DS9701菌株的诱变及其酶学性质的研究
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摘要
聚 3-羟基丁酸(PHB)不仅具有热塑性塑料的性质，还在自然环境中具有生物可降解性，因而成为传统石化塑料的替代品，在工业等领域具有广阔的应用前景，也成为国内外生物材料领域的研究热点。
自然界中的许多种微生物都具有降解PHB的能力，这依赖于分泌一种特殊的胞外PHB解聚酶。本项工作主要是筛选能高效降解PHB的突变株，并对其进行酶学性质的研究，主要结果如下：
1．以青霉(Penicillium.sp)DS9701为出发菌株，通过紫外线诱变分生孢子，采用透明圈初筛和摇瓶培养复筛的方法，获得4个能稳定遗传的PHB解聚酶高产菌株。其中09菌株的酶活是原始菌株的2.3倍。
2．初步研究了02、04、09和14菌株粗酶液的基本性质。02菌株粗酶液的最适反应温度45℃～50℃，温度稳定范围40℃～50℃；最适反应pH 5.0，pH稳定范围4.0-5.0。04菌株粗酶液的最适反应温度50℃，温度稳定范围40℃～50℃；最适反应pH 5.0，pH稳定范围5.0～7.0。09菌株粗酶液的最适反应温度50℃～60℃，温度稳定范围40℃～50℃；最适反应pH 5.0，pH稳定范围5.0～7.0。14菌株粗酶液的最适反应温度40℃，40℃以下稳定；最适反应pH 5.8，pH稳定范围5.0～8.0。
3．经过比较粗酶液的性质后，对09号菌株产生的PHB解聚酶进行了分离纯化，并对其解聚酶的特性进行了探讨。以粗酶液为起始，经硫酸铵分级沉淀、Sephadex G-100凝胶过滤后，分离纯化了该酶，纯化倍数约为37.9，酶活力回收率8.9％。经12.5％SDS-PAGE法测得所分离纯化酶蛋白的相对分子质量约为41.5KD。该酶反应的最适温度和pH分别为50℃和5.0。在温度50℃以下和pH 5.0～6.0范围内稳定。一些金属离子对PHB解聚酶有抑制作用。质谱仪测得酶降解PHB的产物主要是二聚体。
Poly(3-hydroxybutyrate)(PHB) are attracting much commercial and academic interest as substitute for non-degradable petrochemically derived plastics because of their similar material properties to conventional plastics and complete biodegradability under natural environment.
The ability to degrade PHB is widely distributed among bacteria and fungi and depends on the secretion of specific extracellular PHB depolymerase. The aim of this work is to find out the mutant, which can be able to degrade PHB efficiently and to investigate the characteristics of PHB depolymerase. The main results obtained from this work are as follows:
1. The Penicillium.sp DS9701, a strain of degrading PHB, was mutagenized by UV treatment. Through screening a lot of mutants with the method of transparent zones and culture filtrate, the best four were obtained with high-yield of stable PHB depolymerase, named as 02, 04, 09 and 14. The enzyme activity of the 09 mutant was increased to 5.4 u/ml as compared with 2.3 u/ml of the original.
2. Comparative study among the crude extracts of 02, 04, 09 and 14 has been done. The optimum activity of 02 crude extract was observed at the temperature 45-50 and at pH 5.0. The range of temperature stability is from 40 to 50 , the range of pH stability is from 4.0 to 5.0.
The optimum activity of 04 crude extract was observed at the temperature 50 and at pH 5.0. The range of temperature stability is from 40 to 50 , the range of pH stability is from 5.0 to 7.0.
The optimum activity of 09 crude extract was observed at the temperature 50-60 and at pH 5.0. The range of temperature stability is from 40 to 50 , the range of pH stability is from 5.0 to 7.0.
The optimum activity of 14 crude extract was observed at the temperature 40 and at pH 5.8. The range of temperature stability is
below 40 , the range of pH stability is from 5.0 to 8.0.
3. The extracellular PHB depolymerase was purified from 09 by using hydrophobic column chromatography and gel filtration technique in sephadex G-100. The specific activity of the purified enzyme was increased by 37.9 folds over crude extract, and the recovery yield was 8.9%. Its molecular weight was determined running in SDS-PAGE and was found to be 41.5 KDa. The optimum activity of enzyme was observed at the temperature 50 and at pH 5.0. The range of temperature stability is below 50 , the range of pH stability is from 5.0 to 6.0. Different metal ions caused inhibition on the PHB depolymerase activity. Comparative study with DS9701 PHB depolymerase also had been done.
Moreover, the mass spectrum analysis of the hydrolyzed water soluble products of PHB powder after treatment of enzyme was performed and the main product was identified to be dimers.
自然界中的许多种微生物都具有降解PHB的能力，这依赖于分泌一种特殊的胞外PHB解聚酶。本项工作主要是筛选能高效降解PHB的突变株，并对其进行酶学性质的研究，主要结果如下：
1．以青霉(Penicillium.sp)DS9701为出发菌株，通过紫外线诱变分生孢子，采用透明圈初筛和摇瓶培养复筛的方法，获得4个能稳定遗传的PHB解聚酶高产菌株。其中09菌株的酶活是原始菌株的2.3倍。
2．初步研究了02、04、09和14菌株粗酶液的基本性质。02菌株粗酶液的最适反应温度45℃～50℃，温度稳定范围40℃～50℃；最适反应pH 5.0，pH稳定范围4.0-5.0。04菌株粗酶液的最适反应温度50℃，温度稳定范围40℃～50℃；最适反应pH 5.0，pH稳定范围5.0～7.0。09菌株粗酶液的最适反应温度50℃～60℃，温度稳定范围40℃～50℃；最适反应pH 5.0，pH稳定范围5.0～7.0。14菌株粗酶液的最适反应温度40℃，40℃以下稳定；最适反应pH 5.8，pH稳定范围5.0～8.0。
3．经过比较粗酶液的性质后，对09号菌株产生的PHB解聚酶进行了分离纯化，并对其解聚酶的特性进行了探讨。以粗酶液为起始，经硫酸铵分级沉淀、Sephadex G-100凝胶过滤后，分离纯化了该酶，纯化倍数约为37.9，酶活力回收率8.9％。经12.5％SDS-PAGE法测得所分离纯化酶蛋白的相对分子质量约为41.5KD。该酶反应的最适温度和pH分别为50℃和5.0。在温度50℃以下和pH 5.0～6.0范围内稳定。一些金属离子对PHB解聚酶有抑制作用。质谱仪测得酶降解PHB的产物主要是二聚体。
Poly(3-hydroxybutyrate)(PHB) are attracting much commercial and academic interest as substitute for non-degradable petrochemically derived plastics because of their similar material properties to conventional plastics and complete biodegradability under natural environment.
The ability to degrade PHB is widely distributed among bacteria and fungi and depends on the secretion of specific extracellular PHB depolymerase. The aim of this work is to find out the mutant, which can be able to degrade PHB efficiently and to investigate the characteristics of PHB depolymerase. The main results obtained from this work are as follows:
1. The Penicillium.sp DS9701, a strain of degrading PHB, was mutagenized by UV treatment. Through screening a lot of mutants with the method of transparent zones and culture filtrate, the best four were obtained with high-yield of stable PHB depolymerase, named as 02, 04, 09 and 14. The enzyme activity of the 09 mutant was increased to 5.4 u/ml as compared with 2.3 u/ml of the original.
