东方拟无枝酸菌产聚乳酸降解酶和蛋白酶的研究
摘要
随着世界社会经济的发展,由石油等化石资源合成的高分子化合物制品的大量生产、消费及遗弃等所引起的环保问题日趋严重,人们的环境保护意识日趋强烈。为了解决合成树脂和纤维不易被环境分解的问题,生物可降解高分子材料逐渐成为研究的热点,其中聚乳酸被认为是一种最有前途的可降解高分子化合物。与聚乳酸的合成相比,有关其生物降解的研究却相对滞后。PLA在自然界中降解缓慢,已发现的降解微生物及所产降解酶数量种类有限,且具有一定的特殊性,这在一定程度上制约了PLA生物降解机理的阐明,也限制了可控降解材料的开发推广和应用。
本论文的主要工作是从自然环境中筛选得到了一株可以高效降解聚乳酸的菌株。经过菌种鉴定,确定此菌株为放线菌属的东方拟无枝酸菌。研究了不同的诱导物对于该菌产聚乳酸降解酶活性的影响,得出明胶为最佳的诱导物之一。由于明胶也是该菌产生蛋白酶的诱导物,在诱导培养65小时后,其蛋白酶产生量达到最高值,发酵液中蛋白酶活最高可达29,000U/L。虽然蛋白酶酶活和聚乳酸降解酶酶活最高峰出现的时间稍有不同,但两者活性均随诱导时间而增加,说明蛋白酶活性和聚乳酸降解酶活性的产生具有一定的相关性;同时,对发酵粗酶液的基本酶学性质进行了研究,诱导酶系表现出的蛋白酶活性和聚乳酸降解酶活性,均在碱性pH范围内具有较高的活性和稳定性,从而显示聚乳酸降解活性与蛋白酶活性间存在某种内在的联系。
进一步对菌株的胞外酶系进行了分析,纯化得到了五个蛋白酶组分,其中三个具有聚乳酸降解活性,分别为PLAaseⅠPLAaseⅡ和PLAaseⅢ。对粗酶液的以及聚乳酸降解酶组分的底物特异性研究表明,并不是所有的蛋白酶都具有聚乳酸降解酶活性。但从一株菌株当中可以同时分离出三种聚乳酸降解酶组分,说明东方拟无枝酸菌中所产生的聚乳酸降解酶在酶性质上可能存在某种内在联系。探讨了聚乳酸酶解产物的分析方法,从而为进一步研究聚乳酸的酶学降解机制提供分析技术平台。
With the development of the world economy, the environmental condition has been deteriorating by the widespread use of petroleum-derived products, and concerns about environment protection have never been as strong as today. In order to solve the problem of synthetic resin and fiber's strong resistance to degradation when released into the environment, the research of biodegradable plastics has gained enormous attention. PLA is considered to be one of the most promising materials. But compared with research in PLA synthesis, research in the degradation of PLA is relatively lagging behind. PLA is decomposed very slowly in nature, and the distribution of microbials and enzymes capable of accelerating the degradation of PLA is limited. This, to larger extent, also impedes clarifying the mechanism of the PLA degradation and confines the development and application of the controllable degradation of materials such as PLA.
We isolated an actinomycosis strain from our culture collections for its high degradability of PLA. This strain was identified as Amycolatopsis orientali. Further results showed that, among others, geltin was one of the best inducer for inducing the strain to produce PLAases with the enzyme producing peak appearing at 50 hours after incoculation. In accordance with the fact that gelatin usually acts as an protease inducer, the fermented protease activities also reached the highest level of 29,000U/L roughly slightly around 65 hours. In addition, primary-characterization of the crude enzymes from the culture supernatant showed that both protease activities and PLA-degrading activityies were relatively stable within an alkaline pH range (pH 8 to 10 ), implicating a potential relationship between produced protease activity and PLAase activity.
Five protease components were isolated and purified form the fermented culture, with three of them displaying PLA-degrading activities. Characterizing the substrate specificity of both crude purified enzymes revealed that the protease activity and the PLAase activity from the fermented culture was not strictly correlative, namely, not all the proteases have the ability to degrade PLA. But the simultaneous isolation of three PLA-degrading activities from one strain suggested a common feature not being shared by other proteases exist in these enzymes. Finally, methods to analyze the hydrolytic products formed by PLAase-catalyzed reactions,were exploited, which should be very useful for further elucidating the mechanism of enzymatic degradation of PLA.
