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1.
Relationship between Succinate Transport and Production of Extracellular Poly(3-Hydroxybutyrate) Depolymerase in Pseudomonas lemoignei 下载免费PDF全文
The relationship between extracellular poly(3-hydroxybutyrate) (PHB) depolymerase synthesis and the unusual properties of a succinate uptake system was investigated in Pseudomonas lemoignei. Growth on and uptake of succinate were highly pH dependent, with optima at pH 5.6. Above pH 7, growth on and uptake of succinate were strongly reduced with concomitant derepression of PHB depolymerase synthesis. The specific succinate uptake rates were saturable by high concentrations of succinate, and maximal transport rates of 110 nmol/mg of cell protein per min were determined between pH 5.6 and 6.8. The apparent KS0.5 values increased with increasing pH from 0.2 mM succinate at pH 5.6 to more than 10 mM succinate at pH 7.6. The uptake of [14C]succinate was strongly inhibited by several monocarboxylates. Dicarboxylates also inhibited the uptake of succinate but only at pH values near the dissociation constant of the second carboxylate function (pKa2). We conclude that the succinate carrier is specific for the monocarboxylate forms of various carboxylic acids and is not able to utilize the dicarboxylic forms. The inability to take up succinate2− accounts for the carbon starvation of P. lemoignei observed during growth on succinate at pH values above 7. As a consequence the bacteria produce high levels of extracellular PHB depolymerase activity in an effort to escape carbon starvation by utilization of PHB hydrolysis products. 相似文献
2.
Pseudomonas lemoignei is equipped with at least five polyhydroxyalkanoate (PHA) depolymerase structural genes (phaZ1 to phaZ5) which enable the bacterium to utilize extracellular poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate) (PHV), and related polyesters consisting of short-chain-length hxdroxyalkanoates (PHA(SCL)) as the sole sources of carbon and energy. Four genes (phaZ1, phaZ2, phaZ3, and phaZ5) encode PHB depolymerases C, B, D, and A, respectively. It was speculated that the remaining gene, phaZ4, encodes the PHV depolymerase (D. Jendrossek, A. Frisse, A. Behrends, M. Andermann, H. D. Kratzin, T. Stanislawski, and H. G. Schlegel, J. Bacteriol. 177:596-607, 1995). However, in this study, we show that phaZ4 codes for another PHB depolymeraes (i) by disagreement of 5 out of 41 amino acids that had been determined by Edman degradation of the PHV depolymerase and of four endoproteinase GluC-generated internal peptides with the DNA-deduced sequence of phaZ4, (ii) by the lack of immunological reaction of purified recombinant PhaZ4 with PHV depolymerase-specific antibodies, and (iii) by the low activity of the PhaZ4 depolymerase with PHV as a substrate. The true PHV depolymerase-encoding structural gene, phaZ6, was identified by screening a genomic library of P. lemoignei in Escherichia coli for clearing zone formation on PHV agar. The DNA sequence of phaZ6 contained all 41 amino acids of the GluC-generated peptide fragments of the PHV depolymerase. PhaZ6 was expressed and purified from recombinant E. coli and showed immunological identity to the wild-type PHV depolymerase and had high specific activities with PHB and PHV as substrates. To our knowledge, this is the first report on a PHA(SCL) depolymerase gene that is expressed during growth on PHV or odd-numbered carbon sources and that encodes a protein with high PHV depolymerase activity. Amino acid analysis revealed that PhaZ6 (relative molecular mass [M(r)], 43,610 Da) resembles precursors of other extracellular PHA(SCL) depolymerases (28 to 50% identical amino acids). The mature protein (M(r), 41,048) is composed of (i) a large catalytic domain including a catalytic triad of S(136), D(211), and H(269) similar to serine hydrolases; (ii) a linker region highly enriched in threonine residues and other amino acids with hydroxylated or small side chains (Thr-rich region); and (iii) a C-terminal domain similar in sequence to the substrate-binding domain of PHA(SCL) depolymerases. Differences in the codon usage of phaZ6 for some codons from the average codon usage of P. lemoignei indicated that phaZ6 might be derived from other organisms by gene transfer. Multialignment of separate domains of bacterial PHA(SCL) depolymerases suggested that not only complete depolymerase genes but also individual domains might have been exchanged between bacteria during evolution of PHA(SCL) depolymerases. 相似文献
3.
