Characterization of novel thermostable dextranase from Thermotoga lettingae TMO

Biochemical properties of a putative thermostable dextranase gene from Thermotoga lettingae TMO were determined in a recombinant protein (TLDex) expressed in Escherichia coli and purified to sevenfold apparent homogeneity. The 64-kDa protein displayed maximum activity at pH 4.3, and enzyme activity was stable from pH 4.3–10. The optimal temperature was 55–60°C during 15 min incubation, and the half-life of the enzyme was 1.5 h at 65°C. The enzyme showed higher activity against α-(1 → 6) glucan and released isomaltose and isomaltotriose as main products from dextran T2000. An unusual kinetic feature of TLDex was the negative cooperative behavior on the reaction of dextran T2000 cleavage. Enzyme activity was not significantly affected by the presence of metal ions, except for the strong inhibited by 1 mM Fe²⁺ and Ag²⁺. TLDex may prove useful as an enzyme for high temperature sugar milling processes.     

Characterization of a recombinant cellobiose 2-epimerase from <Emphasis Type="BoldItalic">Caldicellulosiruptor saccharolyticus</Emphasis> and its application in the production of mannose from glucose

A putative N-acyl-d-glucosamine 2-epimerase from Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was identified as a cellobiose 2-epimerase by the analysis of the activity for substrates, acid-hydrolyzed products, and amino acid sequence. The cellobiose 2-epimerase was purified with a specific activity of 35 nmol min^–1 mg^–1 for d-glucose with a 47-kDa monomer. The epimerization activity for d-glucose was maximal at pH 7.5 and 75°C. The half-lives of the enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 142, 71, 35, 18, and 4.6 h, respectively. The enzyme catalyzed the epimerization reactions of the aldoses harboring hydroxyl groups oriented in the right-hand configuration at the C2 position and the left-hand configuration at the C3 position, such as d-glucose, d-xylose, l-altrose, l-idose, and l-arabinose, to their C2 epimers, such as d-mannose, d-lyxose, l-allose, l-gulose, and l-ribose, respectively. The enzyme catalyzed also the isomerization reactions. The enzyme exhibited the highest activity for mannose among monosaccharides. Thus, mannose at 75 g l^–1 and fructose at 47.5 g l^–1 were produced from 500 g l^–1 glucose at pH 7.5 and 75°C over 3 h by the enzyme.     

Biochemical characterization of a novel thermostable xyloglucanase from an alkalothermophilic Thermomonospora sp.

Xyloglucanase from an extracellular culture filtrate of alkalothermophilic Thermomonospora sp. was purified to homogeneity with a molecular weight of 144 kDa as determined by SDS-PAGE and exhibited specificity towards xyloglucan with apparent K _m of 1.67 mg/ml. The enzyme was active at a broad range of pH (5–8) and temperatures (40–80°C). The optimum pH and temperature were 7 and 70°C, respectively. The enzyme retained 100% activity at 50°C for 60 h with half-lives of 14 h, 6 h and 7 min at 60, 70 and 80°C, respectively. The kinetics of thermal denaturation revealed that the inactivation at 80°C is due to unfolding of the enzyme as evidenced by the distinct red shift in the wavelength maximum of the fluorescence profile. Xyloglucanase activity was positively modulated in the presence of Zn²⁺, K⁺, cysteine, β-mercaptoethanol and polyols. Thermostability was enhanced in the presence of additives (polyols and glycine) at 80°C. A hydrolysis of 55% for galactoxyloglucan (GXG) from tamarind kernel powder (TKP) was obtained in 12 h at 60°C and 6 h at 70°C using thermostable xyloglucanases, favouring a reduction in process time and enzyme dosage. The enzyme was stable in the presence of commercial detergents (Ariel), indicating its potential as an additive to laundry detergents.     

