scholarly journals Biochemical Characterization, Thermal Stability, and Partial Sequence of a Novel Exo-Polygalacturonase from the Thermophilic Fungus Rhizomucor pusillus A13.36 Obtained by Submerged Cultivation

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Lucas Vinícius Trindade ◽  
Carla Desagiacomo ◽  
Maria de Lourdes Teixeira de Moraes Polizeli ◽  
André Ricardo de Lima Damasio ◽  
Aline Margarete Furuyama Lima ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Biochemical Characterization ◽  
Hydrophobic Interaction Chromatography ◽  
Submerged Cultivation ◽  
Rhizomucor Pusillus ◽  
Irreversible Denaturation ◽  
Optimum Ph ◽  
Sigmoidal Curve ◽  
Hydrolysis Of ◽  
Data Banks

This work reports the production of an exo-polygalacturonase (exo-PG) by Rhizomucor pusillus A13.36 in submerged cultivation (SmC) in a shaker at 45°C for 96 h. A single pectinase was found and purified in order to analyze its thermal stability, by salt precipitation and hydrophobic interaction chromatography. The pectinase has an estimated Mw of approximately 43.5–47 kDa and optimum pH of 4.0 but is stable in pH ranging from 3.5 to 9.5 and has an optimum temperature of 61°C. It presents thermal stability between 30 and 60°C, has 70% activation in the presence of Ca2+, and was tested using citrus pectin with a degree of methyl esterification (DE) of 26%. Ea(d) for irreversible denaturation was 125.5 kJ/mol with positive variations of entropy and enthalpy for that and ΔG(d) values were around 50 kJ/mol. The hydrolysis of polygalacturonate was analyzed by capillary electrophoresis which displayed a pattern of sequential hydrolysis (exo). The partial identification of the primary sequence was done by MS MALDI-TOF and a comparison with data banks showed the highest identity of the sequenced fragments of exo-PG from R. pusillus with an exo-pectinase from Aspergillus fumigatus. Pectin hydrolysis showed a sigmoidal curve for the Michaelis-Menten plot.

scholarly journals INVESTIGATION OF THE FAT FRACTION ENZYMATIC HYDROLYSIS OF THE WASTE FROM PRODUCTION OF HYDROGENATED FAT BY THE LIPASE RHIZOPUS JAPONICUS

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
V. Skliar ◽  
G. Krusir ◽  
V. Zakharchuk ◽  
I. Kovalenko ◽  
T. Shpyrko
Keyword(s):  
Thermal Stability ◽  
Enzymatic Hydrolysis ◽  
Lipase Activity ◽  
Lipolytic Activity ◽  
Complete Loss ◽  
Ph Stability ◽  
Hydrogenated Fat ◽  
Optimum Ph ◽  
Fat Fraction ◽  
Hydrolysis Of

. In the article the conditions of enzymatic hydrolysis of fat fraction of waste from production of hydrogenated fat by the lipase Rhizopus japonicus are considered, namely, the influence of pH of the medium (pH-optimum, pH-stability) and temperature (thermal optimum, thermal stability). The scope of applications of lipases in various branches of the national economy, including for utilization of numerous fatty waste and by-products of oil and fat industry, is disclosed. The main reasons of biotechnological potential of microbial lipases are considered. Objects of research were the lipase Rhizopus japonicus and waste from the demetallization stage of the hydrogenated fat production. Detected, that the optimum pH value for Rhizopus japonicus lipase is 7.0, reducing the pH of the medium from the optimum to pH 6.0 is accompanied by a decrease in activity by 30%, and an increase from 7.0 to 9.0 – decrease by 20%. The maximum activity of the enzyme is observed in the region of physiological values of the temperature. It has been established that the lipase optimal temperature is 40°C. The results of the Rhizopus japonicus lipase stability study showed that incubation of the enzyme at pH 2.5 resulted in a complete loss of lipolytic activity after 30 minutes, and at alkaline pH, the enzyme was more stable. Incubation of lipase Rhizopus japonicus for 30 min at pH 9.0 leads to loss of lipolytic activity by 25% of the maximum, and total loss of activity occurs after 2.5 h. The study of pH-stability of Rhizopus japonicus lipase at an optimal pH of 7 showed that after 60 min of incubation, the enzyme lost 15% of the lipolitical activity, and after 60 min – 50%. Complete loss of Rhizopus japonicus lipase activity at pH 7.0 takes place after 150 minutes of incubation. The results of the study of thermal stability of lipase showed that at a temperature of 40°C and 60°C, the lipase activity remained rather stable for 50 minutes and completely lost after 150 minutes of incubation. At 80°C and 100°C, lipase activity was lost after 40 minutes and 50 minutes of incubation, respectively. The results of the study indicate the prospect of enzymatic hydrolysis of fat fraction of waste by Rhizopus japonicus lipase. The results obtained should be used to improve the processing technology of waste oil and fat industry food and processing industries.

