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研究生:林茹慧
研究生(外文):Ju-Hui Lin
論文名稱:巨型桿菌胞外PHB去聚合酵素之特性分析及蘇力菌胞內PHB去聚合酵素之活化子之純化
論文名稱(外文):Characterization of an extracellular PHB depolymerase of Bacillus megaterium and purification of the activator of Bacillus thuringiensis intracellular PHB depolymerase
指導教授:邵國銓
指導教授(外文):Gwo-Chyuan Shaw
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:119
中文關鍵詞:胞外PHB去聚合酵素胞內PHB去聚合酵素活化子
外文關鍵詞:extracellular PHB depolymeraseintracellular PHB depolymeraseactivator
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中文摘要
Poly(3-hydroxybutyrate) (PHB) 是大部分細菌在營養不均衡的情況下用來儲存碳與能量的物質,細菌可透過胞內PHB去聚合酶 (i-PHBDPs) 或胞外PHB去聚合酶 (e-PHBDPs) 分解PHB以獲得碳源與能量。一般而言,i-PHBDP以及e-PHBDP的專一性受質分別為native以及denatured PHB。相較於格蘭氏陰性菌,目前對格蘭氏陽性菌胞外PHB去聚合酶的了解極少。本研究利用大腸桿菌大量表現QM B1551菌株之胞外PHB去聚合酶PhaZ4重組蛋白,並針對其酵素特性進行分析。結果顯示His-PhaZ4(Full)不僅可結合與分解dPHB以及nPHB,且分解產物大多為3HB單體,除此,His-PhaZ4(Full)可分解來自其他菌種之nPHB granules。更近一步實驗指出,刪除C端之His-PhaZ4-N domain可結合dPHB,但無法結合nPHB,且其水解能力隨His-PhaZ4-N domain濃度增加而提升;相對於His-PhaZ4-N domain,His-PhaZ4-C domain則可以同時結合dPHB及nPHB,故本論文證實PhaZ4為第一個可同時結合與水解nPHB及dPHB之胞外PHB去聚合酶,且其C端區域對PhaZ4結合與水解nPHB granules是不可或缺的。
另外,過去報導指出蘇力菌胞內PHB去聚合酶PhaZ如同Rhodospirillum rubrum胞內PHB去聚合酶PhaZ1,只分解經trypsin活化之nPHB granules,而R. rubrum細胞內存在可取代trypsin功能之activator。本研究發現將野生型以及phaZ突變型蘇力菌培養於含2% glucose之GYS培養液適當時間後,二者菌體皆會堆積大量granules,但細胞粗萃取液皆無法偵測到活化子活性;當進一步將培養液置換成glucose-minus之GYS培養液繼續培養,則野生型蘇力菌之細胞粗萃液具活化子活性,且菌體內granules產生崩解現象,但是phaZ 突變型則體內nPHB granules沒有崩解情形發生,且細胞粗萃取液也無活化子活性。除此之外,硫酸銨鹽沉澱實驗顯示活化子活性主要落於20-40%之飽和硫酸銨鹽溶液析出物。據上述之結果,故本論文推測野生型蘇力菌胞內存在胞內去聚合酶進行去聚合反應所需之活化子。
Abstract
Poly(3-hydroxybutyrate) (PHB) is an intracellular carbon and energy storage material accumulated by various bacteria under imbalanced nutrition. Bacteria can degrade PHB granules by intracellular PHB depolymerases (i-PHBDPs) or extracellular PHB depolymerases (e-PHBDPs) to obtain carbon and energy sources. In general, the specific substrates for i-PHBDP and e-PHBDP are native PHB (nPHB) and denatured PHB (dPHB), respectively. In contrast to those of Gram-negative bacteria, the e-PHBDPs of Gram-positive bacteria are not well investigated. In this study, we overproduced e-PHBDP PhaZ4 of Bacillus megaterium QM B1551 in Escherichia coli and characterized it. The results showed that purified His-PhaZ4(Full) could bind and hydrolyze dPHB and nPHB, generating 3HB monomers as main hydrolytic products. In addition, His-PhaZ4(Full) could also hydrolyze nPHB purified from other bacteria. Further analysis showed that His-PhaZ4-N domain could bind to dPHB but could not bind to nPHB, and the efficiency of dPHB hydrolysis increased with the concentrations of His-PhaZ4-N domain. In contrast to His-PhaZ4-N domain, purified His-PhaZ4-C domain could bind to both nPHB and dPHB. Therefore, we demonstrate for the first time that PhaZ4 is the first e-PHBDP that can hydrolyze both forms of PHB, and the C-terminal region of PhaZ4 is necessary for binding and hydrolyzing nPHB granules.