Cloning,expression and characterization of glycoside hydrolase family 11 endoxylanase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> ARA

An endoxylanase gene, xynA, was cloned from Bacillus pumilus ARA and expressed in Escherichia coli. The open reading frame of the xynA gene was 687 bp encoding a signal peptide and a mature xylanase with a molecular mass of 23 kDa. The enzyme was categorized as a glycosyl hydrolase family 11 member based on the sequence analysis of the putative catalytic domain. The recombinant XynA (Bpu XynA) was purified to homogeneity by Ni–NTA and ion exchange chromatography on DEAE–Sepharose FF. The enzyme exhibited highest activity at pH 6.6 and 50°C. The purified Bpu XynA was stable for at least 2 h at 45°C, and retained over 50% residual activity after being incubated at 60°C for 1 h. The activity of the xylanase was not significantly affected by metal ions and EDTA. The K _m and K _cat /K _m of Bpu XynA for oat-spelt xylan were 5.53 mg/ml and 10.14 ml/mg s at 50°C and pH 6.6. The main product of hydrolysis by Bpu XynA was xylooligosaccharide. The results revealed that the consumption of grass xylan by B. pumilus ARA depended on the synergistic reactions of Bpu XynA and Bpu arabinosidase, and that a typical GH11 xylanase e.g. Tla XynA had capability to remove the side chain of xylan. The properties Bpu XynA make it promising for application in the production of Bifidobacterium growth-promoting factors and in feed industry.     

Purification, immobilization and characterization of linoleic acid isomerase on modified palygorskite

Linoleic acid isomerase from Lactobacillus delbrueckii subsp. bulgaricus 1.1480 was purified by DEAE ion-exchange chromatography and gel filtration chromatography. An overall 5.1% yield and purification of 93-fold were obtained. The molecular weight of the purified protein was ~41 kDa which was analyzed by SDS-PAGE. The purified enzyme was immobilized on palygorskite modified with 3-aminopropyltriethoxysilane. The immobilized enzyme showed an activity of 82 U/g. The optimal temperature and pH for the activity of the free enzyme were 30 °C and pH 6.5, respectively; whereas those for the immobilized enzyme were 35 °C and pH 7.0, respectively. The immobilized enzyme was more stable than the free enzyme at 30–60 °C, and the operational stability result showed that more than 85% of its initial activity was retained after incubation for 3 h. The K _m and V _max values of the immobilized enzyme were found to be 0.0619 mmol l⁻¹ and 0.147 mmol h⁻¹ mg⁻¹, respectively. The immobilized enzyme had high operational stability and retained high enzymatic activity after seven cycles of reuse at 37 °C.     

Overexpression of methyl parathion hydrolase and its application in detoxification of organophosphates

The coding region of mpd gene corresponding to mature methyl parathion hydrolase (MPH) was heterologously overexpressed in Escherichia coli BL21 (DE3) by using pET expression system. The lactose-induced expression yield of MPH is increased 2-fold compared with IPTG as inducer. Furthermore, it was found that specific activity of MPH increased 48% by reducing the induction temperature to 22°C. The addition of 25 mM lactose at 22°C, the MPH activity of fermentation broth had a specific activity of 1.4 × 10⁴ U/mg protein. Plasmid was no significant decrease in the modified medium. The optimal pH and temperature of MPH were 8.0 and 30°C, respectively. Over a period of 5 months, the dried cells showed no significant decrease in the activity of the detoxifying enzymes. The crude enzymes in 50 mM citrate-phosphate buffer (pH 8.0) were able to degrade about 98% of the organophosphate pesticides sprayed on cabbage. The detoxification efficiency was superior to that of the treatments of water, detergent, and a commercially available enzyme product. Additionally, the products of pesticide hydrolysis generated by treatment with the enzyme extract were determined to be virtually nontoxic.     

A highly stable Cu/Zn superoxide dismutase from <Emphasis Type="Italic">Withania somnifera</Emphasis> plant: gene cloning,expression and characterization of the recombinant protein

A gene from Withania somnifera (winter cherry), encoding a highly stable chloroplastic Cu/Zn superoxide dismutase (SOD), was cloned and expressed in Escherichia coli. The recombinant enzyme (specific activity of ~4,200 U mg⁻¹) was purified and characterized. It retained ~90 and ~70% residual activities after 1 h at 80 and 95°C, respectively. At 95°C, thermal inactivation rate constant (K _d) of the enzyme was 2.46 × 10⁻³ min⁻¹ and half-life of heat inactivation was 4.68 h. The enzyme was stable against a broad pH range (2.5–11.0). It also showed a high degree of resistance to detergent, ethanol and protease digestion. This recombinant Cu/Zn SOD could therefore have useful applications.     