2. Comparative study among the crude extracts of 02, 04, 09 and 14 has been done. The optimum activity of 02 crude extract was observed at the temperature 45-50 and at pH 5.0. The range of temperature stability is from 40 to 50 , the range of pH stability is from 4.0 to 5.0.
The optimum activity of 04 crude extract was observed at the temperature 50 and at pH 5.0. The range of temperature stability is from 40 to 50 , the range of pH stability is from 5.0 to 7.0.
The optimum activity of 09 crude extract was observed at the temperature 50-60 and at pH 5.0. The range of temperature stability is from 40 to 50 , the range of pH stability is from 5.0 to 7.0.
The optimum activity of 14 crude extract was observed at the temperature 40 and at pH 5.8. The range of temperature stability is
below 40 , the range of pH stability is from 5.0 to 8.0.
3. The extracellular PHB depolymerase was purified from 09 by using hydrophobic column chromatography and gel filtration technique in sephadex G-100. The specific activity of the purified enzyme was increased by 37.9 folds over crude extract, and the recovery yield was 8.9%. Its molecular weight was determined running in SDS-PAGE and was found to be 41.5 KDa. The optimum activity of enzyme was observed at the temperature 50 and at pH 5.0. The range of temperature stability is below 50 , the range of pH stability is from 5.0 to 6.0. Different metal ions caused inhibition on the PHB depolymerase activity. Comparative study with DS9701 PHB depolymerase also had been done.
Moreover, the mass spectrum analysis of the hydrolyzed water soluble products of PHB powder after treatment of enzyme was performed and the main product was identified to be dimers.
引文
[1].Kalia, V.C., Raizada, N., Sonakya, V., 2000a. Bioplastics. J. Sci. Ind. Res. 59, 433-445.
[2].Johnstone, B., 1990. A throw away answer. Far Eastern Econ. Rev. 147(6), 62-63.
[3].Anderson AJ, Dawes EA. 1990. Occurrence, metabolism, metabolic role, and industrial use of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54:450-72
[4].Doi Y. 1990. Microbial Polyesters. New York: VHC
[5].Madison LL, Huisman GW. 1999. Metabolicengineefing of poly(3-hydroxyalkanoates):from DNA to plastic. Microbiol. Mol. Biol. Rev. 63:21-53
[6].Müller HM, Seebach D. 1993. Poly(hydroxyalkanoates) :a fifth class of physiologicallyimportant organic biopolymers Angew. Chem. Int. Ed. Engl. 32:477-502
[7].Scheper T. 2001. Biopolyesters: Advancesin Biochemical Engineering/Biotechnology. Berlin/Heidelberg: Springer
[8].Steinbüchel A, Doi Y, eds. 2001. Biopolymers, PolyestersⅡ Properties and ChemicalSynthesis. Weinheim: Wiley-VCH.468 pp.
[9].Steinbüchel A, Valentin HE. 1995. Diversity of bacterial polyhydroxyalkanoicacids. FEMS Microbiol. Lett. 128:219-28
[10].Amor SR, Rayment T, Sanders JKM. 1991. Poly(3-hydroxybutyrate) in vivo:NMR and X-ray characterization of theelastomeric state. Macromolecules 24:4583-88
[11].de Koning GJM, Lemstra PJ. 1992. The amorphous state of bacterial poly[(R)-3-hydroxyalkanoate)] in vivo. Polymer 33:3304-6
[12].Chowdhury AA. 1963. Poly-β-hydroxybutters"aure abbauende Bakterien und Exoenzym. Arch. Mikrobiol. 47:167-200
[13].Schirmer A, Jendrossek D, Schlegel HG.1993. Degradation of poly(3-hydroxyoctanoicacid), [P(3HO)], by bacteria. Purificationand properties of a P(3HO) depolymerase of Pseudomonas fluorescensGK13 biovarV. Appl. Environ. Microbiol.
[14].Marchessault RH, Morin FG, Wong S,Saracovan I. 1995.
Artificial granule suspensionsof long side chain poly(3-hydroxyalkanoate). Can. J. Microbiol. 41(Suppl.1): 138-42
[15].Ramsay BA, Saracovan Ⅰ, Ramsay JA,Marchessault RH. 1994. A method forthe isolation of microorganisms producingextracellular long-side-chain poly(βhydroxyalkanoate)depolymerase. J. Environ. Polym. Degrad. 2:1-7
[16].Horowitz DM, Brennan EM, Koon JJ, Gemgross TU. 1999. Novel thermal routeto an amorphous, film-forming polymerlatex. Macromolecules 32:3347-52
[17].Horowitz DM, Sanders JKM. 1995. Biomimetic, amorphous granules of polyhydroxyalkanoates:composition, mobility, and stabilization in vivo by proteins. Can. J. Microbiol. 41(Suppl. 1):115-23
[18].Jendrossek D. 2001. Extracellular PHAdepolymerases: the key enzymes of PHAdegradation. See Ref. 100, pp. 41-83
[19].Briese B-H, Jendrossek D, Schlegel HG. 1994. Degradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by aerobicsewage sludge. FEMS Microbiol. Lett. 117:107-12
[20].Jendrossek D, Knoke Ⅰ, Habibian RB, Steinbtichel A, Schlegel HG. 1993. Degradation of poly(3-hydroxybutyrate), PHB, by bacteria and purification of anovel PHB depolymerase from Comamonassp. J. Environ. Polym. Degrad. 1:53-63
[21].Kasuya K, Takagi K, Ishiwatari S, YoshidaY, Doi Y. 1998. Biodegradabilitiesof various aliphatic polyesters in naturalwaters. Polym. Degrad. Stab. 59:327-32
[22].Ohura T, Aoyagi Y, Takagi K, Yoshida Y, Kasuya K, DoiY. 1999. Biodegradation ofpoly(3-hydroxyalkanoic acids)fibers andisolation of poly(3-hydroxybutyric acid)-degrading microorganisms under aquaticenvironments. Polym. Degrad. Stab. 63:23-2959:1220-27
[23].Schirmer A, Matz C, Jendrossek D. 1995.Substrate specificities of PHA-degradingbacteria and active site studies on the extracellularpoly(3-hydroxyoctanoic acid)[P(3HO)] depolymerase of Pseudomonasfluorescens GK13. Can. J. Microbiol. 41(Suppl.1):170-179
[24].Gonda KE, Jendrossek D, Molitoris HP.2000. Fungal degradation of thermoplasticpolymers under simulated deepsea conditions. Hydrobiologia 426:73-183
[25].Han J-S, Son Y-J, Chang C-S, Kim M-N. 1998. Purification and properties of extracellularpoly(3-hydroxybutyrate) depolymeraseproduced by Penicillium pinophilurn.J. Microbiol. 36:67-73
[26].Matavulj M, Molitoris HP. 1992. Fungal degradation of polyhydroxyalkanoatesand a semiquantitative assay for screeningtheir degradation by terrestrial fungi. FEMS Microbiol. Rev. 103:323-32
[27].Neumeier S. 1994. Abbau thermoplastischerBiopolymere auf Poly-φ-Hydroxyalkanoat-Basis durch terrestrische undmarine Pilze. Diplomarbeit. Univ. Regensburg.99 pp.
