Enzyme Purification

Bromelain is a mixture of proteolytic enzymes derived from the stems and juice of pineapples. It has been extensively used in food industry; for meat tenderization, baking processes, and in prevention of browning of apple juice. Stem bromelain is a highly accepted phytotherapeutic agent. It has anti-tumor and antiinflammatory effects, use in wound healing, as digestive aids, etc. This review aims at presenting a detailed account on the level of achievement on bromelain production from the conventional methods. Specifically, the isolation, purification and the biochemical properties of bromelain are discussed. The study also focused on the current trends and the progress made in the production of recombinant stem bromelain in Escherichia coli. This involved cloning of stem bromelain gene and its expression, optimization of labscale fermentation conditions for E. coli harboring recombinant bromelain as well as the enzyme thermal and storage stability measured using differential scanning calorimetry. It is hoped that this review would provide relevant information to contemporary researchers who are active in the field of bromelain and other related proteases.

The contemporaneously produced alkaline protease and I±-amylase by locally isolated Bacillus methylotrophicus SCJ4 had been purified in two steps and some characteristics of the purified enzymes were studied. Crude enzymes were first salted out using ammonium sulfate, and then concentrated proteins were applied to anion column chromatography Q-sepharose (HiPrep Q FF 16/10 column). The purified alkaline protease has an estimated molecular mass of 24 kDa with maximum enzyme activity (1185U/ml/min) at pH 9.0 and 55ÂoC. This enzyme belongs to serine proteases family with remarkable stability up to 62% and 61.5% toward Sodium Dodecyl Sulfate (SDS) and Hg++ respectively. On the other hand, the purified amylase is a calcium-independent I±-amylase, showing a good stability against SDS up to 73%. The estimated molecular mass was 67.5 kDa with maximum enzyme activity (280U/ml/min) at pH 7.0 and 65ÂoC.

The proteolytic enzyme trypsin was purified from the gastrointestinal tract waste of grass carp Ctenopharyngodon idella (Valenciennes, 1844) with ammonium sulfate fractionation (ASF) followed by DEAE-cellulose chromatography, and Benzamidine Sepharose fast flow column affinity chromatography. Trypsin was purified 26.2-fold with an 11.1% yield. The purified enzyme was active between pH 9.0 and 11.0, and maximal activity of the enzyme was observed at pH 10.0. Highest activity was found at 60°C. The activity was reduced further after reaching the maximum activity point of temperature. The trypsin enzymatic activity was decreased by 40% and 60%, when incubated at 90°C for 30 min. The Km, Kcat, and catalytic efficiency values of purified trypsin were obtained is 0.062 mM and 19.23/s, and 310.16/s/mM. Degree of hydrolysis (DH) of the proteases on muscle protein increased with increase of enzyme concentrations. The enzyme activity was also further inhibited by SBTI, PMSF, and N-α-p-tosyl-L-lysine chloromethyl ketone. The molecular weight of the purified enzyme was obtained to be 20.2 kDa by SDS-PAGE. The study showed that trypsin from grass carp visceral waste of could find use in applications where maximum activity at moderate temperature is desired.

Acidic and alkaline proteases from visceral waste of Cyprinus carpio (Linnaeus, 1758) were extracted, partially purified by ammonium sulphate precipitation followed by dialysis. Their enzyme kinetics and characteristics were studied. The purification fold increased from 1.39 to 2.24 and 1.46 to 2.17 in acidic and alkaline protease respectively along the purification steps. The molecular weights were found in the range of 15-35 kDa and 25-63 kDa respectively in acidic and alkaline proteases. The pH and temperature optima for acidic and alkaline proteases were 4 and 11, and 40°C and 70°C respectively. The protease activity was found decreased by 40 and 60% when incubated at 90°C for 30 min. Both the proteases showed a declining activity of more than 50% at NaCl concentration of 0.5%. Degree of hydrolysis of the proteases on muscle protein increased with increase of enzyme contents. Both soybean trypsin inhibitor and EDTA exhibited high percentage of inhibition when proteases were incu...

Secondary sulfonamides (4a-8h) incorporating acetoxybenzamide, triacetoxybenzamide, hydroxybenzamide, and trihydroxybenzamide and possessing thiazole, pyrimidine, pyridine, isoxazole and thiadiazole groups were synthesized. Lactoperoxidase (LPO, E.C.1.11.1.7), as a natural antibacterial agent, is a peroxidase enzyme secreted from salivary, mammary, and other mucosal glands. In the present study, the in vitro inhibitory effects of some secondary sulfonamide derivatives (4a-8h) were examined against LPO. The obtained results reveal that secondary sulfonamide derivatives (4a-8h) are effective LPO inhibitors. The Ki values of secondary sulfonamide derivatives (4a-8h) were found in the range of 1.096 × 10(-3) to 1203.83 µM against LPO. However, the most effective inhibition was found for N-(sulfathiazole)-3,4,5-triacetoxybenzamide (6a), with Ki values of 1.096 × 10(-3) ± 0.471 × 10(-3) µM as non-competitive inhibition.

Secondary sulfonamides (4a-8h) incorporating acetoxybenzamide, triacetoxybenzamide, hydroxybenzamide, and trihydroxybenzamide and possessing thiazole, pyrimidine, pyridine, isoxazole and thiadiazole groups were synthesized. Lactoperoxidase (LPO, E.C.1.11.1.7), as a natural antibacterial agent, is a peroxidase enzyme secreted from salivary, mammary, and other mucosal glands. In the present study, the in vitro inhibitory effects of some secondary sulfonamide derivatives (4a-8h) were examined against LPO. The obtained results reveal that secondary sulfonamide derivatives (4a-8h) are effective LPO inhibitors. The Ki values of secondary sulfonamide derivatives (4a-8h) were found in the range of 1.096 × 10(-3) to 1203.83 µM against LPO. However, the most effective inhibition was found for N-(sulfathiazole)-3,4,5-triacetoxybenzamide (6a), with Ki values of 1.096 × 10(-3) ± 0.471 × 10(-3) µM as non-competitive inhibition.

