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Background Xylanases have attracted much attention owing to their potential applications. The applicability of xylanases, however, was bottlenecked by their low stabilities at high temperature or extreme pH. The purpose of this work was to enhance the thermostability of a mesophilic xylanase by N-terminal replacement. Results The thermostability of AoXyn11, a mesophilic family 11 xylanase from Aspergillus oryzae, was enhanced by replacing its N-terminal segment with the corresponding one of EvXyn11TS, a hyperthermotolerant family 11 xylanase. A hybrid xylanase with high thermostability, NhXyn1157, was predicted by molecular dynamics (MD) simulation. An NhXyn1157-encoding gene, Nhxyn1157, was then constructed as designed theoretically, and overexpressed in Pichia pastoris. The temperature optimum of recombinant NhXyn1157 (re-NhXyn1157) was 75 °C, much higher than that of re-AoXyn11. Both xylanases were thermostable at 65 and 40 °C, respectively. Additionally, the pH optimum and stability of re-NhXyn1157 were 5.5 and at a range of 4.0–8.5. Its activity was not significantly affected by metal ions tested and EDTA, but strongly inhibited by Mn2+ and Ag+. Conclusion This work obviously enhanced the thermostability of a mesophilic xylanase, making re-NhXyn1157 a promising candidate for industrial processes. It also provided an effective technical strategy for improving thermostabilities of other mesophilic enzymes. © 2013 Society of Chemical Industry

Plant-based lignocellulosic biomass represents a significant global reserve of organic renewable energy, and is considered to have high potential to replace substantial portions of fossil-fuel dependency. Sugars derived from the degradation of lignocellulosic biomass can be utilized in the production of more environmentally-friendly products such as biofuels and numerous other value-added compounds. However, the degradation of the cellulose polysaccharide is complicated by its highly recalcitrant nature resulting from strong intermolecular hydrogen bonds and cross-linkages with other components such as lignin. A variety of bacteria and fungi have evolved the ability to degrade cellulose through the use of cellulases, -glucosidases, and lytic polysaccharide monooxygenases (LPMOs). Such enzymes are thus the focus of intense research for use in biorefineries that aim to convert woody biomass to soluble sugars. Biorefineries utilize a variety of methods, including enzymatic degradation, to purify and isolate various components from biomass. These industrial processes are often characterized by relatively extreme conditions such as high temperatures and acidic or alkaline pH. There is therefore great interest in improving the properties of cellulases for use under these conditions. One approach is the engineering of cellulases with the aim of altering specific properties such pH stability and/or pH optimum. The main aim of this study was to use rational protein engineering in an attempt to optimize a family 6 glycoside hydrolase (GH), mgCel6A, for use at pH 5.0 or lower for more than 24 hours at 60C or higher. The mgCel6A wild-type enzyme was extensively characterized to determine its pH optimum and degree of pH stability on the industrial BALI cellulose substrate, as well as on model substrates. Mutant design aimed at optimizing the pH optimum and stability of mgCel6A was guided by analysis of a homology model of the catalytic domain of the enzyme (mgCel6A∆CBM), a multiple sequence alignment (MSA) of biochemically characterized family 6 GHs, and studies of pH-engineering literature. Mutations were introduced on the surface of the enzyme and in or near the active site. Produced mgCel6A∆CBM enzyme variants (Q152E, L165E, N21D/N46D/A104E/Q152E/L165E/I242E/I271D/Q276E, T44D, D158A, and D158N) were characterized to investigate their pH optima and stability as compared to the wild-type enzyme. Of the six successfully produced mutant enzyme variants, three appeared to have identical stabilities and pH optima compared to that of mgCel6A∆CBM. One mutant variant with additional negative surface charge (N21D/N46D/A104E/Q152E/L165E/I242E/I271D/Q276E) showed a significant decrease in thermal stability. Two mutants lacking an aspartate presumed to raise the pKa of the catalytic acid (D158A and D158N) showed considerable acidic shifts in their pH-activity profiles. Characterization of these mutants indicated that they had obtained pH optima of approximately 5.0 and 5.5, respectively, for 24-hour reactions at 60C with the BALI™ cellulose substrate. However, the activities of these mgCel6A∆CBM variants were drastically reduced as compared to that of the wild-type enzyme, showing that further optimization of these mutants is most likely needed for optimal performance under industrial conditions. Future mutagenesis studies of the D158A and D158N mutants may provide a deeper understanding of the reduced catalytic activity, and may also help in designing modified versions of these mutants with preserved reduced pH optima and concomitantly increased activity. A combination of rational engineering and directed evolution may be advantageous in the further development of mgCel6A∆CBM towards industrial use. Plantebasert lignocellulosisk biomasse utgjør en betydelig global reserve av organisk fornybar energi, og antas å ha stort potensiale til å erstatte mye av det fossile brennstoffet vi i dag er svært avhengige av. Sukker som utvinnes ved nedbryting av lignocellulosisk biomasse kan brukes i produksjonen av mer miljøvennlige produkter slik som biodrivstoff og flere andre foredlede produkter. Nedbryting av cellulose er imidlertid vanskelig grunnet sterke intermolekylære hydrogenbindinger og kryss-linker med andre komponenter slik som lignin. En rekke typer bakterier og sopp har utviklet evnen til å bryte ned cellulose ved hjelp av cellulaser, b-glucosidaser og lytiske polysakkaridmonooksygenaser (LPMOer). Det forskes derfor intenst på disse enzymene for bruk i bioraffinerier hvor målet er å konvertere biomasse til løselige sukkere. Bioraffinerier benytter en rekke metoder, inkludert enzymatisk nedbryting, for å rense og isolere ulike komponenter i biomassen. Disse industrielle prosessene karakteriseres ofte av relativt ekstreme forhold, slik som høye temperaturer og sur eller basisk pH. Det er derfor av stor interesse å kunne forbedre cellulasenes egenskaper til bruk under slike forhold. En tilnærmingsmåte er å utvikle cellulaser med den målsetting å endre spesifikke egenskaper som pH-stabilitet og/eller pH-optimum. Hovedformålet med denne oppgaven har vært å bruke rasjonell protein engineering i et forsøk på å optimalisere en familie 6-glykosid hydrolase (GH), mgCe16A, for bruk ved pH 5.0 eller lavere og ved 60°C eller høyere, i mer enn 24 timer. mgCel6A ble karakterisert for å bestemme villtypeenzymets pH-optimum og grad av pH-stabilitet på det industrielle BALIÔ cellulose-substratet og på modellsubstrater. Mutantdesign med mål om å optimalisere enzymets pH-optimum og stabilitet var basert på analyse av en homologimodell av det katalytiske domenet av enzymet (mgCel6AΔCBM), en multippel sekvenssammenstilling av biokjemiske karakteriserte familie 6-GHer, og pHengineering litteratur. Mutasjoner ble introdusert både på overflaten av enzymet, samt i og i nærheten av det aktive setet. Produserte mgCel6AΔCBM mutanter (Q152E, L165E, N21D/N46D/A104E/Q152E/L165E/I242E/I271D/Q276E, T44D, D158A, og D158N) ble deretter testet for å undersøke deres pH-optimum og stabilitet sammenliknet med villtypeenzymet. Tre av seks produserte mutanter viste like stabiliteter og pH-optima som villtypeenzymet. En av mutantvariantene av mgCe16AΔCBM med flere negative ladninger på overflaten (N21D/N46D/A104E/Q152E/L165E/I242E/I271D/Q276E) viste en betydelig reduksjon i termostabilitet. To andre mutanter som manglet en aspartat (antatt å øke pKa verdien til den katalytiske syren; mutanter D158A og D158N), fikk betydelig lavere pH-optima. Karakterisering av disse mutantene indikerte at de hadde pH-optima på rundt 5.0 og 5.5 i en 24-timers reaksjon ved 60°C på BALIÔ substratet. Imidlertid var aktiviteten til disse mutantvariantene av mgCel6AΔCBM betydelig redusert i forhold til villtypeenzymet. Resultatene indikerer at videre optimalisering av disse mutantvariantene er nødvendig for optimal ytelse ved industrielle betingelser. Fremtidige mutagenesestudier av mutantene D158A og D158N kan gi en dypere forståelse av den reduserte katalytiske aktiviteten, og kan også være til hjelp i design av modifiserte versjoner av disse mutantene med tilsvarende redusert pH-optimum og økt aktivitet. En kombinasjon av rasjonell design og styrt evolusjon (directed evolution) kan være fordelaktig i videre utvikling av mgCel6AΔCBM for industrielle forhold. M-BIOTEK