Recent data on the biosynthesis of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and its regulation in bacteria are reviewed, with special emphasis on the properties and regulation of the relevant enzymes and their genes. Some conditions promoting the synthesis of PHB and PHBV by natural, mutant, and recombinant producers are considered. 相似文献
4.
Assay of Poly(3-Hydroxybutyrate) Depolymerase Activity and Product Determination 总被引:3,自引:0,他引:3 下载免费PDF全文
Two methods for accurate poly(3-hydroxybutyrate) (PHB) depolymerase activity determination and quantitative and qualitative hydrolysis product determination are described. The first method is based on online determination of NaOH consumption rates necessary to neutralize 3-hydroxybutyric acid (3HB) and/or 3HB oligomers produced during the hydrolysis reaction and requires a pH-stat apparatus equipped with a software-controlled microliter pump for rapid and accurate titration. The method is universally suitable for hydrolysis of any type of polyhydroxyalkanoate or other molecules with hydrolyzable ester bonds, allows the determination of hydrolysis rates of as low as 1 nmol/min, and has a dynamic capacity of at least 6 orders of magnitude. By applying this method, specific hydrolysis rates of native PHB granules isolated from Ralstonia eutropha H16 were determined for the first time. The second method was developed for hydrolysis product identification and is based on the derivatization of 3HB oligomers into bromophenacyl derivates and separation by high-performance liquid chromatography. The method allows the separation and quantification of 3HB and 3HB oligomers up to the octamer. The two methods were applied to investigate the hydrolysis of different types of PHB by selected PHB depolymerases. 相似文献
5.
Joana Gangoiti Marta Santos María J. Llama Juan L. Serra 《Applied and environmental microbiology》2010,76(11):3554-3560
The extracellular medium-chain-length polyhydroxyalkanoate (MCL-PHA) depolymerase of Pseudomonas fluorescens GK13 catalyzes the hydrolysis of poly(3-hydroxyoctanoic acid) [P(3HO)]. Based on the strong tendency of the enzyme to interact with hydrophobic materials, a low-cost method which allows the rapid and easy purification and immobilization of the enzyme has been developed. Thus, the extracellular P(3HO) depolymerase present in the culture broth of cells of P. fluorescens GK13 grown on mineral medium supplemented with P(3HO) as the sole carbon and energy source has been tightly adsorbed onto a commercially available polypropylene support (Accurel MP-1000) with high yield and specificity. The activity of the pure enzyme was enhanced by the presence of detergents and organic solvents, and it was retained after treatment with an SDS-denaturing cocktail under both reducing and nonreducing conditions. The time course of the P(3HO) hydrolysis catalyzed by the soluble and immobilized enzyme has been assessed, and the resulting products have been identified. After 24 h of hydrolysis, the dimeric ester of 3-HO [(R)-3-HO-HO] was obtained as the main product of the soluble enzyme. However, the immobilized enzyme catalyzes almost the complete hydrolysis of P(3HO) polymer to (R)-3-HO monomers under the same conditions.Polyhydroxyalkanoates (PHAs) are environmentally friendly polyesters that are biosynthesized by numerous microorganisms during unbalanced growth (3, 32). PHAs show material properties similar to those of conventional plastics, having important