Vanadium-dependent bromoperoxidases from <Emphasis Type="Italic">Gracilaria</Emphasis> algae

Red algae from the Gulf of Thailand were examined for haloperoxidatic activity. Six species, Gracilaria changii, G. edulis, G. firma, G. fisheri, G. salicornia, and G. tenuistipitata, showed bromoperoxidatic activity. Duplicate polyacrylamide electrophoretic gels showed enzyme activity patterns developed by phenol red staining for bromoperoxidatic activity and by 3,3′-diaminobenzidine staining for peroxidatic activity. All algae gave isoenzymic bromoperoxidatic activity bands and peroxidatic activity bands, but there were peroxidatic and bromoperoxidatic activity bands that did not correspond. The bromoperoxidatic activity of the crude enzyme extracts as well as previously dialyzed enzyme solutions was enhanced significantly by incubation with vanadium pentoxide. The three purified bromoperoxidases from G. fisheri contained vanadium, and their relative activities corresponded to the ratio of vanadium to enzyme. In addition, they were not inhibited by H₂O₂. These data confirm that the enzymes are vanadium bromoperoxidases.     

Glucose-6P dehydrogenase in Chlorella sorokiniana (211/8k): an enzyme with unusual characteristics

In Chlorella sorokiniana (211/8k), glucose-6 phosphate dehydrogenase (G6PDH—EC 1.1.1.49) activity is similar in both N-starved cells and nitrate-grown algae when expressed on a PCV basis. A single G6PDH isoform was purified from Chlorella cells grown under different nutrient conditions; the presence of a single G6PDH was confirmed by native gels stained for enzyme activity and by Western blots. The algal G6PDH is recognised only by antibodies raised against higher plants plastidic protein, but not by chloroplastic and cytosolic isoform-specific antisera. Purified G6PDH showed kinetic parameters similar to plastidic isoforms of higher plants, suggesting a different biochemical structure which would confer peculiar regulative properties to the algal G6PDH with respect to higher plants enzymes. The most remarkable property of algal G6PDH is represented by the response to NADPH inhibition. The algal enzyme is less sensitive to NADPH effects compared to higher plants G6PDH: Ki_NADPH is 103 μM for G6PDH from nitrogen-starved C. sorokiniana, similarly to root plastidic P2-G6PDH. In nitrate-grown C. sorokiniana the Ki_NADPH decreased to 48 μM, whereas other kinetic parameters remained unchanged. These results will allow further investigations in order to rule out possible modifications of the enzyme, and/or the expression of a different G6PDH isoform during nitrate assimilation.     

The effect of astaxanthin and cadmium on rat erythrocyte G6PD, 6PGD,GR, and TrxR enzymes activities in vivo and on rat erythrocyte 6PGD enzyme activity in vitro

In this study, the effects of astaxanthin (AST) that belongs to carotenoid family and cadmium (Cd), which is an important heavy metal, on rat erythrocyte G6PD, 6PGD, GR, and TrxR enzyme activities in vivo and on rat erythrocyte 6PGD enzyme activity in vitro were studied. In in vitro studies, 6PGD enzyme was purified from rat erythrocytes with 2′,5′‐ADP Sepharose4B affinity chromatography. Results showed inhibition of enzyme by Cd at IC₅₀; 346.5 μM value and increase of 6PGD enzyme activity by AST. In vivo studies showed an increase in G6PD, 6PGD, and GR enzyme activities (P ? 0.05) and no chance in TrxR enzyme activity by AST. Cd ion inhibited G6PD, 6PGD, and GR enzyme activities (P ? 0.05) and also decreased TrxR enzyme activity (P ? 0.05). AST + Cd group G6PD enzyme activity was statistically low compared with control group (P ? 0.05). 6PGD and TrxR enzyme activities decreased without statistical significance (P ? 0.05); however, GR enzyme activity increased statistically significantly (P ? 0.05).     

