Production,purification and biochemical characterisation of a novel lipase from a newly identified lipolytic bacterium Staphylococcus caprae NCU S6

A novel lipase, SCNL, was isolated from Staphylococcus caprae NCU S6 strain in the study. The lipase was purified to homogeneity with a yield of 6.13% and specific activity of 502.76 U/mg, and its molecular weight was determined to be approximately 87 kDa. SCNL maintained above 80% of its initial activity at a wide range of temperatures (20–50 °C) and pH values (6–11), with an optimal temperature at 40 °C and optimal pH at 9.0 with p-nitrophenyl palmitate as a substrate. SCNL exhibited a higher residual activity than the other staphylococcal lipases in the presence of common enzyme inhibitors and commercial detergents. The lipase activity was enhanced by organic solvents (isooctane, glycerol, DMSO and methanol) and metal ions (Na⁺, Ba²⁺, Ca²⁺, and Mn²⁺). The Km and Vmax values of SCNL were 0.695 mM and 262.66 s⁻¹mM⁻¹, respectively. The enzyme showed a preference for p-NP stearate, tributyrin and canola oil. These biochemical features of SCNL suggested that it may be an excellent novel lipase candidate for industrial and biotechnological applications.     

Partial Optimization of the 5-Terminal Codon Increased a Recombination Porcine Pancreatic Lipase (opPPL) Expression in Pichia pastoris

Pancreatic lipase plays a key role in intestinal digestion of feed fat, and is often deficient in young animals such as weaning piglets. The objective of this study was to express and characterize a partial codon optimized porcine pancreatic lipase (opPPL). A 537 bp cDNA fragment encoding N-terminus amino acid residue of the mature porcine pancreatic lipase was synthesized according to the codon bias of Pichia pastoris and ligated to the full-length porcine pancreatic lipase cDNA fragment. The codon optimized PPL was cloned into the pPICZαA (Invitrogen, Beijing, China) vector. After the resultant opPPL/pPICZαΑ plasmid was transformed into P.pastoris, the over-expressed extracellular opPPL containing a His-tag to the C terminus was purified using Ni Sepharose affinity column (GE Healthcare, Piscataway, NJ, USA), and was characterized against the native enzyme (commercial PPL from porcine pancreas, Sigma). The opPPL exhibited a molecular mass of approximately 52 kDa, and showed optimal temperature (40°C), optimal pH (8.0), K_m (0.041 mM), and V_max (2.008 µmol.mg protein ⁻¹.min⁻¹) similar to those of the commercial enzyme with p-NPP as the substrate. The recombinant enzyme was stable at 60°C, but lost 80% (P<0.05) of its activity after exposure to heat ≥60°C for 20 min. The codon optimization increased opPPL yield for ca 4 folds (146 mg.L⁻¹ vs 36 mg.L⁻¹) and total enzyme activity increased about 5 folds (1900 IU.L⁻¹ vs 367 IU.L⁻¹) compared with those native naPPL/pPICZαΑ tranformant. Comparison of gene copies and mRNA profiles between the two strains indicated the increased rePPL yields may partly be ascribed to the increased protein translational efficiency after codon optimization. In conclusion, we successfully optimized 5-terminal of porcine pancreatic lipase encoding gene and over-expressed the gene in P. pastoris as an extracellular, functional enzyme. The recombination enzyme demonstrates a potential for future use as an animal feed additive for animal improvement.     

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 K_m 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.     

Purification and stability characteristics of an alkaline serine protease from a newly isolated Haloalkaliphilic bacterium sp. AH-6

