 120 [113] Sitrit, Y., Barak, Z., Kapulnik, Y., Oppenheim, A. B., and Chet, I. 1993. Expression of a Serratia marcescens chitinase gene in Rhizobium meliloti during symbiosis on alfalfa roots. Mol. Plant-Microbe Interact. 6: 293-298. [114] Skujins, J. J., Potgieter, H. J., and Alexander, M. 1965. Disolution of fungal cell walls by a treptomycete chitinase and β-(1, 3)-glucanase. Arch. Biochem. Biophys. 111: 358-364. [115] Sundheim, L. 1990. Biocontrol of Fusarium oxysporum with a chitinase encoding gene from Serratia marcescens on a stable plasmid in Pseudomonas. J. Cell. Biochem. 13A: 171. [116] Sundheim, L. 1992. Effect of chitinase encoding genes in biocontrol Pseudomonas spp. In Tjamos, E. C., Papavizas, G. C., and Cook, R. J. (eds): Biological control of plant diseases: progress and challenges for the future. Plenum, New York, 331-333. [117] Takaya, N., Yamazaki, D., Horiuchi, H., Ohta, A., and Takagi, M. 1998. Intracellular chitinase gene from Rhizopus oligosporus: Molecular cloning and characterization. Microbiology 144: 2647-2654. [118] Tanaka, T., Fujiwara, S., Nishikori, S., Fukui, T., Takagi, M., and Imanaka, T. 1999. A unique chitinase with dual active sites and triple substrate binding sites from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1. Appl. Environ. Microbiol. 65: 5338-5344. [119] Tanaka, T., Fukui, T., Atomi, H., and Inamori, T. 2003. Characterization of an exobeta- D-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophlic archaeon Thermococcus kodakaraensis KOD1. J. Bacteriol. 185: 5175-5181. [120] Tanaka, T., Fukui, T., Fujiwara, S., Atomi, H., and Inamori, T. 2004. Concerted action of diacetylchitobiose deacetylase and exo-beta-D-glucosaminidase in a novel chitinolytic pathway in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. J. Biol. Chrm. 279: 30021-30027. [121] Tanaka, T., Fukui, T., and Imanaka, T. 2001. Different cleavage specificities of the dual catalytic domains in chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. J. Biol. Chem. 276: 33629-33635. [122] Tantimavanich, S., Pantuwatana, S., Bhumiratana, A., and Panbangred, W. 1998. Multiple chitinase enzymes from a single gene of Bacillus licheniformis TP-1. J. Ferment. Bioeng. 85: 259-265. 121 [123] Tews, I., Perrakis, A., Oppenheim A. B., Dauter, Z., Wilson, K. S., and Vorgias, C. E. 1996. Bacterial chitobiase structure provides insight into catalytic mechanism and the basis of Tay-Sachs disease. Nat. Struct. Biol. 3: 638-648. [124] Tews, I., van Scheltinga Terwisscha, A. C., Perrakis, A., Wilson, K. S., and Dijkstra, B. W. 1997. Substrate-assisted catalysis unifies 2 families of chitinolytic enzymes. J. Am. Chem. Soc. 119: 7954-7959. [125] Thamthiankul, S., Suan-Ngay, S., Tantimavanich, S., and Panbangred, W. 2001. Chitinase from Bacillus thuringiensis subsp. pakistani. Appl. Microbiol. Biotechnol. 56: 395-401. [126] Tomme, P., Warren, R. A. J., and Gilkes, N. R. 1995. Cellulose hydrolysis by bacteria and fungi. Adv. Microb. Physiol. 37: 1-81. [127] Tomme, P., Gilkes, N. R., Miller, R. C. Jr., Warren, R. A. J., and Kilburn, D. G. 1994. An internal cellulose-binding domain mediates adsorption of an engineered bifunctional xylanase/cellulase. Protein Eng. 7: 117-123. [128] Tronsmo, A. and Harman, G. E. 1993. Detection and quantification of N-acetyl-β-Dglucosaminidase, chitobiosidase, and endochitinase in solutions and on gels. Anal. Biochem. 208: 74-79. [129] Trudel, J. and Asselin, A. 1989. Detection of chitinase activity after polyacrylamide gel electrophoresis. Anal. Biochem. 178: 362-366. [130] Tsujibo, H., Okamoto, T., Hatano, N., Miyamoto, L., Watanabe, T., Mitsutomi, M., and Inamori, Y. 2000. Family 19 chitinases from Streptomyces thermoviolaceus OPC- 520: molecular cloning and characterization. Biosci. Biotechnol. Biochem. 