i-PHBDP PhaZ of B. thuringiensis, like intracellular PhaZ1 of Rhodospirillum rubrum, can only hydrolyze trypsin-activated nPHB. It is known that R. rubrum contains an activator that functions like trypsin. In this study, we found that the wild-type B. thuringiensis and the phaZ mutant cultured in 2% glucose-GYS medium could accumulate massive PHB granules, but they exhibited no activator activity under this growth condition. After being transferred to a fresh glucose-minus GYS medium, the wild-type B. thuringiensis exhibited an activator activity and degradation of nPHB granules occurred. However, in the phaZ mutant, no degradation of nPHB granules occurred and no activator activity was detected. In addition, an activator activity fell within 20-40% saturated ammonium sulfate when ammonium sulfate fractionation was used. According to these findings, we infer that the wild-type B. thuringiensis contains a novel activator important for PHB depolymerization by the intracellular PhaZ.
目錄
圖表之目錄 ---------------------------------------------Ⅳ
中文摘要 ---------------------------------------------Ⅵ
英文摘要 ---------------------------------------------Ⅶ壹、緒論
一、 巨型桿菌 (Bacillus megaterium) 簡介 ------------1
二、 蘇力菌 (Bacillus thuringiensis) 簡介 -----------1
三、 PHA (polyhydroxyalkanoate) 簡介-----------------2
四、 PHB的生合成以及巨型桿菌PHB生合成的相關基因------4
五、 PHA granules -----------------------------------5
六、 PHA之分解---------------------------------------7
七、 胞外PHB去聚合酶 (extracellualr PHB depolymerase, e-PHBDP) ------------------------------------------------9
八、 胞內PHB去聚合酶 (intracellular PHB deploymerase, i-PHBDP) 及其活化子 (activator) ------------------------11
九、 格蘭氏陽性菌之胞外PHB去聚合酶 (e-PHB depolymerase) -----------------------------------------12
十、 蘇力菌胞內PHB分解 -----------------------------13
貳、 實驗方法與材料
一、 染色體DNA的抽取 -------------------------------15
二、 質體DNA的抽取 ----------------------------------15
三、 DNA的回收 -------------------------------------16
四、 大腸桿菌勝任細胞 (competent cells) 的製備 ------16
五、 轉形作用 (Transformation) ----------------------16
1、 大腸桿菌的轉形作用 –熱休克轉形作用 (heat shock transformation )
2、 巨型桿菌的轉形作用 –原生質體轉形作用 (protoplast transformatiom)
六、 蛋白質的表現與純化–鎳離子親和性管柱層析法 (Ni-NTA affinity chromatography) ---------------------------18
七、 全細胞萃取 (whole cell extract ) --------------19
八、 SDS-PAGE 蛋白質電泳分析以及蛋白質定量-----------19
九、 蛋白質純化–重新摺疊 (Refolding) ---------------19
十、 PHB granules之製備 -----------------------------20
1. Native PHB (nPHB) 之製備
2. Denatured PHB (dPHB) 之製備
十一、酵素活性分析 - Turbidity assay (濁度分析)---------21
1. 蘇力菌PhaZ或巨型桿菌PhaZ分解nPHB granules (添加trypsin)
2. 巨型桿菌PhaZ4分解nPHB或dPHB granules (不添加trypsin)
3. 蘇力菌PhaZ之活化子之活性分析
十二、D-3-hydroxybutyrate單體測定------------------------22
十三、結合試驗 (binding assay)---------------------------23
十四、Two-Step RCR---------------------------------------24
十五、BCA蛋白濃度分析以及XylE (catechol 2,3-dioxygenase) 酵素活性分析-----------------------------------------------25
十六、蘇力菌胞內PHB去聚合酶之活化子之純化----------------25
1. 養菌條件
2. 破菌
3. CM column (Merck:Fractogel® TSK CM-650 (M)) 之純化步驟