An alkaline protease secreting Haloalkaliphilic bacterium (Gene bank accession number EU118361) was isolated from the Saurashtra Coast in Western India. The alkaline protease was purified by a single step chromatography on phenyl sepharose 6 FF with 28% yield. The molecular mass was 40 kDa as judged by SDS-PAGE. The enzyme displayed catalysis and stability over pH 8–13, optimally at 9–11. It was stable with 0–4 M NaCl and required 150 mM NaCl for optimum catalysis at 37 °C; however, the salt requirement for optimal catalysis increased with temperature. While crude enzyme was active at 25–80 °C (optimum at 50 °C), the purified enzyme had temperature optimum at 37 °C, which shifted to 80 °C in the presence of 2 M NaCl. The NaCl not only shifted the temperature profile but also enhanced the substrate affinity of the enzyme as reflected by the increase in the catalytic constant (K _cat). The enzyme was also calcium dependent and with 2 mM Ca⁺², the activity reached to maximum at 50 °C. The crude enzyme was highly thermostable (37–90 °C); however, the purified enzyme lost its stability above 50 °C and its half life was enhanced by 30 and sevenfold at 60 °C with 1 M NaCl and 50 mM Ca⁺², respectively. The activity of the enzyme was inhibited by PMSF, indicating its serine type. While the activity was slightly enhanced by Tween-80 (0.2%) and Triton X-100 (0.05%), it marginally decreased with SDS. In addition, the enzyme was highly stable with oxidizing-reducing agents and commercial detergents and was affected by metal ions to varying extent. The study assumes significance due to the enzyme stability under the dual extremities of pH and salt coupled with moderate thermal tolerance. Besides, the facts emerged on the enzyme stability would add to the limited information on this enzyme from Haloalkaliphilic bacteria.     

Low-Temperature Lipase from Psychrotrophic Pseudomonas sp. Strain KB700A

We have previously reported that a psychrotrophic bacterium, Pseudomonas sp. strain KB700A, which displays sigmoidal growth even at −5°C, produced a lipase. A genomic DNA library of strain KB700A was introduced into Escherichia coli TG1, and screening on tributyrin-containing agar plates led to the isolation of the lipase gene. Sequence analysis revealed an open reading frame (KB-lip) consisting of 1,422 nucleotides that encoded a protein (KB-Lip) of 474 amino acids with a molecular mass of 49,924 Da. KB-Lip showed 90% identity with the lipase from Pseudomonas fluorescens and was found to be a member of Subfamily I.3 lipase. Gene expression and purification of the recombinant protein were performed. KB-Lip displayed high lipase activity in the presence of Ca²⁺. Addition of EDTA completely abolished lipase activity, indicating that KB-Lip was a Ca²⁺-dependent lipase. Addition of Mn²⁺ and Sr²⁺ also led to enhancement of lipase activity but to a much lower extent than that produced by Ca²⁺. The optimal pH of KB-Lip was 8 to 8.5. The addition of detergents enhanced the enzyme activity. When p-nitrophenyl esters and triglyceride substrates of various chain-lengths were examined, the lipase displayed highest activity towards C₁₀ acyl groups. We also determined the positional specificity and found that the activity was 20-fold higher toward the 1(3) position than toward the 2 position. The optimal temperature for KB-Lip was 35°C, lower than that for any previously reported Subfamily I.3 lipase. The enzyme was also thermolabile compared to these lipases. Furthermore, KB-Lip displayed higher levels of activity at low temperatures than did other enzymes from Subfamily I.3, indicating that KB-Lip has evolved to function in cold environments, in accordance with the temperature range for growth of its psychrotrophic host, strain KB700A.     

NAD-Linked Isocitrate Dehydrogenase: Isolation, Purification, and Characterization of the Protein from Pea Mitochondria

The NAD⁺-dependent isocitrate dehydrogenase from etiolated pea (Pisum sativum L.) mitochondria was purified more than 200-fold by dye-ligand binding on Matrix Gel Blue A and gel filtration on Superose 6. The enzyme was stabilized during purification by the inclusion of 20% glycerol. In crude matrix extracts, the enzyme activity eluted from Superose 6 with apparent molecular masses of 1400 ± 200, 690 ± 90, and 300 ± 50 kD. During subsequent purification steps the larger molecular mass species disappeared and an additional peak at 94 ± 16 kD was evident. The monomer for the enzyme was tentatively identified at 47 kD by sodium dodecyl-polyacrylamide gel electrophoresis. The NADP⁺-specific isocitrate dehydrogenase activity from mitochondria eluted from Superose 6 at 80 ± 10 kD. About half of the NAD⁺ and NADP⁺-specific enzymes remained bound to the mitochondrial membranes and was not removed by washing. The NAD⁺-dependent isocitrate dehydrogenase showed sigmodial kinetics in response to isocitrate (S_0.5 = 0.3 mm). When the enzyme was aged at 4°C or frozen, the isocitrate response showed less allosterism, but this was partially reversed by the addition of citrate to the reaction medium. The NAD⁺ isocitrate dehydrogenase showed standard Michaelis-Menten kinetics toward NAD⁺ (K_m = 0.2 mm). NADH was a competitive inhibitor (K_i = 0.2 mm) and, unexpectedly, NADPH was a noncompetitive inhibitor (K_i = 0.3 mm). The regulation by NADPH may provide a mechanism for coordination of pyridine nucleotide pools in the mitochondria.     

