Catalytic performance of a highly enantioselective (R)-ester hydrolase from a new isolate Acinetobacter sp. CGMCC 0789

A highly enantioselective (R)-ester hydrolase was partially purified from a newly isolated bacterium, Acinetobacter sp. CGMCC 0789, whose resting cells exhibited a highly enantioselective activity toward the acetate of (4R)-hydroxy-3-methyl-2-(2-propynyl)- cyclopent-2-enone (R-HMPC). The optimum pH and temperature of the partially purified enzyme were 8.0 and 60 °C, respectively. The enantioselectivity of the crude enzyme was increased by 1.2-fold from 16 to 20 when the reaction temperature was raised from 30 to 60 °C. The activity of the crude enzyme was enhanced by 4.1-fold and the enantioselectivity (E-value) was markedly enhanced by 4.3-fold from 16 to 68 upon addition of a cationic detergent, benzethonium chloride [(diisobutyl phenoxyethoxyethyl) dimethyl benzylammoniom chloride]. The hydrolysis of 52 mM (R,S)-HMPC acetate to (R)-HMPC was completed within 8 h, with optical purity of 91.4% ee_p and conversion of 49%.     

Occurrence of ofloxacin ester-hydrolyzing esterase from Bacillus niacini EM001

A Bacillus niacini strain (EM001) producing an ofloxacin ester-enantioselective esterase was isolated from the soil samples collected near Taejon, Korea. The cloned gene showed that the esterase EM001 composed of 495 amino acids corresponding to a relative molecular weight (M_r) of 54,098 kDa. Based on the M_r and the protein sequence, the esterase EM001 was similar to p-nitrobenzyl esterase from Bacillus subtilis with an identity of 41.8%. The optimum temperature and pH of the purified His-tagged enzyme were 45 °C and 9.0, respectively. The purified esterase EM001 hydrolyzed preferably (R)-ofloxacin propyl ester than (S)-form ester at the initial reaction phase with an ee_P of 67% until the conversion rate become up to 35%.     

Biosynthesis and properties of an extracellular metalloprotease from the Antarctic marine bacterium Sphingomonas paucimobilis

An extracellular protease from the marine bacterium Sphingomonas paucimobilis, strain 116, isolated from the stomach of Antarctic krill, Euphausia superba Dana, was purified and characterized. The excretion of protease was maximal at temperatures from 5 to 10°C, i.e. below the temperature optimum for the strain growth (15°C). The highly purified enzyme was a metalloprotease [sensivity to ethylenediaminetetraacetic acid (EDTA)] and showed maximal activity against proteins at 20–30°C and pH 6.5–7.0, and towards N-benzoyl-tyrosine ethyl ester (BzTyrOEt) at pH 8.0. At 0°C the enzyme retained as much as 47% of maximal activity in hydrolysis of urea denatured haemoglobin (Hb) (at pH 7.0), and at −5 and −10°C, 37 and 30%, respectively. The metalloprotease was stable up to 30°C for 15 min and up to 20°C for 60 min. These results indicate that the proteinase from S. paucimobilis 116 is a cold-adapted enzyme.     

Purification and partial characterization of manganese peroxidase from immobilized Phanerochaete chrysosporium

Solid-state culture of the white-rot fungus Phanerochaete chrysosporium BKMF-1767 (ATCC 24725) has been carried out, using an inert support, polystyrene foam. Suitable medium and culture conditions have been chosen to favor the secretion of manganese peroxidase (MnP). The enzyme was isolated and purified from immobilized P. chrysosporium and partially characterized. Partial protein precipitation in crude enzyme was affected using ammonium sulphate, polyethylene glycol, methanol, and ethanol methods. Fractionation of MnP was performed by DEAE-Sepharose ion exchange chromatography followed by Ultragel AcA 54 gel filtration chromatography. This purification attained 23.08% activity yield with a purification factor of 5.8. According to data on gel filtration chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of the enzyme was 45 000±1000 Da. The optimum pH and temperature of purified MnP were 4.5 and 30 °C, respectively. This enzyme was stable in the pH range 4.5–6.0, at 25 °C and also up to 35 °C at pH 4.5 for 1 h incubation period. MnP activity was inhibited by 2 mM NaN₃, ascorbic acid, β-mercaptoethanol and dithreitol. The K_m values of MnP for hydrogen peroxide and 2.6-dimetoxyphenol were 71.4 and 28.57 μM at pH 4.5, respectively. The effects of possible inhibitors and activators of enzyme activity were investigated.     