Substitutions of Thr-103-Ile and Trp-138-Gly in amidase from Pseudomonas aeruginosa are responsible for altered kinetic properties and enzyme instability

Pseudomonas aeruginosa Ph1 is a mutant strain derived from strain AI3. The strain AI3 is able to use acetanilide as a carbon source through a mutation (T103I) in the amiE gene that encodes an aliphatic amidase (EC 3.5.1.4). The mutations in the amiE gene have been identified (Thr103Ile and Trp138Gly) by direct sequencing of PCR-amplified mutant gene from strain Ph1 and confirmed by sequencing the cloned PCR-amplified gene. Site-directed mutagenesis was used to alter the wild-type amidase gene at position 138 for Gly. The wild-type and mutant amidase genes (W138G, T103I-W138G, and T103I) were cloned into an expression vector and these enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide/phenylacetamide followed by gel filtration chromatography. Altered amidases revealed several differences in kinetic properties, namely, in substrate specificity, sensitivity to urea, optimum pH, and enzyme stability, compared with the wild-type enzyme. The W138G enzyme acted on acetamide, acrylamide, phenylacetamide, and p-nitrophenylacetamide, whereas the double mutant (W138G and T103I) amidase acted only on p-nitrophenylacetamide and phenylacetamide. On the other hand, the T103I enzyme acted on p-nitroacetanilide and acetamide. The heat stability of altered enzymes revealed that they were less thermostable than the wild-type enzyme, as the mutant (W138G and W138G-T103I) enzymes exhibited t _1/2 values of 7.0 and 1.5 min at 55°C, respectively. The double substitution T103I and W138G on the amidase molecule was responsible for increased instabiliby due to a conformational change in the enzyme molecule as detected by monoclonal antibodies. This conformational change in altered amidase did not alter its M _r value and monoclonal antibodies reacted differently with the active and inactive T103I-W138G amidase.     

Transfer Action of Cyclodextrin Glycosyltransferase on Starch

The transglycosylation reaction of the cyclodextrin glycosyltransferase from Bacillus megaterium (No. 5 enzyme) and Bacillus macerans (BMA) were examined. No. 5 enzyme was more efficient in transglycosylation reaction than BMA in the every acceptor employed in the present study. The order of the efficient acceptors for No. 5 enzyme was maltose (G2), glucose (Gl), maltotriose (G3) and sucrose (GF). On the other hand, that found for BMA was Gl, G2, GF and G3. The transglycosylation products to glucose formed by the action of No. 5 enzyme on starch were G2, G3, maltotetraose (G4), maltopentaose (G5), maltohexaose (G6) and maltoheptaose (G7) in the order of their quantities, while, in the case of BMA, they were G2, G3, G5, G7=G4 and G6. The larger transglycosylation products to sucrose formed by the action of No. 5 enzyme on starch were maltosylfructose. On the other hand, that formed by the action of BMA was maltoheptaosylfructose.

It was suggested that cyclodextrin glycosyltransferase could transfer the glucosyl residues to an acceptor directly from starch, as well as through cyclodextrin.     

Heterologous expression and characterization of a glucose-stimulated β-glucosidase from the termite <Emphasis Type="Italic">Neotermes koshunensis</Emphasis> in <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

Neotermes koshunensis is a lower termite that secretes endogenous β-glucosidase in the salivary glands. This β-glucosidase (G1NkBG) was successfully expressed in Aspergillus oryzae. G1NkBG was purified to homogeneity from the culture supernatant through ammonium sulfate precipitation and anion exchange, hydrophobic, and gel filtration chromatographies with a 48-fold increase in purity. The molecular mass of the native enzyme appeared as a single band at 60 kDa after gel filtration analysis, indicating that G1NkBG is a monomeric protein. Maximum activity was observed at 50 °C with an optimum pH at 5.0. G1NkBG retained 80% of its maximum activity at temperatures up to 45 °C and lost its activity at temperatures above 55 °C. The enzyme was stable from pH 5.0 to 9.0. G1NkBG was most active towards laminaribiose and p-nitrophenyl-β-d-fucopyranoside. Cellobiose, as well as cello-oligosaccharides, was also well hydrolyzed. The enzyme activity was slightly stimulated by Mn²⁺ and glycerol. The K _m and V _max values were 0.77 mM and 16 U/mg, respectively, against p-nitrophenyl-β-d-glucopyranoside. An unusual finding was that G1NkBG was stimulated by 1.3-fold when glucose was present in the reaction mixture at a concentration of 200 mM. These characteristics, particularly the stimulation of enzyme activity by glucose, make G1NkBG of great interest for biotechnological applications, especially for bioethanol production.     

Action of Cyclodextrin Glycosyltransferase from Bacillus megaterium Strain No. 5 on Starch

The amounts of the cyclodextrins G6, G7 and G8 produced by the action of the enzyme from Bacillus megaterium (No. 5 enzyme) and Bacillus macerans amylase (BMA) on starch-¹⁴C (U) were determined by the calculation of radioactivity. Both fractions of No. 5 enzyme produced the cyclodextrin G6, G7 and G8 in the proportion of 1: 2.4: 1. On the other hand, BMA produced the cyclodextrin G6, G7 and G8 in the proportion of 2.7: 1:1. The cyclodextrin G6 and G8 which are smaller parts of the reaction products by both fractions of No. 5 enzyme were found to be produced directly from starch, not from the redecomposition of cyclodextrin G7. The ratio of the cyclodextrin G6, G7 and G8 were almost constant, regardless of the pH range of the reaction system.