advantages such as biodegradability, apparent biocompatibility, and the ability to be manufactured from renewable resources (6, 38, 39). According to the number of carbon atoms of the side chain of the monomers, PHAs are classified as short-chain-length (SCL) PHAs (3 to 5 carbon atoms) and medium-chain-length (MCL) PHAs (6 to 14 carbon atoms) (16, 17, 32).The ability to degrade extracellular PHA in the environment and to use its degradation products as a source of carbon and energy depends on the release of specific extracellular PHA depolymerases (14, 15, 20). Depending on the depolymerase, as a result of enzymatic PHA degradation, the end products are only monomers, both monomers and dimers, or a mixture of oligomers (16). Enantiomer pure (R)-3-hydroxyalkanoic acid [(R)-3-HA] monomers are very attractive building blocks of interest not only in the biomedical and pharmaceutical fields (9, 10) but also for being used as starting materials to obtain other new polyesters (8). Thus, the development of a cost-effective industrial process for the production of both MCL-PHA depolymerase enzyme and (R)-3-HA monomers is of considerable interest.At present, few extracellular MCL-PHA depolymerases have been purified and characterized (11, 21-24, 33). Traditionally, the purification of microbial depolymerases is achieved by a conventional multistep chromatographic methodology, which includes hydrophobic interaction and size exclusion chromatographies (7, 21, 24, 37). The poly(3-hydroxyoctanoic acid) [P(3HO)] depolymerase from Pseudomonas fluorescens GK13 was the first enzyme purified (37) and characterized at the molecular level (36).Adsorption of lipases on polypropylene supports has been extensively used for large-scale lipase immobilization (18, 25, 28, 29) since it is a simple and economical method. Moreover, the immobilization of enzyme allows its reusability and increases its operational stability and ease of product recovery (1). Accurel MP-1000 is a commercially available hydrophobic, microporous, low-density polypropylene powder that presents a large surface area for adsorption because of its very small particle size (4). This support has been successfully used for adsorption of lipases and esterases with high yield directly from the fermentation broth (2, 13).As lipases, MCL-PHA depolymerases are hydrophobic proteins with a tendency to adsorb to hydrophobic supports. In this study we report a novel method for the purification of the P(3HO) depolymerase from P. fluorescens GK13 by adsorption to a polypropylene support as well as some relevant properties of the enzyme. Moreover, this protocol allows the immobilization of the enzyme directly from the culture broth. The immobilized enzyme degrades completely the P(3HO) polymer and releases 3-hydroxyoctanoic acid [(R)-3-HO]. This is the first report describing the immobilization of an extracellular MCL-PHA depolymerase and its potential use in the production of (R)-3-HO chiral monomers. 相似文献
6.
补料培养提高真养产碱杆菌产3-羟基丁酸和3-羟基戊酸共聚物生产强度的研究 总被引:1,自引:0,他引:1
对Alcaligenes eutrophus进行高密度培养,研究表明在发酵过程中进行有效控制,可以较大幅度地提高3-羟基丁酸和3-羟基戊酸共聚物[P(3HB-co-3HV)]的生产强度。实验中选择使用限氮的方法积累P(3HB-co-3HV),分别采用丙酸和戊酸为3HV前体,对摇瓶种子生长状态,停氮时机对菌体生产P(3HB-co-3HV)的影响以及补酸(3HV前体)策略进行了研究,在6.6L罐中,以葡萄糖为碳源,以丙酸为3HV前体培养50h,细胞干重,PHA产量,PHA含量分别达到149.9g/L,149.9g/L,83.3%(其中3HV组分占PHA的12.4mol%),生产强度达到2.50(g.h^-1.L^-1);以戊酸为3HV前体培养45h,细胞干重,PHA产量,PHA含量分别达到160.2g/L,119.0g/L,74.2%(其中3HV组分占PHA的17.7mol%)生产强度达到2.64(g.h^-1.L^-1)。 相似文献
7.
