Pseudomonas sp.DS0601-fx菌株的选育及其PCL解聚酶酶学性质的研究
|
摘要
聚己内酯(Poly-Caprolactone,PCL),一种可完全降解的半结晶型脂肪族聚酯,近来在医药、农业、环保等方面获得广泛的关注。但其生物降解性研究目前报道较少。
本论文通过采集活性污泥样品,并进行分离、筛选,获得一株可高效降解PCL的细菌菌株DS0601-fx。通过菌株和菌落的形态学观察以及菌株的生理生化特性试验和16S rDNA序列分析比对,初步鉴定该菌株属于假单胞菌属(Pseudomonas sp.)。
在以PCL为唯一碳源的培养基中培养菌株DS0601-fx,对其生长规律、PCL粉末和PCL膜的降解行为进行观察研究,发现DS0601-fx可以在70h以内将PCL膜完全降解,其降解速率趋势与菌株生长趋势趋于一致。
对PCL粉末为唯一碳源的液体培养基进行了优化,发现用PCL粉末培养90h后菌株可以分泌大量蛋白,蛋白具有对PCL的降解能力,对DS0601-fx菌株产生的PCL解聚酶进行进一步分离纯化,通过Sephacryl S-100层析后分离出一种PCL解聚酶。酶的比活力达到900.24 u/mg,纯化倍数为14.24,酶活回收率为2.64%。
对PCL膜降解的SEM照片进行观察,发现解聚酶引起的PCL膜降解存在一定规律,即由表层平面降解到深层立体降解;从非结晶区快速降解到结晶区缓慢降解。
菌株DS0601-fx不仅可以在PCL唯一碳源培养基中良好生长,也可以在其他生物可降解材料PHB和PLA培养基中稳定生长并分泌相应的解聚酶。
Poly-caprolactone(PCL) is a biocompatible ,biodegradable and semi- crystalline polyester proposed for a wide variety of applications in the medical industry, extractive industry and environmental protection in recently years. The biodegradability of PCL has been studied but not reported a lot about research of the PCL de-polymerase.
The strain DS0601-fx which can degrade PCL efficiently was isolated from the soil . According to morphological observation、the physiological and bio-chemical characteristics and 16S rDNA analysis, the strain was identified to belong to Pseudomonas.
The study of the strain growth regulation on the culture medium which use PCL as the sole carbon and energy showed as follows: DS0601-fx can degrade PCL films completely in 70 hours.The trend of the degrade speed is inosculate to the strain growth regulation.
Meliorate the proportion of PCL in culture medium which use PCL as the sole carbon and energy source and found DS0601-fx can abundantly produce the PCL de-polymerase in 90 hour by PCL granule medium. An extra-cellular PCL de-polymerase was purified according to gel filtration technique by Sephacryl S-100. The specific activity of the purified enzyme was 14.24-folds and the recovery yield was 2.64%.
The degradation of PCL films is studied by SEM photos and found there are some formulae in it .The degradation of PCL happened in the surface layer first then deep-seated place. Synchronously the non- crystalline parts degraded rapidly and the crystalline parts slowly degraded at last.
The strain DS0601-fx which can efficient degrade not only PCL but also other biodegradable polyester such as PHB and PLA.
本论文通过采集活性污泥样品,并进行分离、筛选,获得一株可高效降解PCL的细菌菌株DS0601-fx。通过菌株和菌落的形态学观察以及菌株的生理生化特性试验和16S rDNA序列分析比对,初步鉴定该菌株属于假单胞菌属(Pseudomonas sp.)。
在以PCL为唯一碳源的培养基中培养菌株DS0601-fx,对其生长规律、PCL粉末和PCL膜的降解行为进行观察研究,发现DS0601-fx可以在70h以内将PCL膜完全降解,其降解速率趋势与菌株生长趋势趋于一致。
对PCL粉末为唯一碳源的液体培养基进行了优化,发现用PCL粉末培养90h后菌株可以分泌大量蛋白,蛋白具有对PCL的降解能力,对DS0601-fx菌株产生的PCL解聚酶进行进一步分离纯化,通过Sephacryl S-100层析后分离出一种PCL解聚酶。酶的比活力达到900.24 u/mg,纯化倍数为14.24,酶活回收率为2.64%。
对PCL膜降解的SEM照片进行观察,发现解聚酶引起的PCL膜降解存在一定规律,即由表层平面降解到深层立体降解;从非结晶区快速降解到结晶区缓慢降解。
菌株DS0601-fx不仅可以在PCL唯一碳源培养基中良好生长,也可以在其他生物可降解材料PHB和PLA培养基中稳定生长并分泌相应的解聚酶。
Poly-caprolactone(PCL) is a biocompatible ,biodegradable and semi- crystalline polyester proposed for a wide variety of applications in the medical industry, extractive industry and environmental protection in recently years. The biodegradability of PCL has been studied but not reported a lot about research of the PCL de-polymerase.