Characterization of hyperthermostable α-amylase from Geobacillus sp. IIPTN

A newly isolated Geobacillus sp. IIPTN (MTCC 5319) from the hot spring of Uttarakhand's Himalayan region produced a hyperthermostable α-amylase. The microorganism was characterized by biochemical tests and 16S rRNA gene sequencing. The optimal temperature and pH were 60°C and 6.5, respectively, for growth and enzyme production. Although it was able to grow in temperature ranges from 50 to 80°C and pH 5.5–8.5. Maximum enzyme production was in exponential phase with activity 135 U ml⁻¹ at 60°C. Assayed with cassava as substrate, the enzyme displayed optimal activity 192 U ml⁻¹ at pH 5.0 and 80°C. The enzyme was purified to homogeneity with purification fold 82 and specific activity 1,200 U mg⁻¹ protein. The molecular mass of the purified enzyme was 97 KDa. The values of K _m and V _max were 36 mg ml⁻¹ and 222 μmol mg⁻¹ protein min⁻¹, respectively. The amylase was stable over a broad range of temperature from 40°C to 120°C and pH ranges from 5 to 10. The enzyme was stimulated with Mn²⁺, whereas it was inhibited by Hg²⁺, Cu²⁺, Zn²⁺, Mg²⁺, and EDTA, suggesting that it is a metalloenzyme. Besides hyperthermostability, the novelty of this enzyme is resistance against protease.     

Purification and characterization of pectin lyase secreted by <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

The importance of various parameters such as sugarcane juice concentration, pH of the medium, and effects of different solid supports for maximum secretion of pectin lyase from Penicillium citrinum MTCC 8897 has been studied. The enzyme was purified to homogeneity by Sephadex G-100 and DEAE-cellulose chromatography. The molecular mass determined by SDS-PAGE was 31 kDa. The K _m and k _cat values were found to be 1 mg/ml and 76 sec⁻¹, respectively. The optimum pH of the purified pectin lyase was 9.0, though it retains activity in the pH 9.0–12.0 range when exposed for 24 h. The optimum temperature was 50°C, and the pectin lyase was found to be completely stable up to 40°C when exposed for 1 h. The purified pectin lyase was found efficient in retting of Linum usitatissimum, Cannabis sativa, and Crotalaria juncea. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 985–992.     

Co-expression of the lipase and foldase of Pseudomonas aeruginosa to a functional lipase in Escherichia coli

The lipA gene, a structural gene encoding for protein of molecular mass 48 kDa, and lipB gene, encoding for a lipase-specific chaperone with molecular mass of 35 kDa, of Pseudomonas aeruginosa B2264 were co-expressed in heterologous host Escherichia coli BL21 (DE3) to obtain in vivo expression of functional lipase. The recombinant lipase was expressed with histidine tag at its N terminus and was purified to homogeneity using nickel affinity chromatography. The amino acid sequence of LipA and LipB of P. aeruginosa B2264 was 99–100% identical with the corresponding sequence of LipA and LipB of P. aeruginosa LST-03 and P. aeruginosa PA01, but it has less identity with Pseudomonas cepacia (Burkholderia cepacia) as it showed only 37.6% and 23.3% identity with the B. cepacia LipA and LipB sequence, respectively. The molecular mass of the recombinant lipase was found to be 48 kDa. The recombinant lipase exhibited optimal activity at pH 8.0 and 37°C, though it was active between pH 5.0 and pH 9.0 and up to 45°C. K _m and V _max values for recombinant P. aeruginosa lipase were found to be 151.5 ± 29 μM and 217 ± 22.5 μmol min⁻¹ mg⁻¹ protein, respectively.     