WOS: 000395603100023 beta-glucosidase was extracted and purified from Turkish Hacihaliloglu apricot variety and some of its biochemical properties were studied in a two-year study (2008 and 2009). Two isoenzymes (isoenzyme A and B) were obtained upon ammonium sulfate fractionation, ion exchange chromatography using DEAE-Toyopearl 650 M and gel filtration chromatography using Sephadex G-100. The pH optimum was found to be 5.0 for both isoenzymes. The optimum temperature for isoenzyme A activity in both years was found to be 50 degrees C, whereas those for the isoenzyme B in 2008 and 2009 were 50 degrees C and 55 degrees C, respectively. Km values of the isoenzyme A and B were found to be 2.49 and 1.12 mM in 2008, and 2.41 and 1.06 mM in 2009, respectively. The Z values for isoenzyme A in 2008 and 2009 were found to be 20.3 degrees C and 17.9 degrees C, respectively. Those for isoenzyme B in 2008 and 2009 were 21.1 degrees C and 23.0 degrees C, respectively. Ea values for isoenzyme A were 108.1 kjimolK and 1223 kj/molK, while those for isoenzyme B were found to be 103.5 kj/mol K and 95.1 kjimolK in 2008 and 2009, respectively. Of the inhibitors tested, CaCl2 was the most effective. (C) 2017 Elsevier Ltd. All rights reserved. Research Fund of the University of Cukurova, TurkeyCukurova University [ZF2008D4] This study was funded by the Research Fund of the University of Cukurova (ZF2008D4), Turkey.

Con el diseño, construcción y utilización de un biodigestor, de única descarga, se planteó una estrategia de intervención en el aula durante el primer semestre del año 2014, con un grupo de 30 estudiantes de grado undécimo del Instituto Pedagógico Nacional (ipn). En el transcurso de la intervención se estudiaron los conceptos de: cinética química, pH y equilibrio químico, relacionándolo con la velocidad de descomposición de la materia orgánica que conlleva a la producción de biogás, las condiciones de pH óptimas para la supervivencia de las bacterias y los factores que afectan la velocidad de reacción. Dentro de las principales estrategias utilizadas estuvieron las lecturas, tanto académicas como notas informativas,que permitieron orientar discusiones en el campo de las csc (cuestión sociocientifica) y visitas a la granja, lugar en donde se dispuso el biodigestor. Los resultados evidencian logros satisfactorios en los estudiantes, en cuanto a: mejor disposición para la clase de química, desarrollo del pensamiento crítico y profundización en las implicaciones y la búsqueda de posibles soluciones frente al impacto ambiental y social que implica la explotación de gas a gran escala.

(1994). Shifts in the Optimum pH of Rhizopus Glucoamylase Depending on the Reaction Temperatures. Bioscience, Biotechnology, and Biochemistry: Vol. 58, No. 1, pp. 183-184.

Malachite green oxalate dyes are of synthetic origin and their environmental existence is not well understood. They are resistant to direct aerobic bacterial degradation and form potentially carcinogenic aromatic amines. This study shows that applying the oxidative processes of enzymatic treatment with free laccase and sol-gel laccase could lead to dye degradation. The degradation of dye malachite green oxalate using free laccase and sol-gel laccase, respectively were 37% and 13% at 1 hour reaction. The optimum pH for the free laccase and sol-gel laccase respectively were at pH 5 and pH 6. The optimal temperature for degradation of dye by sol-gel laccase was at 40°C. These results showed that free laccase and sol-gel laccase have good performance in the degradation of malachite green oxalate dyes. Pewarna oxalate hijau Malachite adalah sintetik asli dan kewujudan semulajadinya tidak begitu diketahui. Ia bersifat kalis degradasi bakteria aerobik dan membentuk amina aromatik yang berpotensi karsinogen. Kajian ini menunjukkan bahawa penggunaan proses oksidatif untuk rawatan enzim dengan lakase bebas dan lakase sol-gel boleh membawa kepada degradasi pewarna. Degradasi pewarna malachite hijau oxalate yang menggunakan lakase bebas dan lakase sol-gel, masing-masing adalah sebanyak 37% dan 13% pada 1 jam reaksi. pH optimum untuk lakase bebas and lakase sol gel, masing-masing adalah pada pH 5 dan pH 6. Suhu optimum bagi degradasi pewarna oleh lakase sol-gel adalah pada 40°C.

Hydroxide ion inhibits Photosystem II (PSII) activity by extracting Cl– from its binding site in the O2-evolving complex (OEC) under continuous illumination [Critchley, C., et al. (1982) Biochim. Biophys. Acta 682, 436]. The experiments reported here examine whether two subunits of PsbO, the manganese-stabilizing protein, bound to eukaryotic PSII play a role in protecting the OEC against OH– inhibition. The data show that the PSII binding properties of PsbO affect the pH optimum for O2 evolution activity as well as the Cl– affinity of the OEC that decreases with an increasing pH. These results suggest that PsbO functions as a barrier against inhibition of the OEC by OH–. Through facilitation of efficient retention of Cl– in PSII [Popelkova, H., et al. (2008) Biochemistry 47, 12593], PsbO influences the ability of Cl– to resist OH–-induced release from its site in the OEC. Preventing inhibition by OH– allows for normal (short) lifetimes of the S2 and S3 states in darkness [Roose, J. L., et al. (2011) Bioch...

Verticillium dahliae is a fungal plant pathogen that attacks a wide range of plants including anise causing a wilt disease. The pathogen grows slowly on anise seeds using the standard moist blotter (SMB) or deep-freezing blotter (DFB) methods. In these methods saprophytes and fast growing fungi impair the detection of such slow growing fungi. An attempt was carried out to overcome this problem on anise seeds by applying alkaline seed-bed (ASB) technique for detecting slow growing seed-borne fungi. Data showed significant increase in the incidence levels of V. dahliae (3.4 and 4.73%) when alkaline KOH or NaOH technique was applied as compared to 0.2 and 0.7%, in SBM and DFB methods, respectively. Moreover, the ASB technique proved to be an effective option for early detecting the pathogen on anise seeds after 4 days of incubation in compared to other methods. The in vitro study indicated that the alkaline pH is the favored condition by the fungus. However, V. dahliae was able to grow in a wide range of pH (3.5 to 12.5) with a pH optimum of 8 at which the highest growth rate was recorded (0.96 g.L-1/day). The maximum glucose coefficient was achieved at pH 8, while the highest glucose utilization was recorded at pH 12.5. These results suggest that ASB technique is recommended for the detection of the alkalophilic V. dahliae. Key words: Alkaline seed-bed, Pimpinella anisum, seed health, Slow growing fungi,Verticillium wilt.

