Subunit Molecular Mass Research Articles

Biochemical and Spectroscopic Characterization of Highly Stable Photosystem II Supercomplexes from Arabidopsis

Photosystem II (PSII) is a large membrane supercomplex involved in the first step of oxygenic photosynthesis. It is organized as a dimer, with each monomer consisting of more than 20 subunits as well as several cofactors, including chlorophyll and carotenoid pigments, lipids, and ions. The isolation of stable and homogeneous PSII supercomplexes from plants has been a hindrance for their deep structural and functional characterization. In recent years, purification of complexes with different antenna sizes was achieved with mild detergent solubilization of photosynthetic membranes and fractionation on a sucrose gradient, but these preparations were only stable in the cold for a few hours. In this work, we present an improved protocol to obtain plant PSII supercomplexes that are stable for several hours/days at a wide range of temperatures and can be concentrated without degradation. Biochemical and spectroscopic properties of the purified PSII are presented, as well as a study of the complex solubility in the presence of salts. We also tested the impact of a large panel of detergents on PSII stability and found that very few are able to maintain the integrity of PSII. Such new PSII preparation opens the possibility of performing experiments that require room temperature conditions and/or high protein concentrations, and thus it will allow more detailed investigations into the structure and molecular mechanisms that underlie plant PSII function.

English

A lectin was purified from the tuber, Dioscorea mangenotiana in this study. The lectin agglutinated erythrocytes from human and rabbit, and the activity was inhibited by glucose and N-acetyl-D-glucosamine with minimum inhibitory concentration (MIC) of 50 and 25 mM, respectively. The lectin was composed of two isoforms, DML I and DML II. The apparent native molecular mass of DML I was estimated at 51 kDa and the subunit molecular mass at 25 kDa, suggesting a dimeric structure. The lectin was stable up to 90°C and within the physiological pH range. The lectin when incorporated in artificial diet at concentrations of 10 to 160 µg ml-1 and fed ad libitum to the second instar larvae of Eldana saccharina, prolonged the development period and significantly inhibited the pupation and emergence of the insect in a dose-dependent manner. The LC50 calculated based on mortality after 72 h of treatment was 66.6 mg/ml. The activities of hydrolytic enzymes - acid phosphatase, alkaline phosphatase and acetylcholine esterase in the larvae reared on diet containing lethal dose of the lectin were significantly affected as compared to those fed on diet without the lectin. The results showed that DML I has promising anti-insect potential and could be employed in a biotechnological strategy for the pest management. Key words: Lectin, tuber, anti-insect, Dioscorea mangenotiana,Eldana saccharina.

Purification and characterization of two glutathione peroxidases from embryo of the camel tick Hyalomma dromedarii

Two glutathione peroxidase isoenzymes were purified from 24-day old embryos of the camel tick Hyalomma dromedarii and designated tick embryo glutathione peroxidase 1 and 2 (TEGPx1 and TEGPx2). The purification procedure involved ammonium sulfate precipitation, as well as ion exchange and gel filtration column chromatography. Glutathione peroxidase isoenzymes subunit molecular mass was determined by SDS-PAGE to be 36 ± 2 kDa and 59 ± 1.5 kDa for TEGPx1 and TEGPx2, respectively. TEGPx1 isoenzyme exhibited a dimeric structure with native molecular mass of 72 kDa while TEGPx2 was a monomeric protein. TEGPx1 and TEGPx2 displayed their pH optima at 7.6 and 8.2. Both isoenzymes cleaved preferentially H2O2 with K m values of 24 and 49 μM. Iodoacetamide competitively inhibited TEGPx1 with K i value of 0.45 mM and 1.10; phenanthroline competitively inhibited TEGPx2 with K i value of 0.12 mM. These results indicate the presence of two different forms of glutathione peroxidase in the developing camel tick embryos. This finding enhances our knowledge and understanding of the physiology of these ectoparasites and will encourage the development of new and untraditional control methods.

Purification and characterization of the carbonic anhydrase enzyme from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney and inhibition effects of some metal ions on enzyme activity.