Modulation of Mucor miehei lipase properties via directed immobilization on different hetero-functional epoxy resins: Hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid

Purified lipase from Mucor miehei (MML) has been covalently immobilized on different epoxy resins (standard hydrophobic epoxy resins, epoxy-ethylenediamine, epoxy-iminodiacetic acid, epoxy-copper chelates) and adsorbed via interfacial activation on octadecyl-Sepabeads support (fully coated with very hydrophobic octadecyl groups). These immobilized enzyme preparations were used under slightly different conditions (temperature ranging from 4 to 25 °C and pH values from 5 to 7) in the hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid.

Different catalytic properties (activity, specificity, enantioselectivity) were found depending on the particular support used. For example, the epoxy-iminodiacetic acid-Sepabeads gave the most active preparation at pH 7 while, at pH 5, the ethylenediamine-Sepabeads was superior.

More interestingly, the enantiomeric ratio (E) also depends strongly on the immobilized preparation and the conditions employed. Thus, the octadecyl-MML preparation was the only immobilized enzyme derivative which exhibited enantioselectivity towards R isomer (with E values ranging from 5 at 4 °C and pH 7 to 1.2 at pH 5 and 25 °C).

The other immobilized preparations, in contrast, were S selective. Immobilization on iminodiacetic acid-Sepabeads afforded the catalyst with the highest enantioselectivity (E=59 under optimum conditions).     

Improvement of the functional properties of a thermostable lipase from alcaligenes sp. via strong adsorption on hydrophobic supports

Lipase QL from Alcaligenes sp. is a quite thermostable enzyme. For example, it retains 75% of catalytic activity after incubation for 100 h at 55 °C and pH 7.0. Nevertheless, an improvement of the enzyme properties was intended via immobilization by covalent attachment to different activated supports and by adsorption on hydrophobic supports (octadecyl-sepabeads). This latter immobilization technique promotes the most interesting improvement of enzyme properties: (a) the enzyme is hyperactivated after immobilization: the immobilized preparation exhibits a 135% of catalytic activity for the hydrolysis of p-nitrophenyl propionate as compared to the soluble enzyme; (b) the thermal stability of the immobilized enzyme is highly improved: the immobilized preparation exhibits a half-life time of 12 h when incubated at 80 °C, pH 8.5 (a 25-fold stabilizing factor regarding to the soluble enzyme); (c) the optimal temperature was increased from 50 °C (soluble enzyme) up to 70 °C (hydrophobic support enzyme immobilized preparations); (d) the enantioselectivity of the enzyme for the hydrolysis of glycidyl butyrate and its dependence on the experimental conditions was significantly altered. Moreover, because the enzyme becomes reversibly but very strongly adsorbed on these highly hydrophobic supports, the lipase may be desorbed after its inactivation and the support may be reused. Very likely, adsorption occurs via interfacial activation of the lipase on the hydrophobic supports at very low ionic strength. On the other hand, all the covalent immobilization protocols used to immobilize the enzyme hardly improved the properties of the lipase.     

Purification and properties of a maltoheptaose- and maltohexaose-forming amylase produced by Bacillus subtilis US116

A new bacterial strain, identified as Bacillus subtilis US116, was isolated from Tunisian soil and selected for its potential production of an atypical amylase with an industrial interest. The identification was founded on physiological tests and molecular techniques related to the 16S rRNA, 23S rRNA genes and intergenic sequences showing the highest similarity of 98% with regions in the complete genome of Bacillus subtilis 168 (accession no. Z99104). This strain produces an atypical amylase that was purified to homogeneity by a combination of acetone precipitation, size exclusion and ion exchange chromatography. The molecular mass of the enzyme is about 60 kDa as determined by SDS–PAGE. Optimal conditions for the activity of the purified enzyme are pH 6 and 65 °C. The half-life duration is about 3 h at 70 °C and 5 h at 65 °C. This enzyme belongs to the endo-type amylases according to the hydrolytic mode study using Ceralpha and Betamyl methods. It is classified as a maltoheptaose- and maltohexaose-forming amylase since it generates about 30% maltohexaose (DP6) and 20% maltoheptaose (DP7) from starch. Moreover, the minimum length of maltosaccharide cleaved by this enzyme was maltoheptaose.     

