Dual pH Optima Research Articles

Comparison of Type 1 D-3-phosphoglycerate dehydrogenases reveals unique regulation in pathogenic Mycobacteria

D-3-phosphoglycerate dehydrogenases (PGDH) from all organisms catalyze the conversion of D-3-phosphoglycerate to phosphohydroxypyruvate as the first step in the biosynthesis of l-serine. This investigation compares the properties of Type 1 PGDHs from seven different species and demonstrates that conserved residues in the ACT and ASB domains of some allow l-serine to act as a feedback inhibitor at low micromolar concentrations. In addition, the serine sensitivity is dependent on the presence of phosphate ions. These residues are most highly conserved among PGDHs from the actinomycetales family, but only certain pathogenic mycobacteria appear to have the full complement of residues required for high sensitivity to serine. These basic residues are also responsible for the presence of dual pH optima in the acidic region that is also phosphate dependent. Analytical ultracentrifugation analysis demonstrates that the dual pH optima do not require changes in oligomeric state. This study also demonstrates that substrate inhibition is a common feature of Type 1 PGDHs and that it is suppressed by phosphate, indicating that phosphate likely interacts at both the catalytic and regulatory sites. The unique features resulting from the complement of basic residues conserved in pathogenic mycobacteria may impart important metabolic advantages to these organisms.

Production of phytase under solid-state fermentation using Rhizopus oryzae: Novel strain improvement approach and studies on purification and characterization

Present study introduces linseed oil cake as a novel substrate for phytase production by Rhizopus oryzae. Statistical approach was employed to optimize various medium components under solid state fermentation (SSF). An overall 8.41-fold increase in phytase production was achieved at the optimum concentrations (w/w, mannitol, 2.05%; ammonium sulfate, 2.84% and phosphate, 0.38%). Further enhancement by 59% was observed due to a novel strain improvement approach. Purified phytase (∼34kDa) showed optimal temperature of 45°C, dual pH optima at 1.5 and 5.5 and possesses high catalytic efficiency (2.38×106M−1s−1). Characterization study demonstrates the phytase as highly thermostable and resistant to proteolysis, heavy metal ions, etc. Furthermore, an improved HPLC method was introduced to confirm the ability of phytase to degrade phytic acid completely and was found to be an efficient method.

Expression and enzymatic characterization of the soluble recombinant plasmepsin I from Plasmodium falciparum

The plasmepsins are involved in the degradation of host cell hemoglobin during malaria infection. Plasmepsin I (PM I) initiates the degradative process, and has been suggested as an attractive target for the treatment of malaria. The production of active recombinant PM I, however, has been challenging. We report for the first time, the expression and partial characterization of soluble recombinant PM I from Plasmodium falciparum in which a truncated form of PM I (Lys77P-Leu329) (P indicates a propart residues) was fused to thioredoxin in the pET32b(+) vector, Trx-tPM I and expressed in Escherichia coli Rosetta-gami B (DE3)pLysS. The soluble fusion protein was purified from cell culture using a combination of Ni(2+) affinity and gel filtration chromatography and was capable of autocatalytic activation at pH 4.0-5.5, which occurred at Leu116P-Ser117P, seven residues upstream of the native cleavage site (Gly123P-Asn1). The mature tPM I (mtPM I) was capable of hydrolyzing both human hemoglobin with a pH optimum of pH 2.8-4.0 and the synthetic fluorogenic peptide EDANS-CO-CH(2)-CH(2)-CO-ALERMFLSFP-Dap(DABCYL)-OH with a dual pH optima of pH 2.5-3.0 and pH 4.5-5.5. Using the synthetic substrate, mtPM I exhibited kinetic parameters comparable to native PM I.

A Novel Mechanism for Substrate Inhibition in Mycobacterium tuberculosisd-3-Phosphoglycerate Dehydrogenase

Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase undergoes significant inhibition of activity with increasing concentrations of its substrate, hydroxypyruvic acid phosphate. The enzyme also displays an unusual dual pH optimum. A significant decrease in the K(i) for substrate inhibition at pH values corresponding to the valley between these optima is responsible for this phenomena. The change in K(i) has an average pK of approximately 5.8 and involves two functional groups that are protonated and two functional groups that are unprotonated for optimal substrate inhibition to occur. Mutagenesis of positively charged amino acid residues at a putative anion binding site previously revealed by the x-ray structure, produces significant changes in the pH-dependent profile of substrate inhibition. Several single residue mutations eliminate the dual pH optima by reducing substrate inhibition between pH 5 and 7 and a triple mutation was identified that eliminates the substrate inhibition altogether. The mutagenesis data support the conclusion that the anion binding site represents a new allosteric site for the control of enzyme activity and functions in a novel mechanism for substrate inhibition.