Purification and Characterization of α-Amylase from Bacillus licheniformis CUMC305

α-Amylase produced by Bacillus licheniformis CUMC305 was purified 212-fold with a 42% yield through a series of four steps. The purified enzyme was homogeneous as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and discontinuous gel electrophoresis. The purified enzyme showed maximal activity at 90°C and pH 9.0, and 91% of this activity remained at 100°C. The enzyme retained 91, 79, and 71% maximal activity after 3 h of treatment at 60°C, 3 h at 70°C, and 90 min at 80°C, respectively, in the absence of substrate. On the contrary, in the presence of substrate (soluble starch), the α-amylase enzyme was fully stable after a 4-h incubation at 100°C. The enzyme showed 100% stability in the pH range 7 to 9; 95% stability at pH 10; and 84, 74, 68, and 50% stability at pH values of 6, 5, 4, and 3, respectively, after 18 h of treatment. The activation energy for this enzyme was calculated as 5.1 × 10⁵ J/mol. The molecular weight was estimated to be 28,000 by sodium dodecyl sulfate-gel electrophoresis. The relative rates of hydrolysis of soluble starch, amylose, amylopectin, and glycogen were 1.27, 1.8, 1.94, and 2.28 mg/ml, respectively. V_max values for hydrolysis of these substrates were calculated as 0.738, 1.08, 0.8, and 0.5 mg of maltose/ml per min, respectively. Of the cations, Na⁺, Ca²⁺, and Mg²⁺, showed stimulatory effect, whereas Hg²⁺, Cu²⁺, Ni²⁺, Zn²⁺, Ag⁺, Fe²⁺, Co²⁺, Cd²⁺, Al³⁺, and Mn²⁺ were inhibitory. Of the anions, azide, F⁻, SO₃²⁻, SO₄³⁻, S₂O₃²⁻, MoO₄²⁻, and Wo₄²⁻ showed an excitant effect. p-Chloromercuribenzoic acid and sodium iodoacetate were inhibitory, whereas cysteine, reduced glutathione, thiourea, β-mercaptoethanol, and sodium glycerophosphate afforded protection to enzyme activity. α-Amylase was fairly resistant to EDTA treatment at 30°C, but heating at 90°C in presence of EDTA resulted in the complete loss of enzyme activity, which could be recovered partially by the addition of Cu²⁺ and Fe²⁺ but not by the addition of Ca²⁺ or any other divalent ions.     

Na+ Transport by the A1AO-ATP Synthase Purified from Thermococcus onnurineus and Reconstituted into Liposomes

The ATP synthase of many archaea has the conserved sodium ion binding motif in its rotor subunit, implying that these A₁A_O-ATP synthases use Na⁺ as coupling ion. However, this has never been experimentally verified with a purified system. To experimentally address the nature of the coupling ion, we have purified the A₁A_O-ATP synthase from T. onnurineus. It contains nine subunits that are functionally coupled. The enzyme hydrolyzed ATP, CTP, GTP, UTP, and ITP with nearly identical activities of around 40 units/mg of protein and was active over a wide pH range with maximal activity at pH 7. Noteworthy was the temperature profile. ATP hydrolysis was maximal at 80 °C and still retained an activity of 2.5 units/mg of protein at 45 °C. The high activity of the enzyme at 45 °C opened, for the first time, a way to directly measure ion transport in an A₁A_O-ATP synthase. Therefore, the enzyme was reconstituted into liposomes generated from Escherichia coli lipids. These proteoliposomes were still active at 45 °C and coupled ATP hydrolysis to primary and electrogenic Na⁺ transport. This is the first proof of Na⁺ transport by an A₁A_O-ATP synthase and these findings are discussed in light of the distribution of the sodium ion binding motif in archaea and the role of Na⁺ in the bioenergetics of archaea.     