Burkholderia sp. C20 strain isolated from food wastes produces a lipase with hydrolytic activities towards olive oil. Fermentation strategies for efficient production of this Burkholderia lipase were developed using a 5-L bench top bioreactor. Critical factors affecting the fermentative lipase production were examined, including pH, aeration rate, agitation rate, and incubation time. Adjusting the aeration rate from 0.5 to 2 vvm gave an increase in the overall lipase productivity from 0.057 to 0.076 U/(ml h), which was further improved to 0.09 U/(ml h) by adjusting the agitation speed to 100 rpm. The production of Burkholderia lipase followed mixed growth-associated kinetics with a yield coefficient of 524 U/g-dry-cell-weight. The pH optimum for cell growth and lipase production was different at 7.0 and 6.0, respectively. Furthermore, stepwise addition of carbon substrate (i.e., olive oil) enhanced lipase production in both flask and bioreactor experiments.

Adsorption over TiO2 is used efficiently for reactive dyes removal from wastewater. This paper investigates adsorption efficiency for Reactive Red 198 (RR198) removal over TiO2 adsorbent using response surface methodology. The main process parameters considered for optimisation were pH, adsorbent dose, and adsorption time. The experimental scheme was designed according to central composite rotatable design and second order regression model was developed. For regression analysis and ANOVA study, software MINITAB 15 was used. The optimum pH, TiO2 dose, and time were found to be 4.3, 4.3 g L−1, and 32.42 min, respectively. Complete removal was observed. Pareto analysis established pH the most influential parameter. L'adsorption sur TiO2 est utilisee efficacement pour l'elimination des colorants reactifs des eaux usees. Cet article examine l'efficacite de l'adsorption pour l'elimination du rouge reactif 198 (RR198) sur un adsorbant au TiO2 en utilizant la methodologie de surface de reponse. Les parametres principaux du processus consideres pour l'optimization etaient le pH, la dose d'adsorbant et le temps d'adsorption. Le projet experimental a ete concu selon un concept rotatif composite central et un modele de regression de second ordre a ete developpe. Pour l'analyse de regression et l'etude ANOVA, le logiciel MINITAB 15 a ete utilise. Le pH optimum, la dose de TiO2, et le temps etaient de 4.3, 4.3 g L−1, et 32.42 min, respectivement. On a observe une elimination complete. Une analyse de Pareto a etabli que le pH etait le parametre le plus influent.

Xylanases have several industrial uses, particularly in baking, modification of animal feed and in pulp bleaching in the paper industry. Process conditions in kraft pulp bleaching generally favour an enzyme that is active at high pH values. The activities of several glycosyl hydrolase family 11 xylanases reported to be active under alkaline conditions were determined under optimal conditions and found to have optima in the pH 5-6 range. Only one enzyme tested, BadX, was shown to have an alkaline pH optimum. Significant activity at pH values higher than 8 appears often to be the result of excess enzyme added to the reaction mixtures so that substrate is limiting. The different nature of laboratory and industrial substrates needs to be taken into consideration in designing assay conditions. In some cases, significant differences were observed in pH profiles generated using a small-molecule substrate when compared to those generated using xylan. We conclude that small-molecule substrates are not a suitable proxy for determining the pH profiles of family 11 xylanases.

We reported previously that the activities of several glycosyl hydrolase family 11 xylanases claimed to be active under alkaline conditions, were found to have optima in the pH 5-6 range when assayed under optimal conditions. One enzyme, BadX, had enhanced activity at pHs greater than 7 compared to other family 11 xylanases. Gene shuffling between badX and Dictyoglomus thermophilum xynB6 was performed in an attempt to elucidate regions conferring alkaline activity to BadX, and potentially, to increase the alkaline activity of the highly thermophilic XynB6. Segment substitution using degenerate oligonucleotide gene shuffling (DOGS) experiments with combinations of input parental gene fragments from xynB6 and badX was not able to improve the activity of XynB6 at alkaline pH. With one exception, the replacement of a single segment of BadX with the equivalent segment from XynB6 reduced the alkaline activity BadX. The results indicate that it might not be possible to alter significantly the alkaline pH characteristics of family 11 xylanases by one or a few mutations and that family 11 xylanases showing enhanced activity at alkaline pH's require multiple sequence adaptations across the protein.

Aims: To investigate the appropriateness of the extended Lambert–Pearson model (ELPM) to model the effect of pH (as hydrogen and hydroxyl ions) over the whole biokinetic pH range in comparison with other available models. Methods and Results: Data for the effect of pH on microbial growth were obtained from the literature or in-house. Data were examined using several models for pH. Models were compared using the residual mean of squares. Using the ELPM, pH was modelled as hydrogen ions and hydroxyl ions; hence, the model was monotonic in each. The ELPM was able to model data more successfully than the cardinal pH model (CPM) and other models in the majority of cases. Conclusions: Examining the effect of pH as hydrogen and hydroxyl ions has the advantage that the basic form of the ELPM can be retained as each is treated as a distinct antimicrobial effect. With the ELPM, each inhibitor is described by two parameters; from these parameters, the pHmin, pHopt and pHmax can be obtained. Furthermore, the idea of a dose response, absent from other models, becomes important. Significance and Impact of the Study: The CPM is an excellent model for certain situations – where there is a high degree of symmetry between the suboptimal pH and superoptimal pH response and where there are few data points available. The ELPM is more amenable to highly asymmetric behaviour, especially where plateaus of effect around the pH optimum are observed and where the number of data points is not restrictive.