By using the maltooligosaccharides terminated at the reducing end by radioactive glucose, the action of both fractions of No. 5 enzyme and BMA on the maltooligosaccharides were compared with each other. The results showed that both fractions of No. 5 enzyme acted on oligosaccharides larger than maltose, producing the radioactive glucose as the major product from each maltooligosaccharide (G2~G8). On the other hand, BMA acted on oligosaccharides larger than maltotriose, producing the radioactive maltose as the major product.     

Asymmetric Reduction of Iminium Salt with Chiral Dihydropyridines

A gram positive bacterium (strain No. 109) isolated from soil as a producer of cyclodextrinase was identified as Bacillus coagulans. The cyclodextrinase from B. coagulans was purified to a homogeneous state by disc-electrophoresis after Streptomycin treatment, DEAE-Sephadex column chromatography, Ultrogel AcA44 gel filtration and hydroxyapatite column chromatography. The molecular weight of the enzyme was determined to be 6.2}10⁴ by sodium dodecyl-sulfate gel electrophoresis. The isoelectric point of the enzyme was pH 5.0. The enzyme was most active at pH 6.2 and 50°C, and stable up to 45°C at pH 7.0 and in the range of pH 6.0 ~ 7.3 at 40°C on 2 hr incubation. This enzyme hydrolyzed linear maltooligosaccharides (such as maltotetraose (G4), maltopentaose (G5) and maltohexaose (G6)) and α-, β- and α-cyclodextrins (CDs) faster than maltotriose (G3) and short chain amylose ( 18), but did not hydrolyze maltose. The rates of hydrolysis for polysaccharides (such as starch, amylose and amylopectin) were below 1 % as compared to that for β-CD. The Km values for G3, G4, G5, G6, short chain amylose ( 18) and α, β- and γ-CD were 4.5, 4.0,2.3,1.5,1.5,10,2.8 and 0.47 mM, respectively. The products with this enzyme had the α-configulation.     

Lack of correlation between binding of Eco RII methyltransferase to DNA duplexes containing mismatches and the promotion of C to T mutations

The cytosine methyltransferases (MTases) M. HhaIand M. HpaII bind substrates in which the target cytosine is replaced by uracil or thymine, i.e. DNA containing a U:G or a T:G mismatch. We have extended this observation to the EcoRII MTase (M. EcoRII) and determined the apparent K_d for binding. Using a genetic assay we have also tested the possibility that MTase binding to U:G mismatches may interfere with repair of the mismatches and promote C:G to T:A mutations. We have compared two mutants of M. EcoRII that are defective for catalysis by the wild-type enzyme for their ability to bind DNA containing U:G or T:G mismatches and for their ability to promote C to T mutations. We find that although all three proteins are able to bind DNAs with mismatches, only the wild-type enzyme promotes C:G to T:A mutations in vivo. Therefore, the ability of M. EcoRII to bind U:G mismatched duplexes is not sufficient for their mutagenic action in cells. Received: 14 November 1996 / Accepted: 17 February 1997     

Genetic regulation of glucose 6-phosphate dehydrogenase activity in the inbred mouse

The erythrocyte glucose 6-phosphate dehydrogenase activity characteristic of each of 16 inbred mouse strains falls into one of three distinct classes. Strains C57L/J and C57BR/cdJ represent the low activity class: strains A/J and A/HeJ represent the high activity class; other strains have intermediate activities. There is no evidence that structural variation is responsible for the variation in G6PD activity, since partially purified enzyme from each class has the same thermal stability, pH-activity profile, Michaelis constants for G6P and NADP, electrophoretic mobility, and activity using 2-deoxy d-glucose 6-phosphate as substrate. The activities of 6-phosphogluconate dehydrogenase and glucose phosphate isomerase do not differ in erythrocytes of the three G6PD activity classes. Young red cells have higher G6PD activities than old red cells and there is evidence that the intracellular stability of the enzyme is reduced in red cells of strain C57L/J. G6PD activities in kidney and skeletal and cardiac muscle from animals with low red cell G6PD are slightly lower than the activities in kidney and muscle from animals with high red cell G6PD activity. The quantitative differences in red cell G6PD activity are not regulated by X-linked genes, but by alleles at two or more autosomal loci. A simple genetic model is proposed in which alleles at two unlinked, autosomal loci, called Gdr-1 and Gdr-2 regulate G6PD activity in the mouse erythrocyte.     