Purification, characterization and comparison of reptile lysozymes

Cation exchange column chromatography and gel filtration chromatography were used to purify four reptile lysozymes from egg white: SSTL A and SSTL B from soft shelled turtle ( Trionyx sinensis), ASTL from Asiatic soft shelled turtle ( Amyda cartilagenea) and GSTL from green sea turtle ( Chelonia mydas). The molecular masses of the purified reptile lysozymes were estimated to be 14 kDa by SDS-PAGE. Enzyme activity of the four lysozymes could be confirmed by gel zymograms and showed charge differences on native-PAGE. SSTL A, SSTL B and ASTL had sharp pH optima of about pH 6.0, which contrasts with that of GSTL, which showed dual pH optima at about pH 6.0 and pH 8.0. The activities of the reptile lysozymes rapidly decreased within 30 min of incubation at 90 °C except for ASTL, which was more stable. Partial N-terminal amino acid sequencing and peptide mapping strongly suggested that the enzymes were C-type lysozymes. Interestingly, the mature SSTL lysozymes show an extra Gly residue at the N-terminus, which was previously found in soft-shelled turtle lysozyme. The reptile lysozymes showed lytic activity against several species of bacteria, such as Micrococcus luteus and Vibrio cholerae, but showed only weak activity to Pseudomonas aeruginosa and lacked activity towards Aeromonas hydrophila.

Identification of a novel estrogen-sulfating cytosolic SULT from zebrafish: Molecular cloning, expression, characterization, and ontogeny study

By searching the expressed sequence tag database, a zebrafish cDNA encoding a putative cytosolic sulfotransferase (SULT) was identified. Sequence analysis indicated that this zebrafish SULT belongs to the SULT1 cytosolic SULT gene family. The recombinant form of this novel zebrafish SULT, expressed using the pGEX-2TK expression system and purified from transformed BL21 (DE3) Escherichia coli cells, displayed sulfating activities specifically for estrone and 17β-estradiol among various endogenous compounds tested as substrates. The enzyme also exhibited sulfating activities toward some xenobiotic phenolic compounds. This new zebrafish SULT showed dual pH optima, at 6.5 and 10–10.5, with estrone or n-propyl gallate as substrate. Kinetic constants of the sulfation of estrone, 17β-estradiol, and n-propyl gallate were determined. Developmental stage-dependent expression experiments revealed a significant level of expression of this novel zebrafish estrogen-sulfating SULT at the beginning of the hatching period during embryogenesis, which continued throughout the larval stage onto maturity.

Digestive protease characterization, localization and adaptation in blacklip abalone (Haliotis rubraLeach)

Digestive protease in blacklip abalone (Haliotis rubra Leach) from whole gut extracts was found to have dual pH optima at pH 3 and 10 and an optimum temperature of 45 °C. Over the biologically relevant range of pH (5–8), protease activity dropped to a minimum at pH 5 (53% of the maximal rate at pH 3) rising gradually and continuously up to (and beyond) pH 8. Over the biologically relevant range of temperatures (9–24 °C), protease activity increased continuously with activity at 24 °C being 75% higher than activity at 9 °C. Protease digestion was relatively uniform in gut extracts from sections containing gut contents. Digestion thus appears to be significantly extracellular (in the lumen of the gut). Analysis of gut sections washed free of food matter suggests the anatomical origin of protease from activity of gut regions in the order: digestive gland » salivary gland ≈ stomach > crop > intestine > upper oesophagus. Whole gut protease levels did not alter in response to a high protein artificial diet.