RESUMO -O objetivo deste trabalho foi selecionar as condições mais adequadas para a produção de protease utilizando planejamento fatorial pelo Streptomyces parvulus DPUA 1573. As variáveis de produção (concentrações da fonte de carbono-0g/L; 0,5 g/L; 1,0 g/L, de nitrogênio-0,5%; 1,0%; 1,5%; agitação-150, 200 e 250 rpm; temperatura-28, 32 e 37°C) foram determinadas e analisadas por um planejamento fatorial 2 4 tendo como variável resposta produção de protease e biomassa celular. Foi verificado que as variáveis independentes que mais influenciaram na produção foram a temperatura e agitação, sendo o ensaio 3 selecionado (0,5% de farinha de soja, 1,0g/L de glicose em 150 rpm a 28ºC) com 347 U/mL. A maior atividade proteásica foi obtida em 48 horas. Logo, a bactéria filamentosa Streptomyces parvulus DPUA 1573, apresentou atividade proteásica significativa podendo ser inserida no mercado enzimático.

Carbonic anhydrases (CAs, EC 4.2.1.1) had six genetically distinct families described to date in various organisms. There are 16 known CA isoforms in humans. Human CA isoenzymes I and II (hCA I and hCA II) are ubiquitous cytosolic isoforms. Acetylcholine esterase (AChE. EC 3.1.1.7) is a hydrolase that hydrolyzes the neurotransmitter acetylcholine relaying the signal from the nerve. In this study, some trimethoxyindane derivatives were investigated as inhibitors against the cytosolic hCA I and II isoenzymes, and AChE enzyme. Both hCA isozymes were inhibited by trimethoxyindane derivatives in the low nanomolar range. These compounds were good hCA I inhibitors (Kis in the range of 1.66-4.14 nM) and hCA II inhibitors (Kis of 1.37-3.12 nM) and perfect AChE inhibitors (Kis in the range of 1.87-7.53 nM) compared to acetazolamide as CA inhibitor (Ki: 6.76 nM for hCA I and Ki: 5.85 nM for hCA II) and Tacrine as AChE inhibitor (Ki: 7.64 nM).

The contemporaneously produced alkaline protease and α-amylase by locally isolated Bacillus methylotrophicus SCJ4 had been purified in two steps and some characteristics of the purified enzymes were studied. Crude enzymes were first salted out using ammonium sulfate, then concentrated proteins were applied to anion column chromatography Q-sepharose (HiPrep Q FF 16/10 column). The purified alkaline protease has an estimated molecular mass of 24 kDa with maximum enzyme activity (1185U/ml/min) at pH 9.0 and 55ºC. This enzyme belongs to serine proteases family with remarkable stability up to 62% and 61.5% toward Sodium Dodecyl Sulfate (SDS) and Hg ++ , respectively. On the other hand, the purified amylase is a calcium-independent α-amylase, showing a good stability against SDS up to 73%. The estimated molecular mass was 67.5 kDa with maximum enzyme activity (280U/ml/min) at pH 7.0 and 65ºC.

Two low molecular mass endo-l,4-P-D-xylanases from Fusarium oxysporum were purified to homogeneity by gel-filtration and ion-exchange chromatography. They exhibit molecular masses of 20.8 (xylanase I) and 23.5 (xylanase II) kDa, and isoelectric points of 9.5 and 8.45-8.70, respectively. Both xylanases display remarkable pH (9.0) stability. At 40 to 55°C xylanase II is more thermostable than xylanase I but less active on xylan. In contrast to xylanase I, xylanase II is able to hydrolyze 1-O-4-methylumbelliferyl-(~-D-glucopyranosyl)-~-D-xylopyranoside (muxg). Neither of these enzymes hydrolyze xylotriose. They bind on crystalline cellulose but not on insoluble xylan. Analysis of reaction mixtures by high pressure liquid chromatography revealed that both enzymes cleave preferentially the internal glycosidic bonds of xylopentaose and oat spelts xylan. Thus the purified enzymes appeared to be true endo-j?-1,4-xylanases. The amino terminal sequences of xylanases I and II show no homology. Xylanase I shows high similarity with alkaline low molecular mass xylanases of family G/l 1.

Background & Objectives: Thermophilic alpha-amylase can be used in different industries such as starch processing and detergents. This study was performed to isolate alpha-amylase-producing bacteria and characterization of the enzyme. Materials & Methods: After sample collection from Gorooh hot spring in Kerman province, Iran, thermophilic alpha-amylase-producing bacteria were isolated using the starch-agar medium. 16S rDNA sequencing was used to identify the bacterial strain. Characterization of the thermophilic alpha-amylase was performed in the presence of various factors such as pH, temperature, metal ions, chemical compounds, and organic solvents. Also, kinetic parameters of the enzyme were determined in different concentrations of starch. Results: Anoxybacillus gonensis AT23 was identified as the best thermophilic alpha-amylaseproducing strain. The alpha-amylase enzyme showed the optimal activity at pH 5 to 6. Sevenfold increase in the enzyme activity was observed in the presence of NaCl (3M). Mn 2+ and Zn 2+ increased the enzyme activity about 95% and 31%, respectively. Kinetic parameters including K m and V max were estimated about 1.657 mg/ml and 0.0059 mg/ml/min, respectively. Also, enzyme activity was also improved about 2 folds in the presence of organic solvents including n-butanol and 10% cyclohexane. Conclusion: Our results indicated that AT23 alpha-amylase is a halophile and organic solvent-tolerant enzyme. Therefore, it can be used in different industries.