Purification and properties of extracellular poly(3-hydroxybutyrate) depolymerases from Pseudomonas lemoignei 总被引:9,自引:0,他引:9
Extracellular poly(3-hydroxybutyrate) depolymerase was purified from the culture medium of Peudomonas lemoignei and separated into four isozymes (A1, A2, B1 and B2) by CM-Sepharose CL-6B chromatography. The molecular weights of A1 and A2 and those of B1 and B2 were estimated to be 54 000 and 58 000, respectively, by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The isoelectric points of A1, A2, B1 and B2 were found to be approximately pH 9.7, 10.0, 10.0 and 10.6, respectively, by isoelectric focusing. All four enzymes hydrolyzed poly(3-hydroxybutyrate) and oligomeric esters of D-(-)-3-hydroxybutyrate, but showed no activity toward the dimeric ester. Analysis of hydrolytic products of the oligomeric esters with A1 and B2 suggested that the enzymes hydrolyzed mainly the second and third ester bonds from the free hydroxy terminus at different frequencies, depending upon the chain length of the substrates. 相似文献
8.
High-Level Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) by Fed-Batch Culture of Recombinant Escherichia coli 总被引:1,自引:0,他引:1 下载免费PDF全文
Fermentation strategies for production of high concentrations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] with different 3-hydroxyvalerate (3HV) fractions by recombinant Escherichia coli harboring the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes were developed. Fed-batch cultures of recombinant E. coli with the pH-stat feeding strategy facilitated production of high concentrations and high contents of P(3HB-co-3HV) in a chemically defined medium. When a feeding solution was added in order to increase the glucose and propionic acid concentrations to 20 g/liter and 20 mM, respectively, after each feeding, a cell dry weight of 120.3 g/liter and a relatively low P(3HB-co-3HV) content, 42.5 wt%, were obtained. Accumulation of a high residual concentration of propionic acid in the medium was the reason for the low P(3HB-co-3HV) content. An acetic acid induction strategy was used to stimulate the uptake and utilization of propionic acid. When a fed-batch culture and this strategy were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 141.9 g/liter, 88.1 g/liter, 62.1 wt%, and 15.3 mol%, respectively. When an improved nutrient feeding strategy, acetic acid induction, and oleic acid supplementation were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 203.1 g/liter, 158.8 g/liter, 78.2 wt%, and 10.6 mol%, respectively; this resulted in a high level of productivity, 2.88 g of P(3HB-co-3HV)/liter-h. 相似文献
9.
During growth on poly(3-hydroxyvaleric acid), P(3HV), or valerate Pseudomonas lemoignei secretes a P(3HV) depolymerase. This P(3HV) depolymerase was purified from the culture medium of valerate-grown cells by ammonium sulphate precipitation, chromatography on DEAe-sephacel and CM-Sepharose CL 6B. The relative molecular masses of the native as well as the sodium dodecyl sulphate (SDS)-treated enzyme were 53 000 or 54 000, respectively. In contrast to the poly(3-hydroxybutyric acid), P(3HB), depolymerase of Comamonas sp. and P(3HB) depolymerases A and B of P. lemoignei, which are specific for the hydrolysis of P(3HB), the purified P(3HV) depolymerase hydrolysed P(3HB), P(3HV) and co-polymers of 3-hydroxybutyric acid and 3-hydroxyvaleric acid at similar rates. Poly(hydroxyalkanoic acids), consisting of monomers with six and more carbon atoms or substrates characteristic for lipases such as Tween 80 or triolein were not hydrolysed. Maximum activities were measured in 50mm TRIS-HCl buffer, pH 8.0, at 55° C. The apparent K
m values of the purified P(3HV) depolymerase for P(3HB) and P(3HV) were 77 and 65 g polyester/ml, respectively. As the main product of enzymatic hydrolysis of P(3HV), 3-hydroxyvalerate was identified. The depolymerase was insensitive to p-hydroxymercuribenzoate but sensitive to dithioerythritol and phenylmethylsulphonyl fluoride, indicating the absence of active reduced sulphur groups and the presence of essential disulphide bonds and serine residues.