Zusammenfassung Die proteolytischen Fraktionen in den drei verschiedenen Schleimhaut-typen des Schweinemagens (Fundus-, Pylorus- und Cardiadrusenzone) wurden durch Agargelelektrophorese getrennt und nach der Enzym-Substrat-Reaktion mit Hamoglobin auf der Gelplatte qualitativ und quantitativ dargestellt. Die direkte photometrische Ausmessung der Gelplatten fuhrte zu Ergebnissen, die eine Beurteilung der proteolytischen Aktivitat von Schleimhautextrakten auf vergleichender Basis erlauben. Alle untersuchten Extrakte ergaben zwei gut voneinander abgesetzte Pepsinogen-Banden, die in sich jedoch nicht homogen waren, sondern Proteolyse bei mindestens zwei pH-Optima zeigten. Aus den Aktivitatskurven im Bereich pH 1,5–5,0 geht hervor, das in der Magenschleimhaut des Schweins mindestens zwei, wahrscheinlich aber drei Pepsine mit pH-Optima bei pH 1,8–2,0, pH 2,0–2,2 und pH 2,8–3,2 gebildet werden, sowie ein weitgehend alkalistabiles Gastricsin mit Wirkungsoptimum bei pH 3,2–3,4. Die Aktivierung der Enzymvorstufen kann beim pH-Optimum des Enzyms erfolgen, setzt also keine starke Ansauerung des Mageninhalts voraus. Summary Agarelectrophoretic demonstration of proteolytic activity in extracts of the pig gastric mucosa Proteolytic fractions of the three different types of gastric mucosa of the pig (i. e. fundic, pyloric and cardiac) were separated by agar gel electrophoresis. The enzyme-substrate reaction with hemoglobin and subsequent staining were performed directly on the plates. Direct photometric analysis of the agar plates permits quantitative assessment of the proteolytic activities though on a comparative basis only. All mucosal extracts contained two distinct zymogen fractions which, however, were not homogenous showing proteolytic activities with at least two pH optima. Proteolytic activities were studied within the range pH 1.5–5.0, and it appears that gastric mucosa of the pig contains at least two, possibly three different pepsins and one gastricsin. The pH optima were pH 1.8–2.0, pH 2.0–2.2, pH 2.8–3.2 and pH 3.2–3.4. Activation of the zymogens readily occurs at the pH optimum of the respective enzyme, thus strong acidification of gastric contents is not required for proteolytic action. Resume Representation par electrophorese sur agar de l'activite proteolytique d'extraits de la muqueuse stomacale chez le porc On a separe au moyen d'electrophorese sur agar les fractions proteolytiques dans les trois types differents de la muqueuse stomacale du porc (zone des glandes du fond, du pylore et du cardia) et on a procede a une mise en evidence qualitative et quantitative selon la reaction enzyme-substrat avec hemoglobine sur plaque de gelose. La mesure photometrique directe des plaques de gelose dennerent des resultats qui permirent un examen de l'activite proteolytique des extraits de muqueuses sur une base comparable. Tous les extraits examines ont donne deux bandes pepsinogenes bien separees qui n'etaient cependant pas homogenes mais qui montrerent pour le moins une proteolyse par deux pH optima. Il resulta des courbes d'activites dans la marge de pH 1,5–5,0 qu'il y avait production dans la muqueuse stomocale d'au-moins deux, vraisemblablement trois pepsines avec des pH-optima de pH 1,8–2,0, pH 2,0–2,2, pH 2,8–3,2 et d'une gastricsine considerablement alcalistable avec action optimale par pH 3,2–3,4. L'activation des premiers degres de l'enzyme peut so produire au pH optimum de l'enzyme et ne suppose ainsi aucune forte acidification du contenu stomacal. Resumen Representacion electroforetica en agar de la actividad proteolitica en extractos de mucosa gastrica porcina Las fracciones proteoliticas de los tres tipos diferentes de mucosa gastrica de cerdo (zonas fundica, pilorica y cardiaca) se separaron mediante electroforesis en gelosa, representandose cuali y cuantitativamente con arreglo a la reaccion enzima-substrato con hemoglobina en la placa de gelosa. El analisis fotometrico directo de las placas de gelosa condujo a resultados, que permiten la apreciacion cuantitativa de la actividad proteolitica de los extractos de mucosa sobre una base comparativa. Todos los extractos examinados de la mucosa contenian dos fracciones de pepsinogeno bien diferenciadas entre si, las cuales sin embargo no se eran homogenas, sino que mostraban proteolisis al menos en dos optimos de pH. De las curvas de actividad en el margen de pH 1,5–5,0 se desprende que en la mucosa gastrica del cerdo se forman como minimo dos, aunque con toda probabilidad tres pepsinas, con optimos de pH en 1,8–2,0, pH 2,0–2,2 y pH 2,8–3,2, asi como gastricsina alcaliestable en sumo grado con un optimo de actividad a pH 3,2–3,4. La activacion de los zimogenos (fases enzimaticas previas) puede acaecer en el pH optimo del fermento, asi que no se precisa la acidificacion del contenido gastrico.

Jack bean urease has been immobilized on arylamine glass beads (200–400 mesh size, 75–100 A pore size) and its properties compared with soluble enzyme. The binding of urease was 13.71 mg per gram beads. The Km for soluble and immobilized urease for urea was 4.20 mM and 8.81 mM, respectively. Vmax values of urease decreased from 200 to 43.48 μmol of ammonia formed per min per mg protein at 37°C on immobilization. Both pH and buffer ions influenced the activities of soluble as well as immobilized urease. Soluble urease exhibited pH optima at 5.5 and 8.0. However, immobilized urease showed one additional pH optimum at 6.5. In comparison to phosphate buffer, citrate buffer was inhibitory to urease activity. Immobilization of urease on arylamine glass beads resulted in improved thermal, storage and operational stability. Because of inertness of support and stability of immobilized urease, the preparation can find applications in ‘artificial kidney’ and urea estimation in biological fluids viz., blood, milk etc.

Protein glycosylation, a major post-translational modification, plays essential roles in eukaryotic cells. The glycosylation of fungal laccases has been proposed to be the bottleneck for the heterologous production of the enzyme, so it is important to determine its structure and function. We describe here the detailed N-glycosylation profile of Pycnoporus sanguineus laccase and its influence on some of its enzymatic properties. In this enzyme only high mannose structures were found, being those with 5- and 8-mannose units the most abundant. No other type of sugars was found in contrast to other fungal laccases. Enzymatic cleavage of the N-glycans present in the laccase provoked slight changes in the kinetic parameters, in the thermal stability and in the pH optimum of the enzyme.

As to food quality of spruce needles for forest damaging insects. 5. Studies on the starch splitting enzyme (Amylase) in larvae of Gilpinia hercyniae Htg. The starch splitting enzyme (Amylase) was demonstrated in larvae of Gilpinia hercyniae (Fig. 1). The biochemical properties such as substrate concentration, enzyme concentration, activation by Ca++ ions, inhibition by EDTA, pH optimum and sensitivity to temperature (Fig. 2, Tab. 2) were studied. The reaction products due to enzyme action were separated by paper chromatography and compared with standards (Fig. 3). The variation of enzyme activity in 6, 12 and 18 days old larvae was studied (Fig. 4). The results with reference to the various conditions which influence the activity and its variation due to the age of larvae were discussed with respect to other carbohydrate metabolising enzymes such as maltase, gluco- and fructohexokinase. Zusammenfassung Das starkespaltende Enzym (Amylase) wurde in Larven von Gilpinia hercyniae nachgewiesen und seine biochemischen Eigenschaften untersucht. Die Reaktionsprodukte nach Enzymeinwirkung wurden chromatographisch analysiert, die Abhangigkeit der Enzymaktivitat vom Larvenalter nachgewiesen. Die Stellung des Enzyms im abbauenden Kohlenhydratstoffwechsel wird diskutiert.