família. Em especial à minha mãe, por todo esforço, e à minha madrinha Leda e seu marido, Beto, por me acolherem nos meus primeiros passos como universitária. Ao meu namorado, Ronaldo, por me manter sã nesta última etapa, e à sua mãe, D. Fátima, por toda amizade e preocupação comigo. Ao meu orientador, Prof. Dr. Thalles Barbosa Grangeiro, pela paciência, por aceitar minhas ideias, mesmo quando a aplicação parecia difícil, por me ajudar a torná-las plausíveis e por toda orientação. Aos membros da banca examinadora e colegas de laboratório, Simone e Edvar, pela enorme ajuda, por todas as sugestões e pela paciência. À todos os integrantes do Laboratório de Genética Molecular, que permanecem e que já partiram: Ednésio, que me mostrou os primeiros passos na Biologia Molecular e Bioinformática; Juscelino, pela co-orientação durante grande parte da minha permanência no laboratório; Suelen, sempre disposta a ajudar; Mayara e Erlane, pelos cafés com M&M; e a todos os que não foram citados, mas igualmente contribuíram nestes três anos e meio. Aos meus amigos da graduação, João Marcos, Thaís, Átila, Nil, Mayara e Lucas, pelos momentos de risada, pão bola, castanhola e os dias no Pacheco. Ao CNPq e à Capes, pelo financiamento. À todos aqueles que não citei, por esquecimento, mas contribuíram nessa jornada. "DAS UTOPIAS Se as coisas são inatingíveis...ora! Não é motivo para não querê-las... Que tristes os caminhos, se não fora A presença distante das estrelas!" (Mário Quintana) "Somos feitos de poeira estelar." (Carl Sagan) RESUMO O gênero Chromobacterium é composto por Betaproteobactérias Gram-negativas, saprofíticas e de vida livre, com alguns representantes patogênicos. O grupo apresentou significante crescimento entre 2007 e 2017 com a descoberta de dez novas espécies. Chromobacterium violaceum é sua integrante mais estudada e a primeira a ser descrita. A espécie é comumente encontrada em águas e solos de regiões tropicais e subtropicais como saprófita, sendo capaz de utilizar apenas quitina como fonte de energia. A quitina é o segundo biopolímero mais abundante na natureza e pode ser utilizado como fonte de carbono e nitrogênio por diversos organismos, incluindo bactérias. Sua degradação é iniciada por monooxigenases líticas de polissacarídeo (LPMOs), ativas em substrato cristalino. O presente trabalho teve como objetivo a construção, por análises de genômica comparativa, do catabolismo de quitina de Chromobacterium e a expressão de uma LPMO de C. violaceum ATCC 12472. Assim, foi construída uma provável via catabólica em C. violaceum ATCC 12472 a partir daquela descrita para a família Vibrionaceae, das informações obtidas sobre o regulon NagQ na espécie e por análises in silico das localizações subcelulares de cada proteína selecionada. A via foi expandida a nível de gênero pela localização de ortólogos nos 41 genomas de integrantes de Chromobacterium disponíveis no banco de dados do NCBI. Foram encontradas cinco sequências de LPMOs em C. violaceum ATCC 12472: CV0553, CV0554, CV2592, CV3323 e CV3489. Dentre essas, CV0553 e CV2592 tiveram suas sequências codificadoras sintetizadas com códons otimizados e inseridas em plasmídeos pET-Sumo modificados. Os vetores foram introduzidos nas cepas de Escherichia coli BL21(DE3), SHuffle® T7 Express Competent e ArcticExpress (DE3). Das duas proteínas produzidas, apenas rCv2592-Sumo foi encontrada na forma solúvel na fração intracelular dos três hospedeiros analisados. A proteína recombinante foi purificada em matriz de quitina, sendo capaz de degradar α-quitina. Conclui-se que o catabolismo de quitina é conservado em Chromobacterium. Este é um indício da importância da quitina como fonte alternativa de energia no grupo. O gene para o regulador NagQ é bastante conservado no gênero, com sua forma plesiomórfica encontrada em C. haemolyticum e a mais recente em C. violaceum. Por fim, a produção de rCv2592 pode ser uma alternativa eficiente para a produção de derivados de quitina, sendo necessários estudos mais aprofundados sobre o tema.

Human aldo-keto reductase 1B15 (AKR1B15) is a newly discovered enzyme which shares 92% amino acid sequence identity with AKR1B10. While AKR1B10 is a well characterized enzyme with high retinaldehyde reductase activity, involved in the development of several cancer types, the enzymatic activity and physiological role of AKR1B15 are still poorly known. Here, the purified recombinant enzyme has been subjected to substrate specificity characterization, kinetic analysis and inhibitor screening, combined with structural modeling. AKR1B15 is active towards a variety of carbonyl substrates, including retinoids, with lower k cat and K m values than AKR1B10. In contrast to AKR1B10, which strongly prefers alltrans-retinaldehyde, AKR1B15 exhibits superior catalytic efficiency with 9-cis-retinaldehyde, the best substrate found for this enzyme. With ketone and dicarbonyl substrates, AKR1B15 also shows higher catalytic activity than AKR1B10. Several typical AKR inhibitors do not significantly affect AKR1B15 activity. Amino acid substitutions clustered in loops A and C result in a smaller, more hydrophobic and more rigid active site in AKR1B15 compared with the AKR1B10 pocket, consistent with distinct substrate specificity and narrower inhibitor selectivity for AKR1B15.