Correspondence to: D. Jendrossek 相似文献
10.
Roles of Poly(3-Hydroxybutyrate) Depolymerase and 3HB-Oligomer Hydrolase in Bacterial PHB Metabolism
Many poly-3-hydroxybutyrate (PHB)-degrading enzymes have been studied. But biological roles of 3HB-oligomer hydrolases (3HBOHs) and how PHB depolymerases (PHBDPs) and 3HBOHs cooperate in PHB metabolism are not fully elucidated. In this study, several PHBDPs and 3HBOHs from three types of bacteria were purified, and their substrate specificity, kinetic properties, and degradation products were investigated. From the results, PHBDP and 3HBOH seemed to play a role in PHB metabolism in three types of bacteria, as follows: (A) In Ralstonia pickettii T1, an extracellular PHBDP degrades extracellular PHB to various-sized 3HB-oligomers, which an extracellular 3HBOH hydrolyzes to 3HB-monomers. (B) In Acidovorax sp. SA1, an extracellular PHBDP hydrolyzes extracellular PHB to small 3HB-oligomers (dimer and trimer), which an intracellular 3HBOH efficiently degrades to 3HB in the cell. (C) In Ralstonia eutropha H16, an intracellular 3HBOH helps in the degradation of intracellular PHB inclusions by PHBDP. 相似文献
11.
Growth-Associated Production of Poly(3-Hydroxyvalerate) from n-Pentanol by a Methylotrophic Bacterium, Paracoccus denitrificans 下载免费PDF全文
Paracoccus denitrificans accumulated a polyester in its cells during growth on n-pentanol. The composition of the polyester varied during the cultivation: the level of the 3-hydroxyvalerate unit in the polyester increased, and eventually a homopolymeric poly(3-hydroxyvalerate) [P(3HV)] accumulated to an amount 22 to 24% of the cell dry weight. Growth-associated polyester synthesis was considerably affected by n-pentanol when its concentration was controlled at several levels. Maximum accumulation of the polyester was obtained at 0.02% (vol/vol). Physical and mechanical characteristics of the P(3HV) were determined and compared with those of other homo- and copolyesters. The P(3HV) was dextrorotatory and had number-averaged and weight-averaged molecular masses of 128,000 and 888,000 Da, respectively, with a rate of polydispersity of 6.93. The level of tensile strength of the P(3HV) was lower, and its extension to break was higher than that of the poly(3-hydroxybutyrate) homopolyester. 相似文献
12.
Terpe K Kerkhoff K Pluta E Jendrossek D 《Applied and environmental microbiology》1999,65(4):1703-1709
The relationship between extracellular poly(3-hydroxybutyrate) (PHB) depolymerase synthesis and the unusual properties of a succinate uptake system was investigated in Pseudomonas lemoignei. Growth on and uptake of succinate were highly pH dependent, with optima at pH 5.6. Above pH 7, growth on and uptake of succinate were strongly reduced with concomitant derepression of PHB depolymerase synthesis. The specific succinate uptake rates were saturable by high concentrations of succinate, and maximal transport rates of 110 nmol/mg of cell protein per min were determined between pH 5.6 and 6. 8. The apparent KS0.5 values increased with increasing pH from 0.2 mM succinate at pH 5.6 to more than 10 mM succinate at pH 7.6. The uptake of [14C]succinate was strongly inhibited by several monocarboxylates. Dicarboxylates also inhibited the uptake of succinate but only at pH values near the dissociation constant of the second carboxylate function (pKa2). We conclude that the succinate carrier is specific for the monocarboxylate forms of various carboxylic acids and is not able to utilize the dicarboxylic forms. The inability to take up succinate2- accounts for the carbon starvation of P. lemoignei observed during growth on succinate at pH values above 7. As a consequence the bacteria produce high levels of extracellular PHB depolymerase activity in an effort to escape carbon starvation by utilization of PHB hydrolysis products. 相似文献
13.