The strain DS0601-fx which can degrade PCL efficiently was isolated from the soil . According to morphological observation、the physiological and bio-chemical characteristics and 16S rDNA analysis, the strain was identified to belong to Pseudomonas.
The study of the strain growth regulation on the culture medium which use PCL as the sole carbon and energy showed as follows: DS0601-fx can degrade PCL films completely in 70 hours.The trend of the degrade speed is inosculate to the strain growth regulation.
Meliorate the proportion of PCL in culture medium which use PCL as the sole carbon and energy source and found DS0601-fx can abundantly produce the PCL de-polymerase in 90 hour by PCL granule medium. An extra-cellular PCL de-polymerase was purified according to gel filtration technique by Sephacryl S-100. The specific activity of the purified enzyme was 14.24-folds and the recovery yield was 2.64%.
The degradation of PCL films is studied by SEM photos and found there are some formulae in it .The degradation of PCL happened in the surface layer first then deep-seated place. Synchronously the non- crystalline parts degraded rapidly and the crystalline parts slowly degraded at last.
The strain DS0601-fx which can efficient degrade not only PCL but also other biodegradable polyester such as PHB and PLA.
引文
[1]于慧敏,沈忠耀.可生物降解塑料聚-β-羟基丁酸酯(PHB)的研究与发展[J].精细与专用化学品2001,8:11-14
[2]张帅,辛嘉英.生物可降解塑料聚β-羟基丁酸酯的研究进展[J].科技资讯2008 NO.03:7
[3]俞文灿.可降解塑料的应用、研究现状及其发展方向中山大学研究生学刊(自然科学、医学版) 2007.VOL.28, No-1
[4]钱伯章,朱建芳生物可降解塑料发展现状与前景[J]现代化工2008,11 :82-87
[5]贺爱军可降解塑料有待走向工业化[EB/OL] http://www.ntem.com.cn/kjjx2/1225_sd_1.htm, 2002.12
[6]全国塑料制品标准化技术委员会.GB/T20197—2006降解塑料的定义、分类、标志和降解性能要求[S].北京:中国标准出版社,2006.
[7]王东山,黄勇,沈家瑞.生物降解塑料研究进展[J].广州化学, 2001, 26 (4): 38 - 44
[8]Salmoral EM, Gonzalez M E, Mariscal S P. Biodegradable plastic made from bean products [J] Industrial Corps and Production, 2000, 11 (2): 217 - 225
[9]薛林贵,王维国.我国聚β-羟基丁酸的研究进展与展望[J] .微生物学杂志,1999,19(2):52-55.
[10]苏家齐. Biopol -细菌合成塑料[J].化工科技动态, 1995,(2):4
[11]Suresh Kumar. Production of biodegradable plastics from activated sludge generated from a food processing industrial wastewater treatment plant [J]. Bio-resource Technology, 2004, 94 (3): 327-330
[12]Azevedo MC, Reis RL, Claase MB, Grijpma DW, et al. Development of poly-caprolactone/hydroxyapatite composite biomaterials[J]. Mater Sci: Mater Med, 2003 (14):103–107.