4. Hydroxyapatite column (BIO-RAD:Econo-Pac® CHT-II cartridge) 之純化步驟
5. 濃縮
十七、Nile blue A 染色-----------------------------------27
參、 實驗結果
一、巨型桿菌胞外PHB去聚合酶之特性分析
1.1、 His-PhaZ4(Full)蛋白質之表現與純化 -------------28
1.2、 純化之His-PhaZ4(Full) 具有分解巨型桿菌dPHB之能力-----------------------------------------------------------28
1.3、 His-PhaZ4(Full) 可分解巨型桿菌之nPHB granules -------------------------------------------------------------29
1.4、 His-PhaZ4(Full) 可分解蘇力菌之nPHB granules ----30
1.5、 His-PhaZ4(Full) 分解nPHB以及dPHB之產物大多為3HB單體-------------------------------------------------------30
1.6、 PhaZ4之N端以及C端蛋白質之表現與純化 ------------31
1.7、 His-PhaZ4-N domain具有分解dPHB的能力,但無法分解nPHB ---------------------------------------------------32
1.8、 His-PhaZ4(Full) 可結合巨型桿菌 nPHB和dPHB以及蘇力菌nPHB --------------------------------------------------32
1.9、 His-PhaZ4-N domain可結合巨型桿菌dPHB但無法結合nPHB granules -------------------------------------------33
1.10、 His-PhaZ4-C domain具有結合巨型桿菌dPHB以及nPHB之能力-------------------------------------------------------34
1.11、 His-PhaZ4-C domain可結合蘇力菌nPHB granules但N domain無法結合-------------------------------------------34
1.12、 PhaZ4之啟動子之活性分析 -----------------------------------------------------------------------------------35
二、 蘇力菌胞內PHB去聚合酶之活化子之純化
2.1、 蘇力菌胞內PHB去聚合酶 (PhaZ) 需trypsin幫忙才可分解nPHB ----------------------------------------------------36
2.2、 野生型蘇力菌於含有2% glucose 之GYS medium培養後再將培養液置換成glucose-minus GYS medium繼續培養則其細胞粗萃取液具活化子活性而phaZ突變型則無此活性---------------------36
2.3、 野生型蘇力菌於含2% glucose 之GYS medium培養後再將培養液置換成glucose-minus GYS medium繼續培養可見菌內granules出現崩解情形,而phaZ突變型無崩解現象發生 ---------------37
2.4、 純化野生型蘇力菌胞內PHB去聚合酶之活化子---------37
2.5、 活化子活性區落於20-40%之飽和硫酸銨鹽溶液 ------38
肆、 討論 ------------------------------------------40
伍、 圖表 ------------------------------------------46
陸、 參考文獻 ----------------------------------------91
柒、 附錄 --------------------------------------------99




圖表目錄
第一部份:巨型桿菌胞外PHB去聚合酶之特性分析
圖1.1、 PhaZ4重組蛋白質的表現與純化 ----------------------48
圖1.2、 巨型桿菌His-PhaZ4(Full) 分解巨型桿菌dPHB 之活性分析 -------------------------------------------------------49
圖1.3、 巨型桿菌His-PhaZ4(Full) 分解巨型桿菌nPHB granules之活性分析 -------------------------------------------------51
圖1.4、 重新摺疊之His-PhaZ4(Full) 分解巨型桿菌nPHB granules之能力分析------------------------------------------------53
圖1.5、 巨型桿菌His-PhaZ4(Full) 分解蘇力菌nPHB granules之活性分析 ---------------------------------------------------54
圖1.6、 His-PhaZ4(Full) 分解dPHB and nPHB granules產生之3-HB單體量 ---------------------------------------------------56
圖1.7、 PhaZ4之N端以及C端重組蛋白質之表現與純化 ----------------------------------------------------------------------58
圖1.8、 不同濃度之His-PhaZ4-N domain分解巨型桿菌dPHB之活性分析 -------------------------------------------------------59
圖1.9、 不同濃度之His-PhaZ4-N domain分解巨型桿菌nPHB granules之活性分析 ---------------------------------------60
圖1.10、 His-PhaZ4-N domain分解trypsin-activated nPHB granules之活性分析----------------------------------------61
圖1.11、 His-PhaZ4(Full) 和巨型桿菌QM B1551 nPHB granules之結合反應 -------------------------------------------------62