Hui-Ju Chen Shih-Chuan Pan Gwo-Chyuan Shaw 《Applied and environmental microbiology》2009,75(16):5290-5299
A gene that codes for a novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase, designated PhaZ1, has been identified in the genome of Bacillus megaterium. A native PHB (nPHB) granule-binding assay showed that purified soluble PhaZ1 had strong affinity for nPHB granules. Turbidimetric analyses revealed that PhaZ1 could rapidly degrade nPHB granules in vitro without the need for protease pretreatment of the granules to remove surface proteins. Notably, almost all the final hydrolytic products produced from the in vitro degradation of nPHB granules by PhaZ1 were 3-hydroxybutyric acid (3HB) monomers. Unexpectedly, PhaZ1 could also hydrolyze denatured semicrystalline PHB, with the generation of 3HB monomers. The disruption of the phaZ1 gene significantly affected intracellular PHB mobilization during the PHB-degrading stage in B. megaterium, as demonstrated by transmission electron microscopy and the measurement of the PHB content. These results indicate that PhaZ1 is functional in intracellular PHB mobilization in vivo. Some of these features, which are in striking contrast with those of other known nPHB granule-degrading PhaZs, may provide an advantage for B. megaterium PhaZ1 in fermentative production of the biotechnologically valuable chiral compound (R)-3HB.Polyhydroxyalkanoates (PHAs) are a group of polyesters that are produced by numerous bacteria as carbon and energy storage materials in response to nutritional stress (13, 27, 29). Poly(3-hydroxybutyrate) (PHB) is the most common and intensively studied PHA. Intracellular native PHB (nPHB) granules are composed of a hydrophobic PHB core and a surface layer consisting of proteins and phospholipids (13). The PHB of intracellular nPHB granules is in an amorphous state. When intracellular nPHB granules are exposed to extracellular environments due to cell death and lysis, the amorphous PHB is transformed into a denatured semicrystalline state. nPHB granules subjected to physical damage or solvent extraction to remove the surface layer can also crystallize into denatured PHB (dPHB) (13, 15). Artificial PHB (aPHB) granules, in which PHB is in an amorphous state, can be prepared from semicrystalline dPHB and detergents (1, 11, 23, 31).Various extracellular PHB depolymerases (PhaZs) that are secreted by many PHB-degrading bacteria have been demonstrated to specifically degrade dPHB (13, 14, 37). One exception is that PhaZ7, an extracellular PHB depolymerase secreted by Paucimonas lemoignei, displays unusual substrate specificity for amorphous PHB, with 3-hydroxybutyrate (3HB) oligomers as the main products of enzymatic hydrolysis (7). PhaZ7 exhibits no enzymatic activity toward dPHB. So far, a growing number of intracellular PHB depolymerases have been characterized. The intracellular PHB depolymerase PhaZa1 of Ralstonia eutropha (also called Cupriavidus necator) H16 has recently been established to be especially important for the intracellular mobilization of accumulated PHB (42). The main in vitro hydrolytic products of PhaZa1 degradation of amorphous aPHB are 3HB oligomers (31). PhaZd1, another intracellular PHB depolymerase of R. eutropha H16, shows no significant amino acid similarity to PhaZa1. The in vitro hydrolytic products of PhaZd1 degradation of amorphous aPHB are also 3HB oligomers. A 3HB monomer is rarely detected as a hydrolytic product (1). The intracellular PHB depolymerase PhaZ of Paracoccus denitrificans was reported previously to degrade protease-treated nPHB granules in vitro, with the release of 3HB dimers and oligomers as the main hydrolytic products (6). Recently, we have identified a novel intracellular PHB depolymerase from Bacillus thuringiensis serovar “israelensis” (39). The B. thuringiensis PhaZ shows no significant amino acid similarity to any known PHB depolymerase. This PhaZ has strong amorphous PHB-hydrolyzing activity and can release a considerable amount of 3HB monomers by the hydrolysis of trypsin-treated nPHB granules (39). It is of note that purified PhaZd1 from R. eutropha, PhaZ from P. denitrificans, and PhaZ from B. thuringiensis need pretreatment of nPHB granules with protease to remove surface proteins for PHB degradation (1, 6, 39). They show only very little or no activity toward nPHB granules without trypsin pretreatment. It has been demonstrated previously that these intracellular PHB depolymerases cannot hydrolyze dPHB (1, 31, 39).