[28].Jendrossek D. 2001. Microbial degradationof polyesters. See Ref. 89, pp. 293-325
[29].Grochuliski, P., Bouthiller, F., Kazlauskas, R. J., Serreqi, A. N., Schrag, J. D., Ziomek, E. and Cygler, M., Biochemistry, 1994, 33, 3494-3500.
[30].Jendrossek D. 2001. Transfer of [Pseudomanas]lemoignei, a gram-negativerod with restricted catabolic capacity, toPaucimonas gen. nov. with one species, Paucimonas lemoignei comb. nov. Int. J. Syst. Evol. Microbiol. 51:905-8
[31].Klingbeil B, Kroppenstedt R, Jendrossek D. 1996. Taxonomical identification of Streptomyces exfoliatus K10 and characterization of its poly(3-hydroxybutyrate) depolymerase gene. FEMS Microbiol. Lett. 142:215-21
[32].Doi Y, Kitamura S, Abe H. 1995. Microbial synthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Macromolecules 28:4822-28
[33].Jendrossek, D., Frisse, A., Behrends, A., Andermann, M., Kratzin, H. D., Stanislawski, T. and Schlegel, H. G., J Bacteriol., 1994, 2, 75-87.
[34].Kumar A, Gross RA, Jendrossek D. 2000. Poly(3-hydroxybutyrate)-depolymerasefrom Pseudomonas lemoignei: catalysisof
esterifications in organic media, J. Org. Chem. 65:7800-6
[35].Suzuki Y, Taguchi S, Saito S, ToshimaK, Matsumura S, Doi Y. 2001. Involvementof catalytic amino acid residues inenzyme-catalyzed polymerization for thesynthesis of polyesters. Biomacromolecules2:541-44
[36].Saito T, Suzuki K, Yamamoto J, Fukui T, Miwa K, et al. 1989. Cloning, nucleotide PHA DEGRADATION 431 sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis, J. Bacteriol. 171:184-89
[37].Jendrossek D, Backhaus M, Andermann M. 1995. Characterization of the extracellular poly(3-hydroxybutyrate) depolymerase of Comamonas sp. and of its structural gene. Can. J. Microbiol. 41(Suppl.1): 160-69
[38].Kurusu, Y., Kohama, K., Uchida, Y., Saito, T. and Yukawa, H., In Biodegradable Plastics and Polymers, ed. Y. Doi and K. Fukuda, Elsevier Science, Amstrdam, 1994, pp. 357-361.
[39].Zhang, J., Shiraki, M. and Saito, T., J. Bacteriol., 1997, 179, 72-77.
[40].Briese, B. H., Schmidt, B. and Jendrossek, D., J. Environ. Polym. Degrad., 1994, 2, 75-87.
[41].Jendrossek, D., Frisse, A., Behrends, A., Andermann, M., Kratzin, H. D., Stanislawski, T., and Schlegel, H. G., J. Bacteriol., 1995, 177, 596-607.
[42].Jendrossek, D., Muller, B. and Schlegel, H. G., Eur. J. Biochem., 1993, 218, 701-710.
[43].Schirmer A, Jendrossek D. 1994. Molecular characterization of the extracellular poly(3-hydroxyoktanoic acid) [P(3HO)] depolymerase gene of Pseudomonas fluorescens GK13 and of its gene product, J. Bacteriol. 176:7065-73
[44].Martinez, C., de Geus, P., Lauwereys, M., Matthyssens, G. and Gambillau, C., Nature, 1992, 356, 615-618.
[45].Soliday, C. L., Flurkey, W. H., Okita, T. W. and Kolatturkudy, P. E., P. N. A. S., 1984, 81, 3939-3943.
[46].Jaeger K-E, Ransac S, Dijkstra BW, Colson D, van Heuvel M,
Misset O. 1994. Bacterial lipases. FEMS Microbiol. Rev. 15:29-63
[47].Shinohe T, Nojiri M, Saito T, Stanislawski T, 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. 141:103-9
[48].Behrends A, Klingbeil B, Jendrossek D. 1996. Poly(3-hydroxybutyrate) depolymerases bind to their substrate by a C-terminal located substrate binding site. FEMS Microbiol. Lett. 143:191-94
[49].Fukui T, Narikawa T, Miwa K, Shirakura Y, Saito T, Tomita K. 1988. Effect of limited tryptic modification of a bacterial poly(3-hydroxybutyrate) depolymerase on its catalytic activity. Biochim. Biophys. Acta 952:164-71
[50].Little E, Bork P, Doolittle RF. 1994. Tracing the spread of fibronectin type Ⅲ domains in bacterial glycohydrolases, J. Mol. Evol. 39:631-43
[51].Watanabe T, Ito Y, Yamada T, Hashimoto M, Sekine S, Tanaka H. 1994. The roles of the C-terminal domain and type Ⅲ domains of chitinases A1 from Bacillus circulans WL-12 in chitin degradation, J. Bacteriol. 176:4465-72
[52].Gilkes, N. R., Henrissat, B., Kilburn, D. G., Miller, R. C. Jr and Warren, R. A. J., Microbiol. Rev., 1991, 55,303-315.
[53].Shiraki, M., Shimada, T., Tatsumichi, M. and Saito, T., J. Environ. Polym. Degrad., 1995, 3, 13-21.
[54].Jendrossek D, Schirmer A, Handrick R. 1997. Recent advances in characterization of bacterial PHA depolymerases. PHA DEGRADATION 429 In International Symposium on Bacterial Polyhydroxyalkanoates, ed. G Eggink, A Steinb(?)chel, Y Poirier, BWitholt, pp. 89-101. Ottawa, Can.: NRC Res.