[13]张昌辉,赵霞.脂肪族聚酯可降解材料的研究进展[J] .塑料科技2008,Vol36.No.3 84-87
[14]汪群慧,孙晓红,孟令辉等.生物降解性塑料—聚乳酸研究进展[J].塑料工业, 2003,31(5):1-4
[15]Guan J, Sacks MS, Beckman EJ, Wagner WR. Synthesis, characterization, and cytocompatibility of elastomeric, biodegradable poly(ester-urethane)ureas based on poly-caprolactone and putrescine[J].Biomed Mater Res ,2002,61(3):493–503.
[16]孙伯平,赵伟,项素云.可生物降解材料[J].塑料科技, 2004,(2):36-41.
[17]任崇荣任凤梅周正发徐卫兵淀粉基生物降解塑料的研究现状综述及展望[J].塑料制造, 2008,6;86-90
[18]张可喜,付新.淀粉复合材料的研究进展.化学工程师,2006(5):22-24.
[19]黄根龙,周元申.光-生物降解PE塑料配方设计和评价研究[J].现代塑料加工应用,1995,7(3):15-20
[20]黄身岐,林本农,唐健玲.可控光-生物降解塑料薄膜[J].工业化生产及应用研究, 2003,10、11;1-9
[21]艾合麦提·玉素甫,王振斌,朱良.可生物降解材料聚己内酯在医学上的应用进展[J].国外医学生物医学工程分册, 2005,2,Vol.28,No.1
[22]Akihito Ohtaki, Naoki Akakura, Kiyohiko Nakasak, Effects of temperature and inoculum on the degradability of poly-ε-caprolactone during composting[J].Elsevier,1998:279-284.
[23]蒋青锋,陈志良,陈建海,等,聚己内酯理化性质的研究[J].中国医院药学杂志,2006 Mar ,Vol26(3):261-264
[24]William J Bailey et al, Synthesis of Poly-ε-Caprolactone via a Free Radical Mechanism [J].Journal of Polymer Science, 1989, 20 (21).
[25]於秋霞,朱光明,梁国正.聚ε-己内酯的合成、性能及应用进展[J].高分子材料科学与工程,2004,20(5):37-40.
[26]Pushkaraj Joshi, Giridhar Madras, Degradation of poly-caprolactone in supercritical fluids[J], Polymer Degradation and Stability 93 (2008):1901–1908
[27]米红宇.聚己内酯的特性介绍及其应用前景[J].新疆石油学院学报,2002,14(4):72-74.
[28]江涛,吴丽霞.包装材料的发展及生态化研究[J].中国包装,2004.5.
[29]Ana Vidaurre,JoséM. Meseguer Duen?as, Jorge Más Estellés, Isabel Castilla Cortázar Influence of Enzymatic Degradation on Physical Properties of Poly(e-caprolactone) Films and Sponges [J]. Macromol .Symp. 2008(269):38–46
[30]王永亮,易国斌,康正,熊富华等聚己内酯的合成与应用研究进展[J] 2006 ,Vol. 23 No. 3
[31]Sao tone K, Kodaria Y. Makromo l. Chem. 1965, 82:41.
[32]Bixler K J, Calhoun G C,Scholsky K M, et al. Polymerization of epsilon-caprolactone in the presence of carboxylic acids[J]. Polymer Preprints , 1990 ,31 (2) : 4942495.
[33]张其锦(ZHANG Qi-jin) ,王冰(WANG Bing) ,罗筱烈(LUO Xiao-lie) ,等.应用化学, 1994, 11 (2) : 57.
[34]Lisa Saunders Baugh, Joseph A Sissano. Polymerization of methylmet hacrylate and other polar monomers with alkyl-aluminum initiators bearing bidentate and t- ridentate N-and O-donor-ligands[J]. PolymSci Part A:Polym Chem , 2002 ,40 :163321651.
[35]Joseph Jagur-Grodzinski. Functional polymers by living anionic polymerization [J] .J Polym Sci Part A : Polym Chem ,2002 ,40 :211622133.
[36]Kricheldorf H R, Berl M , Scharnagl N. Macromo. lecules, 1988, 24: 286-293.
[37]Stassin F, Halleux O, Jerome R. Macromo lecules, 2001, 34: 775-781.
[38]Fang X, Simone C D, Vaccaro E, et al. Jounal of Polymer Sci: Part A , 2002, 40:2264-2275.