Em 17/03/2016: Anuidade de pedido de patente de invenção no prazo ordinárioDepositadaA presente invenção é relacionada a fatores antimicrobianos extraídos de envoltórios protetores (cascas) de ovos de insetos. A maioria destes fatores compreende peptídeos e/ou proteínas obtidas de tais involuntários e/ou de seus respectivos genes codificantes, em construções gênicas artificiais. São descritas composições microbicidas e processos para sua obtenção, bem como construções gênicas e processos para o controle de pestes

Fungos entomopatogênicos produzem enzimas proteolíticas e são empregados no controle biológico de insetos-pragas. Realizou-se um cultivo de Beauveria bassiana (cepaCG432) a 25ºC/180rpm/5dias/10 6 conídeos/100mL. Após centrifugação, o sobrenadante foi concentrado por ultrafiltração seqüencial e mantido a 4 e 25ºC em pH 7,0 e 9,5. A atividade proteolítica foi relacionada com o teor de peptídeos solúveis resultantes da incubação de 200µL do sobrenadante e 500µL e albumina sérica bovina a 37ºC/30min, adição de 500µL de TCA10% e centrifugação a 1100xg/15min. As enzimas proteolíticas foram estáveis a 25ºC em pH 7,0 durante 10dias, porém demonstraram redução significativa da atividade em pH 9,5 após 3dias.

In this research, optimization of the integrated biodiesel production process composed of transesterification of edible sunflower oil, catalyzed by commercial lipase, with simultaneous extraction of glycerol from the reaction mixture was performed. Deep eutectic solvents (DESs) were used in this integrated process as the reaction and extraction media. For two systems, choline chloride:glycerol (ChCl:Gly) and choline chloride:ethylene glycol (ChCl:EG), respectively, the optimal water content, mass ratio of the phase containing the mixture of reactants (oil and methanol) with an enzyme and a DES phase (mass ratio of phases), and the molar ratio of deep eutectic solvent constituents were determined using response surface methodology (RSM). Experiments performed with ChCl:Gly resulted in a higher biodiesel yield and higher glycerol extraction efficiency, namely, a mass ratio of phases of 1:1, a mass fraction of water of 6.6%, and a molar ratio of the ChCl:Gly of 1:3.5 were determined to...

Conidia of entomopathogenic fungi can penetrate the insect exoskeleton both by mechanic action of the germinative tube and by production of multiple proteases, chitinases, and lipases in response to the composition of the insect cuticle. Therefore the purpose of this work was to produce, to purify, and to characterize the structure of the proteases produced by the fungus Beauveria bassiana CG432, previously activated in adults of coffee berry borer (Hypothenemus hampei) grown under submerged culture conditions. A suspension containing 10 6 activated conidia/mL was inoculated to a culture medium at 28ºC and 150 rpm for 3 days. Protease extracts, were obtained by centrifugation at 8000g for 20 minutes, were fractionated and were concentrated by ultrafiltration using controlled porosity membranes of 100 kDa and 3 kDa, respectively. Gel filtration chromatography on Sephadex G-100 isolated one protein peak (peak II), which contained 56% of residues of the aminoacid aspartic acid, characterized by HPLC using an ODS-C18 reverse phase column. The peak protein showed specific activity 43 times superior when compared to the free-cell extract in bovine serum albumin, subtilisin-like protease activity, and a single protein band reveled by Brilliant Coomassie Blue on a gelatin zimogram PAGE electrophoresis, by native conditions. The homogeneity of peak II was confirmed by revelation of a single band during determination of the isoelectric pH 4.5, but molecular mass determination by PAGE-2D electrophoresis showed 2 bands of 23 and 26 kDa. Proteases were characterized as serine proteases with cysteine residues important to activity, since they had been inhibited by phenylmethylsulphonyl fluoride and p-chlormercuricbenzoic acid. Peak II proteases showed Km of 4x10-4 on subtilisin-like substrate.

In the present study, an extracellular alkali-thermo-tolerant xylanase from Bacillus paramycoides was produced in the presence of an organic solvent. The enzyme was purified by ammonium sulphate precipitation, gel filtration, and ion exchange chromatography, with an overall recovery of 25.9%. The purified enzyme hada 70 kDa molecular weight (MW) confirmed by SDS-PAGE gel analysis. The maximum enzyme activity was reported at 55 °C and pH 7.0. Xylanase activity and stability were improved in the presence of 30% (v/v) n-dodecane, iso-octane, n-decane, and cyclohexane (7 days). The enzyme activity was improved by Co2+, EDTA, and Triton-X-100 while vigorously repressed by Hg2+ and Cu2+. The purified enzyme showed 1.473 mg/mL Km and 654.017 µg/mL/min Vmax values. The distinctive assets of the isolate verified the potential application in the field of biomass conversion into fuel and other industrial processes. Organic solvent-tolerant xylanases can be used for concurrent saccharification ...

In this work, the carbonic anhydrase (CA) enzyme was purified from Kangal Akkaraman sheep in Sivas, Turkey with specific activity value of 6681.57 EU/mg and yield of 14.90% with using affinity column chromatography. For designating the subunit molecular mass and enzyme purity, sodium dodecyl sulfate polyacrylamide gel electrophoresis method was used and single band for this procedure was obtained. The molecular mass of CA enzyme was found as 28.89 kDa. In this study, the optimum temperature and optimum pH were obtained from 30 and 7.5. V max and K m values for p-nitrophenylacetate substrate of the CA were determined from Lineweaver-Burk graphs. Additionally, the inhibitory results of diverse heavy metal ions (Hg + , Fe 2+ , Pb 2+ , Co 2+ , Ag + , and Cu 2+) on sheep were studied. Indeed, CA enzyme activities of Kangal sheep were investigated with using esterase procedure under in vitro conditions. The heavy metal concentrations inhibiting 50% of enzyme activity (IC 50) and K i values were obtained.