Enantioselective production of 2,2-dimethylcyclopropane carboxylic acid from 2,2-dimethylcyclopropane carbonitrile using the nitrile hydratase and amidase of Rhodococcus erythropolis ATCC 25544

The enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid was investigated in 53 Rhodococcus and Pseudomonas related strains. Rhodococcus erythropolis ATCC 25544 was selected as it showed the highest enantioselectivity. The enantioselectivity was due to the amidase activity in a two-step reaction involving nitrile hydratase. The enantiomeric excess of the amidase was highest at pH 7.0 and decreased significantly above 20 °C. For the enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid, the optimum reaction conditions of the cells were determined to be pH 7.0, 20 °C, and 10% (v/v) methanol and were the same as the optimum pH and temperature for the enantioselective conversion by the amidase. Under these conditions, the R. erythropolis ATCC 25544 cells, which harbored nitrile hydratase and amidase enzymes, produced 45 mM (S)-2,2-dimethylcyclopropane carboxylic acid from racemic 100 mM 2,2-dimethylcyclopropane carbonitrile with an 81.8% enantiomeric excess after 64 h.     

Purification and partial characterization of Cu, Zn containing superoxide dismutase from entomogenous fungal species Cordyceps militaris

Cordyceps militaris mycelium produced mainly Cu, Zn containing superoxide dismutase (Cu, Zn-SOD). Cu, Zn-SOD activity was detectable in the culture filtrates, and intracellular Cu, Zn-SOD activity as a proportion protein was highest in early log phase culture. The effects of Cu²⁺, Zn²⁺, Mn²⁺ and Fe²⁺ on enzyme biosynthesis were studied. The Cu, Zn-SOD was isolated and purified to homogeneity from C. militaris mycelium and partially characterized. The purification was performed through four steps: (NH₄)₂SO₄ precipitation, DEAE-sepharose™ fast flow anion-exchange chromatography, CM-650 cation-exchange chromatography, and Sephadex G-100 gel filtration chromatography. The purified enzyme had a molecular weight of 35070 ± 400 Da and consisted of two equal-sized subunits each having a Cu and Zn element. Isoelectric point value of 7.0 was obtained for the purified enzyme. The N-terminal amino acid sequence of the purified enzyme was determined for 12 amino acid residues and the sequences was compared with other Cu, Zn-SODs. The optimum pH of the purified enzyme was obtained to be 8.2–8.8. The purified enzyme remained stable at pH 5.8–9.8, 25 °C and up to 50 °C at pH 7.8 for 1.5 h incubation. The purified enzyme was sensitive to H₂O₂, KCN. 2.5 mM NaN₃, PMSF, Triton X-100, β-mercaptoethanol and DTT showed no significant inhibition effect on the purified enzyme within 5 h incubation period.     

Production, purification and properties of endoglucanase from a newly isolated strain of Mucor circinelloides

A newly isolated strain of the fungus, Mucor circinelloides (NRRL 26519), when grown on lactose, cellobiose, or Sigmacell 50 produces complete cellulase (endoglucanase, cellobiohydrolase, and β-glucosidase) system. The extracellular endoglucanase (EG) was purified to homogeneity from the culture supernatant by ethanol precipitation (75%, v/v), CM Bio-Gel A column chromatography, and Bio-Gel A-0.5 m gel filtration. The purified EG (specific activity 43.33 U/mg protein) was a monomeric protein with a molecular weight of 27 000. The optimum temperature and pH for the action of the enzyme were at 55 °C and 4.0–6.0, respectively. The purified enzyme was fully stable at pH 4.0–7.0 and temperature up to 60 °C. It hydrolysed carboxymethyl cellulose and insoluble cellulose substrates (Avicel, Solka-floc, and Sigmacell 50) to soluble cellodextrins. No glucose, cellobiose, and short chain cellooligosaccarides were formed from these substrates. The purified EG could not degrade oat spelt xylan and larch wood xylan. It bound to Avicell, Solka-floc, and Sigmacell 50 at pH 5.0 and the bound enzyme was released by changing the pH to 8.0. The enzyme activity was enhanced by 27±5 and 44±14% by the addition of 5 mM MgCl₂ and 0.5 mM CoCl₂, respectively, to the reaction mixture. Comparative properties of this enzyme with other fungal EGs are presented.     