[39]刘建飞,沈之荃,孙俊全.稀土乙酰丙酮盐催化聚合ε2己内酯和丙交酯[J] .应用化学,1995 ,12(6) :59-61.
[40]戴炜枫,茹敏良,何月英,郎美东新型官能团化聚己内酯的研究进展[J].高分子通报2008(9):10-18
[41]朱光明,梁国正,徐前永,於秋霞.辐照对聚己内酯结构和性能的影响[J].高分子学报, 2003(5):667-672.
[42]周海峰,朱光明,王业玺聚己内酯的辐射交联及改性研究[J]中国塑料2005,6( 19)
[43]Qian Haitao ,Bei Jiangzhong ,Wang Shenguo. Synthesis ,characerization and degradation of ABA block copolymer of L-lactide andε-caprolactone[J]. Polymer Degradation and Stability , 2000 ,68:4232429.
[44]杨慧娴,刘光烨,李荣勋.聚己内酯(PCL) /苯乙烯2丙烯腈共聚物(SAN)共混体系相容性研究进展[J]塑料2003 ,3(31):46-51
[45]Georgia Chouzouri, Marino Xanthos. In vitro bioactivity and degradation ofpoly-caprolactone composites containing silicate fillers [J]. Acta Biomaterialia 2007(3):745–756
[46]邙志国,薛冬桦,迟惠.变性淀粉用于生物降解材料的研究进展.长春理工大学学报,2007,30(1):105-107.
[47]王彭延,宋存先,施化莲,等.可降解长效抗生育埋植剂的研究[J].生物医学与临床,1997,1(1): 23.
[48]东秀珠,蔡妙英,等.常见细菌系统鉴定手册[M]:第一版.北京:科学出版社,2001.
[49]陈珊,刘东波等.聚β-羟基丁酸酯解聚酶相关性质的研究[J].微生物通报,2002,29(3):50-54.
[50]Tomasi G, Scandola M, Briese B H, et al. Enzymatic degradation of bacterial Poly(ε-hydroxybutyrate) by a depolymerase from Pseudomonas lemoignei[J]. Macromolecules, 1996(29): 507-513.
[51]Yamada K, Mukai L, Doi Y. Enzymatic degradation of poly–hydroxyalkanoates by Pseudomoas pickettii [J]. International Journal of Biological Macromolecules, 1993(25): 215-220.
[52]Shirakura Y, Fukui T, Saito E, et al. Bio-chimicait Biophysica Acta [J]. 1986,880:46-53.
[53]Brucato T L, Wong S S. Archives of Biochemistry and Bio-physics[J]. 1991, 290(2):497-502.
[54]Stinson W, Merrick J M.J Bacteriology [M]. 1974, 119: 152-161.
[55]Foresti M L , Ferreira M L. Synthesis of poly-caprolactone using free/ supported enzymatic and non-enzymatic catalysts[J]. Macromol Rapid Commun , 2004 ,25: 2025-2028.
[56]蒙延峰,温慧颖,李宏飞等聚己内酯的等温与非等温结晶动力学研究高等学校化学学报2006,11,Vol27,No. 11,2198～2203
[2]张帅,辛嘉英.生物可降解塑料聚β-羟基丁酸酯的研究进展[J].科技资讯2008 NO.03:7
[3]俞文灿.可降解塑料的应用、研究现状及其发展方向中山大学研究生学刊(自然科学、医学版) 2007.VOL.28, No-1
[4]钱伯章,朱建芳生物可降解塑料发展现状与前景[J]现代化工2008,11 :82-87
[5]贺爱军可降解塑料有待走向工业化[EB/OL] http://www.ntem.com.cn/kjjx2/1225_sd_1.htm, 2002.12
[6]全国塑料制品标准化技术委员会.GB/T20197—2006降解塑料的定义、分类、标志和降解性能要求[S].北京:中国标准出版社,2006.
[7]王东山,黄勇,沈家瑞.生物降解塑料研究进展[J].广州化学, 2001, 26 (4): 38 - 44
[8]Salmoral EM, Gonzalez M E, Mariscal S P. Biodegradable plastic made from bean products [J] Industrial Corps and Production, 2000, 11 (2): 217 - 225
[9]薛林贵,王维国.我国聚β-羟基丁酸的研究进展与展望[J] .微生物学杂志,1999,19(2):52-55.