[55].Quinteros R, Goodwin S, Lenz RW, Park WH. 1999. Extracellular degradation of medium chain length poly(beta-hydroxyalkanoates) by Comamonas sp. Int. d. Biol. Macromol. 25:135-43
[56].Handrick R, Reinhardt S, Focarete ML,Scandola M, Adamus G, et al. 2001. Anew type of thermoalkalophilic hydrolaseof Paucimonas
lemoignei with high speci-ficity for amorphous polyesters of shortchain-length hydroxyalkanoic acids. J. Biol. Chem. 276:36215-24
[57].Ollis DL, Cheah E, Cygler M, Dijkstra B,Frolow F, et al. 1992. The AE/φ hydrolasefold.Protein Eng. 5:197-211
[58].Delafield FP, DoudoroffM, Palleroni NJ,Lusty C J, Contopoulos R. 1965. Decompositionof poly-β-hydroxybutyrate bypseudomonads. J. Bacteriol. 90:1455-66
[59].M(?)ller B, Jendrossek D. 1993. Purificationand properties of a poly(3-hydroxyvalerate)depolymerase from Pseudomonaslemoignei. Appl. Microbiol. Biotechnol. 38:487-92
[60].Nakayama K, Saito T, Fukui T, ShirakuraY, Tomita K. 1985. Purification andproperties of extracellular poly(3-hydroxybutyrate)depolymerases from Pseudomonaslemoignei. Biochim. Biophys. Acta827:63-72
[61].StinsonMW, Merrick JM. 1974. Extracellularenzyme secretion by Pseudomonaslemoignei. J. Bacteriol. 119:152-61
[62].Terpe K, Kerkhoff K, Pluta E, JendrossekD. 1999. Relationship between succinatetransport and production of extracellularpoly(3-hydroxybutyrate) depolymerase inPseudomonas lemoignei. Appl. Environ. Microbiol. 65:1703-9
[63].Terpe K. 1999. Physiologische und molekularbiologische Untersuchungen zurRegulation extrazellul arer PHB-Depolymerasenin P. lemoignei. PhD thesis. Univ. G(?)ttingen. 158 PP
[64].Foster L JR, Zervas SJ, Lenz RW, FullerRC. 1995. The biodegradation of poly-3-hydroxyalkanoates, PHAs, with longalkyl substituents by Pseudomonas maculicola.Biodegradation 6:67-73
[65].Molitoris KP, Moss ST, de Koning G, JendrossekD. 1996. SEM analysis of polyhydroxyalkanoatedegradation by bacteria. Appl. Microbiol. Biotechnol. 46:570-79
[66].Nobes GAR, Marchessault RH, ChanzyH, Briese BH, Jendrossek D. 1996. Splinteringofpoly(3-hydroxybutyrate) singlecrystals by PHB-depolymerase A from Pseudomonas lemoignei. Macromolecules29: 83 30-3 3
[67]．陈珊，刘东波，夏红梅等，聚3-羟基丁酸酯(PHB)生物降解过程的研究，生物化学与生物物理进展，2002，29(1)，110～113
[68].Focarete ML, Scandola M, Jendrossek D,Adamus G, Sikorska W, Kowalczuk M. 1999. Bioassimilation of oligomers of atacticpoly[(R,S)-3-hydroxybutyrate] by selectedbacterial strains. Macromolecules32:4814-18
[69].Bachmann BM, Seebach D. 1999. Investigationof the enzymatic cleavage of diastereomericoligo(3-hydroxybutanoates)containing two to eight HB units. Amodel for the stereoselectivity of PHB depolymerasefrom Alcaligenes faecalis T1. Macromolecules 32:1777-84
[70].Tanaka, Y., Saito, T., Fukui, T., Tanio, T. and Tomita, T., Eur. J. Biochem, 1981, 118, 177-182.
[71].Abe H, Doi Y. 1996. Enzymatic andenvironmental degradation of racemicpoly (3-hydroxybutyric acid)s with differentstereoregulatoris. Macromolecules29:8683-88
[72].Mergaert J, Schirmer A, Hauben L, MauM, Jendrossek D, Swings J. 1996. Isolationand identification of poly(3-hydroxyvalerate)degrading strains of Pseudomonaslemoignei. Int. J. Syst. Bacteriol. 46:769-73
[73].Jaeger K-E, Steinb(?)chel A, Jendrossek D.1995. Substrate specificities of bacterialpolyhydroxyalkanoate depolymerasesand lipases: bacterial lipases hydrolyzepoly(!-hydroxyalkanoates). Appl. Environ. Microbiol. 61:3113-18
[74].Lemoigne M. 1925. Etudes sur 1'autolyse microbienne—acidification par formation d'acide φ oxybutyrique. Ann. Inst. Pastuer 39:144-55
[75].Macrae RM, Wilkinson JF. 1958. PolyB- hydroxybutyrate metabolism in washed suspensions of Bacillus cereus and Bacillus megaterium. J. Gen. Microbiol. 19:210-22
[76].Hayward AC, Forsyth WGC, Roberts JB. 1959. Synthesis and breakdown of polyβ- hydroxybutyric acid by bacteria, d. Gen. Microbiol. 20:510-18
[77].Doi Y, Segawa A, Kawaguchi Y, Kunioka M. 1990. Cyclic nature
of poly(3-hydroxyalkanoate) metabolism in Alcaligenes eutrophus. FEMS Microbiol. Lett. 67:165-70
[78].Taidi B, Mansfield DA, Anderson AJ. 432 JENDROSSEK 9·HANDRICK 1995. Turnover of poly(3-hydroxybutyrate) (PHB) and its influence on the molecular mass of the polymer accumulated by Alcaligenes eutrophus during batch culture. FEMS Microbiol. Lett. 129:201-6
[79].Choi MH, Yoon SC, Lenz RW. 1999. Production of poly(3-hydroxybutyric acidco- 4-hydroxybutyric acid) and poly(4-hydroxybutyric acid) without subsequent degradation by Hydrogenophaga pseudofiava. Appl. Environ. Microbiol. 65:1570-77
[80].James BW, Mauchline WS, Dennis PJ, Keevil CW, Wait R. 1999. Poly-3-hydroxybutyrate in Legionella pneumophila, an energy source for survival in low-nutrient environments. Appl. Environ. Microbiol. 65:822-27
[81].Handrick R, Reinhard S, Jendrossek D. 428 JENDROSSEK · HANDRICK 2000. Mobilization of poly(3-hydroxybutyrate) in Ralstonia eutropha, d. Bacteriol. 182:5916-18
[82].Griebel RJ, Smith Z, Merrick JM. 1968. Metabolism of poly-β-hydroxybutyrate. I. Purification, composition, and properties of native poly-β-hydroxybutyrate granules from Bacillus megaterium. Biochemistry 7:3676-81
[83].Mayer F, Madkour MH, Pieper-Ftirst U, Wieczorek R, Liebergesell M, Steinb tichel A. 1996. Electron microscopic observations on the macromolecular organization of the boundary layer of bacterial PHA inclusion bodies, d. Gen. Appl. Microbiol. 42:445-55
[84].Griebel RJ, Merrick JM. 1971. Metabolism of poly-β-hydroxybutyrate: effect of mild alkaline extraction on native polyβ- hydroxybutyrate granules, J. Bacteriol. 108:782-89
[85].Merrick JM, DoudoroffM. 1964. Depolymerisation of poly-β-hydroxybutyrate by an intracellular enzyme system, J. Bacteriol. 88:60-71 C, Liebergesell M, et al. 1995. Considerations
[86].Steinbtichel A, Aerts K, BabelW, F(?)llner on the structure and
biochemistry of bacterial polyhydroxyalkanoic acids inclusions. Can. J. Microbiol. 41(Suppl. 1):94-105
[87].York GM, Junker BH, Stubbe JA, Sinskey AJ. 2001. Accumulation of the PhaP phasin of Ralstonia eutropha is dependent on production of polyhydroxybutyrate in cells, J. Bacteriol. 183:4217-26
[88].Saito T, Kobayashi T. 2001. Intracellular degradation of PHAs. See Ref. 100, pp. 23-40
[89].Handrick R, Jendrossek D. 1998. Extracellular and intracellular polyhydroxyalkanoate depolymerases: homologies and differences. In Biochemical Principles and Mechanisms of Biodegradation and Biodegradation of Polymers, ed. A. Steinb(?)chel, pp. 57-67. Weinheim: Wiley-VCH
[90].Merrick JM, Steger R, Dombroski D. 430 JENDROSSEK·HANDRICK 1999. Hydrolysis of native poly(hydroxybutyrate) granules (PHB), crystalline PHB, and artificial amorphous PHB granules by intracellular and extracellular depolymerases. Int. J. Biol. Macromol. 25:129-34
[91].Technow U. 1999. Isolierung und Substratbindedom ane der extrazellul aren PHB Depolymerase A yon Pseudomonas lemoignei aus rekombinanten Escherichia coli und Anreicherung und Charakterisierung des A ktivators im intrazellul aren PHB-Abbau von Rhodospirillum rubrum. Diplomarbeit. Univ. Gottingen. 108 pp.