本论文的主要工作是从自然环境中筛选得到了一株可以高效降解聚乳酸的菌株。经过菌种鉴定,确定此菌株为放线菌属的东方拟无枝酸菌。研究了不同的诱导物对于该菌产聚乳酸降解酶活性的影响,得出明胶为最佳的诱导物之一。由于明胶也是该菌产生蛋白酶的诱导物,在诱导培养65小时后,其蛋白酶产生量达到最高值,发酵液中蛋白酶活最高可达29,000U/L。虽然蛋白酶酶活和聚乳酸降解酶酶活最高峰出现的时间稍有不同,但两者活性均随诱导时间而增加,说明蛋白酶活性和聚乳酸降解酶活性的产生具有一定的相关性;同时,对发酵粗酶液的基本酶学性质进行了研究,诱导酶系表现出的蛋白酶活性和聚乳酸降解酶活性,均在碱性pH范围内具有较高的活性和稳定性,从而显示聚乳酸降解活性与蛋白酶活性间存在某种内在的联系。
进一步对菌株的胞外酶系进行了分析,纯化得到了五个蛋白酶组分,其中三个具有聚乳酸降解活性,分别为PLAaseⅠPLAaseⅡ和PLAaseⅢ。对粗酶液的以及聚乳酸降解酶组分的底物特异性研究表明,并不是所有的蛋白酶都具有聚乳酸降解酶活性。但从一株菌株当中可以同时分离出三种聚乳酸降解酶组分,说明东方拟无枝酸菌中所产生的聚乳酸降解酶在酶性质上可能存在某种内在联系。探讨了聚乳酸酶解产物的分析方法,从而为进一步研究聚乳酸的酶学降解机制提供分析技术平台。
With the development of the world economy, the environmental condition has been deteriorating by the widespread use of petroleum-derived products, and concerns about environment protection have never been as strong as today. In order to solve the problem of synthetic resin and fiber's strong resistance to degradation when released into the environment, the research of biodegradable plastics has gained enormous attention. PLA is considered to be one of the most promising materials. But compared with research in PLA synthesis, research in the degradation of PLA is relatively lagging behind. PLA is decomposed very slowly in nature, and the distribution of microbials and enzymes capable of accelerating the degradation of PLA is limited. This, to larger extent, also impedes clarifying the mechanism of the PLA degradation and confines the development and application of the controllable degradation of materials such as PLA.
We isolated an actinomycosis strain from our culture collections for its high degradability of PLA. This strain was identified as Amycolatopsis orientali. Further results showed that, among others, geltin was one of the best inducer for inducing the strain to produce PLAases with the enzyme producing peak appearing at 50 hours after incoculation. In accordance with the fact that gelatin usually acts as an protease inducer, the fermented protease activities also reached the highest level of 29,000U/L roughly slightly around 65 hours. In addition, primary-characterization of the crude enzymes from the culture supernatant showed that both protease activities and PLA-degrading activityies were relatively stable within an alkaline pH range (pH 8 to 10 ), implicating a potential relationship between produced protease activity and PLAase activity.
Five protease components were isolated and purified form the fermented culture, with three of them displaying PLA-degrading activities. Characterizing the substrate specificity of both crude purified enzymes revealed that the protease activity and the PLAase activity from the fermented culture was not strictly correlative, namely, not all the proteases have the ability to degrade PLA. But the simultaneous isolation of three PLA-degrading activities from one strain suggested a common feature not being shared by other proteases exist in these enzymes. Finally, methods to analyze the hydrolytic products formed by PLAase-catalyzed reactions,were exploited, which should be very useful for further elucidating the mechanism of enzymatic degradation of PLA.
引文
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(3)Maeda.H,Yamagata.Y,Abe.K,Hasegawa.F,Machida.M,Ishioka.R,Gomi.K and Nakajima.T.2005.Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae.Appl Microbiol Biotechnol,67:778-788
(4)Rafael Auras,* Bruce Harte,Susan Selke.An Overview of Polylactides as Packaging Materials Macromol.Biosci.2004,4,835-864
(5)Polylactide-co-glycolida microparticles with surface adsorbed antigens as vaccine delivery systems.Curr Drug Deliv.2006an;3(1):115-20.Review.