(R)-3HB, a biotechnologically valuable chiral compound, has been widely used for syntheses of antibiotics, vitamins, and pheromones (3, 30, 38). One way to produce (R)-3HB is heterologous coexpression of a PHB synthetic operon and a gene encoding an amorphous PHB-degrading PhaZ in Escherichia coli (3, 18, 25, 33, 38). A common problem encountered by this method is that oligomeric and dimeric forms of 3HB often constitute a major portion of the products of enzymatic hydrolysis, thus requiring further hydrolysis by 3HB oligomer hydrolase or heating under alkaline conditions to generate 3HB monomers (3, 18, 25, 33).Bacillus megaterium genes involved in the biosynthesis of nPHB granules have been cloned from strain ATCC 11561 and characterized previously (19, 21, 22). A gene encoding the extracellular PHB depolymerase PhaZ from B. megaterium was recently cloned from strain N-18-25-9 (34). However, little is known about B. megaterium genes involved in the intracellular mobilization of PHB. In this study, we have identified in B. megaterium ATCC 11561 an intracellular PHB depolymerase that could rapidly degrade nPHB granules in vitro without the need for trypsin pretreatment of the nPHB granules. Moreover, almost all the in vitro hydrolytic products released from the degradation of amorphous PHB by this PhaZ were 3HB monomers. This PhaZ could also hydrolyze dPHB with the generation of 3HB monomers. Thus, it appears to be a novel intracellular PHB depolymerase and may have promising potential for biotechnological application in the production of enantiomerically pure (R)-3HB monomers. 相似文献
14.
The ability of succinate to repress the secretion of Pseudomonas lemoignei poly-beta-hydroxybutyrate depolymerase was a function of pH. Repression only occurred when the pH of the medium was 7.0 or less. At a higher pH, lack of sensitivity to succinate concentration may have been due to a limited ability to transport succinate. Actively secreting cultures (at pH 7.4) continued to secrete enzyme for approximately 30 min after the pH was rapidly decreased to pH 6.8, even though sufficient succinate was present to repress enzyme synthesis. Similarly, after the addition of rifampin to secreting cultures, there was a 30-min delay before secretion was inhibited. Evidence is presented which suggests that continued secretion may be the result of depolymerase messenger ribonucleic acid accumulation within the cells. Studies with chloramphenicol indicated that de novo protein synthesis is necessary for the secretion of poly-beta-hydroxybutyrate depolymerase and that exoenzyme is not released from a preformed pool. Studies with various inhibitors of protein synthesis indicated that synthesis of exoenzyme is 5 to 10 times more susceptible to inhibition than is the synthesis of cell-associated proteins. 相似文献
15.
Poly(3-hydroxybutyrate) depolymerase was purified to
homogeneity from the culture filtrate of Paecilomyces lilacinus D218
by column chromatography on CM-Toyopearl 650M and hydroxylapatite. The
molecular weight of the enzyme was estimated to be 48,000 by SDS-PAGE.
Maximal activity was observed near pH 7.0 and 45°C. The
K
m
and V
max values for PHB were 0.13
(mg/ml) and 3750 (U/mg protein), respectively. The enzyme hydrolyzed PHB and
p-nitrophenyl fatty acids but not polycaprolactone and triglycerides.
Received: 29 August 1996 / Accepted: 30 September 1996 相似文献
16.