In this study, the carbonic anhydrase (CA) enzyme was purified from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney with a specific activity of 603.77EU/mg and a yield of 35.5% using Sepharose-4B-l-tyrosine- sulphanilamide affinity column chromatography. For determining the enzyme purity and subunit molecular mass, sodiumdodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed and single band was observed. The molecular mass of subunit was found approximately 29.71kDa. The optimum temperature, activation energy (Ea), activation enthalpy (ΔH) and Q10 values were obtained from Arrhenius plot. Km and Vmax values for p-nitrophenyl acetate of the purified enzyme were calculated from Lineweaver-Burk graphs. In addition, the inhibitory effects of different heavy metal ions (Fe(2+), Pb(2+), Co(2+), Ag(+) and Cu(2+)) on Black Sea trout kidney tissue CA enzyme activities were investigated by using esterase method under in vitro conditions. The heavy metal concentrations inhibiting 50% of enzyme activity (IC50) were obtained. Finally Ki values and inhibition types were calculated from Lineweaver-Burk graphs.

Novel alkalistable α-carbonic anhydrase from the polyextremophilic bacterium Bacillus halodurans: characteristics and applicability in flue gas CO2 sequestration.

The emissions of CO2 into the atmosphere have been constantly rising due to anthropogenic activities, which have led to global warming and climate change. Among various methods proposed for mitigating CO2 levels in the atmosphere, carbonic anhydrase (CA)-mediated carbon sequestration represents a greener and safer approach to capture and convert it into stable mineral carbonates. Despite the fact that CA is an extremely efficient metalloenzyme that catalyzes the hydration of CO2 (CO2 + H2O ↔ HCO3 (-) + H(+)) with a kcat of ∼10(6)s(-1), a thermostable, and alkalistable CA is desirable for the process to take place efficiently. The purified CA from alkaliphilic, moderately thermophilic, and halotolerant Bacillus halodurans TSLV1 (BhCA) is a homodimeric enzyme with a subunit molecular mass of ~37kDa with stability in a broad pH range between 6.0 and 11.0. It has a moderate thermostability with a T1/2 of 24.0 ± 1.0min at 60°C. Based on the sensitivity of CA to specific inhibitors, BhCA is an α-CA; this has been confirmed by nucleotide/amino acid sequence analysis. This has a unique property of stimulation by SO4 (2-), and it remains unaffected by SO3 (2-), NOx, and most other components present in the flue gas. BhCA is highly efficient in accelerating the mineralization of CO2 as compared to commercial bovine carbonic anhydrase (BCA) and is also efficient in the sequestration of CO2 from the exhaust of petrol driven car, thus, a useful biocatalyst for sequestering CO2 from flue gas.

Highly Active and Stable Large Catalase Isolated from a Hydrocarbon Degrading Aspergillus terreus MTCC 6324.

A hydrocarbon degrading Aspergillus terreus MTCC 6324 produces a high level of extremely active and stable cellular large catalase (CAT) during growth on n-hexadecane to combat the oxidative stress caused by the hydrocarbon degrading metabolic machinery inside the cell. A 160-fold purification with specific activity of around 66 × 105 U mg−1 protein was achieved. The native protein molecular mass was 368 ± 5 kDa with subunit molecular mass of nearly 90 kDa, which indicates that the native CAT protein is a homotetramer. The isoelectric pH (pI) of the purified CAT was 4.2. BLAST aligned peptide mass fragments of CAT protein showed its highest similarity with the catalase B protein from other fungal sources. CAT was active in a broad range of pH 4 to 12 and temperature 25°C to 90°C. The catalytic efficiency (K cat/K m) of 4.7 × 108 M−1 s−1 within the studied substrate range and alkaline pH stability (half-life, t 1/2 at pH 12~15 months) of CAT are considerably higher than most of the extensively studied catalases from different sources. The storage stability (t 1/2) of CAT at physiological pH 7.5 and 4°C was nearly 30 months. The haem was identified as haem b by electrospray ionization tandem mass spectroscopy (ESI-MS/MS).