An Unusual Oxygen-Sensitive, Iron- and Zinc-Containing Alcohol Dehydrogenase from the Hyperthermophilic Archaeon Pyrococcus furiosus

Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100°C by the fermentation of peptides and carbohydrates to produce acetate, CO₂, and H₂, together with minor amounts of ethanol. The organism also generates H₂S in the presence of elemental sulfur (S⁰). Cell extracts contained NADP-dependent alcohol dehydrogenase activity (0.2 to 0.5 U/mg) with ethanol as the substrate, the specific activity of which was comparable in cells grown with and without S⁰. The enzyme was purified by multistep column chromatography. It has a subunit molecular weight of 48,000 ± 1,000, appears to be a homohexamer, and contains iron (~1.0 g-atom/subunit) and zinc (~1.0 g-atom/subunit) as determined by chemical analysis and plasma emission spectroscopy. Neither other metals nor acid-labile sulfur was detected. Analysis using electron paramagnetic resonance spectroscopy indicated that the iron was present as low-spin Fe(II). The enzyme is oxygen sensitive and has a half-life in air of about 1 h at 23°C. It is stable under anaerobic conditions even at high temperature, with half-lives at 85 and 95°C of 160 and 7 h, respectively. The optimum pH for ethanol oxidation was between 9.4 and 10.2 (at 80°C), and the apparent K_ms (at 80°C) for ethanol, acetaldehyde, NADP, and NAD were 29.4, 0.17, 0.071, and 20 mM, respectively. P. furiosus alcohol dehydrogenase utilizes a range of alcohols and aldehydes, including ethanol, 2-phenylethanol, tryptophol, 1,3-propanediol, acetaldehyde, phenylacetaldehyde, and methyl glyoxal. Kinetic analyses indicated a marked preference for catalyzing aldehyde reduction with NADPH as the electron donor. Accordingly, the proposed physiological role of this unusual alcohol dehydrogenase is in the production of alcohols. This reaction simultaneously disposes of excess reducing equivalents and removes toxic aldehydes, both of which are products of fermentation.     

Recombinant production and biochemical characterization of a hyperthermostable α-glucan/maltodextrin phosphorylase from Pyrococcusfuriosus

Alpha-glucan phosphorylase catalyzes the reversible cleavage of α-1-4-linked glucose polymers into α-D-glucose-1-phosphate. We report the recombinant production of an α-glucan/maltodextrin phosphorylase (PF1535) from a hyperthermophilic archaeon, Pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. The apparent 98 kDa recombinant enzyme was active over a broad range of temperatures and pH, with optimal activity at 80 °C and pH 6.5–7. This archaeal protein retained its complete activity after 24 h at 80 °C in Tris-HCl buffer. Unlike other previously reported phosphorylases, the Ni-affinity column purified enzyme showed broad substrate specificity in both the synthesis and degradation of maltooligosaccharides. In the synthetic direction of the enzymatic reaction, the lowest oligosaccharide required for the chain elongation was maltose. In the degradative direction, the archaeal enzyme can produce glucose-1-phosphate from maltotriose or longer maltooligosaccharides including both glycogen and starch. The specific activity of the enzyme at 80 °C in the presence of 10 mM maltoheptaose and at 10 mg ml^–1 glycogen concentration was 52 U mg^–1 and 31 U mg^–1, respectively. The apparent Michaelis constant and maximum velocity for inorganic phosphate were 31 ± 2 mM and 0.60 ± 0.02 mM min^–1 µg^–1, respectively. An initial velocity study of the enzymatic reaction indicated a sequential bi-bi catalytic mechanism. Unlike the more widely studied mammalian glycogen phosphorylase, the Pyrococcus enzyme is active in the absence of added AMP.     

Effect of ATPase inhibitors on cell potential and k influx in corn roots

Experiments were performed to determine the effect of plasmalemma ATPase inhibitors on cell potentials (Ψ) and K⁺ (⁸⁶Rb) influx of corn root tissue over a wide range of K⁺ activity. N,N′Dicyclohexylcarbodiimide (DCCD), oligomycin, and diethylstilbestrol (DES) pretreatment greatly reduced active K⁺ influx and depolarized Ψ at low, but not at high, K⁺ activity (K°). More comprehensive studies with DCCD and anoxia showed nearly complete inhibition of the active component of K⁺ influx over a wide range of K°, with no effect on the apparent permeability constant. DCCD had no effect on the electrogenic component of the cell potential (Ψ_p) above 0.2 millimolar K°. Net proton efflux was rapidly reduced 80 to 90% by DCCD. Since tissue ATP content and respiration were only slightly affected by the DCCD-pretreatment, the inhibitions of active K⁺ influx and Ψ_p at low K° can be attributed to inhibition of the plasmalemma ATPase.     