[10]苏家齐. Biopol -细菌合成塑料[J].化工科技动态, 1995,(2):4
[11]Suresh Kumar. Production of biodegradable plastics from activated sludge generated from a food processing industrial wastewater treatment plant [J]. Bio-resource Technology, 2004, 94 (3): 327-330
[12]Azevedo MC, Reis RL, Claase MB, Grijpma DW, et al. Development of poly-caprolactone/hydroxyapatite composite biomaterials[J]. Mater Sci: Mater Med, 2003 (14):103–107.
[13]张昌辉,赵霞.脂肪族聚酯可降解材料的研究进展[J] .塑料科技2008,Vol36.No.3 84-87
[14]汪群慧,孙晓红,孟令辉等.生物降解性塑料—聚乳酸研究进展[J].塑料工业, 2003,31(5):1-4
[15]Guan J, Sacks MS, Beckman EJ, Wagner WR. Synthesis, characterization, and cytocompatibility of elastomeric, biodegradable poly(ester-urethane)ureas based on poly-caprolactone and putrescine[J].Biomed Mater Res ,2002,61(3):493–503.
[16]孙伯平,赵伟,项素云.可生物降解材料[J].塑料科技, 2004,(2):36-41.
[17]任崇荣任凤梅周正发徐卫兵淀粉基生物降解塑料的研究现状综述及展望[J].塑料制造, 2008,6;86-90
[18]张可喜,付新.淀粉复合材料的研究进展.化学工程师,2006(5):22-24.
[19]黄根龙,周元申.光-生物降解PE塑料配方设计和评价研究[J].现代塑料加工应用,1995,7(3):15-20
[20]黄身岐,林本农,唐健玲.可控光-生物降解塑料薄膜[J].工业化生产及应用研究, 2003,10、11;1-9
[21]艾合麦提·玉素甫,王振斌,朱良.可生物降解材料聚己内酯在医学上的应用进展[J].国外医学生物医学工程分册, 2005,2,Vol.28,No.1
[22]Akihito Ohtaki, Naoki Akakura, Kiyohiko Nakasak, Effects of temperature and inoculum on the degradability of poly-ε-caprolactone during composting[J].Elsevier,1998:279-284.
[23]蒋青锋,陈志良,陈建海,等,聚己内酯理化性质的研究[J].中国医院药学杂志,2006 Mar ,Vol26(3):261-264
[24]William J Bailey et al, Synthesis of Poly-ε-Caprolactone via a Free Radical Mechanism [J].Journal of Polymer Science, 1989, 20 (21).
[25]於秋霞,朱光明,梁国正.聚ε-己内酯的合成、性能及应用进展[J].高分子材料科学与工程,2004,20(5):37-40.
[26]Pushkaraj Joshi, Giridhar Madras, Degradation of poly-caprolactone in supercritical fluids[J], Polymer Degradation and Stability 93 (2008):1901–1908
[27]米红宇.聚己内酯的特性介绍及其应用前景[J].新疆石油学院学报,2002,14(4):72-74.
[28]江涛,吴丽霞.包装材料的发展及生态化研究[J].中国包装,2004.5.
[29]Ana Vidaurre,JoséM. Meseguer Duen?as, Jorge Más Estellés, Isabel Castilla Cortázar Influence of Enzymatic Degradation on Physical Properties of Poly(e-caprolactone) Films and Sponges [J]. Macromol .Symp. 2008(269):38–46
[30]王永亮,易国斌,康正,熊富华等聚己内酯的合成与应用研究进展[J] 2006 ,Vol. 23 No. 3
[31]Sao tone K, Kodaria Y. Makromo l. Chem. 1965, 82:41.
[32]Bixler K J, Calhoun G C,Scholsky K M, et al. Polymerization of epsilon-caprolactone in the presence of carboxylic acids[J]. Polymer Preprints , 1990 ,31 (2) : 4942495.