Purification and biological characterization of a halophilic thermostable protease from <Emphasis Type="Italic">Haloferax lucentensis</Emphasis> VKMM 007

An extremely halophilic archaeon Haloferax lucentensis VKMM 007, isolated from a solar saltern, was found to produce a protease. This extracellular enzyme consisted of a single polypeptide chain of 57.8 kDa as determined by SDS–PAGE and was purified by a combination of ultrafiltration, bacitracin–Sepharose affinity chromatography and Sephadex G-100 gel filtration. The purified protein was stable in a wide range of temperatures (20–70°C), NaCl concentrations (0.85–5.13 M) and pH (5.0–9.0) with maximal activity observed at 60°C, 4.3 M NaCl and pH 8.0. Proteolytic activity was enhanced by Ca²⁺, K⁺, Mg²⁺, Na⁺, and Fe²⁺ ions and the protein was classified as a trypsin-like serine protease. Further assays indicated highest degree of specificity when hemoglobin was used as an enzyme substrate. Most importantly, the proteolytic activity remained stable or only marginally inhibited in the presence of various polar and non-polar solvents, surfactants and reducing agents thus emphasizing the biotechnological potential of this novel halophilic protease.     

Purification and characterization of a highly selective sucrose isomerase from Erwinia rhapontici NX-5

A highly selective sucrose isomerase (SIase) was purified to homogeneity from the cell-free extract of Erwinia rhapontici NX-5 with a recovery of 27.7% and a fold purification of 213.6. The purified SIase showed a high specific activity of 427.1 U mg⁻¹ with molecular weight of 65.6 kDa. The K _m for sucrose was 222 mM while V _max was 546 U mg⁻¹. The optimum pH and temperature for SIase activity were 6.0 and 30 °C, respectively. The purified SIase was stable in the temperature range of 10–40 °C and retained 65% of the enzyme activity after 2 weeks’ storage at 30 °C. The SIase activity was enhanced by Mg²⁺ and Mn²⁺, inhibited by Ca²⁺, Cu²⁺, Zn²⁺, and Co²⁺, completely inhibited by Hg²⁺ and Ag²⁺. The purified SIase was strongly inhibited by SDS, while partially inhibited by dimethylformamide, tetrahydrofuran, and PMSF. Additionally, glucose and fructose acted as competitive inhibitors for purified SIase.     

Substrate specificity characterization of a thermostable keratinase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> KS-1

A feather-degrading strain of Pseudomonas aeruginosa KS-1 was used in the present study. Its crude cell-free fermentation broth completely degraded chicken feather within 12 h, in the absence of disulphide reductase activity. Keratinase from its extracellular broth was purified and characterized, assuming that it would be a potential β-keratin-degrading enzyme with prospective applications in degradation of β-plaques of prions. The keratinase was purified by using Q-Sepharose anion exchange chromatography and its molecular weight, as determined by SDS–PAGE analysis, was 45 kDa. It was an alkaline, serine protease with pH and temperature optima of 9 and 60°C, respectively. The enzyme was highly thermostable with a t _1/2 > 2 h at 80°C and had a very high K to C (keratinolytic to caseinolytic) ratio of 2.5. Besides feather keratin, it also hydrolyzed a variety of other complex substrates including fibrin, gelatin and meat protein. Its activity on synthetic substrates revealed that it efficiently cleaves them in the order phenylalanine > lysine > alanine > leucine p-nitroanilides. It also cleaved insulin B chain between Val¹²-Glu¹³, Ala¹⁴-Leu¹⁵, Gly²⁰-Glu²¹ and Arg²²-Gly²³ residues.     

Purification and characterization of a cold active alkaline protease from <Emphasis Type="Italic">Stenotrophomonas</Emphasis> sp., isolated from Kashmir,India

A Psychrotolerant alkaline protease producing bacterium IIIM-ST045 was isolated from a soil sample collected from the Thajiwas glacier of Kashmir, India and identified as Stenotrophomonas sp. on the basis of its biochemical properties and 16S ribosomal gene sequencing. The strain could grow well within a temperature range of 4–37°C however, showed optimum growth at 15°C. The strain was found to over-produce proteases when it was grown in media containing lactose as carbon source (157.50 U mg⁻¹). The maximum specific enzyme activity (398 U mg⁻¹) was obtained using soya oil as nitrogen source, however, the inorganic nitrogen sources urea, ammonium chloride and ammonium sulphate showed the lowest production of 38.9, 62.2 and 57.9 U mg⁻¹. The enzyme was purified to 18.45 folds and the molecular weight of the partially purified protease was estimated to be ~55 kDa by SDS-PAGE analysis. The protease activity increased as the increase in enzyme concentration while as the optimum enzyme activity was found when casein (1% w/v) was used as substrate. The enzyme was highly active over a wide range of pH from 6.5 to 12.0 showing optimum activity at pH 10.0. The optimum temperature for the enzyme was 15°C. Proteolytic activity reduced gradually with higher temperatures with a decrease of 56% at 40°C. The purified enzyme was checked for the removal of protein containing tea stains using a silk cloth within a temperature range of 10–60°C. The best washing efficiency results obtained at low temperatures indicate that the enzyme may be used for cold washing purposes of delicate fabrics that otherwise are vulnerable to high temperatures.     