Mozambique Tilapia (Tilapia mossambica) viscera extracts were prepared using water, KCl solution and EDTA homogenization. The extract with the highest protease specific activities of 1687.5 and 2800 IU/mg protein, respectively. The enzyme was active at the optimum pH (4.0) and temperature (45 °C) when casein was used as a substrate. The enzyme showed the highest activity and purification when precipitated at 0-80% ammonium sulphate effectively improved specific activity of enzyme. Thus, the results revealed that active digestive enzymes could be prepared from tilapia viscera and can be used to digest the waste build-up in the pond and which greatly increase the clarity of the water by hydrolysing the proteinacious waste materials that build up. This research was carried out for the utilization of fish by-products for producing enzymes and also to the reduction of waste disposal problems.

Trypsin inhibitor was purified from the viscera except for the hepatopancreas of Japanese common squid (Todarodes pacificus) by gel filtration and anion-exchange chromatography. Final inhibitor preparation was homogeneous as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be approximately 6,000 Da. The N-terminal amino acid sequence of the squid trypsin inhibitor was determined as ANEATXVYKGKTYKKGEGF. The purified inhibitor was heat stable up to 150 min at 80C at pH 8.0. The inhibitory activity was also retained at pH 2.0 and 37C for 180 min. The squid trypsin inhibitor was active against walleye pollock trypsin as well as porcine pancreatic trypsin. The squid trypsin inhibitor showed hypoglycemic effect on blood glucose levels of GK rats, a model of type 2 diabetes. PRACTICAL APPLICATIONS In this study, we purified a trypsin inhibitor from the viscera except for hepatopancreas of the Japanese common squid (Todarodes pacificus). The squid trypsin inhibitor was acid and heat stable peptide. So, it was thought that the squid trypsin inhibitor is useful for biochemical regent and medicine of acute inflammation of human pancreas like soybean trypsin inhibitor and bovine aplotinin. Moreover, the squid trypsin inhibitor showed hypoglycemic effect on blood glucose levels of GK rats, a model of type 2 diabetes. Therefore, it may be useful for prevention of diabetes.

α-Amylases (E.C 3.2.1.1) hydrolyse starch into smaller moieties such as maltose and glucose by breaking α-1,4-glycosidic linkages. The application of α-amylases in various industries has made the large-scale productions of these enzymes crucial. Thermostable α-amylase that catalyses starch degradation at the temperatures higher than 50 °C is favourable in harsh industrial applications. Due to ease in genetic manipulation and bulk production, this enzyme is most preferably produced by microorganisms. Bacillus sp. and Escherichia coli are commonly used microbial expression hosts for α-amylases (30 to 205 kDa in molecular weight). These amylases can be purified using ultrafiltration, salt precipitation, dialysis, and column chromatography. Recently, affinity column chromatography has shown the most promising result where the recovery rate was 38 to 60% and purification up to 13.2-fold. Microbial thermostable α-amylases have the optimum temperature and pH ranging from 50 °C to 100 °C and 5.0 to 10.5, respectively. These enzymes have high specificity towards potato starch, wheat starch, amylose, and amylopectin. EDTA (1 mM) gave the highest inhibitory effect (79%), but Ca 2+ (5 mM) was the most effective co-factor with 155%. This review provides insight regarding thermostable α-amylases obtained from microbial sources for industrial applications.

In the present research, 30 xylanase producers were isolated by serial dilution and spread plate method from waste water samples of paper and pulp industry, Kupwad MIDC, Sangli (MH), India. This isolates were named as AG1, AG2 up to AG30. This all 30 isolates were screened for xylanase production by primary and screening. In the primary screening, AG12 showed 1.8mm zone of hydrolysis therefore AG12 were selected for Solid State fermentation at 25℃. AG12 showed highest xylanase production on 5 th days of incubation period with 44.12 µg/ml/Mol activity. Further, xylanase were extracted and purified by Ammonium sulphate precipitation and dialysis with 20 Mol/ml/min and 12.04 (U/ml) respectively. The crude Xylanase were characterized by using different parameters such as temperature ranges from 10-50°c (optima-30°c), pH ranges from 4-10 (optima-pH 8.0).

A thermostable and detergent-stable α-amylase from a newly isolated Anoxybacillus sp. AH1 was purifi ed and characterized. Maximum enzyme production (1874.8 U/mL) was obtained at 24 h of incubation. The amylase was purifi ed by using Sephadex G-75 gel fi ltration, aft er which an 18-fold increase in specifi c activity and a yield of 9 % were achieved. The molecular mass of the purifi ed enzyme was estimated at 85 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature values of the enzyme were 7.0 and 60 °C, respectively. The enzyme was highly stable in the presence of 30 % glycerol, retaining 85 % of its original activity at 60 °C within 120 min. K m and v max values were 0.102 μmol and 0.929 μmol/min, respectively, using Lineweaver-Burk plot. The enzyme activity was increased by various detergents, but it was signifi cantly inhibited in the presence of urea. Mg 2+ and Ca 2+ also signifi cantly activated α-amylase, while Zn 2+ , Cu 2+ and metal ion chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline (phen) greatly inhibited the enzyme activity. α-Amylase activity was enhanced by β-mercaptoethanol (β-ME) and dithiothreitol (DTT) to a great extent, but inhibited by p-chloromercuribenzoic acid (PCMB). Iodoacetamide (IAA) and N-ethylmaleimide (NEM) had a slight, whereas phenylmethylsulfonyl fl uoride (PMSF) had a strong inhibitory eff ect on the amylase activity.