[33]张其锦(ZHANG Qi-jin) ,王冰(WANG Bing) ,罗筱烈(LUO Xiao-lie) ,等.应用化学, 1994, 11 (2) : 57.
[34]Lisa Saunders Baugh, Joseph A Sissano. Polymerization of methylmet hacrylate and other polar monomers with alkyl-aluminum initiators bearing bidentate and t- ridentate N-and O-donor-ligands[J]. PolymSci Part A:Polym Chem , 2002 ,40 :163321651.
[35]Joseph Jagur-Grodzinski. Functional polymers by living anionic polymerization [J] .J Polym Sci Part A : Polym Chem ,2002 ,40 :211622133.
[36]Kricheldorf H R, Berl M , Scharnagl N. Macromo. lecules, 1988, 24: 286-293.
[37]Stassin F, Halleux O, Jerome R. Macromo lecules, 2001, 34: 775-781.
[38]Fang X, Simone C D, Vaccaro E, et al. Jounal of Polymer Sci: Part A , 2002, 40:2264-2275.
[39]刘建飞,沈之荃,孙俊全.稀土乙酰丙酮盐催化聚合ε2己内酯和丙交酯[J] .应用化学,1995 ,12(6) :59-61.
[40]戴炜枫,茹敏良,何月英,郎美东新型官能团化聚己内酯的研究进展[J].高分子通报2008(9):10-18
[41]朱光明,梁国正,徐前永,於秋霞.辐照对聚己内酯结构和性能的影响[J].高分子学报, 2003(5):667-672.
[42]周海峰,朱光明,王业玺聚己内酯的辐射交联及改性研究[J]中国塑料2005,6( 19)
[43]Qian Haitao ,Bei Jiangzhong ,Wang Shenguo. Synthesis ,characerization and degradation of ABA block copolymer of L-lactide andε-caprolactone[J]. Polymer Degradation and Stability , 2000 ,68:4232429.
[44]杨慧娴,刘光烨,李荣勋.聚己内酯(PCL) /苯乙烯2丙烯腈共聚物(SAN)共混体系相容性研究进展[J]塑料2003 ,3(31):46-51
[45]Georgia Chouzouri, Marino Xanthos. In vitro bioactivity and degradation ofpoly-caprolactone composites containing silicate fillers [J]. Acta Biomaterialia 2007(3):745–756
[46]邙志国,薛冬桦,迟惠.变性淀粉用于生物降解材料的研究进展.长春理工大学学报,2007,30(1):105-107.
[47]王彭延,宋存先,施化莲,等.可降解长效抗生育埋植剂的研究[J].生物医学与临床,1997,1(1): 23.
[48]东秀珠,蔡妙英,等.常见细菌系统鉴定手册[M]:第一版.北京:科学出版社,2001.
[49]陈珊,刘东波等.聚β-羟基丁酸酯解聚酶相关性质的研究[J].微生物通报,2002,29(3):50-54.
[50]Tomasi G, Scandola M, Briese B H, et al. Enzymatic degradation of bacterial Poly(ε-hydroxybutyrate) by a depolymerase from Pseudomonas lemoignei[J]. Macromolecules, 1996(29): 507-513.
[51]Yamada K, Mukai L, Doi Y. Enzymatic degradation of poly–hydroxyalkanoates by Pseudomoas pickettii [J]. International Journal of Biological Macromolecules, 1993(25): 215-220.
[52]Shirakura Y, Fukui T, Saito E, et al. Bio-chimicait Biophysica Acta [J]. 1986,880:46-53.
[53]Brucato T L, Wong S S. Archives of Biochemistry and Bio-physics[J]. 1991, 290(2):497-502.
[54]Stinson W, Merrick J M.J Bacteriology [M]. 1974, 119: 152-161.
[55]Foresti M L , Ferreira M L. Synthesis of poly-caprolactone using free/ supported enzymatic and non-enzymatic catalysts[J]. Macromol Rapid Commun , 2004 ,25: 2025-2028.
[56]蒙延峰,温慧颖,李宏飞等聚己内酯的等温与非等温结晶动力学研究高等学校化学学报2006,11,Vol27,No. 11,2198～2203