Purification and Partial Characterization of a Prolyl-Dipeptidyl Aminopeptidase from Lactobacillus helveticus CNRZ 32

X-prolyl-dipeptidyl aminopeptidase, which hydrolyzed Gly-Pro-p-nitroanilide (relative activity [RA] = 100%) and Arg-Pro-p-nitroanilide (RA, 130%), was purified to homogeneity from the cell extract of Lactobacillus helveticus CNRZ 32. The enzyme also hydrolyzed Ala-Pro-Gly (RA, 11%) and Ala-Ala-p-nitroanilide (RA, 2%) but was not active on Ala-Leu-Ala, dipeptides, and endopeptidase and carboxypeptidase substrates. The enzyme was purified 145-fold by streptomycin sulfate precipitation, ammonium sulfate fractionation, and a series of column chromatographies on DEAE-cellulose, arginine-Sepharose 4B, and glycyl-prolyl-AH-Sepharose 4B. The purified enzyme appeared as a single band on native polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoreses and had a molecular weight of 72,000. Optima for activity by the purified enzyme were pH 7.0 and 40°C. The enzyme was incubated at 40°C for 15 min with various metal ions. It was activated by Mg²⁺ (2.5 mM), Ca²⁺ (0.1 to 2.5 mM), Na⁺ (10 to 50 mM), and K⁺ (10 to 50 mM) and was inhibited by Hg²⁺ (0.1 to 2.5 mM), Cu²⁺ (0.1 to 2.5 mM), and Zn²⁺ (0.1 to 2.5 mM). Enzyme activity was partially inhibited by EDTA (1.0 mM, 20 h at 40°C), 1,10-phenanthroline (1.0 mM, 15 min at 40°C), phenylmethylsulfonyl fluoride (1.0 mM), N-ethylmaleimide (1.0 mM), and iodoacetate (1.0 mM). It was completely inhibited by diisopropyl fluorophosphate (1.0 mM, 2 h at 40°C) and p-chloromercuribenzoate (1.0 mM, 15 min at 40°C). The enzyme was not affected by dithioerythritol (1.0 to 10 mM).     

Temperature-dependence of activation and inhibition of rat-brain adenosine triphosphatase activated by sodium and potassium ions

1. The adenosine-triphosphatase activity of rat-brain microsomes was measured between 0° and 37°. The stimulatory effect of Na⁺ plus K⁺ on the Mg²⁺-dependent adenosine-triphosphatase activity decreased sharply with decreasing temperature and became negligible at 0°. An Arrhenius plot drawn from the experimental data showed two discontinuities: one at about 6° and the other at about 20°. 2. The increment in activity induced by Na⁺ plus K⁺ was more sensitive to oligomycin at lower than at higher temperatures, but the opposite was observed for ouabain. The action of oligomycin showed a biphasic character, since below a certain concentration it caused slight activation of Na⁺-plus-K⁺-activated adenosine triphosphatase. 3. Where oligomycin increased the activity of the enzyme, it also enhanced the accumulation of an acid-precipitable phosphorylated compound formed through the transfer of the γ-phosphate group of [³²P]ATP to the enzyme system. Stimulatory concentrations of oligomycin did not interfere with K⁺-mediated dephosphorylation of the intermediate, though high concentrations of oligomycin counteracted the effect of K⁺. 4. The temperature profile of K⁺-stimulated microsomal phosphatase qualitatively resembled that of microsomal adenosine triphosphatase.     

Analysis of Comparative Sequence and Genomic Data to Verify Phylogenetic Relationship and Explore a New Subfamily of Bacterial Lipases

Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca²⁺-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter P_lac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65°C and retained ≥ 97% activity after incubation at 50°C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents.     