The alkaline-induced unfolding and the salt-induced refolding of high molecular mass and low molecular mass goat brain cystatins under high pH conditions have been monitored by enzyme inhibitory activity, intrinsic and extrinsic fluorescence, and CD measurements. An increase in pH to 14.0 at low ionic strength was accompanied by complete loss of the activity of both cystatins. The maximum activity for HMGBC was observed at pH 8.0 whereas pH 7.0 was pH optimum for LMGBC. The results obtained for HMGBC indicate that, at low ionic strength, an increase in pH enhanced the extent of unfolding of the enzyme to the maximum unfolded state at pH 14.0. The unfolding occurred via a simple native to unfolded state transition. As for LMGBC, at low ionic concentration a pH increase from 7 to 10 caused unfolding of the cystatin, while at pH 12, we observed the formation of a compact intermediate state. Thus, the alkaline-induced LMGBC unfolding occurs via three step transition from native to intermediate, and, then, to unfolded state. At pH 12, an increase in the ionic strength by addition of salts (KCl/Na2SO4) resulted in the folding of both alkaline-denatured cystatins. The salt-induced refolded inhibitors (HMGBC/LMGBC) also showed significantly higher activity compared to the unfolded forms in presence of salts. Na2SO4 was observed to be more efficient in refolding the alkaline-unfolded cystatins at low concentrations (1 M) as compared to the KCl (3 M).

The chlorination and oxidation activity of chloroperoxidase (CPO) was enhanced 32 and 20% by chitosan, and oxidation activity 24% by thioglycolic acid, respectively. The pH optimum of chlorination reaction shifted 3 units to a less acidic environment by chitosan (from 2.75 to 5.75). The kinetic parameters indicated both the affinity and specificity of CPO to substrate were improved.

The effect of carbonate concentration, pH of the medium, and illumination intensity on the major physiological characteristics (growth rate and the intensities of CO2 assimilation and oxygen photoproduction) of the natronophilic cyanobacterium Euhalothece sp. Z-M001 have been studied. It was established that the investigated microorganism has at least two transport systems (TS) for CO2, which differ in both the pH optimum and substrate affinity: TS I has a pHopt 9.4–9.5 and a K S 0.5 of 13–17 mM, whereas TS II has a pHopt 9.9–10.2 and a K S 0.5 of 600–800 mM. The substrate affinity of these transport systems is several orders of magnitude lower than the substrate affinity of the transport systems of freshwater cyanobacteria. It is suggested that they are unique for extremely alkaliphilic cyanobacteria and reflect their adaptation to the seasonal cycles of the lake hydrochemistry.

The effects of pH on the performance of anoxic sulfide oxidizing (ASO) reactor were evaluated. Performance was investigated under various operational conditions at influent pH range of 4-11. At the influent pH of 7-7.5 during loading tests and HRT tests, the sulfide oxidation was partial. In general, the amount of sulfate formed decreased with the increasing sulfide and nitrite loadings. The bacterial communities in ASO reactors were more sensitive to acidic pH compared with alkaline pH, as nitrite and sulfide removal rates dropped significantly when exposed to acidic pH 3. High dissolved bisulfide ions, nitrite and excess of sulfate (>300 mg/L) might have inhibited the sulfide oxidation under highly acidic and alkaline conditions in the ASO reactor. Based on sulfide and nitrite removal efficiencies, the ASO reactor can be operated in a wide range of pH, i.e. 5-11.

A method is described for the identification of patients and carriers of Gaucher's disease, using leukocytes from a small volume of blood. The fluorogenic substrate, 4-methylumbelliferyl-beta-D-glucopyranoside, was assayed in the presence of pure sodium taurocholate (2.5 mg/ml) and Triton X-100 (2.0 mg/ml). Some commercial brands of pure sodium taurocholate were satisfactory for this purpose. The pH optimum for controls, Gaucher disease carriers and Gaucher disease patients was 5.4 using citrate-phosphate buffer. Although leukocytes prepared from only a small amount of blood (2-8 ml) are required, there is sufficient quantity for measuring other lysosomal enzymes as controls. Using this method, 12 patients with all types of Gaucher's disease and 12 obligate heterozygotes were identified. Carrier status was predicted in six other family members and ruled out in six others. Eight unaffected people married to Gaucher carriers or Gaucher patients were predicted to be non-carriers of Gaucher's disease, thereby ruling out children affected with Gaucher's disease in that mating.

Biofilms are the moving, constantly changing, heterogeneous bacterial communities. Microorganisms growing in a biofilm are highly resistant to both antimicrobial agents and immune mechanisms. Objective of this paper was to study the effect of extracellular and membrane-bound proteases B. subtilis on the formation of biofilms in liquid media. The strains B. subtilis 168 (wild type) and B. subtilis BRB14 (protease-deficit strain) were examined on the ability to form biofilms in liquid medium. The LB and 1% glucose LB media were used as rich culture medium, and the synthetic E-medium was chosen as poor culture medium. A decreased formation of biofilms by 20% was observed during growth in the glucose-containing LB medium as compared with the glucose-free LB medium. The level of biofilm formation in the synthetic E-medium was 7 times higher than the total level in both LB media. Under identical culture conditions, the ability of mutant strain B. subtilis to form biofilms was 10%-15% higher than the level of biofilm formation by a wild strain. By the 48th hour of growth, the level of biofilm content in the culture of both strains reached maximum and declined significantly by the 72nd hour of growth. pH-Optimum of biofilm formation by both strains B. subtilis was 7.4. The temperature optimum for the formation of biofilms for both strains B. subtilis also ranged from 22°C to 45°C. The results of the studies allow us to conclude that the extracellular and membrane-bound proteinases B. subtilis, deleted in the mutant strain, do not play a key role in the formation of biofilm, however, their effect is more significant during growth of bacteria in a nutrient-rich environment rather than in synthetic one.

Struvite (MgNH 4 PO 4 .6H 2 O) is one of phosphate minerals, commonly forms into aqueous solutions. It can be precipitated as mineral deposits for optimization of phosphate recovery based on the pH optimum, molar ratio and temperature levels. This paper presents results of a study on the struvite precipitation under the influence of pH variation, at optimized molar ratio and temperature, which were calculated from an experimental design methodology. Based on the methodology, a laboratory prepared struvite, made by mixing solutions to NH 4 OH, MgCl 2 and H 3 PO 4 for a molar ratio of 1: 2: 1 in a 500 mL volume of batch stirred crystallizer at room temperature. The crystallization was done at 200 rpm and the pH variation was adjusted to 8, 9 and 10 with KOH for a time of 70 minutes. The resulting crystals were filtered and dried at room temperature for 48 h and subsequently stored for further analysis. Material characterisasion of the crystals was conducted using XRPD Rietveld method of mineralogical composition. SEM equipped by EDX was employed for investigation of morphology and elemental composition of the crystals obtained. During the experiment, struvite crystals were firstly nucleated and subsequently developed at major value. The increase in pH is assumed to convert some of the struvite phase into struvite (K) and minor sylvite (KCl). It demonstrates that Visual MINTEQ can be employed to estimate the mineral formation out the synthetic solutions.

© 2016, Springer Science+Business Media New York.The ability to form biofilms in natural isolate Bacillus subtilis 168 and mutants with deleted genes of regulatory proteins AbrB, DegU, CcpA, and SpoOA, constructed on its basis, was investigated to elucidate the pathways regulating biofilm formation in B. subtilis. The B. subtilis 168 wild-type forms a biofilms in the liquid medium with maximum at 48th hour of culture growth. pH optimum for the biofilm formation in the wild-type strain is in the range of 7.4–8.0. Temperature optimum was in the range of 22 to 45 °C. The level of biofilm formation for all regulatory mutants was lower than that in the wild-type for 40–50 %. Temperature and pH optima for the mutant strains are the same as for the wild-type strain—7.4–8 pH and temperature of 22–45 °C.

