Characterization of highly stable extracellular lipase from the extremely halophilic archaeon Halolamina sp

Enzymes of the archaea living in extreme environments are resistant to the challenging conditions. Lipase is among the important enzymes used in the industry and agriculture. In this study, the extracellular lipase from extremely halophilic archaeon Halolamina sp. was characterized for the first time. Optimum temperature for the enzyme activity was determined as 70oC, optimum pH was 7.0, and the optimum salt concentration was 3.6 M. Additionally, more than 70% of the enzyme activity was remained between pH 3.0-10.0 for 48 h as well as incubation of the enzyme at 70oC for 30 min increased its activity for 44%, and no activity loss was observed after incubation at 80oC. Also, presence of the metals increased the enzyme activity up to 88%. The enzyme was highly resistant to the organic solvents acetone, methanol, and DMSO while strong inhibition was caused by n-butanol. Among the detergents, the enzyme kept its activity substantially in the presence of SDS; however, other detergents caused inhibition of the enzyme activity. This characterization study showed that the lipase from the haloarchaeon Halolamina sp. is highly stable at the wide ranges of temperature and pH values as well as in the presence of diverse inhibitors. This enzyme is promising to be used in biotechnological applications.

Preformulation of a liquid dosage formulation of captopril for pediatric use: drug-excipient compatibility and stability studies

Currently, medications used in children are typically modified from pharmaceutical dosage forms designed for adults. Captopril is widely adapted to liquid formulations for use in hospitals. Its stability in the aqueous medium is reduced since it undergoes oxidation producing captopril disulfide (its main metabolite). The aim of this formulation study was to suggest favorable conditions for the development of a stable captopril formulation. The compatibility between the drug and excipients was evaluated by differential scanning calorimetry analysis (DSC). For studies in solution, different formulations were prepared according to a factorial design varying EDTA concentration, water purity and pH. The resultant formulations were stored at 60°C and analyzed over a twelve-day period using HPLC. The DSC curves obtained suggested, although not conclusive to elucidation, interactions of captopril with citric acid and sucralose. The stability study of these solutions revealed that the variables significantly influenced captopril content, which degraded at zero order kinetics and rates differing by a factor of up to 7 times, where pH proved the most influential factor. Interactions between variables were observed. Therefore, development of a stable captopril formulation is feasible provided EDTA and a buffering agent is used at suitable concentrations (0.08% and pH 3.85).

Stability enhancement of urethanase from Lysinibacillus fusiformis by site-directed mutagenesis / 生物工程学报

Ethyl carbamate as a potential carcinogen commonly exists in traditional fermented foods and beverages. Enzymatic removal of ethyl carbamate from fermented foods and beverages is an efficient and safe method. In this study, we mutated urethanase from Lysinibacillus fusiformis SC02 on the Q328 site through computer aided design approaches. The half-life of resulting mutants Q328C and Q328V was detected to be 7.46 and 1.96 folds higher than that of the original enzyme, and Q328R presented better thermal-tolerance than the original urethanase when incubated at high temperature. The tolerance of Q328C to ethanol and acid also increased when compared with that of the original enzyme. The stability and tolerance to acid and ethanol of urethanase could be improved by modification on its Q328 site.

Actividad enzimática colinesterasa en muestras de sangre humana: efecto de las condiciones de almacenamiento / Cholinesterase enzyme activity in human blood samples: effects of the store conditions

Introducción: La determinación de la actividad enzimática colinesterasa (ChE) es el principal biomarcador de efecto de la exposición a los plaguicidas organofosforados y carbamatos. Por lo tanto, la estabilidad de la actividad de las ChEs en muestras de sangre es un parámetro pre-analítico importante que necesita ser considerado en términos de la seguridad diagnóstica. Objetivo: Determinar el efecto del tiempo y de la temperatura de almacenamiento sobre la actividad de las ChEs en muestras de sangre humana. Metodología: Muestras de sangre entera y suspensiones de eritrocitos (eritrocitos + solución salina 0.9% proporción 1:1) fueron almacenados a -20°C, 4°C y 25°C. Las determinaciones enzimáticas se realizaron una hora después de la toma de la muestra y se repitieron entre el día 1 hasta el día 90. La actividad enzimática ChE total y Acetil-ChE se determinaron respectivamente mediante el método colorimétrico de Limperos & Ranta y mediante el método potenciométrico de Michel. Resultados: La máxima estabilidad de la actividad ChE total se observó a -20°C hasta por 60 días, además, dicha estabilidad perduró hasta por el tiempo máximo del estudio a 4°C para la Acetil-ChE. Una considerable disminución de la actividad Acetil-ChE se observó después de los días 7 a 25°C y 4 a -20°C. Conclusión: Considerando la seguridad diagnóstica, nosotros recomendamos almacenar las muestras de sangre entera a -20°C por un tiempo máximo de 30 días para la determinación de la actividad ChE total y la suspensión de eritrocitos en 0.9% de NaCl a 4°C por 14 días máximo para la determinación de la Acetil-ChE.