[92].Foster L JR, Lenz RW, Fuller RC. 1994. Quantitative determination of intracellular depolymerase activity in Pseudomonas oleovorans inclusions containing poly-3-hydroxyalkanoates with long alkyl substituents. FEMS Microbiol. Lett. 118:279-82
[93].Foster LJR, Stuart ES, Tehrani A, Lenz RW, Fuller RC. 1996. Intracellular depolymerase and polyhydroxyoctanoate granule integrity in Pseudomonas oleovorans. Int. J. Biol. Macromol. 19:177-83 B, Witholt B. 1991. Metabolism
[94].HuismanGW,Wonink E, Meima R, Terpstra of poly(3-hydroxyalkanoates) (PHA) by Pseudomonas oleovorans. J. Biol. Chem. 266:2191-98
[95].Ruiz JA, Lopez NI, Fernandez RO, Mendez BS. 2001. Polyhydroxyalkanoate degradation is associated with nucleotide accumulation and enhances stress resistance and survival of Pseudomonas oleovorans in naturalwater microcosms. Appl. Environ. Microbiol. 67:225-30
[96].Stuart ES, Foster L JR, Lenz RW, Fuller RC. 1996. Intracellular depolymerase functionality and location in Pseudomonas oleovorans inclusions containing polyhydroxyalkanoate. Int. J. Biol. Macromol. 19:171-76
[97].Brandl H, Bachofen R, Mayer J, Degradation and applications of polyhydroxyalkanoates. Can J Microbiol. 1995,41 (suppl. 1): 143～153
[98] 陈珊，刘东波，，夏红梅等，一株可降解聚β-羟基丁酸酯的真菌[J]．菌物系统2001，(3)：423～425
[99]．陈珊，刘东波，夏红梅等，降解聚β-羟基丁酸酯的真菌的研究[J]，东北师大学报自然科学版，2002，34(3)：13-17
[100]．陈珊，刘东波，夏红梅等，聚β-羟基丁酸酯解聚酶相关性质的研究，微生物学通报，2002，29(3)：50～54
[101]．陈珊，刘东波，夏红梅等，聚β-羟基丁酸酯解聚酶的纯化及性质，分子科学学报，2002，18(4)：220～225
[102].Yamada K, Mukai K, Doi Y. Enzymatic degradation of poly(hydroxyalkanoates) by Pseu#omonas pickettii.[J] Iht J Biol Macromol, 1993. 15:215～220.
[103].Tomasi G, Scandola M, Holger B et al. Enzymatic degradation of bacterial poly(3-hydroxybutyrate) by a depolymerase from Pseudomonas lemoignei. Macromolecules, 1996. 29: 507～508.
[104]．陶文沂 工业微生物生理与遗传育种学 北京：中国轻工业出版社，1997
[105]．章名春 工业微生物诱变育种 北京：科学出版社，1984
[106].Horowitz DM, Sanders J K M. J Am Chem Soc, 1994, 116(7): 2695-2697
[107].Shirakura Y, Fukui T, Saito T, et al. Biochimica et Biophysica
Acta, 1986, 880:46～53
[108].Brucato T L, Wong S S. Archives of Biochemistry and Biophysics, 1991,290(2): 497～502
[109].Stinson M W, Merrick J M. J Bacteriology, 1974, 119: 152～161
[110].Lowry O H, Rosebrough N J, Fart A L, et al, [J]. J Bio Chem, 1951, 193:265～275
[111]．萨姆布鲁克E F弗里奇，T 曼尼阿蒂斯．分子克隆实验指南，第二版，北京：科学出版社，1992．
[2].Johnstone, B., 1990. A throw away answer. Far Eastern Econ. Rev. 147(6), 62-63.
[3].Anderson AJ, Dawes EA. 1990. Occurrence, metabolism, metabolic role, and industrial use of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54:450-72
[4].Doi Y. 1990. Microbial Polyesters. New York: VHC
[5].Madison LL, Huisman GW. 1999. Metabolicengineefing of poly(3-hydroxyalkanoates):from DNA to plastic. Microbiol. Mol. Biol. Rev. 63:21-53
[6].Müller HM, Seebach D. 1993. Poly(hydroxyalkanoates) :a fifth class of physiologicallyimportant organic biopolymers Angew. Chem. Int. Ed. Engl. 32:477-502
[7].Scheper T. 2001. Biopolyesters: Advancesin Biochemical Engineering/Biotechnology. Berlin/Heidelberg: Springer
[8].Steinbüchel A, Doi Y, eds. 2001. Biopolymers, PolyestersⅡ Properties and ChemicalSynthesis. Weinheim: Wiley-VCH.468 pp.
[9].Steinbüchel A, Valentin HE. 1995. Diversity of bacterial polyhydroxyalkanoicacids. FEMS Microbiol. Lett. 128:219-28
[10].Amor SR, Rayment T, Sanders JKM. 1991. Poly(3-hydroxybutyrate) in vivo:NMR and X-ray characterization of theelastomeric state. Macromolecules 24:4583-88
[11].de Koning GJM, Lemstra PJ. 1992. The amorphous state of bacterial poly[(R)-3-hydroxyalkanoate)] in vivo. Polymer 33:3304-6
[12].Chowdhury AA. 1963. Poly-β-hydroxybutters"aure abbauende Bakterien und Exoenzym. Arch. Mikrobiol. 47:167-200
[13].Schirmer A, Jendrossek D, Schlegel HG.1993. Degradation of poly(3-hydroxyoctanoicacid), [P(3HO)], by bacteria. Purificationand properties of a P(3HO) depolymerase of Pseudomonas fluorescensGK13 biovarV. Appl. Environ. Microbiol.
[14].Marchessault RH, Morin FG, Wong S,Saracovan I. 1995.