Purification and properties of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase expressed in recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH₄)₂SO₄ fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5 and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature stability was up to 45 °C. Metal ions, such as, Mn²⁺ and K⁺ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg²⁺ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu²⁺ and Ag²⁺) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K _m of 120 and 330 μM and V _max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively.     

Characterization of an organic solvent-tolerant α-amylase from a halophilic isolate, <Emphasis Type="Italic">Thalassobacillus</Emphasis> sp. LY18

A halophilic isolate Thalassobacillus sp. LY18 producing extracellular amylase was isolated from the saline soil of Yuncheng Salt Lake, China. Production of the enzyme was synchronized with bacterial growth and reached a maximum level during the early stationary phase. The amylase was purified to homogeneity with a molecular mass of 31 kDa. Major products of soluble starch hydrolysis were maltose and maltotriose, indicating an α-amylase activity. Optimal enzyme activity was found to be at 70°C, pH 9.0, and 10 % NaCl. The α-amylase was highly stable over broad temperature (30–90°C), pH (6.0–12.0), and NaCl concentration (0–20 %) ranges, showing excellent thermostable, alkalistable, and halotolerant nature. The enzyme was stimulated by Ca²⁺, but greatly inhibited by EDTA, indicating it was a metalloenzyme. Complete inhibition by diethyl pyrocarbonate and β-mercaptoethanol revealed that histidine residue and disulfide bond were essential for enzyme catalysis. The surfactants tested had no significant effects on the amylase activity. Furthermore, it showed high activity and stability in the presence of water-insoluble organic solvents with log P _ow ≥ 2.13.     

Partial Purification and Characterization of Fibrinolytic Enzymes from Yellow Mealworm

Two proteins with fibrinolytic activity were partially purified from yellow mealworm (Tenebrio molitor) by ammonium sulfate precipitation between 30 and 70% saturation, gel filtration on Sephacryl-S200-HR, ion exchange chromatography on DEAE-Sepharose-FF and metal chelate on Cu–HiTrap–IMAC–FF, but the enzymes had not been completely separated from each other. The two partially purified fibrinolytic enzymes were designated as TMFE-I and TMFE-II (Tenebrio molitor fibrinolytic enzyme) with molecular weights of 27.5 and 24.9 kDa by SDS-PAGE individually. The partially purified solution of TMFE-I and TMFE-II was considerably stable in the range of pH 5–10 and characterized by pH optimum of the enzymatic activity at 8.0. Thermal stability of TMFE was excellent at 45°C and below. The K _M value was 0.26 mM for amidolysis of Bz–Arg–pNA. According to inhibitor analysis by fibrin plate method, phenylmethylsulfonyl fluoride and tosyl-lysine chloromethyl ketone inactivated TMFE almost completely, but trans-(epoxysuccinyl)-l-leucylamino-4-guanidinobutane (E-64) and EDTA had little effect on their fibrinolytic activity. According to metal ion analysis by fibrin plate method, the effect of metal ions on activity of TMFE showed a great difference. Na⁺, K⁺ and Zn²⁺ had little effect on the activity of TMFE. Mg²⁺ and Cu²⁺ showed inhibition effect on the fibrinolytic activity of TMFE, but Ca²⁺ increased the fibrinolytic activity of TMFE at final concentration varying from 0 to 30 mM.     