Purification and characterization of a mycelial mucin specific lectin from Aspergillus panamensis with potent mitogenic and antibacterial activity

A lectin was isolated from Aspergillus panamensis mycelia using single-step affinity chromatography on porcine stomach mucin coupled Sepharose 4B. A purification fold of 19.2 with 62.5% yield was achieved by this method. Gel filtration chromatography showed the native molecular mass of 78kDa and the subunit molecular mass of 39kDa was confirmed by 10% SDS-PAGE and MALDI-TOF analysis. Isoelectric focussing of native A. panamensis lectin revealed it to be an acidic protein with pI 4.5. Carbohydrate content of purified lectin was estimated to be 4.95%. All database search of N-terminal amino acid sequences revealed no homology of the purified lectin with previously reported microbial, viral and animal lectins except 29kDa subunit of heterodimeric Vigna sesquipedalis lectin. The lectin from this fungal source is a homodimer. Lectin was fairly thermostable with pH optima of 6.5–8.0 and had no requirement of divalent metal cations for its activity. Maximum mitogenic activity toward mouse spleen cells was exhibited at 100μg/mL concentration of purified lectin. Results of the antimicrobial assay showed that the lectin had potent antibacterial activity against Bacillus cereus, Staphylococcous aureus, Pseudomonas aeruginosa and Escherichia coli.

Purification and partial characterization of novel penicillin V acylase from Acinetobacter sp. AP24 isolated from Loktak Lake, an Indo-Burma biodiversity hotspot

ABSTRACTMembers of the bacterial genus Acinetobacter have attracted great attention over the past few decades, on account of their various biotechnological applications and clinical implications. In this study, we are reporting the first experimental penicillin V acylase (PVA) activity from this genus. Penicillin acylases are pharmaceutically important enzymes widely used in the synthesis of semisynthetic beta-lactam antibiotics. The bacterium, identified as Acinetobacter sp. AP24, was isolated from the water of Loktak Lake (Manipur, India), an Indo-Burma biodiversity hotspot. PVA production was increased threefold in an optimized medium with 0.2% sodium glutamate and 1% glucose as nitrogen and carbon sources respectively, after 24 hr of fermentation at 28°C and pH 7.0 with shaking at 180 rpm. The enzyme was purified to homogeneity by cation-exchange chromatography using SP-sepharose resin. The PVA is a homotetramer with subunit molecular mass of 34 kD. The enzyme was highly specific toward penicillin V with optimal hydrolytic activity at 40°C and pH 7.5. The enzyme was stable from pH 5.0 to 9.0 at 25 °C for 2 hr. The enzyme retained 75% activity after 1 hr of incubation at 40°C at pH 7.5.

Application of termite hindgut metagenome derived carboxyl ester hydrolases in the modification of cephalosporin substrates

In the pharmaceutical industry, de-acetylated cephalosporins are highly valuable starting materials for producing semi-synthetic β-lactam antibiotics. In this study a fosmid metagenome library from termite hindgut symbionts was screened for carboxyl ester hydrolases capable of de-acetylating cephalosporins. Recombinant Escherichia coli clones with esterolytic phenotypes on tributyrin agar plates were selected and further tested for de-acetylating activity against Cephalothin and 7-aminocephalosporanic acid (7-ACA). Two clones displaying de-acetylating activity were sequenced and the corresponding two carboxyl ester hydrolase encoding genes (axeA and axeB) belonging to the carbohydrate esterase family 7 (CE7) were identified. The primary structure of both the axeA and axeB revealed the presence of G-X-S-X-G sequence motif and respective subunit molecular masses of 40kDa. In addition to de-acetylating cephalosporin based molecules, the two enzymes were also shown to be true esterases based on their preferences for short chain length fatty acid esters.

Role of Active Site Residues on Catalytic Activity of Catalase with Oxidase Activity from Scytalidium Thermophilum

Scytalidium thermophilum produces a catalase with phenol oxidase activity (CATPO) that catalyses the dismutation of hydrogen peroxide (H2O2) to dioxygen and water and also oxidizes several phenolic compounds in the absence of hydrogen peroxide. It comprises 717 amino acids with a 19 amino acid signal sequence, and a 17 amino acid prosequence. It is a homotetrameric protein of molecular mass 320 kDa and subunit molecular mass 80 kDa. Although catalases have been studied for many years, a peroxide independent oxidative activity of catalases has recently been recognized. There are a great number of reports available describing the structural and biochemical characterization of catalases. However basic questions related to substrate and product flow remain unanswered, particularly related to the oxidase activity. The goals of our current studies are to investigate the main and lateral channels known that connect the deeply buried active site to the exterior of the enzyme. We have introduced a number of mutations into these regions and analyzed their specific activities.