Artificial granule suspensionsof long side chain poly(3-hydroxyalkanoate). Can. J. Microbiol. 41(Suppl.1): 138-42
[15].Ramsay BA, Saracovan Ⅰ, Ramsay JA,Marchessault RH. 1994. A method forthe isolation of microorganisms producingextracellular long-side-chain poly(βhydroxyalkanoate)depolymerase. J. Environ. Polym. Degrad. 2:1-7
[16].Horowitz DM, Brennan EM, Koon JJ, Gemgross TU. 1999. Novel thermal routeto an amorphous, film-forming polymerlatex. Macromolecules 32:3347-52
[17].Horowitz DM, Sanders JKM. 1995. Biomimetic, amorphous granules of polyhydroxyalkanoates:composition, mobility, and stabilization in vivo by proteins. Can. J. Microbiol. 41(Suppl. 1):115-23
[18].Jendrossek D. 2001. Extracellular PHAdepolymerases: the key enzymes of PHAdegradation. See Ref. 100, pp. 41-83
[19].Briese B-H, Jendrossek D, Schlegel HG. 1994. Degradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by aerobicsewage sludge. FEMS Microbiol. Lett. 117:107-12
[20].Jendrossek D, Knoke Ⅰ, Habibian RB, Steinbtichel A, Schlegel HG. 1993. Degradation of poly(3-hydroxybutyrate), PHB, by bacteria and purification of anovel PHB depolymerase from Comamonassp. J. Environ. Polym. Degrad. 1:53-63
[21].Kasuya K, Takagi K, Ishiwatari S, YoshidaY, Doi Y. 1998. Biodegradabilitiesof various aliphatic polyesters in naturalwaters. Polym. Degrad. Stab. 59:327-32
[22].Ohura T, Aoyagi Y, Takagi K, Yoshida Y, Kasuya K, DoiY. 1999. Biodegradation ofpoly(3-hydroxyalkanoic acids)fibers andisolation of poly(3-hydroxybutyric acid)-degrading microorganisms under aquaticenvironments. Polym. Degrad. Stab. 63:23-2959:1220-27
[23].Schirmer A, Matz C, Jendrossek D. 1995.Substrate specificities of PHA-degradingbacteria and active site studies on the extracellularpoly(3-hydroxyoctanoic acid)[P(3HO)] depolymerase of Pseudomonasfluorescens GK13. Can. J. Microbiol. 41(Suppl.1):170-179
[24].Gonda KE, Jendrossek D, Molitoris HP.2000. Fungal degradation of thermoplasticpolymers under simulated deepsea conditions. Hydrobiologia 426:73-183
[25].Han J-S, Son Y-J, Chang C-S, Kim M-N. 1998. Purification and properties of extracellularpoly(3-hydroxybutyrate) depolymeraseproduced by Penicillium pinophilurn.J. Microbiol. 36:67-73
[26].Matavulj M, Molitoris HP. 1992. Fungal degradation of polyhydroxyalkanoatesand a semiquantitative assay for screeningtheir degradation by terrestrial fungi. FEMS Microbiol. Rev. 103:323-32
[27].Neumeier S. 1994. Abbau thermoplastischerBiopolymere auf Poly-φ-Hydroxyalkanoat-Basis durch terrestrische undmarine Pilze. Diplomarbeit. Univ. Regensburg.99 pp.
[28].Jendrossek D. 2001. Microbial degradationof polyesters. See Ref. 89, pp. 293-325
[29].Grochuliski, P., Bouthiller, F., Kazlauskas, R. J., Serreqi, A. N., Schrag, J. D., Ziomek, E. and Cygler, M., Biochemistry, 1994, 33, 3494-3500.
[30].Jendrossek D. 2001. Transfer of [Pseudomanas]lemoignei, a gram-negativerod with restricted catabolic capacity, toPaucimonas gen. nov. with one species, Paucimonas lemoignei comb. nov. Int. J. Syst. Evol. Microbiol. 51:905-8
[31].Klingbeil B, Kroppenstedt R, Jendrossek D. 1996. Taxonomical identification of Streptomyces exfoliatus K10 and characterization of its poly(3-hydroxybutyrate) depolymerase gene. FEMS Microbiol. Lett. 142:215-21
[32].Doi Y, Kitamura S, Abe H. 1995. Microbial synthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Macromolecules 28:4822-28
[33].Jendrossek, D., Frisse, A., Behrends, A., Andermann, M., Kratzin, H. D., Stanislawski, T. and Schlegel, H. G., J Bacteriol., 1994, 2, 75-87.
[34].Kumar A, Gross RA, Jendrossek D. 2000. Poly(3-hydroxybutyrate)-depolymerasefrom Pseudomonas lemoignei: catalysisof
esterifications in organic media, J. Org. Chem. 65:7800-6
[35].Suzuki Y, Taguchi S, Saito S, ToshimaK, Matsumura S, Doi Y. 2001. Involvementof catalytic amino acid residues inenzyme-catalyzed polymerization for thesynthesis of polyesters. Biomacromolecules2:541-44
[36].Saito T, Suzuki K, Yamamoto J, Fukui T, Miwa K, et al. 1989. Cloning, nucleotide PHA DEGRADATION 431 sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis, J. Bacteriol. 171:184-89
[37].Jendrossek D, Backhaus M, Andermann M. 1995. Characterization of the extracellular poly(3-hydroxybutyrate) depolymerase of Comamonas sp. and of its structural gene. Can. J. Microbiol. 41(Suppl.1): 160-69
[38].Kurusu, Y., Kohama, K., Uchida, Y., Saito, T. and Yukawa, H., In Biodegradable Plastics and Polymers, ed. Y. Doi and K. Fukuda, Elsevier Science, Amstrdam, 1994, pp. 357-361.
[39].Zhang, J., Shiraki, M. and Saito, T., J. Bacteriol., 1997, 179, 72-77.
[40].Briese, B. H., Schmidt, B. and Jendrossek, D., J. Environ. Polym. Degrad., 1994, 2, 75-87.
[41].Jendrossek, D., Frisse, A., Behrends, A., Andermann, M., Kratzin, H. D., Stanislawski, T., and Schlegel, H. G., J. Bacteriol., 1995, 177, 596-607.
[42].Jendrossek, D., Muller, B. and Schlegel, H. G., Eur. J. Biochem., 1993, 218, 701-710.
[43].Schirmer A, Jendrossek D. 1994. Molecular characterization of the extracellular poly(3-hydroxyoktanoic acid) [P(3HO)] depolymerase gene of Pseudomonas fluorescens GK13 and of its gene product, J. Bacteriol. 176:7065-73
[44].Martinez, C., de Geus, P., Lauwereys, M., Matthyssens, G. and Gambillau, C., Nature, 1992, 356, 615-618.
[45].Soliday, C. L., Flurkey, W. H., Okita, T. W. and Kolatturkudy, P. E., P. N. A. S., 1984, 81, 3939-3943.