The effects of experimental variables for the electrochemical reduction of carbon dioxide by Carbon Monoxide Dehydrogenase (CODH) were investigated. It shows the pH optimum at 6.3 where the feasibility of electro-chemical reduction and the stability of CODH compromise each other. The optimum temperature for the reduction was at where the enzyme shows the optimum activity although the solubility of carbon dioxide decreases as increasing temperature.

The furthering of scientific knowledge depends in part upon the reproducibility of experimental results. When experimental conditions are not set with sufficient precision, the resulting background noise often leads to poorly reproduced and even faulty experiments. An example of the catastrophic consequences of this background noise can be found in the design of strategies for the development of solutions aimed at preventing garlic paste greening, where reported results are contradictory. To avoid such consequences, this paper presents a two-step strategy based on the concept of experimental design. In the first step, the critical factors inherent to the problem are identified, using a 2(III)(7-4) Plackett-Burman experimental design, from a list of seven apparent critical factors (ACF); subsequently, the critical factors thus identified are considered as the factors to be optimized (FO), and optimization is performed using a Box and Wilson experimental design to identify the stationary point of the system. Optimal conditions for preventing garlic greening are examined after analysis of the complex process of green-pigment development, which involves both chemical and enzymatic reactions and is strongly influenced by pH, with an overall pH optimum of 4.5. The critical step in the greening process is the synthesis of thiosulfinates (allicin) from cysteine sulfoxides (alliin). Cysteine inhibits the greening process at this critical stage; no greening precursors are formed in the presence of around 1% cysteine. However, the optimal conditions for greening prevention are very sensitive both to the type of garlic and to manufacturing conditions. This suggests that optimal solutions for garlic greening prevention should be sought on a case-by-case basis, using the strategy presented here.

Glucose isomerase ex Lactobacillus brevis was successfully immobilised on microcrystalline cellulose, using the transition metal-link method. Immobilisation could be performed over a pH range of 5 to 9, and usually resulted in an apparent specific activity increase. The immobilised glucose isomerase generally displayed properties similar to those of the soluble enzyme, with the exception of the following differences: (i) a pH optimum at pH = 6, an acid shift of 0.5 units on immobilisation; (ii) an optimum reaction temperature at 50 °C, lower than that for the soluble enzyme; (iii) on incubation at 4 °C, a retention of 53% of the initial specific activity, when stored in 0.02 M, pH = 7, Tris buffer, after 8 weeks, compared with an apparent activation of the soluble enzyme after 10 and 19 weeks' storage. Storage properties of the immobilised enzyme at 4 °C in Tris were apparently improved by the presence of Mn++ and Co++, although associated with some protein release. Storage at 4 °C in water alone, as opposed to Tris, resulted in a more rapid activity loss.

Well-defined amphiphilic block copolymers poly(styrene-b-acrylic acid) (PS-b-PAA) with controlled block length were synthesized using atom transfer radical polymerization (ATRP). Pectinase enzyme was immobilized on the well-defined amphiphilic block copolymers PS-b-PAA. The carboxyl groups on the amphiphilic PS-b-PAA diblock copolymers present a very simple, mild, and time-saving process for enzyme immobilization. Various characteristics of immobilized pectinase such as the pH and temperature stability, thermal stability, and storage stability were valuated. Among them the pH optimum and temperature optimum of free and immobilized pectinase were found to be pH 6.0 and 65 degrees C.

In this study, Macrolepiota mastoidea, a wild edible mushroom, was evaluated for its polyphenol oxidase potential. Native electrophoresis, stained by l-dihydroxyphenylalanine, of the crude extracts from this species showed two bands having Rf values of 0.38 (minor) and 0.50 (major), supporting a polyphenol oxidase potential. The crude extracts showed monophenolase activity against 3-(4-hydroxyphenyl)-propionic acid and diphenolase activity against 4-methylcatechol as substrates. Monophenolase and diphenolase activities of enzyme extract prepared from M. mastoidea showed pH optimum values at pH 6.0 and pH 4.0, respectively. The extracts retained about 100% and 60% of their original monophenolase and diphenolase activities at their optimum pH values, respectively. It was estimated from thermodynamic data that M. mastoidea had a thermostable monophenolase activity. Thiourea and ascorbic acid were highly potential inhibitors for monophenolase, and ascorbic acid and sodium metabisulphite for diphenolase activity. It is clear from the present results that the enzyme extracts prepared from M. mastoidea possess polyphenol oxidase activities with interesting properties.

Summary1. Sulfated mucopolysaccharides inhibit several lysosomal glycosidases. The activity of s-galactosidase is especially sensitive to inhibition by such polyanionic molecules.2. The abnormal kinetics and the abnormal pH optimum of s-galactosidase in patients with different types of mucopolysaccharidoses are very similar to what is seen in a mixture of normal liver homogenate and of mucopolysaccharides.3. The isoenzyme distribution of several lysosomal glycosidases is abnormal in the liver of mucopolysaccharidosis and in a mixture of normal liver extracts and mucopolysaccharides.4. Sedimentation and solubility properties of lysosomal enzymes in mucopolysaccharidosis also are abnormal. The same abnormalities are found in a mixture of normal liver homogenate and chondroitin sulfate.5. The low s-galactosidase activity in liver extracts of patients can be restored in vitro by adding high amounts of albumine.6. A comprehensive mechanism is described which takes into account most abnormalities in mucopolysaccharidoses: the low s-galactosidase, simultaneous storage of water-soluble GAG’s and water-insoluble galactolipids, the presence of abnormal lysosomal "isoenzymes," the high activity of several glycosidases and the clinical signs such as mental deficiency and skeletal malformations.

β-Alanine synthase (EC 3.5.1.6) catalyzes the conversion of N-carbamyl-β-alanine to β-alanine, ammonia and CO2. The enzyme has been purified to apparent homogeneity from calf liver. The molecular size, pH optimum and substrate specificity have been determined. Sequence alignment of β-alanine synthases with N-carbamyl-D-amino acid amidohydrolase from Agrobacter sp. revealed the conservation of a catalytically important triad Glu-Lys-Cys, most likely involved in the breakdown of N-carbamyl-β-alanine.

Phosphomonoeaterase (PMEase) and phosphodiesterase (PDEase) activity was studied in the cyanobacterial cultures of Chroococcidiopsis isolated from two diverse cryptoendolithic habitats of Antarctic and Arizona. Because this organism is found within the rocks it appears that phosphorus metab- olism by alkaline phosphatase activity is a key factor to sustain growth of these organisms in that state, there being no other source of external P except in bound form in the rocks. The main findings in this paper show that specific pH and temperature regulate the PMEase and PDEase activities studied with different substrates in isolated of Chroococcidiopsis 1 and 2 although values were vastly different. The pH and tem- perature optima for phosphatase activity (PMEase and PDEase) of Chroococcidiopsis 1 and 2 were 9.5, 20 °C and 8.5, 40 °C respectively. It needs mentioning here that although the pH optimum for the enzyme activities in the Antarctic rock samples was the same i.e. 9.5 there was a striking difference in the tem- perature optimum in which maximum activity of both enzymes were recorded at 5 °C. A very crucial role of light and dark conditions were important for the enzyme activity and differed to a significant extent when compared with naturally occurring organisms in the Antarctic rocks. Low light fluxes of about 8 µmol photon m -2 s -1 showed higher PMEase and PDEase activity then total dark conditions in the Chroococcidiopsis -1 culture. However under natural conditions when this organism is found within the rocks 8-µmol photon m -2 s -1 was found to be inhibitory and dark conditions gave higher PMEase and PDEase activity. Arizona rock samples containing Chroococcidiopsis -2 however did not show dark stim- ulation. Increase in light intensity from 8 µmol photon m -2 s -1 to 60 µmol photon m -2 s -1 maintained in the cul- ture room increased the PMEase and PDEase activity of both cultures. The unique light and temperature responses for PMEase and PDEase activities in Chroococcidiopsis-1 found within the Antarctic rocks are unique. It points to some change in the cells probably by producing some cryoprotectant which protects the enzymes from becoming non functional at low temperatures. It also indicates that exposure to light fluxes as low as 8 µmol photon m -2 s