A nicotinamide mononucleotide adenylyltransferase with unique adenylyl group donor specificity from a hyperthermophilic archaeon, Pyrococcus horikoshii OT-3

A gene encoding a nicotinamide mononucleotide (NMN) adenylyltransferase (NMNAT, EC 2.7.7.1) homologue was identified via genome sequencing in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3. The gene encoded a protein of 186 amino acids with a molecular weight of 21,391. The deduced amino acid sequence of the gene showed 59% identities to the NMNAT from Methanococcus jannaschii. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified to homogeneity. Characterization of the enzyme revealed that it is an extremely thermostable NMNAT; the activity was not lost after incubation at 80 °C for 30 min. The native molecular mass was estimated to be 77 kDa. The K_m values for ATP and NMN were calculated to be 0.056 and 0.061 mM, respectively. The optimum temperature of the reaction was estimated to be around 90 °C. The adenylyl group donor specificity was examined by high-performance liquid chromatography (HPLC). At 70 °C, ATP was a prominent donor. However, above 80 °C, a relatively small, but significant, NMNAT activity was detected when ATP was replaced by ADP or AMP in the reaction mixture. To date, an NMNAT that utilizes ADP or AMP as an adenylyl group donor has not been found. The present study provides interesting information in which a di- or mono-phosphate nucleotide can be utilized by adenylyltransferase at high temperature.     

Production, purification and characterization of an endopolygalacturonase from Mucor rouxii NRRL 1894

An extracellular polygalacturonase (PGase) from Mucor rouxii NRRL 1894 was purified to homogeneity by two chromatographic steps using CM-Sepharose and Superdex 75. The purified enzyme was a monomer with a molecular weight of 43100 Da and a pI of 6. The PGase was optimally active at 35 °C and at pH 4.5. It was stable up to 30 °C and stability of PGase decrease rapidly above 60 °C. The extent of hydrolysis of different pectins was decreased with increasing of degrees of esterification. Except Mn²⁺, all the examined metal cations showed inhibitory effects on the enzyme activity. The apparent K_m and V_max values for hydrolyze of polygalacturonic acid (PGA) were 1.88 mg/ml and 0.045 μmol/ml/min, respectively. The enzyme released a series of oligogalacturonates from polygalacturonic acid indicating that it had an endo-action. Its N-terminal sequence showed homologies with the endopolygalacturonase from the psychrophilic fungus Mucor flavus.     

Biocatalytic preparation of (S)-phenyl glycidyl ether using newly isolated Bacillus megaterium ECU1001

A bacterial strain (ECU1001) capable of utilizing phenyl glycidyl ether as sole carbon source and energy source was isolated from soil samples through two steps of screening and was identified as a Bacillus megaterium. The epoxide hydrolase from Bacillus megaterium ECU1001 was biosynthesized in parallel with cell growth and a maximum activity of 31.0 U/l was reached after 30 h of culture when the biomass (DCW) was 9.1 g/l. A temperature of 35°C and pH 8.0 were optimal for the bioconversion. The lyophilized whole cells of Bacillus megaterium ECU1001 could preferentially hydrolyze the (R)-enantiomer of phenyl glycidyl ether, yeilding (S)-epoxide and (R)-diol with high enantioselectivity (E=47.8). The (S)-enantiomer of the epoxide remained in the reaction mixture with >99.5% ee (enantiomeric excess) at a conversion of 55.9%. The substrate concentration could be increased up to 60 mM without affecting the ee and (S)-phenyl glycidyl ether could be obtained with an optical purity of 100% ee and 25.6% yield. Therefore, the method is potentially useful for the preparative resolution of epoxides.     

Recombinant expression, characterization, and pulp prebleaching property of a Phanerochaete chrysosporium endo-β-1,4-mannanase