(6)Bioabsorbable self-reinforced plates and screws in raniomaxillofacial surgery.Biomed Mater Eng.2004;14(4):517-24.Review.
(7)Biodegradable polyester elastomers in tissue engineering.Expert Opin Biol Whet.2004 Jun;4(6):801-12.Review.
(8)Bioabsorbable scaffold for in situ bone regeneration.Biomed Pharmaeother.2006 Sep;60(8):386-92.Epub 2006 Aug 11.
(9)Erwin T.H.Vinka,Karl R.Ra bagob,David A.Classnerb,Patrick R.Gruberb Applications of life cycle assessment to NatureWorksTM polylactide(PLA)production Polymer Degradation and Stability 80(2003)403-419
(10)James Lunt.Large-scale production,properties and commercial applications of polylactic acid polymers.Polymer Degradation and Stability 59(1998) 145-152,
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(13) Nishida, H., and Y. Tokiwa. 1993. Distribution of poly (β -hydroxybutyrate) and poly (ε -caprolactone) aerobic degrading microorganisms in different environments. J. Environ. Polym. Degrad. 1:227-233.
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(18) Sakai, K. , H. Kawano, A. Iwami, M. Nakamura, and M. Moriguchi. 2001. Isolation of a thermophilic poly-L-lactide degrading bacterium from compost and its enzymatic characterization. J. Biosci. Bioeng. 92:298-300.
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(21)Ikura Y,Kudo T(1999)Isolation of a microorganism capable of degrading poly(L-lactide).J.Gen.Appl.Microbiol.45:247-251.
(22)Tokiwa Y,Konno M,Nishida H(1999)Isolation of silk degrading microorganisms and its poly(L-lactide)degradability.Chem.Lett.1999:355-356.
(23)Nakamura K,Tomita T,Abe N,Kamio Y(2001)Purification and characterization of an extracellular poly(L-lactic acid)depolymerase from a soil isolate,Amycolatopsis sp.strain K104-1.Appl.Environ.Microbiol.67:345-353.
(24)Jarerat A,Tokiwa Y,Tanaka H(2003)Poly(L-lactide)degradation by Kibdelosporangium aridum.Biotechnol.Lett.25:2035-2038.
(25)Jarerat A,Tokiwa Y(2003)Degradation of poly(L-lactide)by Saccharothrix waywayandensis.Biotechnol.Lett.25:401-404.
(26)Pranamuda H,Tokiwa Y(1999)Degradation of poly(L-lactide)by strains belonging to genus Amycolatopsis.Biotechnol.Lett.21:901-905.
(27)Jarerat A,Pranamuda H,Tokiwa Y(2002)Poly(L-lactide)-degrading activity in various actinomycetes.Macromol.Biosci.2:420-428.
(28)Williams DF(1981)Enzymatic hydrolysis of polylactic acid.Eng.Med.10:5-7
(29)Hideto Tsuji,Shinya Miyauchi.Poly(1-lactide):Ⅵ Effects of crystallinity on enzymatic hydrolysis of poly(1-lactide)without free amorphous region.Polymer Degradation and Stability 71(2001) 415-424
(30)Oda Y,Yonetsu A,Urakami T,Tonomura K(2000)Degradation of polylactide by commercial proteases.J.Polym.Environ.8:29-32.
(31)Hyun-A Lim,Takao Raku,Yutaka Tokiwa.Hydrolysis of polyesters by serine proteases.Biotechnology Letters(2005)27:459-464
(32)Nakayama A,Kawasaki N,Aiba S,Maeda Y,Arvanitoyannis I,Yamamoto N(1998)Synthesis and biodegradability of novel copolyesters containing γ-butyrolactone units.Folymer39:1213- 1222.
(33)Yukie Akutsu-Shigeno,Teerawat Teeraphatpornchai,Kamonluck Toshiaki Nakajima-Kambe Cloning and Sequencing of a Poly(DL-Lactic Acid)Depolymerase Gene from Paenibacillus amylolyticus Strain TB-13and Its Functional Expression in Escherichia coli APPLIED AND ENVIRONMENTAL MICROBIOLOGY,May 2003,p.2498-2504 Vol.69,No.5
(34)Pranamuda H,Tsuchii A,Tokiwa Y(2001)Poly(L-lactide)-degrading enzyme produced by Amycolatopsis sp.Macromol.Biosci.1:25-29.