Growth parameters (temperature and pH) were determined for collection cultures of aerobic heterotrophic bacteria. Analysis of the experimental data with the use of the Rosso model made it possible to calculate the extreme values of temperature and pH permissive for culture growth. The examined cultures were subdivided into three groups with respect to their growth temperature and pH. The first group is represented by the cultures with minimum, maximum, and optimal growth temperatures of < 20, 60-64, and 38-40 degrees C, respectively, and with the optimal growth pH 8.0-8.5. Bacteria of the second group are true alkalithermophilic organisms with a temperature optimum of 45-50 degrees C and pH optimum of 8.5-9.0. The third group includes a culture of a thermophilic alkalitolerant bacterium.

It has been shown that a commercially available pectinase, Pectinex™, hydrolyzes chitin fairly efficiently with a K m of 2.2 mg ml −1 of chitin and V max equal to 6.5 nmol min −1 mg −1 . The pH optimum range (5.0–6.0) and temperature optimum range (55–65 °C) for this unusual activity are more in agreement with the corresponding known values for chitinases rather than the values observed in case of its pectinase activity. The half lives (of chitinolytic activity) of 81 and 76 min at 55 and 65 °C indicate that the chitinolytic activity survives much better that the pectinase activity of the molecule. The occurrence of a carboxyl group in both pectinases and chitinases suggests that similar active site designs may be responsible for this unusual and useful activity of Pectinex™.

The major cellulase secreted by the filamentous fungus Trichoderma reesei is cellobiohydrolase Cel7A. Its three‐dimensional structure has been solved and various mutant enzymes produced. In order to study the potential use of T. reesei Cel7A in the alkaline pH range, the thermal stability of Cel7A was studied as a function of pH with the wild‐type and two mutant enzymes using different spectroscopic methods. Tryptophan fluorescence and CD measurements of the wild‐type enzyme show an optimal thermostability between pH 3.5–5.6 (Tm, 62 ± 2°C), at which the highest enzymatic activity is also observed, and a gradual decrease in the stability at more alkaline pH values. A soluble substrate, cellotetraose, was shown to stabilize the protein fold both at optimal and alkaline pH. In addition, unfolding of the Cel7A enzyme and the release of the substrate seem to coincide at both acidic and alkaline pH, demonstrated by a change in the fluorescence emission maximum. CD measurements were used to show that the five point mutations (E223S/A224H/L225V/T226A/D262G) that together result in a more alkaline pH optimum [Becker, D., Braet, C., Brumer, H., III, Claeyssens, M., Divne, C., Fagerström, R.B., Harris, M., Jones, T.A., Kleywegt, G.J., Koivula, A., et al. (2001) Biochem. J.356, 19–30], destabilize the protein fold both at acidic and alkaline pH when compared with the wild‐type enzyme. In addition, an interesting time‐dependent fluorescence change, which was not observed by CD, was detected for the pH mutant. Our data show that in order to engineer more alkaline pH cellulases, a combination of mutations should be found, which both shift the pH optimum and at the same time improve the thermal stability at alkaline pH range.

본 논문에서는 산화 환원 효소의 최적 pH를 예측해 보았다. Boltzman 분배를 이용하여 어떤 아미노산의 side chain이 물에서 발견될수 있는 상대적 확률이나 물에 대한 상대적 친화력을 구하였으며 p $K_R$ 을 이용해 protonated 아미노산의 양과 deprotonated 아미소산의 양을 계산하였다. 효소의 최적 pH는 아미노산의 side chain이 물에서 발견될수 있는 상대적 확률과 protonated 또는 deprotonated된 아미노산 양의 곱인 유효 protonated된 양과 유효 depotonated된 양이 같아지는 pH로 예측하였다. 문헌 값과 비교해 보았을 때 예측치는 상당히 일치하는 경향을 보였으며 이 결과 로 효소 자체의 전도도가 생물학적 기능에 있어 매우 중요한 역할을 하는 것을 알 수있다. 【For various oxidoreductases, the optimum pHs of the enzymes can be calculated using the rule based on proton transfer. Relative probability of a certain amino acid side chain to be in the water, or the relative affinity to the water was calculated using Boltzman distribution. Also, the protonated and deprotonated portions of a certain amino acid side chain were calculated using p $K_R$ of that and the effective protonated and deprotonated protions were the product of relative probability and the protonated and deproteonated protions. Where the total effective protonated portion was equal to the effective deprotonated portion of amino acid side chains, it was expected that oxidoreductases have max-imum activities. The optimum pHs calculated by our rule were compared with the experimental results.】

Summary Phytate-degrading enzymes catalyse the step-wise release of phosphate from phytate, the principle storage form of phosphorus in plant seeds and pollen.They are widespread in nature, occurring in plants and micro-organisms, as well as in some animal tissues. Phytate-degrading enzymes have been studied intensively in recent years because of the great interest in such enzymes for reducing phytate content in animal feed and food for human consumption.Phytate-degrading enzymes are also of interest for producing defined breakdown products of phytate for kinetic and physiological studies.Certain myo-inositol phosphates have been proposed to have novel metabolic effects and therefore, the physiological role of different myo-inositol phosphates is presently undergoing extensive research.Generally, phytase behaves like a monomeric enzyme with molecular masses between 40 and 70 kDa.Up to now, two main types of phytate-degrading enzymes have been identified; acid phytate-degrading enzymes with an pH optimum around pH 5 and alkaline phytate-degrading enzymes with an pH optimum around pH 8.Most of the so far described phytate-degrading enzymes belong to the acidic type, and their optimal pH ranges from 4.5 to 6.0. This review summarises the molecular features as well as catalytic properties of phytate-degrading enzymes and also discusses enzymatic phytate degradation.