A Phanerochaete chrysosporium cDNA predicted to encode endo-1,4-β-d-mannanase, man5D, was cloned and expressed in Aspergillus niger. The coding region of the gene man5D was predicted to contain, in order from the N-terminal: a secretory signal peptide, cellulose-binding domain, linker region, and glycosyl hydrolase family 5 catalytic site. The enzyme was purified from culture filtrate of A. niger transformants that carried the recombinant man5D. Recombinant Man5D had an apparent molecular size of about 65 kDa by SDS-PAGE, and optimal activity at pH 4.0–6.0 and 60 °C. It was stable from pH 4.0 to 8.0 and up to 60 °C. The enzyme showed affinity for Avicel cellulose, suggesting that the predicted cellulose-binding domain is biologically functional. The specific activities of Man5D on mannan, galactomannan, and glucomannan at pH 5 and 60 °C ranged from 160 to 460 μmol/(min mg), with apparent K_m values from 0.54 to 2.3 mg/mL. Product analysis results indicated that Man5D catalyzes endo-cleavage, and appears to have substantial transglycosylase activity. When used to treat softwood kraft pulp, Man5D hydrolyzed mainly glucomannan and exhibited a positive effect as a prebleaching agent. Compared to a commercial prebleaching with xylanase, the prebleaching effect of Man5D was weaker but with reduced loss of fibre yield as determined by the release of solubilized sugars.     

Cloning, expression and characterization of a novel thermal stable and short-chain alcohol tolerant lipase from Burkholderia cepacia strain G63

A lipase gene lipA and its chaperone gene lipB were cloned from Burkholderia cepacia strain G63. The lipA was composed of 1092 bp, encoding 363 amino acid residues, and the lipB composed of 1035 bp, corresponding to 344 amino acid residues. The significant amino acid similarity with Pseudomonas cepacia lipase revealed that this enzyme could be classified into the lipolytic subfamily I.2. The lipA and lipB genes were cloned into pBBR1Tp vector and conjugated into B. cepacia strains G63 with the help of pRK2013. The recombinant strain was fermented in 10 l bioreactor and the lipase was purified by a combination of ammonium sulfate fractionation, DEAE ion-exchange chromatography and gel filtration. The purified lipase kept stable at a temperature range of 40–70 °C. After incubated at 70 °C, the optimal temperature of this enzyme, for 10 h it remained 86.1% of its activity. The enzyme was also highly tolerant to a series of organic solution. Incubated in 50% methanol solution up to 48 h, the enzyme still kept 98.3% of its activity. The transesterification activity of soybean oil to fatty acid methyl esters (FAMEs) reached 87.8% after 72 h, indicating that it is a potential biocatalyzer for biodiesel production.     

Uracil-DNA N-glycosylase (UNG) from the marine, psychrophilic bacterium Vibrio salmonicida shows cold adapted features: A comparative analysis to Vibrio cholerae uracil-DNA N-glycosylase

The genes encoding uracil-DNA N-glycosylase (UNG) from the marine, psychrophilic bacterium Vibrio salmonicida and the mesophilic counterpart Vibrio cholerae have been cloned and expressed in Escherichia coli. The purified proteins have been characterized in order to reveal possible cold adapted features of the V. salmonicida UNG (vsUNG) compared to the V. cholerae UNG (vcUNG). Characterization experiments demonstrated that both enzymes possessed the highest activities at pH 7.0–7.5 and at salt concentrations in the range of 25–50 mM NaCl. Temperature optima for activity were determined to approximately 30 °C for vsUNG and 50 °C for vcUNG. Temperature stability of the enzymes was compared at 4 °C and 37 °C, and vsUNG was found to be more temperature labile than vcUNG. Kinetic studies performed at three different temperatures, 15 °C, 22 °C and 37 °C, demonstrated higher catalytic efficiency for vsUNG compared to vcUNG due to lower K_M-values. The increased substrate affinity of vsUNG is probably caused by an increased number of positively charged residues in the DNA-binding site of the enzyme compared to vcUNG. Thus, activity and stability measurements reveal typical cold adapted features of vsUNG.     

Lipase-catalyzed production of optically active (S)-flurbiprofen in aqueous phase reaction system containing chiral succinyl β-cyclodextrin

The lipase-catalyzed production of optically active (S)-flurbiprofen was carried out in a dispersion reaction-system induced by chiral succinyl β-cyclodextrin (suβ-CD). The optimal reaction conditions were 500 mM (R,S)-flurbiprofen ethyl ester ((R,S)-FEE), 600 units of Candida rugosa lipase per 1 mmol of (R,S)-FEE, and 1000 mM suβ-CD at 37 °C for 72 h. An extremely high enantiomeric excess of 0.98 and conversion yield of 0.48 were achieved in the dispersed aqueous phase reaction system containing chiral suβ-CD added as a dispenser and chiral selector. The inclusion complex formability of the immiscible substrate (S)- and (R)-form of FEE with suβ-CD was compared using a phase-solubility diagram, DSC, and ¹H NMR. (S)-Isomer formed a more stable and selective inclusion complex with chiral suβ-CD. It was hydrolyzed much more selectively by lipase from C. rugosa, due to the selective structural modification through inclusion complexation with chiral suβ-CD.     