(35)Kazuo Masaki,Numbi Ramudu Kamini,Hiroko Ikeda,and Haruyuki Iefuji.Cutinase-Like Enzyme from the Yeast Cryptococcus sp.Strain S-2 Hydrolyzes Polylactic Acid and Other Biodegradable Plastics.APPLIED AND ENVIRONMENTAL MICROBIOLOGY,Nov.2005,p.7548-7550 Vol.71,No.11
(36)Emiko Matsuda,Naoki Abe,Hideyuki Tamakawa,Jun Kaneko,and Yoshiyuki Kamio.Gene Cloning and Molecular Characterization of an Extracellular Poly(L-Lactic Acid)Depolymerase from Amycolatopsis sp.Strain K104-1.JOURNAL OF BACTERIOLOGY,Nov.2005,p.7333-7340 Vol.187,No.21
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(2)Initiated by the 1st International Conference on Bio-based Polymers (ICBP 2003),November 12-14 2003,Saitama,Japan.The Sustainability of Nature Works TM Polylactide Polymers and Ingeo TM Polylactide Fibersa:an Update of the Future.Macromol.Biosci.2004.4.551-564.
(3)Maeda.H,Yamagata.Y,Abe.K,Hasegawa.F,Machida.M,Ishioka.R,Gomi.K and Nakajima.T.2005.Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae.Appl Microbiol Biotechnol,67:778-788
(4)Rafael Auras,* Bruce Harte,Susan Selke.An Overview of Polylactides as Packaging Materials Macromol.Biosci.2004,4,835-864
(5)Polylactide-co-glycolida microparticles with surface adsorbed antigens as vaccine delivery systems.Curr Drug Deliv.2006an;3(1):115-20.Review.
(6)Bioabsorbable self-reinforced plates and screws in raniomaxillofacial surgery.Biomed Mater Eng.2004;14(4):517-24.Review.
(7)Biodegradable polyester elastomers in tissue engineering.Expert Opin Biol Whet.2004 Jun;4(6):801-12.Review.
(8)Bioabsorbable scaffold for in situ bone regeneration.Biomed Pharmaeother.2006 Sep;60(8):386-92.Epub 2006 Aug 11.
(9)Erwin T.H.Vinka,Karl R.Ra bagob,David A.Classnerb,Patrick R.Gruberb Applications of life cycle assessment to NatureWorksTM polylactide(PLA)production Polymer Degradation and Stability 80(2003)403-419
(10)James Lunt.Large-scale production,properties and commercial applications of polylactic acid polymers.Polymer Degradation and Stability 59(1998) 145-152,
(11) Vesna Simic .Valerie Girardon ,Nicolas Spassky ,Liliane G, Hubert-Pfalzgraf and Andrzej Duda. Ring-opening polymerization of lactides initiated with yttrium Tris-isopropoxyethoxide . .Polymer Degradation and Stability 59(1998) 227-229.
(12) Pranamuda, H., Y. Tokiwa, and H. Tanaka. 1997. Polylactide degradation an Amycolatopsis sp. Appl. Environ. Microbiol. 3:1637-1640.
(13) Nishida, H., and Y. Tokiwa. 1993. Distribution of poly (β -hydroxybutyrate) and poly (ε -caprolactone) aerobic degrading microorganisms in different environments. J. Environ. Polym. Degrad. 1:227-233.
(14) Pranamuda H, Tokiwa Y, Tanaka H (1995) Microbial degradation of an aliphatic polyester with a high melting point, poly(tetramethylene succinate). Appl. Environ. Microbiol. 61: 1828-1832.
(15) A. TORRES, S. M. LI,S. ROUSSOS, M. VERT . Screening of Microorganisms for Biodegradation of Poly(Lactic Acid) and Lactic Acid-Containing Polymers . APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1996, p. 2393 - 2397
(16) Amnat Jarerat, Yutaka Tokiwa. Degradation of Poly(1-lactide) by a Fungus. Macromol. Biosci. 2001, 1, 136-140
(17) KOSUKE TOMITA, * YUTAKA KUROKI, AND KOTA NAGAI Isolation of Thermophiles Degrading Poly(L-Lactic Acid) , JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 87, No. 6, 752-755. 1999
(18) Sakai, K. , H. Kawano, A. Iwami, M. Nakamura, and M. Moriguchi. 2001. Isolation of a thermophilic poly-L-lactide degrading bacterium from compost and its enzymatic characterization. J. Biosci. Bioeng. 92:298-300.