Improved <Emphasis Type="Italic">A. faecalis</Emphasis> Penicillin Amidase Mutant Retains the Thermodynamic and pH Stability of the Wild Type Enzyme

Penicillin amidase from Alacaligenes faecalis is an attractive biocatalyst for hydrolysis of penicillin G for production of 6-aminopenicillanic acid, which is used in the synthesis of semi-synthetic β-lactam antibiotics. Recently a mutant of this enzyme with extended C-terminus of the A-chain comprising parts of the connecting linker peptide was constructed. Its turnover number for the hydrolysis of penicillin G was 140 s⁻¹, about twice of the value for the wild-type enzyme (80 s⁻¹). At the same time the specificity constant was improved about three-fold. The wild-type and the mutant enzymes showed similar pH stability suggesting that the linker peptide fragment covalently attached to the A-chain does not alter the electrostatic interactions in the protein core. Although the global stability of A. faecalis wild-type enzyme and the T206GS213G variant does not differ, the presence of the linker fragment stabilizes the domains interface, as evidenced by the monophasic transition of the mutant enzyme from folded to unfolded state during urea-induced denaturation. The high stability and activity of the mutant enzyme provides a rationale to use it as a biocatalyst in the industrial processes, where the enzyme must be more robust to fluctuations in the operational conditions.     

Development and characterization of a potent producer of penicillin G amidase by mutagenization

Summary The penicillin G amidase (PGA) activity of a parent strain of E. coli (PCSIR-102) was enhanced by chemical mutagenization with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). After screening and optimization, a penicillinase deficient mutant (MNNG-37) was isolated and found effective for the production of penicillin G amidase as compared to the parent strain of E. coli (PCSIR-102). Penicillin G amidase activity of MNNG-37 appeared during an early stage of growth, whereas PCSIR-102 did not exhibit PGA activity due to the presence of penicillinase enzyme which inhibits the activity of enzyme PGA. However, MNNG-37 gave a three-fold increase in enzyme activity (231 IU mg⁻¹) as compared to PCSIR-102 (77 IU mg⁻¹) in medium containing 0.15 and 0.1% concentrations of phenylacetic acid, respectively which was added after 6 h of cultivation. The difference in K _m values of the enzyme produced by parent strain PCSIR-102 (0.26 mM) and mutant strain MNNG-37 (0.20 mM) is significant (1.3-fold increase in K _m value) which may show the superiority of the latter in terms of better enzyme properties.     

Regulation of the pentose phosphate pathway at fertilization in sea urchin eggs

Summary

After fertilization of sea urchin eggs, there is a rapid increase in cellular levels of NADPH, a metabolite utilized in a variety of biosynthetic reactions during early development. Recent studies have shown that a dramatic increase in the activity of the pentose phosphate shunt occurs in vivo shortly after fertilization, consistent with the hypothesis mat this metabolic pathway is a major supplier of NADPH in sea urchin zygotes. One mechanism that may account, in part, for this increase in pentose shunt activity is the dissociation of glucose-6-phosphate dehydrogenase (G6PDH), the first enzyme of the shunt, from cell structural elements. In vitro, G6PDH is associated with the insoluble matrix obtained from homogenates of unfertilized eggs, and in this state, the enzyme is inhibited. Within minutes of fertilization, G6PDH is released as an active, soluble enzyme. A similar solubilization and activation of G6PDH occurs after fertilization of eggs of other marine invertebrates and in mammalian cells in culture stimulated by growth factors. The occurrence of this phenomenon in such diverse cell types, in response to different stimuli, suggests that the redistribution of G6PDH between insoluble and soluble locations may be involved in the regulation of the pentose phosphate shunt during cell activation in general.     