圖1.12、 His-PhaZ4(Full) 和蘇力菌 nPHB granules之結合反應 ------------------------------------------------------------63
圖1.13、 His-PhaZ4(Full) 和巨型桿菌QM B1551 dPHB之結合反應 -----------------------------------------------------------64
圖1.14、 His-PhaZ4-N domain和巨型桿菌QM B1551 dPHB之結合反應 -------------------------------------------------------65
圖1.15、 His-PhaZ4-N domain和巨型桿菌QM B1551 nPHB granules之結合反應------------------------------------------------66
圖1.16、 His-PhaZ4-N domain 和trypsin-activated nPHB granules之結合反應 ---------------------------------------67
圖1.17、 His-PhaZ4-C domain與巨型桿菌QM B1551 nPHB granules以及dPHB之結合反應 ---------------------------------------68
圖1.18、 His-PhaZ4-N domain及C domain和蘇力菌 nPHB granules之結合反應------------------------------------------------69
圖1.19、 分析巨型桿菌phaZ4啟動子活性表現區域 -------------70
圖1.20、 突變phaZ4 可能的-10啟動子區域 -------------------71

第二部份:純化蘇力菌胞內PHB去聚合酶之活化子
圖2.1、 蘇力菌His-PhaZ分解nPHB granules之活性分析 --------75
圖2.2 、野生型以及phaZ突變型蘇力菌細胞粗萃取液之活化子活性分析 -------------------------------------------------------76
圖2.3、 野生型蘇力菌於添加2% glucose的GYS medium 培養16小時後以Nile blue A進行螢光染色,觀察菌體內granules堆積情形 --------------------------------------------------------------78
圖2.4、 野生型蘇力菌培養於含2% glucose之GYS medium 16小時再將培養液置換成glucose-minus GYS medium繼續培養4小時後之菌體內granules堆積情形 --------------------------------------------------------------------------------------------------79
圖2.5、 利用CM column純化野生型蘇力菌細胞粗萃取液之活化子 ------------------------------------------------------------80
圖2.6、 將CM column fraction #26- #33以及 #38- #48再次以hydroxyapatite column進行純化- ---------------------------81
圖2.7、利用硫酸銨鹽劃分CM column殘留液之活化子活性區域 ---87
圖2.8、利用硫酸銨鹽劃分野生型蘇力菌細胞粗萃取液之活化子活性區域 -----------------------------------------------------89
陸、參考文獻
Abe, T., Kobayashi, T., and Saito, T. (2005). Properties of a novel intracellular poly(3-hydroxybutyrate) depolymerase with high specific activity (PhaZd) in Wautersia eutropha H16. J Bacteriol 187, 6982-6990.
Agaisse, H., and Lereclus, D. (1995). How does Bacillus thuringiensis produce so much insecticidal crystal protein? J Bacteriol 177, 6027-6032.
Anderson, A.J., and Dawes, E.A. (1990). Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54, 450-472.
Barnard, G.N., and Sanders, J.K. (1989). The poly-beta-hydroxybutyrate granule in vivo. A new insight based on NMR spectroscopy of whole cells. J Biol Chem 264, 3286-3291.
Behrends, A., Klingbeil, B., and Jendrossek, D. (1996). Poly(3-hydroxybutyrate) depolymerases bind to their substrate by a C-terminal located substrate binding site. FEMS Microbiol Lett 143, 191-194.
Brandl, H., Gross, R.A., Lenz, R.W., and Fuller, R.C. (1990). Plastics from bacteria and for bacteria: poly(beta-hydroxyalkanoates) as natural, biocompatible, and biode -gradable polyesters. Adv Biochem Eng Biotechnol 41, 77-93.
Fukui, T., Ito, M., and Tomita, K. (1982). Purification and characterization of acetoacetyl-CoA synthetase from Zoogloea ramigera I-16-M. Eur J Biochem 127, 423- 428.