espanolSe logro purificar tripsina del hepatopancreas de Dosidicus gigas por fraccionacion con sultafo de amonio (30-70% de saturacion), cromatografia de filtracion en gel, afinidad e intercambio ionico. El peso molecular de la tripsina fue de 23,5 kDa, segun la electroforesis SDS-PAGE y mostro una sola banda en el zymograma. La enzima mostro maxima actividad a pH 8,5 y 40oC, usando BAPNA como sustrato. La actividad de la enzima se afecto de manera efectiva por el metil fenil sulfonil fluoruro (PMSF) (98%) y por Nα-p-tosil-L-lisina clorometil cetona (TLCK) (99%). Ademas la actividad de la enzima fue afectada por los suguientes iones en orden decreciente: Hg2+, Fe2+, Cu2+, Li1+, Mg2+, K1+, Mn2+, Ca2+. Por otro lado la actividad de la enzima disminuyo de manera progresiva a medida que aumento la concentracion de NaCl de 0 a 30%. Los valores de Km y kcat fueron de 0,085 ± 1,45 mM y 1,76 ± 0,12s-1, respectivamente. Segun los resultados obtenidos se sugiere que la enzima presenta potenci...

A thermostable and detergent-stable α-amylase from a newly isolated Anoxybacillus sp. AH1 was purifi ed and characterized. Maximum enzyme production (1874.8 U/mL) was obtained at 24 h of incubation. The amylase was purifi ed by using Sephadex G-75 gel fi ltration, aft er which an 18-fold increase in specifi c activity and a yield of 9 % were achieved. The molecular mass of the purifi ed enzyme was estimated at 85 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature values of the enzyme were 7.0 and 60 °C, respectively. The enzyme was highly stable in the presence of 30 % glycerol, retaining 85 % of its original activity at 60 °C within 120 min. K m and v max values were 0.102 μmol and 0.929 μmol/min, respectively, using Lineweaver-Burk plot. The enzyme activity was increased by various detergents, but it was signifi cantly inhibited in the presence of urea. Mg 2+ and Ca 2+ also signifi cantly activated α-amylase, while Zn 2+ , Cu 2+ and metal ion chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline (phen) greatly inhibited the enzyme activity. α-Amylase activity was enhanced by β-mercaptoethanol (β-ME) and dithiothreitol (DTT) to a great extent, but inhibited by p-chloromercuribenzoic acid (PCMB). Iodoacetamide (IAA) and N-ethylmaleimide (NEM) had a slight, whereas phenylmethylsulfonyl fl uoride (PMSF) had a strong inhibitory eff ect on the amylase activity.

The enzyme responsible for the hydrolysis of phosphonoacetic acid, a non-biogenic C-P compound, was purified to electrophoretic homogeneity from a wild-type strain of Penicillium oxalicum. A 50-fold enrichment was obtained by a combination of anion exchange, hydrophobic interaction and MonoQ-fast protein liquid chromatography, with a yield of one-third of the initial activity. A characterization of the protein showed both similarities and differences with respect to the well-characterized bacterial counterpart. The fungal phosphonoacetate hydrolase is a 43-kDa monomeric protein showing low affinity toward its substrate and high sensitivity to even mildly acidic pH values. Enzyme activity neither required nor was stimulated by the presence of divalent cations. Polyclonal antibodies were raised in mouse against the purified protein, allowing the study of enzyme induction as a function of the phosphate status of the cell. Peptide mass mapping led to the determination of about 20% of the primary structure. Despite the biochemical differences, amino acid alignment showed a high degree of similarity of the fungal hydrolase with the few sequences available to date for the bacterial enzyme. The possible physiological role of a phosphonoacetate hydrolase is discussed.

A1-Pyrroline-5-carboxylate (P5C) dehydrogenase (EC 1.5.1.1 2), the second enzyme in the proline catabolic pathway and a catalyst for the oxidation of P5C to glutamate, was purified from cultured potato (Solanum tuberosum L. var Desiree) cells. Homogeneous enzyme preparations were obtained by a three-step procedure that used anion-exchange, adsorption, and substrate elution chromatography. A 1600-fold purification was achieved, with a recovery of one-third of the initial activity. lhe purified enzyme was characterized with respect to structural, kinetic, and biochemical properties. It appeared to be an a-4 tetramer with subunits of an apparent molecular mass of about 60 kD and had a mildly acidic isoelectric point value. Potato P5C dehydrogenase had Michaelis constant values of 0.11 and 0.46 mM for NAD+ and PSC, respectively. Although NAD+ was the preferred electron acceptor, NADP+ also yielded an unusually high rate, and thus was found to serve as a substrate. Maximal activity was observed at pH values in the 7.3 to 8.3 range, and was progressively inhibited by chloride ions, a finding that strengthens recent suggestions that hyperosmotic stress negatively modulates in vivo proline oxidation.

The enzyme responsible for the hydrolysis of phosphonoacetic acid, a non-biogenic C-P compound, was purified to electrophoretic homogeneity from a wild-type strain of Penicillium oxalicum. A 50-fold enrichment was obtained by a combination of anion exchange, hydrophobic interaction and MonoQ-fast protein liquid chromatography, with a yield of one-third of the initial activity. A characterization of the protein showed both similarities and differences with respect to the well-characterized bacterial counterpart. The fungal phosphonoacetate hydrolase is a 43-kDa monomeric protein showing low affinity toward its substrate and high sensitivity to even mildly acidic pH values. Enzyme activity neither required nor was stimulated by the presence of divalent cations. Polyclonal antibodies were raised in mouse against the purified protein, allowing the study of enzyme induction as a function of the phosphate status of the cell. Peptide mass mapping led to the determination of about 20% of the primary structure. Despite the biochemical differences, amino acid alignment showed a high degree of similarity of the fungal hydrolase with the few sequences available to date for the bacterial enzyme. The possible physiological role of a phosphonoacetate hydrolase is discussed.