Degradation of naphthalene by thermophilic bacteria <Emphasis Type="Italic">via</Emphasis> a pathway,through protocatechuic acid

A number of thermophilic bacteria capable of utilizing naphthalene as a sole source of carbon were isolated from a high-temperature oilfield in Lithuania. These isolates were able to utilize several other aromatic compounds, such as anthracene, benzene, phenol, benzene-1, 3-diol, protocatechuic acid as well. Thermophilic isolate G27 ascribed to Geobacillus genus was found to have a high aromatic compound degrading capacity. Spectrophotometric determination of enzyme activities in cell-free extracts revealed that the last aromatic ring fission enzyme in naphthalene biotransformation by Geobacillus sp. G27 was inducible via protocatechuate 3, 4-dioxygenase; no protocatechuate 4, 5-dioxygenase, protocatechuate 2, 3-dioxygenase activities were detected. Intermediates such as o-phthalic and protocatechuic acids detected in culture supernatant confirmed that the metabolism of naphthalene by Geobacillus sp. G27 can proceed through protocatechuic acid via ortho-cleavage pathway and thus differs from the pathways known for mesophilic bacteria.     

Studies on Transglycosidation to Vitamin B6 by Microorganisms

The enzyme activity to synthesize pyridoxine glucoside was demonstrated in intact cells and cell extracts of genera, Sarcina and Micrococcus. The isolated and identified strain, Micrococcus sp. No. 431 was found to have high activity of this enzyme in its cell extract.

The enzyme activity reached to a maximum after 20 hr of cultivation.

The enzyme which synthesized pyridoxine glucoside via transglucosidation from sucrose to pyridoxine was purified from Micrococcus sp. No. 431 by means of ammonium sulfate fractionation, DEAE-Sephadex, hydroxylapatite and Sephadex G–100 column chromatographies. The enzyme was purified about 354–fold and confirmed to be homogenous in polyacrylamide-gel electrophoresis and ultracentrifugation.     

Purification and Some Properties of Dipeptidyl Carboxypeptidase from Bacillus pumilus

An intracellular protease from a bacterium, Bacillus pumilus HL721, was purified about 5000-fold by Chromatography with a Q-Sepharose Fast Flow column, TSK-gel HA-1000 glass column, and TSK-gel G3000SWXL column using Bz-Gly-Ala-Pro as a substrate. The enzyme was the most active at pH around 7.5 and stable from 4.5 and 8.0. The enzyme activity was inhibited by Cu²⁺, EDTA, N-ethylmaleimide, o-phenanthroline, and p-chloromercuribenzoic acid. The molecular weight of the enzyme was 155,000 by gel filtration. The enzyme removed dipeptide from the carboxyl end of some peptides used as substrates. From these results the enzyme seems to be a dipeptidyl carboxypeptidase.     

Isolation of Phytolacain G and Its Inhibition Measured by Affinity Labeling

A cysteine protease, phytolacain G, was purified to homogeneity from unripe fruits of pokeweed (Phytolacca americana). The apparent molecular mass of the purified phytolacain G was 25.5 kDa. The caseinolytic activity of the enzyme was completely inhibited by a synthetic peptide containing an S-(3-nitro-2-pyridinesulfenyl) group (Npys). The inhibitory activity of this compound against phytolacain G resembled that for papain.     

Purification and characterization of the glucoside 3-dehydrogenase produced by a newly isolated Stenotrophomonas maltrophilia CCTCC M 204024

A soluble glucoside 3-dehydrogenase (G3DH) from Stenotrophomonas maltrophilia CCTCC M 204024, recently isolated from wheat soil in our laboratory, was purified to 37.4-fold with a yield of 24.7% and was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 66 kDa. 2,6-Dichlorophenolindophenol (DCPIP) and ferricyanide were able to act as artificial electron acceptors for the enzyme. The optimal pH of G3DH was in the range of 6.0–7.0 in the presence of DCPIP. The enzyme was stable in the pH range of 4.4–10.6 and was sensitive to heat. G3DH exhibited extremely broad substrate specificity by converting many sugars to their corresponding 3-ketoglucosides. They produced a characteristic spectrum by alkaline treatment with a peak at 340 nm. The apparent K _m values for validoxylamine A and d-glucose were 8.3 and 1.1 mM, respectively. Cu²⁺, Ag²⁺, and Hg₂Cl₂ inhibited the activity of G3DH.     

The identification of the potato cyst nematodes Globodera rostochiensis and G. pallida by isoelectric focusing of proteins on polyacrylamide gels

Isoelectric focusing on thin layers of polyacrylamide gel was used to separate proteins from the potato cyst-nematodes Globodera rostochiensis and G. pallida. General protein patterns could be used to identify the two species, even from. single cysts. Staining for the enzyme phosphoglucomutase (PGM) also produced species specific patterns and the presence of PGM variation among G. pallida populations was revealed. The potential of isolectric focusing as a routine research and advisory tool in nematology is discussed.