(19) Teeraphatpornchai, T. , T. Nakajima-Kambe, Y. Shigeno-Akutsu, M. Nakayama,N.Nomura,T.Nakahara,and H.Uchiyama.2003.Isolation and characterization of a bacterium that degrades various polyester-based biodegradable plastics.Biotechnol.Lett.25:23-28.
(20)Yutaka Tokiwa,Amnat Jarerat.giodegradation of poly(L-lactide)Biorechnology Letters 26:771-777,2004.
(21)Ikura Y,Kudo T(1999)Isolation of a microorganism capable of degrading poly(L-lactide).J.Gen.Appl.Microbiol.45:247-251.
(22)Tokiwa Y,Konno M,Nishida H(1999)Isolation of silk degrading microorganisms and its poly(L-lactide)degradability.Chem.Lett.1999:355-356.
(23)Nakamura K,Tomita T,Abe N,Kamio Y(2001)Purification and characterization of an extracellular poly(L-lactic acid)depolymerase from a soil isolate,Amycolatopsis sp.strain K104-1.Appl.Environ.Microbiol.67:345-353.
(24)Jarerat A,Tokiwa Y,Tanaka H(2003)Poly(L-lactide)degradation by Kibdelosporangium aridum.Biotechnol.Lett.25:2035-2038.
(25)Jarerat A,Tokiwa Y(2003)Degradation of poly(L-lactide)by Saccharothrix waywayandensis.Biotechnol.Lett.25:401-404.
(26)Pranamuda H,Tokiwa Y(1999)Degradation of poly(L-lactide)by strains belonging to genus Amycolatopsis.Biotechnol.Lett.21:901-905.
(27)Jarerat A,Pranamuda H,Tokiwa Y(2002)Poly(L-lactide)-degrading activity in various actinomycetes.Macromol.Biosci.2:420-428.
(28)Williams DF(1981)Enzymatic hydrolysis of polylactic acid.Eng.Med.10:5-7
(29)Hideto Tsuji,Shinya Miyauchi.Poly(1-lactide):Ⅵ Effects of crystallinity on enzymatic hydrolysis of poly(1-lactide)without free amorphous region.Polymer Degradation and Stability 71(2001) 415-424
(30)Oda Y,Yonetsu A,Urakami T,Tonomura K(2000)Degradation of polylactide by commercial proteases.J.Polym.Environ.8:29-32.
(31)Hyun-A Lim,Takao Raku,Yutaka Tokiwa.Hydrolysis of polyesters by serine proteases.Biotechnology Letters(2005)27:459-464
(32)Nakayama A,Kawasaki N,Aiba S,Maeda Y,Arvanitoyannis I,Yamamoto N(1998)Synthesis and biodegradability of novel copolyesters containing γ-butyrolactone units.Folymer39:1213- 1222.
(33)Yukie Akutsu-Shigeno,Teerawat Teeraphatpornchai,Kamonluck Toshiaki Nakajima-Kambe Cloning and Sequencing of a Poly(DL-Lactic Acid)Depolymerase Gene from Paenibacillus amylolyticus Strain TB-13and Its Functional Expression in Escherichia coli APPLIED AND ENVIRONMENTAL MICROBIOLOGY,May 2003,p.2498-2504 Vol.69,No.5
(34)Pranamuda H,Tsuchii A,Tokiwa Y(2001)Poly(L-lactide)-degrading enzyme produced by Amycolatopsis sp.Macromol.Biosci.1:25-29.
(35)Kazuo Masaki,Numbi Ramudu Kamini,Hiroko Ikeda,and Haruyuki Iefuji.Cutinase-Like Enzyme from the Yeast Cryptococcus sp.Strain S-2 Hydrolyzes Polylactic Acid and Other Biodegradable Plastics.APPLIED AND ENVIRONMENTAL MICROBIOLOGY,Nov.2005,p.7548-7550 Vol.71,No.11
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