scholarly journals Chain extension of ribonucleic acid by enzymes from rat liver cytoplasm

1968 ◽  
Vol 109 (4) ◽  
pp. 485-494 ◽  
Author(s):  
N. M. Wilkie ◽  
R. M. S. Smellie
Keyword(s):  
Rat Liver ◽  
Alkaline Hydrolysis ◽  
Actinomycin D ◽  
Chain Extension ◽  
Supernatant Fraction ◽  
Inorganic Pyrophosphate ◽  
Optimum Ph ◽  
Microsome Fraction ◽  
Hydrolysis Of ◽  
Generating System

1. The 105000g supernatant fraction of rat liver catalyses the incorporation of ribonucleotides from ribonucleoside triphosphates into polyribonucleotide material. The reaction requires Mg2+ ions and is enhanced by the addition of an ATP-generating system and RNA, ATP, UTP and CTP but not GTP are utilized in this reaction. In the case of UTP, the product is predominantly a homopolymer containing 2–3 uridine residues, and there is evidence that these may be added to the 3′-hydroxyl ends of RNA or oligoribonucleotide primers. 2. The microsome fraction of rat liver incorporates ribonucleotides from ATP, GTP, CTP and UTP into polyribonucleotide material. This reaction requires Mg2+ ions and is enhanced slightly by the addition of an ATP-generating system, and by RNA but not DNA. Supplementation of the reaction mixture with the three complementary ribonucleoside 5′-triphosphates greatly increases the utilization of a single labelled ribonucleoside 5′-triphosphate. The optimum pH is in the range 7·0–8·5, and the reaction is strongly inhibited by inorganic pyrophosphate and to a much smaller degree by inorganic orthophosphate. It is not inhibited by actinomycin D or by deoxyribonuclease. In experiments with [32P]UTP in the absence of ATP, GTP and CTP, 80–90% of 32P was recovered in UMP-2′ or −3′ after alkaline hydrolysis of the reaction product. When the reaction mixture was supplemented with ATP, GTP and CTP, however, about 40% of the 32P was recovered in nucleotides other than UMP-2′ or −3′. Although the reactions seem to lead predominantly to the synthesis of homopolymers, the possibility of some formation of some heteropolymer is not completely excluded.

CHOLESTEROL ESTER HYDROLYSIS BY HOMOGENATES OF WHOLE TESTIS, SEMINIFEROUS TUBULES AND INTERSTITIAL CELLS OF MATURE RATS

1977 ◽  
Vol 75 (2) ◽  
pp. 235-243 ◽  
Author(s):  
J. P. RENSTON ◽  
T. J. IHRIG ◽  
R. H. RENSTON ◽  
B. GONDOS ◽  
R. J. MORIN
Keyword(s):  
Cholesterol Ester ◽  
Incubation Temperature ◽  
Hydrolytic Activity ◽  
Interstitial Cells ◽  
Seminiferous Tubules ◽  
Cholesterol Ester Hydrolase ◽  
Ester Hydrolase ◽  
Optimum Ph ◽  
Hormone Biosynthesis ◽  
Hydrolysis Of