Introduction: Determination of cholinesterase (ChE) enzyme activity is the main biomarker of exposure to pesticides organophosphorus and carbamate. Therefore, the enzyme stability of ChEsin blood samples is an important pre-analytical factor to take into account in the diagnosis. Objective: To determine the effect of storage time and temperature on ChEs enzyme activity in human blood samples. Methodology: Whole-blood samples and erythrocyte suspensions (erythrocyte + 0.9% saline solution; ratio 1:1) were stored at -20°C, 4°C and 25°C. Enzyme activity measurements were performed at one hour after the blood samples have been obtained and then were repeated between days 1 and 90. Total ChE and Acetyl-ChE activities were determined using the Limperos & Ranta colorimetric method and the potentiometric method of Michel respectively. Results: The maximum stability of the total ChE enzyme activity was achieved at -20°C for 60 days and, in the case of Acetyl-ChE, at 4°C for the time the study was conducted. A decrease of Acetyl-ChE activity was shown after 7 days at 25°C and 4 days at -20°C. Conclusion: In terms of diagnosis, we recommend that in order to measure the total ChE activity the wholeblood samples should be stored at -20°C for 30 days, whereas to measure the Acetyl-ChE activity the erythrocyte suspensions in 0.9% NaCl at 4°C for 14 days.

Chemical modification of Aspergillus niger β-glucosidase and its catalytic properties

Aspergillus niger β-glucosidase was modified by covalent coupling to periodate activated polysaccharides (glycosylation). The conjugated enzyme to activated starch showed the highest specific activity (128.5 U/mg protein). Compared to the native enzyme, the conjugated form exhibited: a higher optimal reaction temperature, a lower Ea (activation energy), a higher Km (Michaelis constant) and Vmax (maximal reaction rate), and improved thermal stability. The calculated t1/2 (half-life) values of heat in-activation at 60 °C and 70 °C were 245.7 and 54.5 min respectively, whereas at these temperatures the native enzyme was less stable (t1/2 of 200.0 and 49.5 min respectively). The conjugated enzyme retained 32.3 and 29.7%, respectively from its initial activity in presence of 5 mM Sodium Dodecyl Sulphate (SDS) and p-Chloro Mercuri Benzoate (p-CMB), while the native enzyme showed a remarkable loss of activity (retained activity 1.61 and 13.7%, respectively). The present work has established the potential of glycosylation to enhance the catalytic properties of β-glucosidase enzyme, making this enzyme potentially feasible for biotechnological applications.

Bordonein-L, a new L-amino acid oxidase from Crotalus durissus terrificus snake venom: isolation, preliminary characterization and enzyme stability

Background Crotalus durissus terrificus venom (CdtV) is one of the most studied snake venoms in Brazil. Despite presenting several well known proteins, its L-amino acid oxidase (LAAO) has not been studied previously. This study aimed to isolate, characterize and evaluate the enzyme stability of bordonein-L, an LAAO from CdtV.Methods The enzyme was isolated through cation exchange, gel filtration and affinity chromatography, followed by a reversed-phase fast protein liquid chromatography to confirm its purity. Subsequently, its N-terminal amino acid sequence was determined by Edman degradation. The enzyme activity and stability were evaluated by a microplate colorimetric assay and the molecular mass was estimated by SDS-PAGE using periodic acid-Schiff staining and determined by mass spectrometry.Results The first 39 N-terminal amino acid residues exhibited high identity with other snake venom L-amino acid oxidases. Bordonein-L is a homodimer glycoprotein of approximately 101 kDa evaluated by gel filtration. Its monomer presents around 53 kDa estimated by SDS-PAGE and 58,702 Da determined by MALDI-TOF mass spectrometry. The enzyme exhibited maximum activity at pH 7.0 and lost about 50 % of its activity after five days of storage at 4 °C. Bordonein-L's activity was higher than the control when stored in 2.8 % mannitol or 8.5 % sucrose.Conclusions This research is pioneering in its isolation, characterization and enzyme stability evaluation of an LAAO from CdtV, denominated bordonein-L. These results are important because they increase the knowledge about stabilization of LAAOs, aiming to increase their shelf life. Since the maintenance of enzymatic activity after long periods of storage is essential to enable their biotechnological use as well as their functional studies.(AU)

Bordonein-L, a new L-amino acid oxidase from Crotalus durissus terrificus snake venom: isolation, preliminary characterization and enzyme stability