Gerngross, T.U., Reilly, P., Stubbe, J., Sinskey, A.J., and Peoples, O.P. (1993). Immunocytochemical analysis of poly-beta-hydroxybutyrate (PHB) synthase in Alcaligenes eutrophus H16: localization of the synthase enzyme at the surface of PHB granules. J Bacteriol 175, 5289-5293.
Griebel, R., Smith, Z., and Merrick, J.M. (1968). Metabolism of poly-beta- hydroxybutyrate. I. Purification, composition, and properties of native poly-beta- hydroxy -butyrate granules from Bacillus megaterium. Biochemistry 7, 3676-3681.
Griebel, R.J., and Merrick, J.M. (1971). Metabolism of poly-b -hydroxybutyrate: effect of mild alkaline extraction on native poly-b -hydroxybutyrate granules. J Bacteriol 108, 782-789.
Gross, R.A., and Kalra, B. (2002). Biodegradable polymers for the environment. Science 297, 803-807.
Handrick, R., Reinhardt, S., Focarete, M.L., Scandola, M., Adamus, G., Kowalczuk, M., and Jendrossek, D. (2001). A new type of thermoalkalophilic hydrolase of Paucimonas lemoignei with high specificity for amorphous polyesters of short chain- length hydroxyalkanoic acids. J Biol Chem 276, 36215-36224.
Handrick, R., Reinhardt, S., and Jendrossek, D. (2000). Mobilization of poly(3-hydroxybutyrate) in Ralstonia eutropha. J Bacteriol 182, 5916-5918.
Handrick, R., Reinhardt, S., Kimmig, P., and Jendrossek, D. (2004a). The "intracellular" poly(3-hydroxybutyrate) (PHB) depolymerase of Rhodospirillum rubrum is a periplasm-located protein with specificity for native PHB and with structural similarity to extracellular PHB depolymerases. J Bacteriol 186, 7243-7253.
Handrick, R., Reinhardt, S., Schultheiss, D., Reichart, T., Schuler, D., Jendrossek, V., and Jendrossek, D. (2004b). Unraveling the function of the Rhodospirillum rubrum activator of polyhydroxybutyrate (PHB) degradation: the activator is a PHB- granule- bound protein (phasin). J Bacteriol 186, 2466-2475.
Handrick, R., Technow, U., Reichart, T., Reinhardt, S., Sander, T., and Jendrossek, D. (2004c). The activator of the Rhodospirillum rubrum PHB depolymerase is a polypeptide that is extremely resistant to high temperature (121 degrees C) and other physical or chemical stresses. FEMS Microbiol Lett 230, 265-274.
He, J., Chen, S., and Yu, Z. (2002). Determination of poly-beta-hydroxybutyric acid in Bacillus thuringiensis by capillary zone electrophoresis with indirect ultraviolet absorbance detection. J Chromatogr A 973, 197-202.
Hisano, T., Kasuya, K., Tezuka, Y., Ishii, N., Kobayashi, T., Shiraki, M., Oroudjev, E., Hansma, H., Iwata, T., Doi, Y., et al. (2006). The crystal structure of polyhydroxybuty -rate depolymerase from Penicillium funiculosum provides insights into the recogni -tion and degradation of biopolyesters. J Mol Biol 356, 993-1004.
Hrafnsdottir, S., Nichols, J.W., and Menon, A.K. (1997). Transbilayer movement of fluorescent phospholipids in Bacillus megaterium membrane vesicles. Biochemistry 36, 4969-4978.
Huisman, G.W., de Leeuw, O., Eggink, G., and Witholt, B. (1989). Synthesis of poly-3-hydroxyalkanoates is a common feature of fluorescent pseudomonads. Appl Environ Microbiol 55, 1949-1954.
Jaeger, K.E., Ransac, S., Dijkstra, B.W., Colson, C., van Heuvel, M., and Misset, O. (1994). Bacterial lipases. FEMS Microbiol Rev 15, 29-63.
Jendrossek, D. (2009). Polyhydroxyalkanoate (PHA) Granules Are Complex Subcellular Organelles (Carbonosomes). J Bacteriol.
Jendrossek, D., Backhaus, M., and Andermann, M. (1995a). Characterization of the extracellular poly(3-hydroxybutyrate) depolymerase of Comamonas sp. and of its structural gene. Can J Microbiol 41 Suppl 1, 160-169.