The characteristics and localization of a cholesterol ester hydrolase enzyme in homogenates of whole testis and in isolated seminiferous tubules and interstitial cells of mature rats have been investigated. Hydrolysis of cholesteryl [1-14C]oleate occurred at an optimum pH of 7·0 was linearly related to time up to 5–6 h of incubation and increased linearly up to 0·25 mg protein/incubation. Hydrolytic activity was inhibited by increasing the incubation temperature from 29 to 41 °C and by sonication. Cholesterol ester hydrolase activity/mg protein was three times greater in homogenates of seminiferous tubules than in interstitial cells. Cholesterol ester hydrolase may function to provide precursors for use in seminiferous tubular steroid hormone biosynthesis or germ cell maturation.

scholarly journals Properties of a new fungal b-galactosidase with potential application in the dairy industry

1999 ◽  
Vol 30 (3) ◽  
pp. 265-271 ◽  
Author(s):  
Rubens Cruz ◽  
Vinícius D'Arcádia Cruz ◽  
Juliana Gisele Belote ◽  
Marcelo de Oliveira Khenayfes ◽  
Claudia Dorta ◽  
...  
Keyword(s):  
Hydrolytic Activity ◽  
Industrial Applications ◽  
Transferase Activity ◽  
Optimum Temperature ◽  
Milk Whey ◽  
Beneficial Effects ◽  
Optimum Ph ◽  
Semi Solid ◽  
Good Productivity ◽  
Hydrolysis Of

<FONT FACE="Symbol">b</font>-Galactosidase or <FONT FACE="Symbol">b</font>-D-galactoside-galactohydrolase (EC. 3.2.1.23) is an important enzyme industrially used for the hydrolysis of lactose from milk and milk whey for several applications. Lately, the importance of this enzyme was enhanced by its galactosyltransferase activity, which is responsible for the synthesis of transgalactosylated oligosaccharides (TOS) that act as functional foods, with several beneficial effects on consumers. Penicillium simplicissimum, a strain isolated from soil, when grown in semi-solid medium showed good productivity of <FONT FACE="Symbol">b</font>-galactosidase with galactosyltransferase activity. The optimum pH for hydrolysis was in the 4.04.6 range and the optimum pH for galactosyltransferase activity was in the 6.07.0 range. The optimum temperature for hydrolysis and transferase activity was 55-60°C and 50°C, respectively, and the enzyme showed high thermostability for the hydrolytic activity. The enzyme showed a potential for several industrial applications such as removal of 67% of the lactose from milk and 84% of the lactose from milk whey when incubated at their original pH (4.5 and 6.34, respectively) under optimum temperature conditions. When incubated with a 40% lactose solution in 150 mM McIlvaine buffer, pH 4.5, at 55°C the enzyme converted 86.5% of the lactose to its component monosaccharides. When incubated with a 60% lactose solution in the same buffer but at pH 6.5 and 50°C, the enzyme can synthetize up to 30.5% TOS, with 39.5% lactose and 30% monosaccharides remaining in the preparation.

scholarly journals Influence of Water Activity on Protease Adsorbed on Biochar in Organic Solvents

2017 ◽  
Vol 6 (4) ◽  
pp. 96 ◽  
Author(s):  
Hidetaka Noritomi ◽  
Jumpei Nishigami ◽  
Nobuyuki Endo ◽  
Satoru Kato ◽  
Katsumi Uchiyama
Keyword(s):  
Thermal Stability ◽  
Catalytic Activity ◽  
Organic Solvents ◽  
Water Activity ◽  
Initial Rate ◽  
Butyl Ester ◽  
Nitrogen Atmosphere ◽  
Bamboo Charcoal ◽  
Influence Of Water ◽  
Hydrolysis Of