64: 2445- 2453. [131] van Roey, P., Rao, V., Plummer, T. H., and Tarentino, A. L. 1994. Crystal structure of endo-β-N-acetylglucosaminidases F1, and α/β-barrel enzyme adapted for a complex substrate. Biochemistry 33: 13989-13996. [132] van Scheltinga Terwisscha, A. C., Armand, S., Kalk, K. H., Isogai, A., Henrissat, B., and Dijkstra, B. W. 1995. Stereochemistry of chitin hydrolysis by a plant chitinase/lysozyme and x-ray structure of a complex with allosamidin. Evidence for substrate assisted catalysis. Biochemistry 34: 15619-15623. [133] van Scheltinga Terwisscha, A. C., Hennig, M., and Dijkstra, B. W. 1996. The 1.8 A resolution structure of hevamine, a plant chitinase/lysozyme, and analysis of the conserved sequence and structure motifs of glycosyl hydrolase family 18. J. Mol. Biol. 122 262: 243-257. [134] van Scheltinga Terwisscha, A. C., Kalk, K. H., Beintema, J. J., and Dijkstra, B. W. 1994. Crystal structure of hevamine, a plant defense protein with chitinase and lysozyme activity, and its complex with an inhibitor. Structure 2: 1181-1189. [135] Vazquez-Garciduenas, S., Leal-Morales, C. A., and Herrera-Estrella, A. 1998. Analysis of the β-1, 3-glucanolytic system of the biocontrol agent Trichoderma harzianum. Appl. Environ. Microbiol. 64: 1442-1446. [136] Wang, F., Xiao, X., Saito, A., and Schrempf, H. 2002. Streptomyces olivaceoviridis possesses a phophotransferase system that mediates specific, phosphoenolpyruvatedependent uptake of N-acetylglucosamine. Mol. Genet. Genomics 268: 344-351. [137] Wang, F. P., Li, Q., Zhou, Y., Li, M. G., and Xiao, X. 2003. The C-terminal module of Chi1 from Aeromonas caviae CB101 has a function in substrate binding and hydrolysis. Proteins 53: 908-916. [138] Wang, S. Y., Moyne, A. L., Thottappilly, G., Wu, S. J., Locy, R. D., and Singh, N. K. 2001. Purification and characterization of a Bacillus cereus exochitinase. Enzyme Microbial Technol. 28: 492-498. [139] Warren, R. A. J. 1996. Microbial hydrolysis of polysaccharides. Annu. Rev. Microbiol. 50: 183-212. [140] Watanabe, T., Ito, Y., Yamada, T., Hashimoto, M., Sekine, S., and Tanaka, H. 1994. The roles of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation. J. Bacteriol. 176: 4465-4472. [141] Watanabe, T., Kobori, K., Miyashita, K., Fujii, T., Sakai, H., Uchida, M., and Tanaka, H. 1993. Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity. J. Biol. Chem. 268: 18567-18572. [142] Watanabe, T., Oyanagi, W., Suzuki, K., Ohnishi, K., and Tanaka, H. 1992. Structure of the gene encoding chitinase D of Bacillus circulans WL-12 and possible homology of the enzyme to other prokaryotic chitinases and class III plant chitinases. J. Bacteriol. 174: 408-414. [143] Watanabe, T., Oyanagi, W., Suzuki, K., and Tanaka, H. 1990a. Chitinase system of Bacillus circulans WL-12 and importance of chitinase A1 in chitin degradation. J. Bacteriol. 172: 4017-4022. [144] Watanabe, T., Suzuki, K., Oyanagi, W., Ohnishi, K., and Tanaka, H. 1990b. Gene 123 cloning of chitinase A1 from Bacillus circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type III homology units of fibronectin. J. Biol. Chem. 265: 15659-15665. [145] Weidling, R. 1932. Trichoderma lignorum as a parasite of other fungi. Phytopathology 22: 837-845. [146] Xiao, X., Wang, F., Saito, A., Majka, J., Schlosser, A., and Schrempf, H. 2002. The novel Streptomyces olivaceoviridis ABC transporter Ngc mediates uptake of Nacetylglucosamine and N, N’-diacetylchitobiose. Mol. Genet. Genomics 267: 429-439. [147] Xu, G. Y., Ong, E., Gilkes, N. R., Kilburn, D. G., Muhandiram, D. R., Harris-Brendts, M., Carver, J. P., Kay, L. E., and Harvey, T. S. 1995. Solution structure of a cellulosebinding domain from Cellulomonas fimi by nuclear magnetic resonance spectroscopy. Biochemistry 34: 6993-7009. [148] Yamada, S., Ohashi, E., Agata, N., and Venkateswaran, K. 1999. Cloning and nucleotide sequence analysis of gyrB of Bacillus cereus, B. thuringiensis, B. mycoides, and B. anthracis and their application to the detection of B. cereus in rice. Appl. Environ. Microbiol. 65: 1483-1490. [149] Zeltins, A., and Schrempf, H. 1995. Visualization of α-chitin with a specific chitinbinding protein (CHB1) from Streptomyces olivaceoviridis. Anal. Biochem. 231: 287- 294. [150] Zeltins, A., and Schrempf, H. 1997. Specific interaction of the Streptomyces chitinbinding protein CHB1 with α-chitin: the role of individual tryptophan residues. Eur. J. Biochem. 246: 557-564.	en
item.openairecristype	http://purl.org/coar/resource_type/c_1843	-
item.fulltext	with fulltext	-
item.grantfulltext	open	-
item.cerifentitytype	Products	-
item.openairetype	other	-
item.languageiso639-1	en_US	-
Appears in Collections:	植物病理與微生物學系

Files in This Item:

File	Description	Size	Format
ntu-95-F89633001-1.pdf		23.53 kB	Adobe PDF	View/Open

Show simple item record

Page view(s)

checked on Jun 22, 2020

Download(s) 10

checked on Jun 22, 2020

Google Scholar^TM

Check

Files in This Item:

Page view(s)

Download(s) 10

Google ScholarTM

Google Scholar^TM