Jendrossek, D., Frisse, A., Behrends, A., Andermann, M., Kratzin, H.D., Stanislawski, T., and Schlegel, H.G. (1995b). Biochemical and molecular characterization of the Pseudomonas lemoignei polyhydroxyalkanoate depolymerase system. J Bacteriol 177, 596-607.
Jendrossek, D., and Handrick, R. (2002). Microbial degradation of polyhydroxy- alkanoates. Annu Rev Microbiol 56, 403-432.
Jendrossek, D., Muller, B., and Schlegel, H.G. (1993). Cloning and characterization of the poly(hydroxyalkanoic acid) depolymerase gene locus, phaZ1, of Pseudomonas lemoignei and its gene product. Eur J Biochem 218, 701-710.
Jendrossek, D., Schirmer, A., and Schlegel, H.G. (1996). Biodegradation of polyhydroxyalkanoic acids. Appl Microbiol Biotechnol 46, 451-463.
Jurasek, L., and Marchessault, R.H. (2002). The role of phasins in the morphogenesis of poly(3-hydroxybutyrate) granules. Biomacromolecules 3, 256-261.
Kasuya, K., Inoue, Y., Tanaka, T., Akehata, T., Iwata, T., Fukui, T., and Doi, Y. (1997). Biochemical and molecular characterization of the polyhydroxybutyrate depolymerase of Comamonas acidovorans YM1609, isolated from freshwater. Appl Environ Microbiol 63, 4844-4852.
Kasuya, K., Ohura, T., Masuda, K., and Doi, Y. (1999). Substrate and binding specificities of bacterial polyhydroxybutyrate depolymerases. Int J Biol Macromol 24, 329- 336.
Kikkawa, Y., Fujita, M., Hiraishi, T., Yoshimoto, M., and Doi, Y. (2004). Direct observation of poly(3-hydroxybutyrate) depolymerase adsorbed on polyester thin film by atomic force microscopy. Biomacromolecules 5, 1642-1646.
Kita, K., Ishimaru, K., Teraoka, M., Yanase, H., and Kato, N. (1995). Properties of poly(3-hydroxybutyrate) depolymerase from a marine bacterium, Alcaligenes faecalis AE122. Appl Environ Microbiol 61, 1727-1730.
Klingbeil, B., Kroppenstedt, R.M., and Jendrossek, D. (1996). Taxonomic identification of Streptomyces exfoliatus K10 and characterization of its poly(3-hydroxybutyrate) depolymerase gene. FEMS Microbiol Lett 142, 215-221.
Kobayashi, T., Shiraki, M., Abe, T., Sugiyama, A., and Saito, T. (2003). Purification and properties of an intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZ2) in Ralstonia eutropha H16 and its identification as a novel intracellular poly(3-hydroxybutyrate) depolymerase. J Bacteriol 185, 3485-3490.
Kobayashi, T., Uchino, K., Abe, T., Yamazaki, Y., and Saito, T. (2005). Novel intracellular 3-hydroxybutyrate-oligomer hydrolase in Wautersia eutropha H16. J Bacteriol 187, 5129-5135.
Lee, S.Y. (1996). Bacterial polyhydroxyalkanoates. Biotechnol Bioeng 49, 1-14.
Lee, T.R., Lin, J.S., Wang, S.S., and Shaw, G.C. (2004). PhaQ, a new class of poly-beta-hydroxybutyrate (phb)-responsive repressor, regulates phaQ and phaP (phasin) expression in Bacillus megaterium through interaction with PHB. J Bacteriol 186, 3015-3021.
Luengo, J.M., Garcia, B., Sandoval, A., Naharro, G., and Olivera, E.R. (2003). Bioplastics from microorganisms. Curr Opin Microbiol 6, 251-260.
Madison, L.L., and Huisman, G.W. (1999). Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol Mol Biol Rev 63, 21-53.
Maehara, A., Taguchi, S., Nishiyama, T., Yamane, T., and Doi, Y. (2002). A repressor protein, PhaR, regulates polyhydroxyalkanoate (PHA) synthesis via its direct interaction with PHA. J Bacteriol 184, 3992-4002.