Background Crotalus durissus terrificus venom (CdtV) is one of the most studied snake venoms in Brazil. Despite presenting several well known proteins, its L-amino acid oxidase (LAAO) has not been studied previously. This study aimed to isolate, characterize and evaluate the enzyme stability of bordonein-L, an LAAO from CdtV.Methods The enzyme was isolated through cation exchange, gel filtration and affinity chromatography, followed by a reversed-phase fast protein liquid chromatography to confirm its purity. Subsequently, its N-terminal amino acid sequence was determined by Edman degradation. The enzyme activity and stability were evaluated by a microplate colorimetric assay and the molecular mass was estimated by SDS-PAGE using periodic acid-Schiff staining and determined by mass spectrometry.Results The first 39 N-terminal amino acid residues exhibited high identity with other snake venom L-amino acid oxidases. Bordonein-L is a homodimer glycoprotein of approximately 101 kDa evaluated by gel filtration. Its monomer presents around 53 kDa estimated by SDS-PAGE and 58,702 Da determined by MALDI-TOF mass spectrometry. The enzyme exhibited maximum activity at pH 7.0 and lost about 50 % of its activity after five days of storage at 4 °C. Bordonein-Ls activity was higher than the control when stored in 2.8 % mannitol or 8.5 % sucrose.Conclusions This research is pioneering in its isolation, characterization and enzyme stability evaluation of an LAAO from CdtV, denominated bordonein-L. These results are important because they increase the knowledge about stabilization of LAAOs, aiming to increase their shelf life. Since the maintenance of enzymatic activity after long periods of storage is essential to enable their biotechnological use as well as their functional studies.

The influence of pH, polyethylene glycol and polyacrylic acid on the stability of stem bromelain

Enzyme stability is critical in biotechnology, pharmaceutical and cosmetic industries. Investigations on this subject have drawn attention because of its practical application. Bromelain is a thiol-endopeptidase, obtained from pineapple (Ananas comosus), known for its clinical and therapeutic applications, particularly to selective burn debridement and improvement of antibiotic action and anti-inflammatory activities. To date, the use of bromelain in pharmacological or industrial applications is limited, due to commercial availability, costs, and sensitivity to pH and temperature. Therefore, a better understanding of enzyme stability would be of great interest. The aim of this study was to evaluate bromelain activity and stability in several pH (2.0 to 8.0) and in polyethylene glycol and polyacrylic acid solutions. We observed that bromelain was able to maintain its stability at pH 5.0 for the temperatures studied. PEG solutions increased bromelain stability, but PAA solutions had the opposite effect.

Estabilidade de enzimas é uma questão fundamental em indústrias biotecnológicas, farmacêuticas e cosméticas. As investigações sobre o assunto têm chamado a atenção por sua aplicação prática. A bromelina é uma tiol-endopeptidase, obtida a partir do abacaxi (Ananas comosus). É conhecida por suas aplicações clínicas e terapêuticas, especialmente para desbridamento seletivo de queimaduras, melhoria de ações antibiótica e de atividades anti-inflamatórias. Até o momento, a utilização da bromelina em aplicações farmacológicas industriais é limitada, devido à disponibilidade comercial, os custos, a sensibilidade ao pH e temperatura. Portanto, a maior compreensão da estabilidade desta enzima seria de grande interesse. O objetivo deste estudo foi avaliar a estabilidade da atividade da bromelina em vários pH (2,0 a 8,0) e em soluções de polietilenoglicol e de ácido poliacrílico. Observamos que a bromelina foi capaz de manter a sua estabilidade em pH 5.0, em todas as temperaturas estudadas. Soluções de PEG aumentaram a estabilidade da bromelina, enquanto que soluções de PAA obtiveram efeito oposto.

Long-Term Stability of Selected Serum Biomarkers in Leftover Samples / 임상검사와정도관리

BACKGROUND: The purpose of this study was to assess the quality of long-term-stored leftover blood samples, and to evaluate the long-term stability of selected serum biomarkers such as proteins, enzymes, electrolytes, and tumour markers. METHODS: Stored blood samples were transferred to our biobank after being used to conduct tests for routine medical examinations in one health care institution, and were preserved at or below -70degrees C for 4 years. We analysed 24 biomarkers whose levels had been reported 4 years ago and tested them using the same analyser, reagents, and methods by utilizing an ADVIA Centaur Immunoassay System (Siemens Healthcare Diagnostics, USA) or an ADVIA 2400 Chemistry System (Siemens, USA). RESULTS: A total of 15 out of the 24 tested biomarkers showed significant differences in paired Student t-tests (P0.975). Two biomarkers, creatinine and rheumatoid arthritis factor, showed no significant differences but were poorly correlated with previously analysed data. Aspartate aminotransferase, alanine aminotransferase, hepatitis B virus (HBV) surface antigen, and insulin levels were discordant according to their reference ranges. A total of 3 biomarkers, C-reactive protein, cancer antigen 125, and HBV surface antibody, showed no significant differences and good correlations without discordant data. CONCLUSIONS: Our findings showed that long-term storage for more than 4 years can result in a considerable bias for variable biomarkers. Only 3 of the 24 biomarkers evaluated were found to be stable biomarkers. Long-term storage of leftover samples is not recommended for most chemical analyses.