[46].Jaeger K-E, Ransac S, Dijkstra BW, Colson D, van Heuvel M,
Misset O. 1994. Bacterial lipases. FEMS Microbiol. Rev. 15:29-63
[47].Shinohe T, Nojiri M, Saito T, Stanislawski T, 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. 141:103-9
[48].Behrends A, Klingbeil B, Jendrossek D. 1996. Poly(3-hydroxybutyrate) depolymerases bind to their substrate by a C-terminal located substrate binding site. FEMS Microbiol. Lett. 143:191-94
[49].Fukui T, Narikawa T, Miwa K, Shirakura Y, Saito T, Tomita K. 1988. Effect of limited tryptic modification of a bacterial poly(3-hydroxybutyrate) depolymerase on its catalytic activity. Biochim. Biophys. Acta 952:164-71
[50].Little E, Bork P, Doolittle RF. 1994. Tracing the spread of fibronectin type Ⅲ domains in bacterial glycohydrolases, J. Mol. Evol. 39:631-43
[51].Watanabe T, Ito Y, Yamada T, Hashimoto M, Sekine S, Tanaka H. 1994. The roles of the C-terminal domain and type Ⅲ domains of chitinases A1 from Bacillus circulans WL-12 in chitin degradation, J. Bacteriol. 176:4465-72
[52].Gilkes, N. R., Henrissat, B., Kilburn, D. G., Miller, R. C. Jr and Warren, R. A. J., Microbiol. Rev., 1991, 55,303-315.
[53].Shiraki, M., Shimada, T., Tatsumichi, M. and Saito, T., J. Environ. Polym. Degrad., 1995, 3, 13-21.
[54].Jendrossek D, Schirmer A, Handrick R. 1997. Recent advances in characterization of bacterial PHA depolymerases. PHA DEGRADATION 429 In International Symposium on Bacterial Polyhydroxyalkanoates, ed. G Eggink, A Steinb(?)chel, Y Poirier, BWitholt, pp. 89-101. Ottawa, Can.: NRC Res.
[55].Quinteros R, Goodwin S, Lenz RW, Park WH. 1999. Extracellular degradation of medium chain length poly(beta-hydroxyalkanoates) by Comamonas sp. Int. d. Biol. Macromol. 25:135-43
[56].Handrick R, Reinhardt S, Focarete ML,Scandola M, Adamus G, et al. 2001. Anew type of thermoalkalophilic hydrolaseof Paucimonas
lemoignei with high speci-ficity for amorphous polyesters of shortchain-length hydroxyalkanoic acids. J. Biol. Chem. 276:36215-24
[57].Ollis DL, Cheah E, Cygler M, Dijkstra B,Frolow F, et al. 1992. The AE/φ hydrolasefold.Protein Eng. 5:197-211
[58].Delafield FP, DoudoroffM, Palleroni NJ,Lusty C J, Contopoulos R. 1965. Decompositionof poly-β-hydroxybutyrate bypseudomonads. J. Bacteriol. 90:1455-66
[59].M(?)ller B, Jendrossek D. 1993. Purificationand properties of a poly(3-hydroxyvalerate)depolymerase from Pseudomonaslemoignei. Appl. Microbiol. Biotechnol. 38:487-92
[60].Nakayama K, Saito T, Fukui T, ShirakuraY, Tomita K. 1985. Purification andproperties of extracellular poly(3-hydroxybutyrate)depolymerases from Pseudomonaslemoignei. Biochim. Biophys. Acta827:63-72
[61].StinsonMW, Merrick JM. 1974. Extracellularenzyme secretion by Pseudomonaslemoignei. J. Bacteriol. 119:152-61
[62].Terpe K, Kerkhoff K, Pluta E, JendrossekD. 1999. Relationship between succinatetransport and production of extracellularpoly(3-hydroxybutyrate) depolymerase inPseudomonas lemoignei. Appl. Environ. Microbiol. 65:1703-9
[63].Terpe K. 1999. Physiologische und molekularbiologische Untersuchungen zurRegulation extrazellul arer PHB-Depolymerasenin P. lemoignei. PhD thesis. Univ. G(?)ttingen. 158 PP
[64].Foster L JR, Zervas SJ, Lenz RW, FullerRC. 1995. The biodegradation of poly-3-hydroxyalkanoates, PHAs, with longalkyl substituents by Pseudomonas maculicola.Biodegradation 6:67-73
[65].Molitoris KP, Moss ST, de Koning G, JendrossekD. 1996. SEM analysis of polyhydroxyalkanoatedegradation by bacteria. Appl. Microbiol. Biotechnol. 46:570-79
[66].Nobes GAR, Marchessault RH, ChanzyH, Briese BH, Jendrossek D. 1996. Splinteringofpoly(3-hydroxybutyrate) singlecrystals by PHB-depolymerase A from Pseudomonas lemoignei. Macromolecules29: 83 30-3 3
[67]．陈珊，刘东波，夏红梅等，聚3-羟基丁酸酯(PHB)生物降解过程的研究，生物化学与生物物理进展，2002，29(1)，110～113
[68].Focarete ML, Scandola M, Jendrossek D,Adamus G, Sikorska W, Kowalczuk M. 1999. Bioassimilation of oligomers of atacticpoly[(R,S)-3-hydroxybutyrate] by selectedbacterial strains. Macromolecules32:4814-18
[69].Bachmann BM, Seebach D. 1999. Investigationof the enzymatic cleavage of diastereomericoligo(3-hydroxybutanoates)containing two to eight HB units. Amodel for the stereoselectivity of PHB depolymerasefrom Alcaligenes faecalis T1. Macromolecules 32:1777-84
[70].Tanaka, Y., Saito, T., Fukui, T., Tanio, T. and Tomita, T., Eur. J. Biochem, 1981, 118, 177-182.