The ability of anti-MBP Atlantic salmon antibodies to bind to MBP was investigated using ELISA under a range of pHs (3–10) and osmolarities (100–1000 mOsml −1 ) to determine the optimum binding conditions required for Atlantic salmon antibodies. The pH optimum was between pH 7–8 with pH 7·4 giving the highest level of antibody binding. However, the decrease in optical density was in excess of 60% at pHs 6·0 and 9·0 and in excess of 99% at pHs of 3·0 and 10·0. The optimum osmolarity required for antibody binding was in the range 100–400 mOsml −1 with 330 mOsml −1 giving the highest level of antibody binding. Above 400 mOsml −1 there was a rapid decrease in antibody binding with the OD dropping by over 50% at all dilutions. This decrease was maintained at all osmolarities over 400 mOsml −1

Has conducted research on the effects of methanol extract of leaves papaya (Carica papaya L.) Against Lipase Enzyme Activity. This study aimed to obtain information in a methanol extract inhibits lipase activity. The method used is Complete Random Design (RAL), with goal attainment is done by determining the incubation time lipase enzyme, pH optimum lipase enzyme, and maximum substrate concentration lipase enzyme. And determining the effect of the methanol extract to inhibit the lipase enzyme activity. The results showed that the best incubation time of lipase obtained by time of 120 minutes, the optimum pH 7 and the maximum substrate concentration of 4%. With the highest activity were obtained 16.94 mol /ml.menit, 13.33 mol / ml.menit, and 12.89 mol / ml.menit. The methanol extract of papaya leaves can inhibit the lipase enzyme activity the best concentration of 2% with the acquisition activity of 3.67 mol/ml.menit.Keyword: papaya, lipase, incubation time, optimum pH, substrate concentration.

Two exopolyphosphatases (polyphosphatase I and II; EC 3.6.1.11) from Microlunatus phosphovorus isolated from an activated sludge, release orthophosphate from inorganic polyphosphates and have been detected and characterized for the first time. Polyphosphatase I has a molecular mass of 93 kDa, pH optimum of 4.5, does not require K + for its activity and is stimulated by divalent cations. Polyphosphatase II has a molecular mass of 55 kDa, pH optimum of 7.5 and displays its optimal activity in the presence of K + and divalent cations. The content of polyphosphatase I increases during growth, while polyphosphatase II varies only slightly.

Phosphatase activities and environmental features were characterized for 12 terrestrial and aquatic mosses in upland northern England, along with four species sampled from subarctic Sweden. Phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) activities of shoot tips were measured using para -nitrophenyl phosphate ( p -NPP) and bis- p NPP. All species showed PMEase activity, but not all showed PDEase activity. The mean pH optimum was 5·0 for PMEase and 5·7 for PDEase. The kinetic parameters K m and V max were calculated from three linear transformations of the Michaelis‐Menten equation. The mean K m values of the mosses ranged between 77 and 468 µ M for PMEase and 26 and 414 µ M for PDEase. The corresponding V max values were 0·6‐205 µ µ µ mol p NP g − 1 DW h − 1 for PMEase and 1·4‐110 µ mol pNP g − − − 1 DW h − 1 for PDEase. Mosses from Sweden displayed greater K m and smaller V max values than those from England. The aquatics Fontinalis antipyretica and Rhynchostegium riparioides displayed two-phase kinetics for PMEase and PDEase, with K m and V max being dependent on substrate concentration. Staining suggested that PMEase activity was located in the cell wall of most mosses. Phosphatase assays provide a rapid method for screening environmental nutrient status and a standard procedure is recommended.

Uptake of 14 C-labelled glutamine by seven Australian Pisolithus isolates (representing two species) was investigated over the concentration range 0.5 mmol m −3 -20 mol m −3 . Total uptake did not conform to simple Michaelis-Menten kinetics over this concentration range. At concentrations above 0.5 mol m −3 much of the glutamine uptake appeared to be diffusion-like. At concentrations below 0.5 mol m −3 , subtraction of uptake in the presence of 2,4-dinitrophenol from total uptake revealed an active component that followed Michaelis-Menten kinetics. Estimated K m and V max values for active glutamine uptake by all isolates were in the ranges 4.0 - 210 mmol m −3 and 80 - 637 nmol g −1 d wt. min −1 respectively. pH optima for glutamine uptake were in the ranges 4.5-6.0, 3.7-5.0 or 3.0-4.5, depending on the isolate. While intraspecific variation was observed, there were no apparent relationships between the two Pisolithus species and either kinetic parameters for glutamine uptake or the pH optimum for the process.

Peptides, quinolones, diuretics and peptide hormones are important substances of biomedical interest. In this work a model describing the effect of pH on retention in liquid chromatography (LC) is established and tested for these compounds using an octadecylsilica column. The suggested model uses the pH value measured in the hydro-organic mixture used as mobile phase instead of the pH value in water and takes into account the effect of the activity coefficients. The proposed equations permit the prediction of the pH optimum using a minimum number of measurements and also permit the determination of the acidity constants of the compounds considered in the medium used as mobile phase. Moreover, these equations can be combined with the previously derived equations, that relate the retention with the solvent composition of the mobile phase, to establish a general model that relates the elution behaviour of the solute with the significant mobile phase properties: composition, pH and ionic strength.

Phospholipase D from Dacus carota (carrot) was purified by subjecting it to three-phase partitioning. The single step of three phase partitioning led to 13-fold purification with an activity recovery of 72%. SDS-PAGE analysis showed a single band with minimum molecular weight corresponding to nearly 60 kDa. The purified enzyme had a pH optimum in the range of 6.0–6.5 and was unstable above 30°C. Kinetic studies showed a K m value of 9.5 mM and a V max of 0.35 mL min -1 . The enzyme purified by three-phase partitioning was found to resolve into two isoenzymes on a DEAE-cellulose column.

Soil extractable Al, F and Zn concentrations decreased whereas extractable Ca, Cu, K, Mg, Na and P concentrations increased when the soil pH was raised from 3 to 6. These trends led to a decrease in growth of tea seedlings as determined by measurements of relative dry weight gain (RDW), relative leaf number gain (RLN) and relative leaf area gain (RLA). Tea seedlings of both 'large-leafed' and 'small-leafed' varieties grown in soils at pH 3 and 3.5 were the tallest and healthiest, while those at pH 6 died after 3 months. The large-leafed variety showed higher growth rates than the small-leafed variety. The highest (p Le pH optimum des sols pour les plants de the se situe entre 5 et 5,6. Les sols ayant un pH a partir de 6,5 demandent un ajustement avec des materiaux acides avant que le the ne soit plante. Dans les plantations de the, le sol acidifie provoque une augmentation du prelevement d'aluminium et du fluor. Le principal objectif de cette etude est de determiner l'effet du pH du sol sur l'absorption d' aluminium et de fluor par le the (Camellia sinensis). De l'acide sulfurique et de la soude sont appliques au sol pour determiner les effets du pH.

Zymograms of a crude protein extract from S. cerevisiae strain SCPP containing endopolygalacturonase were studied and compared to the purified enzyme by determining their physico-chemical properties. The results obtained with crude extract were similar to those of the purified enzyme. The endopolygalacturonase from both sources displayed a pH optimum between 3.0 and 4.0, and was active at temperatures between 4 and 50°C on a large panel of substrates. These characteristics make this S. cerevisiae endopolygalacturonase an attractive tool for the beverage industry. Journal of Industrial Microbiology & Biotechnology (2000) 24, 296–300.

Because enzymes usually require distinct pH conditions for maximum activity, an approach of spontaneous pH shift of solution was devised to derive multiple reactions in a sequence. The two enzymes selected, horseradish peroxidase (HRP) and β-galactosidase (GAL), had dissimilar optimal pH levels, i.e., 5.1 and 7.0, respectively. In a solution, HRP initially reacted at a lower pH range and the GAL reaction was consecutively carried out at a higher range in the presence of a third enzyme, urease, which caused an increase in pH. Under optimal conditions, the multiple system provided comparable performances with those of single reactions.