We have found that the organic solvent-resistance of Alpha-chymotrypsin (Alpha-CT) is enhanced by adsorbing Alpha-CT onto bamboo charcoal powder (BCP), which is obtained by pyrolyzing bamboo waste under nitrogen atmosphere, and is markedly dependent on the thermodynamic water activity (aw) in organic solvents. When BCP-adsorbed　Alpha-CT was immersed in acetonitrile at an appropriate water activity, it effectively enhanced the transesterification of N-acetyl-L-tyrosine ethyl ester (N-Ac-Tyr-OEt) with n-butanol (BuOH) to produce N-acetyl-L-tyrosine butyl ester (N-Ac-Tyr-OBu), compared to the hydrolysis of N-Ac-Tyr-OEt with water to give N-acetyl-L-tyrosine (N-Ac-Tyr-OH). When the water activity was 0.28, the initial rate of transesterification catalyzed by BCP-adsorbed Alpha-CT was about sixty times greater than that catalyzed by free Alpha-CT. Regarding the reaction selectivity which is defined as a ratio of the initial rate of transesterification to that of hydrolysis, BCP-adsorbed α-CT was much superior to free Alpha-CT. The catalytic activity of BCP-adsorbed Alpha-CT was markedly dependent on the reaction temperature. Furthermore, concerning the thermal stability at 50 oC, the half-life of BCP-adsorbed Alpha-CT exhibited 3.8-fold, compared to that of free Alpha-CT.

scholarly journals Biosynthesis, purification, characterization and immobilization of laccase from Lithothelium sp.

Chemija ◽  
2020 ◽  
Vol 31 (3) ◽  
Author(s):  
Ingrida Radveikienė ◽  
Ingrida Pilotaitė ◽  
Rimgailė Dainytė ◽  
Regina Vidžiūnaitė
Keyword(s):  
Residual Activity ◽  
Catalytic Efficiency ◽  
Hydrophobic Interaction Chromatography ◽  
Metal Salts ◽  
Biotechnological Applications ◽  
Affinity Constants ◽  
Sds Page ◽  
Wide Range ◽  
Optimum Ph ◽  
Sulphate Salts

Novel fungal laccase isoenzymes (namely L95-1 and L95-2) produced by the Ascomycete Lithothelium sp. isolated from the forest soil were purified. However, only one of them was characterized, because the other isoenzyme lost its activity during purification. Extracellular L95-1 laccase was purified 30-fold using ion-exchange and hydrophobic interaction chromatography, with an overall yield of 88%. The molecular mass of purified L95-1 was estimated to be 85 kDa by SDS-PAGE analysis. L95-1 laccase was stable at temperature 4–22°C and pH 6.0–6.5. The substrate specificity of L95-1 laccase was examined with various compounds. Determined affinity constants (KM) varied in a wide range of 3.7–2020.0 µM, whereas catalytic efficiency constants (kcat/KM) covered a range of 0.008–1.9 µM–1 s–1. The optimum pH for most substrates varied in a range from pH 5.0 to 6.0. Sodium azide and fluoride strongly inhibited L95-1 activity, whereas sulphate salts inhibited weakly. The laccase was immobilized on the Fe3O4 nanoparticles and characterized. Residual activity remained at 20% after ten cycles of ABTS oxidation reaction. The immobilized laccase showed higher tolerance to various metal salts. The properties of L95-1 laccase make it potentially useful in the biotechnological applications.

scholarly journals Alkaline ceramidase catalyzes the hydrolysis of ceramides via a catalytic mechanism shared by Zn2+-dependent amidases

2018 ◽  
Author(s):  
Jae Kyo Yi ◽  
Ruijuan Xu ◽  
Lina M. Obeid ◽  
Yusuf A. Hannun ◽  
Michael V. Airola ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Biochemical Characterization ◽  
Trichostatin A ◽  
Membrane Lipid ◽  
Yeast Cells ◽  
Adiponectin Receptors ◽  
Wild Type Enzyme ◽  
Zinc Coordination ◽  
Hydrolysis Of ◽  
Insight Into