V. A. Zhuikov A. P. Bonartsev T. K. Makhina V. L. Myshkina V. V. Voinova G. A. Bonartseva K. V. Shaitan 《Biophysics》2018,63(2):169-176
The hydrolytic degradation of polymer films of poly(3-hydroxybutyrate) of different molecular weights and its copolymers with 3-hydroxyvalerate (9 mol % 3-hydroxyvalerate in the poly(3-hydroxybutyrate) chain) of different molecular weights was studied in model conditions in vitro. The changes in the physicochemical properties of the polymers were investigated using different analytical techniques: viscometry, differential scanning calorimetry, gravimetrical method, and water contact angle measurement for polymers. The data showed that in a period of 6 months the weight of polymer films decreased insignificantly. The molecular weight of the samples was reduced significantly; the largest decline (up to 80% of the initial molecular weight of the polymer) was observed in the high-molecular-weight poly(3-hydroxybutyrate). The surface of all investigated polymers became more hydrophilic. In this work, we focus on a mathematical model that can be used for the analysis of the kinetics of hydrolytic degradation of poly(3-hydroxyaklannoate)s by noncatalytic and autocatalytic hydrolysis mechanisms. It was also shown that the degree of crystallinity of some polymers changes differently during degradation in vitro. Thus, the studied polymers can be used to develop biodegradable medical devices such that they can perform their functions for a long period of time. 相似文献
17.
Byoung-In Sang Won-Kwon Lee Katsutoshi Hori Hajime Unno 《World journal of microbiology & biotechnology》2006,22(1):51-57
Summary Poly(3-hydroxybutyrate) [P(3HB)] depolymerase was purified from a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]-degrading fungus, Paecilomyces lilacinus F4-5 by hydrophobic and ion exchange column chromatography, and showed a molecular mass of 45 kDa. The optimum temperature
and pH of the P(3HB) depolymerase were 50 °C and 7.0, respectively. The enzyme was stable for at least 30 min at temperatures
below 40 °C, while the activity abruptly decreased over 55 °C. Enzymatic P(3HB-co-3HV) degradation showed a similar degradation pattern to that of film overlaid by fungal hyphae. It reflects that the fungal
degradation of P(3HB-co-3HV) in soil is mainly caused by extracellular depolymerases. 相似文献
18.
19.
J Han F Zhang J Hou X Liu M Li H Liu L Cai B Zhang Y Chen J Zhou S Hu H Xiang 《Journal of bacteriology》2012,194(16):4463-4464
Haloferax mediterranei, an extremely halophilic archaeon, has shown promise for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from unrelated cheap carbon sources. Here we report the complete genome (3,904,707 bp) of H. mediterranei CGMCC 1.2087, consisting of one chromosome and three megaplasmids. 相似文献
20.
Enzymatic degradation processes of microbial copolyesters, poly(3-hydroxybutyrate-co-3-hydroxyvalerate): P(3HB-co-3HV) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate): P(3HB-co-4HB), were studied by the weight loss (erosion) of copolyester films. These studies employed three extracellular depolymerases which degrade poly(3-hydroxybutyrate): P(3HB). Two enzymes were purified from the culture supernatant of Pseudomonas lemoignei and one from Alcaligenes faecalis T1. The rate of enzymatic degradation of microbial copolyester films with various compositions showed an almost similar tendency to three different P(3HB) depolymerases, and decreased in the following order: P(3HB-co-4HB) greater than P(3HB) greater than P(3HB-co-3HV). An inhibitory protein of P(3HB) depolymerases in the succinate culture medium of P. lemoignei was isolated and characterized. The molecular weight of P(3HB) depolymerase inhibitor was 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This inhibitor of a single polypeptide chain may reversibly bind the serine residues at the active site of P(3HB) depolymerase. This inhibitory protein was not induced in the culture medium when P. lemoignei was grown on P(3HB) as the sole carbon source. 相似文献