Thermostable invertases from Paecylomyces variotii produced under submerged and solid-state fermentation using agroindustrial residues

The filamentous fungus Paecylomices variotii was able to produce high levels of cell extract and extracellular invertases when grown under submerged fermentation (SbmF) and solid-state fermentation, using agroindustrial products or residues as substrates, mainly soy bran and wheat bran, at 40°C for 72 h and 96 h, respectively. Addition of glucose or fructose (≥1%; w/v) in SbmF inhibited enzyme production, while the addition of 1% (w/v) peptone as organic nitrogen source enhanced the production by 3.7-fold. However, 1% (w/v) (NH₄)₂HPO₄ inhibited enzyme production around 80%. The extracellular form was purified until electrophoretic homogeneity (10.5-fold with 33% recovery) by DEAE-Fractogel and Sephacryl S-200 chromatography. The enzyme is a monomer with molecular mass of 102 kDa estimated by SDS–PAGE with carbohydrate content of 53.6%. Optima of temperature and pH for both, extracellular and cell extract invertases, were 60°C and 4.0–4.5, respectively. Both invertases were stable for 1 h at 60°C with half-lives of 10 min at 70°C. Mg²⁺, Ba²⁺ and Mn²⁺ activated both extracellular and cell extract invertases from P. variotii. The kinetic parameters K_m and V_max for the purified extracellular enzyme corresponded to 2.5 mM and 481 U/mg prot⁻¹, respectively.     

Extracellular production of <Emphasis Type="Italic">Streptomyces exfoliatus</Emphasis> poly(3-hydroxybutyrate) depolymerase in <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. T104: determination of optimal biocatalyst conditions

The phaZ _Sex gene encoding poly(3-hydroxybutyrate) depolymerase from Streptomyces exfoliatus has been successfully cloned and expressed in Rhodococcus sp. T104 for the first time. Likewise, the recombinant enzyme was efficiently produced as an extracellular active form and purified to homogeneity by two hydrophobic chromatographic steps. MALDI-TOF analysis showed that the native enzyme is a monomer. Circular dichroism studies have revealed a secondary structure showing 25.6% α-helix, 21.4% β-sheet, 17.1% β-turns, and 35.2% random coil, with a midpoint transition temperature (T _m) of 55.8 °C. Magnesium and calcium ions enhanced the enzyme activity, whereas manganese inhibited it. EDTA moderately decreased the activity, and the enzyme was completely deactivated at 3 M NaCl. Chemical modification studies indicated the presence of the catalytic triad serine–histidine–carboxylic acid in the active site. High-performance liquid chromatography (HPLC)–mass spectrometry (MS) analysis of PHB products of enzymatic hydrolysis showed monomers and dimers of 3-hydroxybutyric acid, demonstrating that PHB depolymerase is an exo-hydrolase. Addition of methyl-β-cyclodextrin simultaneously increased the activity as well as preserved the enzyme during lyophilization. Finally, thermoinactivation studies showed that the enzyme is highly stable at 40 °C. All these features support the potential industrial application of this recombinant enzyme in the production of (R)-3-hydroxyalkanoic acid derivatives as well as in the degradation of bioplastics.     

Purification and characterization of a new L-methioninase from solid cultures of <Emphasis Type="Italic">Aspergillus flavipes</Emphasis>

L-Methioninase was purified to electrophoretic homogeneity from cultures of Aspergillus flavipes using anion-exchange and gel filtration chromatography by 12.1 fold compared to the crude enzyme preparation. The purified enzyme had a molecular mass of 47 kDa under denaturing conditions and an isoelectric point of 5.8 with no structural glycosyl residues. The enzyme had optimum activity at pH 7.8 and pH stability from 6.8–8.0 at 35°C. The enzyme appeared to be catalytically stable below 40°C. The enzyme activity was strongly inhibited by DL-propargylglycine, hydroxylamine, PMSF, 2-mercaptoethanol, Hg⁺, Cu²⁺, and Fe²⁺, with slight inhibition by Triton X-₁₀₀. A flavipes L-methioninase has a higher catalytic affinity towards L-methionine (Km, 6.5 mM and Kcat, 14.1 S⁻¹) followed by a relative demethiolating activity to L-homo-cysteine (Km, 12 mM and Kcat, 9.3 S⁻¹). The enzyme has two absorption maxima at 280 and 420 nm, typical of other PLP-enzymes. Apo-L-methioninase has the ability to reconstitute its structural catalytic state completely upon addition of 0.15 mM PLP. L-Methioninase has neither an appreciable effect on liver function, platelet aggregation, nor hemolysis of human blood. The purified L-methioninase from solid cultures of A. flavipes displayed unique biochemical and catalytic properties over the currently applied Pseudomonad enzyme.