Objective: Oral administration of bromelain as an anti-inflammation therapy still faces several challenges, such as the risk of contact with the gastric fluid and its low absorption rate. Therefore, bromelain isolated from the pineapple core will be formulated as a topical base in a nanoemulsion to increase its stability, activity, and ability to penetrate the skin. Methods: Bromelain was isolated from pineapple core using ammonium sulfate precipitation and dialysis, and then the isolated fraction was loaded into topical nanoemulsion for subsequent evaluation of its characteristics, stability, enzymatic activity, and for in vitro study of penetration using Franz diffusion cell. Results: The highest specific activity of bromelain fractions found in ammonium sulfate concentration is around 20–50%. After being dialyzed, the bromelain fraction showed an increase in specific activity 2.78-fold as compared to crude extract. The characteristics of bromelain nanoemulsion showed a globule si...

This work aimed to determine the physicochemical and biochemical characterization of the intestinal trypsin from beluga (Huso huso) and sevruga (Acipenser stellatus), two highly valuable sturgeon species, by a series of assays. According to the results obtained from casein-zymogram and inhibitory activity staining method, indicating the existence of trypsin in the intestinal crude extract of both species, molecular weight of the enzyme was estimated to be 27.5 and 29.5 kDa in sevruga and beluga, respectively. Optimum pH and temperature of both trypsins were recorded at 8.5 and 55°C by BAPNA (a speci c substrate), respectively. The stability of both trypsins was well preserved at pH from 6.0 to11.0 and temperatures of up to 50°C. TLCK and SBTI, two speci c trypsin inhibitors, showed a signi cant inhibitory effect on the enzymatic activity of both trypsins (P < 0.05). The enzyme activity was signi cantly increased in the presence of Ca + 2 and surfactants and decreased by oxidizing agents, Cu + 2 , Zn + 2 and Co + 2 (P < 0.05). However, univalent ions Na + and K + did not show any signi cant effect on the activity of both trypsins (P > 0.05). Therefore, the results of our study can contribute to the clear understanding of the intestinal trypsin activity in beluga and sevruga under evaluated experimental conditions.

Bromelain is a mixture of enzymes from the pineapple that contains, among other components, various closely related proteases. It has a wide industrial and therapeutic applications. The present research extracted bromelain from the stem and fruit of fully repined pineapple plants cultivated in Nigeria by homogenization in an aqueous buffer and purified by ammonium sulfate precipitation and DEAE-cellulose chromatography. Enzyme activities were determined using N α-Carbobenzoxy-Lysine p-Nitrophenyl Ester (LNPE) as substrate, spectrophotometrically. Total protein was estimated using Bradford method. The results obtained, showed that stem bromelain (SBM) was better suited for the purification procedure adopted with the percentage yield of 463% and purification fold of 4.64 over fruit bromelan (FBM) which showed a percentage yield and purification fold of 0.23 and 23.4%, respectively. Keywords: cysteine proteases, bromelain, stem bromelain, fruit bromelain, LNPE, bioremediation

A sericicultura e uma fonte de renda para a agricultura familiar que gera recursos para muitos produtores, principalmente no estado do Parana. Contudo ela vem sendo comprometida por diversos patogenos, entre estes, podemos destacar os fungo, como no caso de Metarhizium anisopliae, que e amplamente utilizado no controle biologico de insetos pragas, mas tambem pode infectar o B. mori. Assim, tendo em vista a importância de se realizarem estudos desta relacao, o presente trabalho objetivou analisar a patologia do tegumento de larvas de B. mori de quinto instar submetidas a inoculacao pelo M. anisopliae, uma vez que e a via de infeccao deste fungo. Para isso, larvas foram inoculadas com o M. anisopliae procedendo-se a analise sintomatologica e o processamento histologico em diferentes tempos posinoculacao (d.p.i). As larvas mostraram-se susceptiveis a partir do segundo d.p.i., apresentando sintomas caracteristicos de infeccao por fungos: reducao na alimentacao e no tamanho do corpo do i...

During adrenal steroidogenesis the competition between 3␤-hydroxysteroid dehydrogenase/ 5-4 isomerase (3␤HSD) and cytochrome P450 17␣-hydroxylase/17,20 lyase (CYP17A1) for 5 steroid intermediates greatly influences steroidogenic output. Cytochrome-b 5 (Cyt-b 5), a small electron transfer hemoprotein, known to augment the lyase activity of CYP17A1, has been shown to alter the steroidogenic outcome of this competition. In this study, the influence of Cyt-b 5 on 3␤HSD activity was investigated. In COS-1 cells, Cyt-b 5 was shown to significantly increase the activity of both caprine and ovine 3␤HSD towards pregnenolone, 17-OH pregnenolone and dehydroepiandrosterone in a substrate and species specific manner. Furthermore, kinetic studies revealed Cyt-b 5 to have no influence on the K m values while significantly increasing the V max values of ovine 3␤HSD for all its respective substrates. In addition, the activity of ovine 3␤HSD in microsomal preparations was significantly influenced by the addition of either purified Cyt-b 5 or anti-Cyt-b 5 IgG. The results presented in this study indicate that Cyt-b 5 augments 3␤HSD activity and represents the first documentation of such augmentation in any species.