Maehara, A., Ueda, S., Nakano, H., and Yamane, T. (1999). Analyses of a polyhydroxyalkanoic acid granule-associated 16-kilodalton protein and its putative regulator in the pha locus of Paracoccus denitrificans. J Bacteriol 181, 2914-2921.
McCool, G.J., and Cannon, M.C. (1999). Polyhydroxyalkanoate inclusion body-associated proteins and coding region in Bacillus megaterium. J Bacteriol 181, 585- 592.
McCool, G.J., and Cannon, M.C. (2001). PhaC and PhaR are required for polyhydroxyalkanoic acid synthase activity in Bacillus megaterium. J Bacteriol 183, 4235- 4243.
Merrick, J.M., and Doudoroff, M. (1964). Depolymerization of Poly-Beta- Hydroxybutyrate by Intracellular Enzyme System. J Bacteriol 88, 60-71.
Merrick, J.M., Steger, R., and Dombroski, D. (1999). Hydrolysis of native poly(hydroxybutyrate) granules (PHB), crystalline PHB, and artificial amorphous PHB granules by intracellular and extracellular depolymerases. Int J Biol Macromol 25, 129-134.
Mukai, K., Yamada, K., and Doi, Y. (1992). Extracellular poly(hydroxyalkanoate) depolymerases and their inhibitor from Pseudomonas lemoignei. Int J Biol Macromol 14, 235-239.
Nojiri, M., and Saito, T. (1997). Structure and function of poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1. J Bacteriol 179, 6965-6970.
Numata, K., Yamashita, K., Fujita, M., Tsuge, T., Kasuya, K., Iwata, T., Doi, Y., and Abe, H. (2007). Adsorption and hydrolysis reactions of poly(hydroxybutyric acid) depolymerases secreted from Ralstonia pickettii T1 and Penicillium funiculosum onto poly[(R)-3-hydroxybutyric acid]. Biomacromolecules 8, 2276-2281.
Ohura, T., Kasuya, K.I., and Doi, Y. (1999). Cloning and characterization of the polyhydroxybutyrate depolymerase gene of Pseudomonas stutzeri and analysis of the function of substrate-binding domains. Appl Environ Microbiol 65, 189-197.
Ostle, A.G., and Holt, J.G. (1982). Nile blue A as a fluorescent stain for poly-beta- hydroxybutyrate. Appl Environ Microbiol 44, 238-241.
Raux, E., Lanois, A., Warren, M.J., Rambach, A., and Thermes, C. (1998). Cobalamin (vitamin B12) biosynthesis: identification and characterization of a Bacillus megaterium cobI operon. Biochem J 335 ( Pt 1), 159-166.
Reddy, C.S., Ghai, R., Rashmi, and Kalia, V.C. (2003). Polyhydroxyalkanoates: an overview. Bioresour Technol 87, 137-146.
Rehm, B.H. (2003). Polyester synthases: natural catalysts for plastics. Biochem J 376, 15-33.
Saegusa, H., Shiraki, M., Kanai, C., and Saito, T. (2001). Cloning of an intracellular Poly[D(-)-3-Hydroxybutyrate] depolymerase gene from Ralstonia eutropha H16 and characterization of the gene product. J Bacteriol 183, 94-100.
Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D.R., and Dean, D.H. (1998). Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62, 775-806.
Schultheiss, D., Handrick, R., Jendrossek, D., Hanzlik, M., and Schuler, D. (2005). The presumptive magnetosome protein Mms16 is a poly(3-hydroxybutyrate) granule- bound protein (phasin) in Magnetospirillum gryphiswaldense. J Bacteriol 187, 2416- 2425.
Senior, P.J., and Dawes, E.A. (1973). The regulation of poly-beta-hydroxybutyrate metabolism in Azotobacter beijerinckii. Biochem J 134, 225-238.
Shinohe, T., Nojiri, M., Saito, T., Stanislawski, T., and Jendrossek, D. (1996). Determination of the active sites serine of the poly (3-hydroxybutyrate) depolymerases of Pseudomonas lemoignei (PhaZ5) and of Alcaligenes faecalis. FEMS Microbiol Lett 141, 103-109.
Shirakura, Y., Fukui, T., Saito, T., Okamoto, Y., Narikawa, T., Koide, K., Tomita, K., Takemasa, T., and Masamune, S. (1986). Degradation of poly(3-hydroxybutyrate) by poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1. Biochim Biophys Acta 880, 46-53.