Effect of preincubation temperature on in vitro light saturated photosystem I activity in thylakoids isolated from cold hardened and nonhardened rye

Thylakoids isolated from winter rye (Secale cereale L. cv Muskateer) grown at 5°C or 20°C were compared with respect to their capacity to exhibit an increase in light saturated rates of photosystem I (PSI) electron transport (ascorbate/dichlorophenolindophenol → methylviologen) after dark preincubation at temperatures between 0 and 60°C. Thylakoids isolated in the presence or absence of Na⁺/Mg²⁺ from 20°C grown rye exhibited transient, 40 to 60% increases in light saturated rates of PSI activity at all preincubation temperatures between 5 and 60°C. This increase in PSI activity appeared to occur independently of the electron donor employed. The capacity to exhibit this in vitro induced increase in PSI activity was examined during biogenesis of rye thylakoids under intermittent light conditions at 20°C. Only after exposure to 48 cycles (1 cycle = 118 minutes dark + 2 min light) of intermittent light did rye thylakoids exhibit an increase in light saturated rates of PSI activity even though PSI activity could be detected after 24 cycles. In contrast to thylakoids from 20°C grown rye, thylakoids isolated from 5°C grown rye in the presence of Na⁺/Mg²⁺ exhibited no increase in light saturated PSI activity after preincubation at any temperature between 0 and 60°C. This was not due to damage to PSI electron transport in thylakoids isolated from 5°C grown plants since light saturated PSI activity was 60% higher in 5°C thylakoids than 20°C thylakoids prior to in vitro dark preincubation. However, a two-fold increase in light saturated PSI activity of 5°C thylakoids could be observed after dark preincubation only when 5°C thylakoids were initially isolated in the absence of Na⁺/Mg²⁺. We suggest that 5°C rye thylakoids, isolated in the presence of these cations, exhibit light saturated PSI electron transport which may be closer to the maximum rate attainable in vitro than 20°C thylakoids and hence cannot be increased further by dark preincubation.     

Purification and characterization of the extracellular lipase Lip2 from Yarrowia lipolytica

An extracellular lipase from Yarrowia lipolytica (YlLip2) has been purified by ion exchange chromatography on Q sepharose FF, followed by hydrophobic interaction chromatography on butyl sepharose FF. SDS-PAGE showed that the molecular weight of this lipase is about 38 kDa. N-terminal amino acid sequencing and MALDI-TOF mass spectral analysis showed that this lipase is encoded by gene LIP2 (GenBank accession no. AJ012632). Enzymatic deglycosylation showed that this lipase is a glycosylated protein which contains about 12% sugar. The corresponding deglycosylated lipase remained 88% specific activity of untreated lipase. There was a high amino acid sequence identity (91%) between YlLip2 and Candida deformans lipase CdLip1 (GenBank accession no. AJ428393). The optima temperature and pH for the purified lipase was 40 °C and 8.0, respectively. The lipase showed a preference for long chain fatty acid methyl esters (C12–C16), with the highest activity toward methyl myristate (C14). Lipase activity was stimulated by Ca²⁺ and Mg²⁺ and inhibited by Zn²⁺, Ni²⁺ and Cu²⁺, whereas EDTA had no effect on its activity. A 0.1% of Tween 80 and Span 65 increased slightly the enzyme activity and SDS inhibited it.     

A Novel Type II NAD+-Specific Isocitrate Dehydrogenase from the Marine Bacterium Congregibacter litoralis KT71

In most living organisms, isocitrate dehydrogenases (IDHs) convert isocitrate into ɑ-ketoglutarate (ɑ-KG). Phylogenetic analyses divide the IDH protein family into two subgroups: types I and II. Based on cofactor usage, IDHs are either NAD⁺-specific (NAD-IDH) or NADP⁺-specific (NADP-IDH); NADP-IDH evolved from NAD-IDH. Type I IDHs include NAD-IDHs and NADP-IDHs; however, no type II NAD-IDHs have been reported to date. This study reports a novel type II NAD-IDH from the marine bacterium Congregibacter litoralis KT71 (ClIDH, GenBank accession no. EAQ96042). His-tagged recombinant ClIDH was produced in Escherichia coli and purified; the recombinant enzyme was NAD⁺-specific and showed no detectable activity with NADP⁺. The K _m values of the enzyme for NAD⁺ were 262.6±7.4 μM or 309.1±11.2 μM with Mg²⁺ or Mn²⁺ as the divalent cation, respectively. The coenzyme specificity of a ClIDH Asp487Arg/Leu488His mutant was altered, and the preference of the mutant for NADP⁺ was approximately 24-fold higher than that for NAD⁺, suggesting that ClIDH is an NAD⁺-specific ancestral enzyme in the type II IDH subgroup. Gel filtration and analytical ultracentrifugation analyses revealed the homohexameric structure of ClIDH, which is the first IDH hexamer discovered thus far. A 163-amino acid segment of CIIDH is essential to maintain its polymerization structure and activity, as a truncated version lacking this region forms a non-functional monomer. ClIDH was dependent on divalent cations, the most effective being Mn²⁺. The maximal activity of purified recombinant ClIDH was achieved at 35°C and pH 7.5, and a heat inactivation experiment showed that a 20-min incubation at 33°C caused a 50% loss of ClIDH activity. The discovery of a NAD⁺-specific, type II IDH fills a gap in the current classification of IDHs, and sheds light on the evolution of type II IDHs.     