[71].Abe H, Doi Y. 1996. Enzymatic andenvironmental degradation of racemicpoly (3-hydroxybutyric acid)s with differentstereoregulatoris. Macromolecules29:8683-88
[72].Mergaert J, Schirmer A, Hauben L, MauM, Jendrossek D, Swings J. 1996. Isolationand identification of poly(3-hydroxyvalerate)degrading strains of Pseudomonaslemoignei. Int. J. Syst. Bacteriol. 46:769-73
[73].Jaeger K-E, Steinb(?)chel A, Jendrossek D.1995. Substrate specificities of bacterialpolyhydroxyalkanoate depolymerasesand lipases: bacterial lipases hydrolyzepoly(!-hydroxyalkanoates). Appl. Environ. Microbiol. 61:3113-18
[74].Lemoigne M. 1925. Etudes sur 1'autolyse microbienne—acidification par formation d'acide φ oxybutyrique. Ann. Inst. Pastuer 39:144-55
[75].Macrae RM, Wilkinson JF. 1958. PolyB- hydroxybutyrate metabolism in washed suspensions of Bacillus cereus and Bacillus megaterium. J. Gen. Microbiol. 19:210-22
[76].Hayward AC, Forsyth WGC, Roberts JB. 1959. Synthesis and breakdown of polyβ- hydroxybutyric acid by bacteria, d. Gen. Microbiol. 20:510-18
[77].Doi Y, Segawa A, Kawaguchi Y, Kunioka M. 1990. Cyclic nature
of poly(3-hydroxyalkanoate) metabolism in Alcaligenes eutrophus. FEMS Microbiol. Lett. 67:165-70
[78].Taidi B, Mansfield DA, Anderson AJ. 432 JENDROSSEK 9·HANDRICK 1995. Turnover of poly(3-hydroxybutyrate) (PHB) and its influence on the molecular mass of the polymer accumulated by Alcaligenes eutrophus during batch culture. FEMS Microbiol. Lett. 129:201-6
[79].Choi MH, Yoon SC, Lenz RW. 1999. Production of poly(3-hydroxybutyric acidco- 4-hydroxybutyric acid) and poly(4-hydroxybutyric acid) without subsequent degradation by Hydrogenophaga pseudofiava. Appl. Environ. Microbiol. 65:1570-77
[80].James BW, Mauchline WS, Dennis PJ, Keevil CW, Wait R. 1999. Poly-3-hydroxybutyrate in Legionella pneumophila, an energy source for survival in low-nutrient environments. Appl. Environ. Microbiol. 65:822-27
[81].Handrick R, Reinhard S, Jendrossek D. 428 JENDROSSEK · HANDRICK 2000. Mobilization of poly(3-hydroxybutyrate) in Ralstonia eutropha, d. Bacteriol. 182:5916-18
[82].Griebel RJ, Smith Z, Merrick JM. 1968. Metabolism of poly-β-hydroxybutyrate. I. Purification, composition, and properties of native poly-β-hydroxybutyrate granules from Bacillus megaterium. Biochemistry 7:3676-81
[83].Mayer F, Madkour MH, Pieper-Ftirst U, Wieczorek R, Liebergesell M, Steinb tichel A. 1996. Electron microscopic observations on the macromolecular organization of the boundary layer of bacterial PHA inclusion bodies, d. Gen. Appl. Microbiol. 42:445-55
[84].Griebel RJ, Merrick JM. 1971. Metabolism of poly-β-hydroxybutyrate: effect of mild alkaline extraction on native polyβ- hydroxybutyrate granules, J. Bacteriol. 108:782-89
[85].Merrick JM, DoudoroffM. 1964. Depolymerisation of poly-β-hydroxybutyrate by an intracellular enzyme system, J. Bacteriol. 88:60-71 C, Liebergesell M, et al. 1995. Considerations
[86].Steinbtichel A, Aerts K, BabelW, F(?)llner on the structure and
biochemistry of bacterial polyhydroxyalkanoic acids inclusions. Can. J. Microbiol. 41(Suppl. 1):94-105
[87].York GM, Junker BH, Stubbe JA, Sinskey AJ. 2001. Accumulation of the PhaP phasin of Ralstonia eutropha is dependent on production of polyhydroxybutyrate in cells, J. Bacteriol. 183:4217-26
[88].Saito T, Kobayashi T. 2001. Intracellular degradation of PHAs. See Ref. 100, pp. 23-40
[89].Handrick R, Jendrossek D. 1998. Extracellular and intracellular polyhydroxyalkanoate depolymerases: homologies and differences. In Biochemical Principles and Mechanisms of Biodegradation and Biodegradation of Polymers, ed. A. Steinb(?)chel, pp. 57-67. Weinheim: Wiley-VCH
[90].Merrick JM, Steger R, Dombroski D. 430 JENDROSSEK·HANDRICK 1999. Hydrolysis of native poly(hydroxybutyrate) granules (PHB), crystalline PHB, and artificial amorphous PHB granules by intracellular and extracellular depolymerases. Int. J. Biol. Macromol. 25:129-34
[91].Technow U. 1999. Isolierung und Substratbindedom ane der extrazellul aren PHB Depolymerase A yon Pseudomonas lemoignei aus rekombinanten Escherichia coli und Anreicherung und Charakterisierung des A ktivators im intrazellul aren PHB-Abbau von Rhodospirillum rubrum. Diplomarbeit. Univ. Gottingen. 108 pp.
[92].Foster L JR, Lenz RW, Fuller RC. 1994. Quantitative determination of intracellular depolymerase activity in Pseudomonas oleovorans inclusions containing poly-3-hydroxyalkanoates with long alkyl substituents. FEMS Microbiol. Lett. 118:279-82
[93].Foster LJR, Stuart ES, Tehrani A, Lenz RW, Fuller RC. 1996. Intracellular depolymerase and polyhydroxyoctanoate granule integrity in Pseudomonas oleovorans. Int. J. Biol. Macromol. 19:177-83 B, Witholt B. 1991. Metabolism
[94].HuismanGW,Wonink E, Meima R, Terpstra of poly(3-hydroxyalkanoates) (PHA) by Pseudomonas oleovorans. J. Biol. Chem. 266:2191-98
[95].Ruiz JA, Lopez NI, Fernandez RO, Mendez BS. 2001. Polyhydroxyalkanoate degradation is associated with nucleotide accumulation and enhances stress resistance and survival of Pseudomonas oleovorans in naturalwater microcosms. Appl. Environ. Microbiol. 67:225-30
[96].Stuart ES, Foster L JR, Lenz RW, Fuller RC. 1996. Intracellular depolymerase functionality and location in Pseudomonas oleovorans inclusions containing polyhydroxyalkanoate. Int. J. Biol. Macromol. 19:171-76
[97].Brandl H, Bachofen R, Mayer J, Degradation and applications of polyhydroxyalkanoates. Can J Microbiol. 1995,41 (suppl. 1): 143～153
[98] 陈珊，刘东波，，夏红梅等，一株可降解聚β-羟基丁酸酯的真菌[J]．菌物系统2001，(3)：423～425
[99]．陈珊，刘东波，夏红梅等，降解聚β-羟基丁酸酯的真菌的研究[J]，东北师大学报自然科学版，2002，34(3)：13-17
[100]．陈珊，刘东波，夏红梅等，聚β-羟基丁酸酯解聚酶相关性质的研究，微生物学通报，2002，29(3)：50～54
[101]．陈珊，刘东波，夏红梅等，聚β-羟基丁酸酯解聚酶的纯化及性质，分子科学学报，2002，18(4)：220～225
[102].Yamada K, Mukai K, Doi Y. Enzymatic degradation of poly(hydroxyalkanoates) by Pseu#omonas pickettii.[J] Iht J Biol Macromol, 1993. 15:215～220.
[103].Tomasi G, Scandola M, Holger B et al. Enzymatic degradation of bacterial poly(3-hydroxybutyrate) by a depolymerase from Pseudomonas lemoignei. Macromolecules, 1996. 29: 507～508.
[104]．陶文沂 工业微生物生理与遗传育种学 北京：中国轻工业出版社，1997
[105]．章名春 工业微生物诱变育种 北京：科学出版社，1984
[106].Horowitz DM, Sanders J K M. J Am Chem Soc, 1994, 116(7): 2695-2697
[107].Shirakura Y, Fukui T, Saito T, et al. Biochimica et Biophysica
Acta, 1986, 880:46～53
[108].Brucato T L, Wong S S. Archives of Biochemistry and Biophysics, 1991,290(2): 497～502
[109].Stinson M W, Merrick J M. J Bacteriology, 1974, 119: 152～161
[110].Lowry O H, Rosebrough N J, Fart A L, et al, [J]. J Bio Chem, 1951, 193:265～275
[111]．萨姆布鲁克E F弗里奇，T 曼尼阿蒂斯．分子克隆实验指南，第二版，北京：科学出版社，1992．