Purification and partial characterization of serine fibrinolytic enzyme from Bacillus megaterium KSK-07 isolated from kishk, a traditional Egyptian fermented food

A potent fibrinolytic enzyme from Bacillus megaterium KSK-07 isolated from an Egyptian fermented food, kishk, has been purified to electrophoretic homogeneity by gel filtration and anion-exchange chromatography. Purification increased its specific activity to 1124-fold with a recovery of 23%. The subunit molecular mass of the purified enzyme was estimated to be 28.5 kDa by SDS-PAGE. The optimal reaction temperature, pH and pI values for chymotrypsin from B. megaterium KSK-07 were 50°C, 8.0, and 10.0, respectively. Enzyme hydrolyzed not only fibrin but also several synthetic substrates, particularly 3-carbomethoxypropionyl-L-arginyl-L-prolyl-L-tyrosine p-nitroaniline hydrochloride (MeO-Suc-Arg-Pro-Tyr-pNA-HCl). In addition, PMSF can completely inhibit its fibrinolytic activity. These results indicated that chymotrypsin from B. megaterium KSK-07 is a chymotrypsin-family serine protease. Its apparent KM, Vmax and Kcat for the synthetic substrate L-succinyl-L-phenylalanine p-nitroanilide (N-Suc-Phe-pNA) were 0.61 mM, 10.2 μmoles mg−1min−1, and 56.7 s−1, respectively. It demonstrated direct action upon blood clots in vitro and prolonged the blood clotting time to 1.9-fold. The enzyme could not degrade collagen suggesting this enzyme be an effective thrombolytic agent with high specificity to fibrin and non-specificity to other plasma proteins.

Two strictly polyphosphate-dependent gluco(manno)kinases from diazotrophic Cyanobacteria with potential to phosphorylate hexoses from polyphosphates.

The single-copy genes encoding putative polyphosphate-glucose phosphotransferases (PPGK, EC 2.7.1.63) from two nitrogen-fixing Cyanobacteria, Nostoc sp. PCC7120 and Nostoc punctiforme PCC73102, were cloned and functionally characterized. In contrast to their actinobacterial counterparts, the cyanobacterial PPGKs have shown the ability to phosphorylate glucose using strictly inorganic polyphosphates (polyP) as phosphoryl donors. This has proven to be an economically attractive reagent in contrast to the more costly ATP. Cyanobacterial PPGKs had a higher affinity for medium-long-sized polyP (greater than ten phosphoryl residues). Thus, longer polyP resulted in higher catalytic efficiency. Also in contrast to most their homologs in Actinobacteria, both cyanobacterial PPGKs exhibited a modest but significant polyP-mannokinase activity as well. Specific activities were in the range of 180-230 and 2-3 μmol min(-1) mg(-1) with glucose and mannose as substrates, respectively. No polyP-fructokinase activity was detected. Cyanobacterial PPGKs required a divalent metal cofactor and exhibited alkaline pH optima (approx. 9.0) and a remarkable thermostability (optimum temperature, 45 °C). The preference for Mg(2+) was noted with an affinity constant of 1.3 mM. Both recombinant PPGKs are homodimers with a subunit molecular mass of ca. 27 kDa. Based on database searches and experimental data from Southern blots and activity assays, closely related PPGK homologs appear to be widespread among unicellular and filamentous mostly nitrogen-fixing Cyanobacteria. Overall, these findings indicate that polyP may be metabolized in these photosynthetic prokaryotes to yield glucose (or mannose) 6-phosphate. They also provide evidence for a novel group-specific subfamily of strictly polyP-dependent gluco(manno)kinases with ancestral features and high biotechnological potential, capable of efficiently using polyP as an alternative and cheap source of energy-rich phosphate instead of costly ATP. Finally, these results could shed new light on the evolutionary origin of sugar kinases.

Expression, purification, and characterization of recombinant mangrove glutamine synthetase.