Production and characterization of endo-polygalacturonase from Aspergillus niger in solid-state fermentation in double-surface bioreactor

Endo-polygalacturonase (endo-PG) production by Aspergillus niger T0005/007-2 in solid medium with 170 mm of height was evaluated in a cylindrical double surface bioreactor in 96-h experiments. Cell concentration close to 92 mg.g -¹ dm (mg per g of dry medium) in the standard condition (static) was achieved, whereas in tests under forced aeration of 1.4 and 2.8 L.min-1. Kg-1 mm (L of air per minute per Kg of moist medium) and with the central shaft fungal biomass attained approximately 100 mg.g-1 dm. Superior endo-PG activity was obtained with the central-shaft system, 78 U.g-1 dm (units per g of dry medium). Forced aeration and pressure pulse showed no positive effect on the production of endo-PG, 45 U.g-1 dm and 28 U.g-1 dm, respectively. None of the conditions evaluated was efficient for medium temperature control. Endo-PG was stable up to 40ºC. The activity decreased in 50 percent after 120 minutes at 50ºC, which is a temperature normally found during this process.

Caracterização cinética da enzima polifenol oxidase, usando extrato bruto da casca de banana nanica (Musa acuminata) / Kinetic characterization of enzyme polyphenol oxidase, utilizing crude of extract of pell of stunded banana (Musa acuminata)

Introdução - Extrato bruto da casca de banana nanica (Musa acuminata); melhor fonte de enzima polifenol oxidase (PFO) [EC.1.14.18.1] foi estudado como material biocatalítico para a oxidação aeróbica de substratos fenólicos. Material e Métodos - Foi estudada a extração da PFO dessa fonte. A atividade da enzima PFO e proteína total foram determinadas nesse extrato. O estudo da quantidade e do tempo de contato do polímero SB-100 com esse extrato foi realizado. A caracterização cinética da PFO foi determinada com temperatura ótima, estabilidade ao calor, pH ótimo de atividade e de estabilidade. Os valores obtidos foram comparados com a PFO da polpa de banana nanica. Resultados - Os resultados mostraram que a quantidade de polímero SB-100 foi maior usando o extrato da casca de banana nanica do que para a polpa, mas o tempo de contato não houve alteração. O pH de atividade (pH 6,0), de estabilidade (pH 6,5) e a temperatura ótima (30°C), não apresentaram variações comparando os dois extratos utilizados. Conclusões - Verificou-se, portanto que o uso do extrato bruto da casca de banana nanica apresentou maior atividade enzimática de PFO do que para a polpa da fruta; enquanto a caracterização cinética praticamente não variou nos dois extratos. O que se pode concluir é que, para a construção do biossensor a casca da banana é melhor que a polpa por apresentar maior atividade da enzima. A seguir, um biossensor será construído, para detecção de fenólicos e os resultados serão comparados com os da polpa.

Introduction - Crude extract of the pell of stunded banana (Musa acuminata); the best source enzyme polyphenol oxidase (PPO) [EC.1.14.18.1] was studied as material biocatalitic for the aerobic phenolic substratum oxidation. Material and Methods - The extraction of the PPO of this source was studied. The activity of enzyme PPO and total protein had been determined in this extract. The study of the amount and the contact time of polymer SB-100 with this extract was done. The kinetic characterization of the PPO was determined with great temperature, stability to the heat, great pH of activity and stability. The gotten values had been compared with the PPO of the pulp of stunded banana. Results - The results had shown that the amount of polymer SB-100 was bigger used in the extract of the pell of stunded banana than the pulp, but the contact time did not have any alteration. The pH of activity (pH 6,0), of stability (pH 6,5) and the great temperature (30°C), had not presented variations comparing the two used extracts. Conclusions - It was verified, therefore that the use of the crude extract of the pell of stunded banana presented greater enzimatic activity of PPO than in the pulp of fruit; while the kinetic characterization practically did not vary in two extracts. We can conclude that for the construction of the biosensor the pell of the banana is better than the pulp for presenting greater activity of the enzyme. Next, a biosensor will be constructed, for detection of phenolics and the results will be compared with ones the pulp.