Lipase from Pseudomonas aeruginosa LP602: biochemical properties and application for wastewater treatment

Lipase from Pseudomonas aeruginosa LP602, a bacterial strain isolated from a domestic wastewater sample, was preliminarily characterized. The enzyme exhibited maximum lipolytic activity at pH 8.0 where it was also stably maintained. At 55°C, the lipase had the highest activity but not stability. The enzyme was insensitive to EDTA and to many ions tested except Zn²⁺. It was sensitive to SDS but not to Tween-20, Tween-80 or Triton X-100. The enzyme was active towards a number of commercial food grade fats and oils. A suitable medium formula for lipase production was MMP containing 6.25% whey as a carbon source, 1% soybean oil as inducer and 0.5% yeast extract supplement. The culture was fed with glucose to a final concentration of 0.1% at the 15th hour of incubation. Lipase production under this condition was 3.5 U ml⁻¹. Both P. aeruginosa LP602 cells and the lipase were shown to be usable for lipid-rich wastewater treatment. Received 21 April 1998/ Accepted in revised form 6 August 1998     

Properties of Solubilized Microsomal Lipase from Germinating Brassica napus

Lipase (triacylglycerol acylhydrolase [EC 3.1.1.3.]) was extracted from the microsomal fraction of cotyledons of dark grown seedlings of Canola (Brassica napus L. cv Westar) by treatment with Triton X-100. The enzyme was partially purified by chromatography on Sephacryl S-300 and DEAE Bio-Gel and was stable when stored at −20°C in 50% (v/v) glycerol. The lipase aggregated readily but the distribution of species present in solution could be controlled by nonionic detergents. A species with an apparent M_r of about 250,000 was obtained by gel filtration chromatography in the presence of 1% (v/v) Triton X-100. Lipase activity was optimal near neutral pH, and the reaction approached maximum velocity at a concentration of 0.5 to 1 millimolar emulsified triolein. The reaction rate responded linearly to temperature up to about 40°C and the hydrolytic process had an activation energy of 18 kilocalories per mole. Microsomal lipase lost about 20% and 80% activity when heat-treated for 1 hour at 40°C and 60°C, respectively. At appropriate concentrations, the detergents Triton X-100, n-octyl-β-d-glucopyranoside, (3-[(3-cholamidopropyl-O-dimethylammonio]-1-propanesulfonate, cetyl trimethylammonium bromide, and sodium dodecyl sulfate all inhibited lipase activity. n-Octyl-β-d-glucopyranoside, however, was stimulatory in the 2 to 8 millimolar concentration range. The inhibitory effects of Triton X-100 were reversible.     

Warm growth temperatures decrease soybean cholinephosphotransferase activity

The activity of cytidine 5′-diphosphate (CDP) choline: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) in developing soybean (Glycine max L. var Williams 82) seeds was 3 to 5 times higher in cotyledons grown at 20°C than in those grown at 35°C. Some characteristics of the enzyme from cotyledons cultured at 20 and 35°C were compared. In preparations from both growth temperatures, the enzyme showed a pH optimum of 7, K_m of 7.0 micromolar for CDP-choline, and an optimum assay temperature of 45°C. Both enzyme preparations were stimulated by increasing concentrations of Mg²⁺ or Mn²⁺, up to 10 millimolar and 50 micromolar, respectively, though Mn²⁺ produced lower activities than Mg²⁺. Enzymes from both 20 and 35°C show the same specificity for exogenous diacylglycerol. No metabolic effectors were detected by addition of heat treated extracts to the assay mixture. The above findings suggest that the higher enzyme activity at 20°C can be attributed to a higher level of the enzyme rather than to the involvement of isozymes or metabolic effectors. Enzyme activity decreased rapidly during culture at 35°C, indicating a rapid turnover of the enzyme. The level of temperature modulation was found to be a function of seed developmental stage.