Improved xylanase production from a haloalkalophilic Staphylococcus sp. SG-13 using inexpensive agricultural residues

The production of an alkali-stable xylanase, with dual pH optima, from haloalkalophilic Staphylococcus sp. SG-13 has been enhanced using agro-residues in submerged fermentation and a biphasic growth system. The agro-residues such as wheat bran, sugarcane bagasse, corncobs and poplar wood when used as sole carbon source, improved the xylanase yield by five-fold as compared to xylose and xylan. Staphylococcus sp. SG-13 also produced equally good amounts of xylanase when grown simply in deionized water (pH 8.0) supplemented with agro-residues as sole carbon source. In the biphasic growth system (lower layer containing agricultural residue set in agar medium with liquid medium above it), the prime substrate, wheat bran (1% w/v), resulted in maximum xylanase production of 4525 U l−1 (pH 7.5) and 4540 U l−1 (pH 9.2) at an agar: broth ratio of 4.0 after 48 h of incubation at 37 °C under static conditions. In general, the cost-effective agro-residues were found to be more suitable inducers for xylanase production over expensive substrates like xylan.

Isolation, purification and characterization of xylanasefrom Staphylococcus sp. SG-13 and its application in biobleaching of kraft pulp.

A haloalkalophilic Staphylococcus sp. SG-13 produced an alkalistable xylanase in wheat bran medium. A 12-fold purification was achieved by using standard purification techniques. The purified xylanase exhibited a dual pH optima of 7.5 and 9.2. The optimum temperature for enzyme activity was 50 degrees C. The enzyme was stable at 50 degrees C for more than 4 h. The xylanase exhibited Km and Vmax values of 4 mg ml-1, 90 micromol min-1 per mg for birchwood xylan and 7 mg ml-1, 55 micromol min-1 per mg for oatspelt xylan, respectively. The substrate binding affinity of xylanase was more for oatspelt xylan but birchwood xylan was hydrolysed more rapidly. The xylanase activity was stimulated by Fe2+, Ni2+, Cu2+ and dithiothreitol up to 60% and was strongly inhibited in the presence of Co2+, Hg2+, Pb2+, phenyl methane sulphonyl fluoride, ethylenediaminetetraacetic acid, and acetic anhydride up to 100%. The xylanase dose of 1.8 U g-1 moisture free pulp, exhibited bleach boosting of kraft pulps optimally at pH 9.5-10.0 and 50 degrees C after 4 h of reaction time. Pretreatment of pulp with xylanase and its subsequent treatment with 8% hypochlorite, reduced the kappa number by 30%, enhanced the brightness and viscosity by 11% and 1.8%, respectively, and improved the paper properties such as tensile strength and burst factor up to 10% and 17%, respectively.

A lipase preparation with dual pH optima, wide temperature optima and broad substrate specificity for multiple applications

Optimised procedures for the production and recovery of lipase at 25 litre scale by submerged fermentation using aRhizopus species isolated from petroleum-soaked soil are developed. These lipase preparations are characterized by dual pH optima (4.0 and 8.0), wide temperature optima (over 80% activity between 40–57°C), broad specificity for substrates, high degree of tolerance for common salts and excellent stability during usage as well as storage.

Immunoaffinity purification and characterization of vacuolar H+ATPase from bovine kidney.

Vacuolar proton-translocating ATPase from bovine kidney was purified in one step by immunoprecipitation and immunoaffinity chromatography using an immobilized anti-H+ATPase monoclonal antibody. The monoclonal antibody affinity matrix coprecipitated polypeptides with Mr of 70,000, a cluster at 56,000, 45,000, 42,000, 38,000, 33,000, 31,000, 15,000, 14,000, and 12,000 from solubilized bovine kidney microsomal membranes, a pattern that was unaffected by different detergent washing conditions. A nearly identical pattern of polypeptides was observed in H+ATPase partially purified by an entirely independent method. The immunoaffinity purified H+ATPase had reconstitutively active ATP-induced acidification and potential generation that was inhibited by N-ethylamaleimide. The purified enzyme had specific activities as high as 3.1 mumol/min/mg protein, dual pH optima at 6.5 and 7.2, and a Km for ATP of 150 microM. The substrate preference was ATP greater than ITP much greater than UTP greater than GTP greater than CTP. The affinity purified H+ATPase was stimulated by phosphatidyl glycerol greater than phosphatidyl inositol much greater than phosphatidyl choline greater than phosphatidyl serine. The immunoaffinity purified enzyme did not require monovalent anions or cations for activity, and the divalent cation preference for activity was Mn, Mg much greater than Ca greater than Co much greater than Sr, Ba. The enzyme was not inhibited by ouabain, azide, or vanadate, but had kappa 1/2 inhibitory concentrations of 22.2 microM for N-ethylmaleimide, 14.9 microM for NBD-Cl, 4.9 microM for N,N'-dicyclohexylcarbodiimide, 13.8 microM for 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, and 315 microM for Zn, values close to those for inhibition of proton transport in the native vesicles. The affinity purified kidney enzyme has similarities to but also significant differences in structural and enzymatic properties from those reported for other vacuolar H+ATPase.