To expand our knowledge about the relationship of nitrogen use efficiency and glutamine synthetase (GS) activity in the mangrove plant, a cytosolic GS gene from Avicennia marina has been heterologously expressed in and purified from Escherichia coli. Synthesis of the mangrove GS enzyme in E. coli was demonstrated by functional genetic complementation of a GS deficient mutant. The subunit molecular mass of GSI was ~40kDa. Optimal conditions for biosynthetic activity were found to be 35°C at pH 7.5. The Mg(2+)-dependent biosynthetic activity was strongly inhibited by Ni(2+), Zn(2+), and Al(3+), whereas was enhanced by Co(2+). The apparent K m values of AmGLN1 for the substrates in the biosynthetic assay were 3.15mM for glutamate, and 2.54mM for ATP, 2.80mM for NH4 (+) respectively. The low affinity kinetics of AmGLN1 apparently participates in glutamine synthesis under the ammonium excess conditions.

Inter-Ring Communication Is Dispensable in the Reaction Cycle of Group II Chaperonins

Chaperonins are ubiquitous molecular chaperones with the subunit molecular mass of 60kDa. They exist as double-ring oligomers with central cavities. An ATP-dependent conformational change of the cavity induces the folding of an unfolded protein that is captured in the cavity. In the group I chaperonins, which are present in eubacteria and eukaryotic organelles, inter-ring communication takes important role for the reaction cycle. However, there has been limited study on the inter-ring communication in the group II chaperonins that exist in archaea and the eukaryotic cytosol. In this study, we have constructed the asymmetric ring complex of a group II chaperonin using circular permutated covalent mutants. Although one ring of the asymmetric ring complex lacks ATPase or ATP binding activity, the other wild-type ring undergoes an ATP-dependent conformational change and maintains protein-folding activity. The results clearly demonstrate that inter-ring communication is dispensable in the reaction cycle of group II chaperonins.

Chloromuconolactone dehalogenase ClcF of actinobacteria

This work investigated the distribution of the clcF gene in actinobacteria isolated from different ecotopes. The gene encodes chloromuconolactone dehalogenase (CMLD) ClcF, the enzyme found to date in only one representative of Gram‐positive bacteria, Rhodococcus opacus 1CP, adapted to 2‐chlorophenol (2CP). Using primers specific to the clcF gene, from the DNA matrix of rhodococcal strains closely related to species Rhodococcus wratislaviensis (P1, P12, P13, P20, G10, KT112, KT723, BO1) we obtained PCR products whose nucleotide sequences were 100% identical to that of the clcF gene from strain R. opacus 1CP. CMLDs isolated from the biomass of strains Rhodococcus spp. G10 and P1 grown on 2CP did not differ by their subunit molecular mass deduced from the known amino acid sequence of the clcF gene from the ClcF of strain R. opacus 1CP. Matrix‐assisted laser dissociation/ionization time‐of‐flight mass spectrometry showed the presence of a peak with m/z 11,194–11,196 Da both in whole cells and in protein solutions with a ClcF activity. Thus, we have first time shown the distribution of ClcF among actinobacteria isolated from geographically distant habitats.

Discovery of a cell wall porin in the mycolic‐acid‐containing actinomycete Dietzia marisDSM 43672

The cell wall of the Gram-positive mycolic-acid-containing actinomycete Dietziamaris DSM 43672 was found to contain a pore-forming protein, as observed from reconstitution experiments with artificial lipid bilayer experiments in the presence of cell wall extracts. The cell wall porin was purified to homogeneity using different biochemical methods and had an apparent molecular mass of about 120 kDa on tricine-containing SDS/PAGE. The 120 kDa protein dissociated into subunits with a molecular mass of about 35 kDa when it was heated to 100°C in 8m urea. The 120 kDa protein, here named PorADm , formed ion-permeable channels in lipid bilayer membranes with a high single-channel conductance of about 5.8 nS in 1m KCl. Asymmetric addition of PorADm to lipid bilayer membranes resulted in an asymmetric voltage dependence. Zero-current membrane potential measurements with different salt solutions suggested that the porin of D.maris is cation-selective because of negative charges localized at the channel mouth. Analysis of the single-channel conductance using non-electrolytes with known hydrodynamic radii indicated that the diameter of the cell wall channel is about 2nm. The channel characteristics of the cell wall porin of D.maris are compared with those of other members of the mycolata. They share some common features because they are composed of small molecular mass subunits and form large and water-filled channels. The porin was subjected to protein analysis by mass spectrometry but its sequence had no significant homology to any known porin sequences.