Thermostable glutamate dehydrogenase from a commensal thermophile, Symbiobacterium toebii; overproduction, characterization, and application

A gene encoding glutamate dehydrogenase (GDH) was found in the genome sequence of a commensal thermophile, Symbiobacterium toebii. The amino acid sequence deduced from the gdh I of S. toebii was well conserved with other thermostable GDHs. The gdh I which encodes GDH consisting of 409 amino acids was cloned and expressed in E. coli DH5 under the control of a highly constitutive expression (HCE) promoter in a pHCE system. The recombinant GDH was expressed without addition of any inducers in a soluble form. The molecular mass of the GDH was estimated to be 263 kDa by Superose 6 HR gel filtration chromatography and 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicating that the GDH was composed of hexameric form. The optimal temperature and pH of the purified enzyme were 60 °C and 9.0, respectively, and the purified GDH retained more than 75% of its original activity after an incubation at 70 °C for 30 min. Although NADP(H) was the preferred cofactor, S. toebii GDH was able to utilize either NADP(H) or NAD(H) as coenzyme.     

Purification and characterization of an endocellulase from the thermophilic fungus Chaetomium thermophilum CT2

Chaetomium thermophilum CT2 produced endocellulases at 50 °C, when grown on 2% microcrystalline cellulose, 1% soluble starch, and 0.4% yeast extract medium. A major endocellulase component was purified to homogeneity by fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE-Sepharose, Phenyl-Sepharose hydrophobic interaction chromatography and gel filtration on Sephacryl S-100. The molecular weight of the enzyme was estimated to be 67.8 kDa and the enzyme was found to be a glycoprotein containing 18.9% carbohydrate. The K_m of the purified enzyme for carboxymethyl cellulose, sodium salt (CMC), was 4.6 mg ml⁻¹. The enzyme displayed highest activity towards CMC and significantly lower activities towards phosphoric acid swollen cellulose and filter paper. The activity was enhanced in the presence of Na⁺, K⁺ and Ca²⁺ but inhibited by Hg²⁺, Zn²⁺, Ag⁺, Mn²⁺, Ba²⁺, Fe²⁺, Cu²⁺, Mg²⁺ and NH₄⁺. Optimum activity was at 60 °C and pH 4.0. The enzyme was stable over 60 min incubation at 60 °C and half-life at 70, 80 and 90 °C was approximately 45, 24 and 7 min, respectively.     

Extraction of polyhydroxyalkanoate from Sinorhizobium meliloti cells using Microbispora sp. culture and its enzymes

Sinorhizobium meliloti produced 50% polyhydroxyalkanoate (PHA) in the biomass in the presence of sucrose as carbon substrate. Isolation of the intracellular PHA was achieved through a secondary fermentation involving a cell lytic actinomycetes species namely Microbispora sp. without further supplementation of nutrients to the S. meliloti fermented broth, at 30 °C, 150 rpm up to 72 h. Microbispora sp. cells that showed pelleted growth was removed by filtration and the released polymer contained in the filtrate was extracted by chloroform or an admixture of Triton X 100 (0.6%) a surfactant and ethylene diamine tetra acetic acid (EDTA) a chelating agent. Yield of PHA obtained was 49, 41 and 7% of biomass weight after 24, 48 and 72 h of lytic culture fermentation, respectively. Corresponding recovery of the polymer was 94, 82 and 15% of 90% purity. Alternatively Microbispora sp. lytic enzyme was obtained by its cultivation in nutrient broth with S. meliloti cells as substrate and the supernatant was used for the hydrolysis of the PHA containing biomass to release PHA. A620 lytic activity value for the broth was 200 at 72 h. The enzyme showed optimized activity at 50 °C, pH 7 and this was used to hydrolyze 5 g/l of thermally inactivated biomass of S. meliloti to recover 94% of total PHA present in the cells and the polymer produced was 92% pure. Decreased cell lytic activity in the presence of soluble protein added in the form of bovine serum albumin indicated that the hydrolytic activity may be due to proteases. The polymer was characterized by GC, NMR and DSC and was found to be polyhydroxybutyrate-co-hydroxyvalerate (97:3 mol%) with a melt temperature of 169 °C.