Characterization of cathepsin D from the skeletal muscle of fresh water fish, Tilapia mossambica.

Cathepsin D (EC. 3.4.4.23) from the skeletal muscle of fresh water fish Tilapia mossambica was purified 114-fold with 14% recovery by employing conventional gel chromatography procedures. The purified enzyme appeared to be homogeneous by Polyacrylamide gel electrophoresis. The apparent molecular weight of the enzyme was found to be 38,000 by Sephadex G–100 column chromatography. The enzyme exhibited dual pH optima at pH 2.8 and 3.8 when denatured hemoglobin was used as the substrate. However, the degradation of endogenous sarcoplasmic proteins occurred maximally at pH 5.0. The optimum temperature of enzyme assay was 50°C. The enzyme displayed heat stability at 60°C in presence of substrate, while in the absence of substrate it lost 95% of activity during incubation for 60 min at 60°C. Sulphydryl compounds and divalent metal ions had no effect on the enzyme activity. However NaCl at 10% concentration inactivated the enzyme almost completely.

Reduction of benzydamine N-oxide by rat liver xanthine oxidase.

Evidence is presented that a purified xanthine oxidase preparation, like milk xanthine oxidase, is responsible for the xanthine-dependent reduction of benzydamine N-oxide. Rat liver preparations contain enzymes which catalyze the anaerobic reduction of benzydamine N-oxide by either reduced nicotinamide adenine dinucleotide (phosphate) NAD (P) H or xanthine. In crude enzyme preparations, the enzymatic reduction seems to involve xanthine oxidase and/or cytochrome P-450 because allopurinol, an inhibitor of xanthine oxidase, and n-octylamine, an inhibitor of aliphatic tertiary amine N-oxide reductase (cytochrome P-450) block the reduction of benzydamine N-oxide in crude enzyme preparations of rat liver. Moreover, the enzymatic system exhibited dual pH optima of 7.4 and 9.0 for reductions dependent on NADPH and xanthine, respectively. An enzyme preparation which did not contain cytochrome P-450 was isolated from rat liver and purified 186-fold in terms of xanthine oxidase activity. Xanthine oxidase activity and xanthine-dependent benzydamine N-oxide reduction activity were copurified in parallel.

Characterization of arylsulfatase isoenzymes from Pseudomonas aeruginosa.

A method is described for the separation and isolation of two electrophoretically distinct arylsulfatases (arylsulfatase EC.3.1.6.1) from Pseudomonas aeruginosa.Characterization of the enzymes revealed that they were type I arylsulfatases, with similar kinetic properties. They differed, however, in respect to charge, pH optima for substrate hydrolysis, and activation by anions. In addition, the enzymes displayed dual pH optima curves with both substrates used and the curious property of a shift in pH optima with varied p-nitrophenyl sulfate concentrations.

Lipolytic activity of insect tissues and its significance in lipid transport

Manometric techniques allowed the study of esterase activity in several tissues from H. cecropia and P. americana when triacetin or tributyrin were employed as substrates. These techniques were too insensitive to detect true lipase activity (hydrolysis of long-chain fatty acid glycerides). A new lipase radioassay using C 14-triolein as substrate uncovered lipase activity in the fat body and flight muscle of the moth and in the muscle, midgut, and fat body of the cockroach. The digestive lipase exhibited dual pH optima while a single pH optimum was found for fat body. The fact that flight-muscle lipase hydrolyses diglycerides at a rate five times that of triglycerides suggests that lipid transported from the fat body, in the form of protein-bound diglyceride, enters the flight muscle as diglyceride where the lipase makes the fatty acids available for sarcosomal oxidation.