Purification and characterization of an oxygen-evolving photosystem II from Leptolyngbya sp. strain O-77

A new cyanobacterium of strain O-77 was isolated from a hot spring at Aso-Kuju National Park, Kumamoto, Japan. According to the phylogenetic analysis determined by 16S rRNA gene sequence, the strain O-77 belongs to the genus Leptolyngbya, classifying into filamentous non-heterocystous cyanobacteria. The strain O-77 showed the thermophilic behavior with optimal growth temperature of 55°C. Moreover, we have purified and characterized the oxygen-evolving photosystem II (PSII) from the strain O-77. The O2-evolving activity of the purified PSII from strain O-77 (PSIIO77) was 1275 ± 255 μmol O2 (mg Chl a)(-1) h(-1). Based on the results of MALDI-TOF mass spectrometry and urea-SDS-PAGE analysis, the purified PSIIO77 was composite of the typical PSII components of CP47, CP43, PsbO, D2, D1, PsbV, PsbQ, PsbU, and several low molecular mass subunits. Visible absorption and 77 K fluorescence spectra of the purified PSIIO77 were almost identical to those of other purified PSIIs from cyanobacteria. This report provides the successful example for the purification and characterization of an active PSII from thermophilic, filamentous non-heterocystous cyanobacteria.

Purification and Characterization of a Zinc-Dependent Cinnamyl Alcohol Dehydrogenase from Leucaena leucocephala, a Tree Legume

A cinnamyl alcohol dehydrogenase (CAD) from the secondary xylem of Leucaena leucocephala has been purified to homogeneity through successive steps of ammonium sulfate fractionation, DEAE cellulose, Sephadex G-75, and Blue Sepharose CL-6B affinity column chromatographies. CAD was purified to 514.2 folds with overall recovery of 13% and specific activity of 812. 5nkat/mg. Native and subunit molecular masses of the purified enzyme were found to be ∼76 and ∼38kDa, respectively, suggesting it to be a homodimer. The enzyme exhibited highest catalytic efficiency (Kcat/Km 3.75μM(-1)s(-1)) with cinnamyl aldehyde among all the substrates investigated. The pH and temperature optima of the purified CAD were pH8.8 and 40°C, respectively. The enzyme activity was enhanced in the presence of 2.0mM Mg(2+), while Zn(2+) at the same concentration exerted an inhibitory effect. The inclusion of 2.0mM EDTA in the assay system activated the enzyme. The enzyme was inhibited with caffeic acid and ferulic acid in a concentration-dependent manner, while no inhibition was observed with salicylic acid. Peptide mass analysis of the purified CAD by MALDI-TOF showed a significant homology to alcohol dehydrogenases of MDR superfamily.

Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones

A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the C=O bond of allylic aldehydes/ketones to the corresponding α,β-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (S)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg(-1) for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg(-1) using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower Km values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP(+), suggesting the nature of being an aldehyde reductase.

EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF COLD-ADAPTED INORGANIC PYROPHOSPHATASE FROM PSYCHROPHILIC Shewanella sp. AS-11

In the presence of divalent cations, inorganic pyrophosphatase is activated to hydrolyze inorganic pyrophosphate to inorganic phosphate. Here, we clone, express, purify, and characterize inorganic pyrophosphatase from the psychrophilic Shewanella sp. AS-11 (Sh-PPase). The recombinant Sh-PPase was expressed in Escherichia coli BL21 (DE3) at 20°C using pET16b as an expression vector and purified from the cell extracts by a combination of ammonium sulfate fractionation and anion-exchange chromatography. Sh-PPase was found to be a family II PPase with a subunit molecular mass of 34 kD that preferentially utilizes Mn2+ over Mg2+ ions for activity. The functional characteristics of Sh-PPase, such as activity, temperature dependency, and thermal inactivation, were greatly influenced by manganese ions. Manganese ion activation increased the enzyme's activity at low temperatures; therefore, it was required to gain the cold-adapted characteristics of Sh-PPase.