ABSTRACTHuman alkaline ceramidase 3 (ACER3) is one of three alkaline ceramidases (ACERs) that catalyze the conversion of ceramide to sphingosine. ACERs are the members of the CREST superfamily of integral-membrane lipid hydrolases, including the adiponectin receptors which play roles in energy metabolism. All CREST members conserve a set of three Histidine, one Aspartate, and one Serine residue. However, the structural and catalytic roles for these residues are unclear. Here, we use ACER3 as a prototype enzyme to gain insight into this unique class of enzymes. Recombinant ACER3 was expressed in yeast cells that lack endogenous ceramidase activity, and microsomes were used for biochemical characterization. Six point mutantions of the conserved CREST motif were developed that are predicted to form a Zn-dependent active site based on homology with the human adiponectin receptors, whose crystal structures were recently determined. Five mutations completely lost their activity, except for S77A, which showed a 600-fold decrease compared with the wild-type enzyme. The activity of S77C mutation was pH sensitive, with neutral pH partially recovering ACER3 activity. This suggested a role for S77 in stabilizing the oxyanion of the transition state and differs from the proposed role in Zinc coordination for the adiponectin receptors (Vasiliauskaité-Brooks et. al., Nature, 2017). Together, these data suggest ACER3 is a Zn2+-dependent amidase that uses a catalytic mechanism for ceramide hydrolysis that is similar to other soluble Zn-based amidases. Consistent with this mechanism, ACER3 was specifically inhibited by trichostatin A, an HDAC inhibitor, which is a strong chelator of Zinc.

scholarly journals Production of Recombinant Trichoderma reesei Cellobiohydrolase II in a New Expression System Based on Wickerhamomyces anomalus

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Dennis J. Díaz-Rincón ◽  
Ivonne Duque ◽  
Erika Osorio ◽  
Alexander Rodríguez-López ◽  
Angela Espejo-Mojica ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Recombinant Expression ◽  
Expression System ◽  
Enzymatic Extract ◽  
Native Strain ◽  
Wickerhamomyces Anomalus ◽  
Optimum Ph ◽  
Hydrolysis Of Cellulose ◽  
Cellobiohydrolase Ii ◽  
Hydrolysis Of

Cellulase is a family of at least three groups of enzymes that participate in the sequential hydrolysis of cellulose. Recombinant expression of cellulases might allow reducing their production times and increasing the low proteins concentrations obtained with filamentous fungi. In this study, we describe the production of Trichoderma reesei cellobiohydrolase II (CBHII) in a native strain of Wickerhamomyces anomalus. Recombinant CBHII was expressed in W. anomalus 54-A reaching enzyme activity values of up to 14.5 U L−1. The enzyme extract showed optimum pH and temperature of 5.0–6.0 and 40°C, respectively. Enzyme kinetic parameters (KM of 2.73 mM and Vmax of 23.1 µM min−1) were between the ranges of values reported for other CBHII enzymes. Finally, the results showed that an enzymatic extract of W. anomalus 54-A carrying the recombinant T. reesei CBHII allows production of reducing sugars similar to that of a crude extract from cellulolytic fungi. These results show the first report on the use of W. anomalus as a host to produce recombinant proteins. In addition, recombinant T. reesei CBHII enzyme could potentially be used in the degradation of lignocellulosic residues to produce bioethanol, based on its pH and temperature activity profile.

scholarly journals Immobilization of Isolated Lipase From Moldy Copra(Aspergillus Oryzae)

2011 ◽  
Vol 8 (2) ◽  
pp. 896-902
Author(s):  
Seniwati Dali ◽  
A. B. D. Rauf Patong ◽  
M. Noor Jalaluddin ◽  
Pirman ◽  
Baharuddin Hamzah
Keyword(s):  
Thermal Stability ◽  
Aspergillus Oryzae ◽  
Immobilized Lipase ◽  
Optimum Temperature ◽  
Adsorption Method ◽  
Ph Optimum ◽  
Optimum Ph ◽  
Recovery Technique ◽  
Free Lipase

Enzyme immobilization is a recovery technique that has been studied in several years, using support as a media to help enzyme dissolutions to the reaction substrate. Immobilization method used in this study was adsorption method, using specific lipase fromAspergillus oryzae. Lipase was partially purified from the culture supernatant ofAspergillus oryzae. Enzyme was immobilized by adsorbed on silica gel. Studies on free and immobilized lipase systems for determination of optimum pH, optimum temperature, thermal stability and reusability were carried out. The results showed that free lipase had optimum pH 8,2 and optimum temperature 35 °C while the immobilized lipase had optimum 8,2 and optimum temperature 45 °C. The thermal stability of the immobilized lipase, relative to that of the free lipase, was markedly increased. The immobilized lipase can be reused for at least six times.

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