Slepecky, R.A., and Law, J.H. (1961). Synthesis and degradation od poly-beta-hydroxybutyric acid in connection with sporulation od Bacillus megaterium. J Bacteriol 82, 37-42.
Steinbuchel, A., and Hein, S. (2001). Biochemical and molecular basis of microbial synthesis of polyhydroxyalkanoates in microorganisms. Adv Biochem Eng Biotechnol 71, 81-123.
Suga, K., Shiba, Y., Sorai, T., Shioya, S., and Ishimura, F. (1990). Reaction kinetics and mechanism of immobilized penicillin acylase from Bacillus megaterium. Ann N Y Acad Sci 613, 808-815.
Sugiyama, A., Kobayashi, T., Shiraki, M., and Saito, T. (2004). Roles of poly(3-hydroxybutyrate) depolymerase and 3HB-oligomer hydrolase in bacterial PHB metabolism. Curr Microbiol 48, 424-427.
Takaku, H., Kimoto, A., Kodaira, S., Nashimoto, M., and Takagi, M. (2006). Isolation of a Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterium from compost, and cloning and characterization of a gene encoding PHB depolymerase of Bacillus megaterium N-18-25-9. FEMS Microbiol Lett 264, 152-159.
Tanio, T., Fukui, T., Shirakura, Y., Saito, T., Tomita, K., Kaiho, T., and Masamune, S. (1982). An extracellular poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis. Eur J Biochem 124, 71-77.
Tokiwa, Y., and Calabia, B.P. (2004). Degradation of microbial polyesters. Biotechnol Lett 26, 1181-1189.
Tseng, C.L., Chen, H.J., and Shaw, G.C. (2006). Identification and characterization of the Bacillus thuringiensis phaZ gene, encoding new intracellular poly-3- hydroxybutyrate depolymerase. J Bacteriol 188, 7592-7599.
Uchino, K., Katsumata, Y., Takeda, T., Arai, H., Shiraki, M., and Saito, T. (2007). Purification and molecular cloning of an intracellular 3-hydroxybutyrate-oligomer hydrolase from Paucimonas lemoignei. J Biosci Bioeng 104, 224-226.
Valappil, S.P., Boccaccini, A.R., Bucke, C., and Roy, I. (2007). Polyhydroxyalkanoates in Gram-positive bacteria: insights from the genera Bacillus and Streptomyces. Antonie Van Leeuwenhoek 91, 1-17.
Vary, P.S., Biedendieck, R., Fuerch, T., Meinhardt, F., Rohde, M., Deckwer, W.D., and Jahn, D. (2007). Bacillus megaterium--from simple soil bacterium to industrial protein production host. Appl Microbiol Biotechnol 76, 957-967.
Wieczorek, R., Pries, A., Steinbuchel, A., and Mayer, F. (1995). Analysis of a 24-kilodalton protein associated with the polyhydroxyalkanoic acid granules in Alcaligenes eutrophus. J Bacteriol 177, 2425-2435.
Yamada, M., Yamashita, K., Wakuda, A., Ichimura, K., Maehara, A., Maeda, M., and Taguchi, S. (2007). Autoregulator protein PhaR for biosynthesis of polyhydroxybutyrate [P(3HB)] possibly has two separate domains that bind to the target DNA and P(3HB): Functional mapping of amino acid residues responsible for DNA binding. J Bacteriol 189, 1118-1127.
York, G.M., Lupberger, J., Tian, J., Lawrence, A.G., Stubbe, J., and Sinskey, A.J. (2003). Ralstonia eutropha H16 encodes two and possibly three intracellular Poly[D-(-)-3-hydroxybutyrate] depolymerase genes. J Bacteriol 185, 3788-3794.
York, G.M., Stubbe, J., and Sinskey, A.J. (2002). The Ralstonia eutropha PhaR protein couples synthesis of the PhaP phasin to the presence of polyhydroxybutyrate in cells and promotes polyhydroxybutyrate production. J Bacteriol 184, 59-66.
Zinn, M., Witholt, B., and Egli, T. (2001). Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate. Adv Drug Deliv Rev 53, 5-21.
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