Bromelain is a concoction of sulfhydryl proteolytic enzymes. Depending upon the site of extraction it can be regarded as either stem bromelain (SBM) (EC 3.4.22.32) or fruit bromelain (FBM) (EC 3.4.22.33). Bromelain remain enzymatic active over a broad spectrumand endure a range of pH (5.5 to 8.0) and temperature (35.5 to 71 ºC). It is one of the extensively investigated proteolytic enzyme owing to its astonishing applications in various industries. This necessitated employing a strategy that result in highest purified bromelain in less steps and lowest cost. Use of modernistic approach such as membrane filtration, reverse micellar systems, aqueous two phase extraction and chromatographic techniques have shown promise in this regard. Besides its industrial applications, bromelain has been widely utilized as a potential phytomedical compound. Some of its reported actions include inhibition of platelet aggregation, anti-edematous, anti-thrombotic, anti-inflammatory, modulation of cytokines and immunity, skin debridement and fibrinolytic activity. It also assist digestion, enhance absorption of other drugs and is a potential postoperatively agent that promote wound healing and reduce postsurgical discomfort and swelling.

Immobilization and purification of enzymes are usual requirements for their industrial use. Both purification and immobilization have a common factor: they use a solid activated support. Using a support for enzyme purification means having mild conditions for enzyme release and a selective enzyme–support interaction is interesting. When using a support for immobilization, however, enzyme desorption is a problem. The improvement of enzyme features through immobilization is a usual objective (e.g., stability, selectivity). Thus, a support designed for enzyme purification and a support designed for enzyme immobilization may differ significantly. In this review, we will focus our attention on the requirements of a support surface to produce the desired objectives. The ideal physical properties of the matrix, the properties of the introduced reactive groups, the best surface activation degree to reach the desired objective, and the properties of the reactive groups will be discussed.

This article reviews the information about fructosyltransferases, the enzymes producing important prebiotic compounds, fructooligosaccharides. The chemical structure, safety and physiological effects, preparation and application of fructooligosaccharides are discussed. Plant and microbial sources of fructosyltransferases are presented. A detailed description is devoted to the mechanisms of action, substrate specificities, and regioselectivities of fructosyltransferases. Their physicochemical and catalytic properties are also discussed. Finally, the methods of purification of fructosyltransferases reported in literature are summarized in respect to the yield and factor of purification.

Enzymatic fructosylation of organic acceptors other than saccharides brings new possibilities to synthesize molecules that do not exist in nature. The introduction of fructosyl moiety may lead to glycosides possessing enhanced physicochemical and bioactive properties which could be useful in the pharmaceutical and cosmetic industry. In this work, the regioselective synthesis of tyrosol β‐d‐fructofuranoside (TF) catalyzed by β‐fructofuranosidase is investigated. In the first step, 32 commercial enzyme preparations are screened for fructoside‐hydrolyzing activity. The most active preparations are subsequently examined for fructofuranosyl transfer from sucrose to tyrosol. The best candidate, Novozym 188, is chosen to study the effect of reaction conditions on the product formation in a batch reactor. The effects of substrate concentration, temperature, pH, time, and enzyme dosage on the concentration of TF produced are studied using the design of experiments methodology. The maximal pr...

This article reviews the information about fructosyltransferases, the enzymes producing important prebiotic compounds, fructooligosaccharides. The chemical structure, safety and physiological effects, preparation and application of fructooligosaccharides are discussed. Plant and microbial sources of fructosyltransferases are presented. A detailed description is devoted to the mechanisms of action, substrate specificities, and regioselectivities of fructosyltransferases. Their physicochemical and catalytic properties are also discussed. Finally, the methods of purification of fructosyltransferases reported in literature are summarized in respect to the yield and factor of purification.

The catalytic properties of Seqenzym® FT, a fungal fructosyltransferase heterologously expressed in yeasts, were investigated at a temperature of 55 °C and pH 5.5. The initial rate measurements showed that the transfructosylation rate was only slightly inhibited by sucrose above the concentration of 1.5 M. A rather low level of hydrolytic side activity was observed even at sucrose concentrations as low as 0.25 M. In progress curve experiments, the mass yield of fructooligosaccharides (FOS) reached a maximum value of 57% at this sucrose concentration, although it dropped to about 35% later on. At high initial sucrose concentrations up to 2 M, the FOS yield reached a maximum value of approximately 63% at a sucrose conversion of approximately 90%. Although neither the yield nor the conversion changed much later on, the progress of the reaction was manifested by the gradual depletion of shorter chain FOS, 1-kestose and nystose, and the accumulation of 1-β-fructofuranosyl nystose. At ini...

and a highly extraordinary stable macromolecule (Kokwe et al., 2023). In order to produce keratinase, keratinous materials such as chicken feathers, hooves, and hair are needed as substrate materials (Gupta and Ramnani, 2006). Moreover, many types of non keratinous substrates can also act as keratinase inducers such as casein, skimmed milk, gelatin and soybean meal (Casarin et al., 2008).

Here we provide evidence for a critical role of PP2As (protein phosphatase 2As) in the transformation of Trypanosoma cruzi. In axenic medium at pH 5.0, trypomastigotes rapidly transform into amastigotes, a process blocked by okadaic acid, a potent PP2A inhibitor, at concentrations as low as 0.1 μM. 1-Norokadaone, an inactive okadaic acid analogue, did not affect the transformation. Electron microscopy studies indicated that okadaic acid-treated trypomastigotes had not undergone ultrastructural modifications, reinforcing the idea that PP2A inhibits transformation. Using a microcystin–Sepharose affinity column we purified the native T. cruzi PP2A. The enzyme displayed activity against 32P-labelled phosphorylase a that was inhibited in a dose-dependent manner by okadaic acid. The protein was also submitted to MS and, from the peptides obtained, degenerate primers were used to clone a novel T. cruzi PP2A enzyme by PCR. The isolated gene encodes a protein of 303 amino acids, termed TcPP2...