Partial Purification Research Articles

Utilization of orange pulp and corn steep liquor for L-methioninase production by Wickerhamomyces subpelliculosus

Background and objective L-methioninase has attracted much attention with respect to its proposed applications in both pharmaceuticals and food industry. The aim of this study was to develop an economic medium formulation using agro-industrial by-products as substrates for large-scale production of L-methioninase. Materials and methods Identification of a high L-methioninase-producing yeast isolate was carried out using 18S rRNA molecular technique. Screening of various agro-industrial by-products and optimization of different process parameters were investigated. Partial purification and characterization of a crude enzyme were carried out. Results and discussion A high L-methioninase-producing yeast isolate was phylogenetically identified as Wickerhamomyces subpelliculosus. Among different agro-industrial by-products tested, orange pulp supported maximum enzyme production (94.08 U/ml) followed by cane and beet molasses. In addition, corn steep liquor (CSL) gave high enzyme level (141.12 U/ml) and could be used as an inexpensive alternate for yeast extract. The optimum growth conditions were found to be orange pulp 30% (w/v), CSL 4% (v/v), CaCl2 0.05%, and KH2PO4 0.05% (w/v) at pH 6.0 after 48 h of incubation. This developed medium formulation increased L-methioninase production (161.95 U/ml) by twofold compared with that obtained by the Czapek–Dox’s medium (73.92 U/ml). Crude enzyme was partially purified by heat treatment at 70°C with 2.9 purification fold. The enzyme activity was optimal at temperature 60°C and pH 7.0. The results showed that a mixed formulation of orange pulp and CSL can be used as an effective and economic substrate for the production of L-methioninase by W. subpelliculosus.

Enhanced production and biochemical characterization of a thermostable amylase from thermophilic bacterium Geobacillus icigianus BITSNS038

The current study focuses on optimizing, characterizing, and purifying a thermostable amylase from thermophilic bacteria Geobacillus icigianus (BITSNS038). The amylase was produced optimally at 18–24 h of incubation in the presence of starch and tryptone at 7.5 and 3.0 g/L, respectively (pH 7.0, 70°C, and 150 rpm). Km , V max values for starch were 2.17 mg/mL and 4.16 U/mL respectively. The enzyme showed excellent thermal stability at 70°C, retaining 62.5% residual activity after 8 h and a reduced deactivation rate constant (kd ) of 0.001. When tested for its efficacy in starch hydrolysis, it showed 34.5% hydrolysis of corn starch slurry and antibiofilm activity, but no antimicrobial activity was noticed. The ultrafiltration led to partial purification of amylase showed 2.67-fold purification with a molecular weight in the range of 45 and 66 kDa. This is probably the first-ever report on a thermostable amylase from G. icigianus and its extensive potential uses.

Characterization of the antimicrobial activity produced by Bacillus sp. isolated from wetland sediment.

Bacteria of the genus Bacillus sp. present the potential for inhibiting various pathogens, making them a promising starting point in the search for new antimicrobial substances. In this study, bacteria were isolated from sediment samples from humid areas of a Natural Conservation Unit in the state of Rio Grande do Sul, Brazil. The isolate Bacillus sp. sed 1.4 was selected for production of antimicrobial activity, and was characterized by MALDI-TOF and 16S rDNA sequencing. Phylogenetic analysis showed that Bacillus sed 1.4 was closely related to Bacillus altitudinis and Bacillus pumilus. The cell-free supernatant was partially purified using ammonium sulfate precipitation, gel filtration chromatography (Sephadex G-200) and an ultrafiltration membrane. Partial purification resulted in specific activity of 769.23 AU/mg, with a molecular mass of approximately 148 kDa. This antimicrobial substance showed stability at 100°C for 5 min, and was inactivated by proteolytic enzymes. An antimicrobial effect against Listeria species was observed. Considering the importance of the Listeria genus in the area of food safety, this antimicrobial activity should be further explored, specifically in the field of dairy products and with a focus on food biopreservation studies.

Antimicrobial Activity and Identification of Gene Encoding Enterocin Enterococcus faecalis K2B1 Isolated from Toraja's Belang Buffalo Milk

Background: Enterocin in Enterococcus is coded by enterocin encoding genes namely A, B, P and L50A / B. The purpose of this study was to identify enterocin gene encoding enterococcus faecalis K2B1 probiotic candidate from Belang Toraja buffalo milk and antimicrobial activity to S. typhi. Methods: identification of enterocin gene encoding using ent A, B, P and L50A / B, partial purification using ammonium sulfate on 80 % concentration and antimicrobial activity against to Salmonella typhi using disk diffusion method. The results of PCR amplification are then sequenced and BLASTX on NCBI. Result: Antimicrobial activity of Precipitate and crude against S. typhi are 193 and 201 respectively. Identification gene encoding enterocin shows that Ent A, B and P cannot be amplified and only EntL50A / B can be amplified with a sequence size of 86 bp. The sequence of enterocin encoding genes in E. faecalis K2B1 has 94% similarity with hypothetical protein EB34_00789 E. faecalis on GenBank with accession number RBR60004.1 Conclusion: EntL50A / B E. faecalis K2B1 has a size of 86 bp and is 94% identical to the hypothetical protein EB34_00789 and Enterocin can be used as antimicrobial or bio preservative. 
 

Production, Partial Purification and Characterization of Two α-Amylase Isoforms from Saccharomyces cerevisiae strain YOP 1/2-2 Isolated from Tchapalo (Côte d’Ivoire)

Amylases play an important role in biotechnology and find applications in several industrial fields such as pharmaceutical, food, paper, cosmetics and detergents. Thus, it appears necessary to identify new sources of amylase, especially from microbial origin, due to the low energy consumption, cost-effective, high metabolic diversity, rapid cell growth, non-toxic and eco-friendly characteristics. In the present report, we carried out the production and partial purification of α-amylase by Saccharomyces cerevisiae strains isolated from Tchapalo, a traditional alcoholic beverage of Côte d'Ivoire. Five fungal isolates were screened initially for α-amylase production by using method of wells on Yeast Extract Peptone Dextrose Agar medium, a complete medium for yeast growth. One step DEAE-Sepharose Fast Flow was achieved for partial purification of α-amylase obtained. Among yeasts, isolate S. cerevisiae YOP 1/2-2 was able to provoke starch hydrolysis halo of 15.067±0.12 mm on starch agar plate after 48 h of incubation at 30°C. The partial purification of resulting enzyme showed two protein peaks, designated α-amylase 1 (AMY1) and α-amylase 2 (AMY2) with amylolytic activity and specific activities of 1.57-1.58 U/mg protein. Both isoforms (AMY1 and AMY2) were thermostable with optimal activity at 50 and 55°C, respectively, and at pH ranged from 4.5 to 5.3 in 0.1 M sodium acetate buffer. EDTA and Cd2+ strongly inhibited α-amylase activity by 72-75% and 64-65%, respectively, whereas cations Ca2+ and Mn2+ showed 85-99% and 71% increased amylolytic activity, respectively. All these properties show the potential uses of α-amylases from S. cerevisiae in the industrial transformation of starch.

Isolation and partial Purification and for amylase from diabetic patients and some kinetic properties

Amylase is an enzyme which is secreted from pancreas, and salivary glands (α-Amylase EC3.2.1.1), there is a small amount of this enzyme presents in the blood ranging between 100–300 I.U/L, when this percentage in the blood would increase,it means that the above organs are ill. The study included apartial purification of this enzyme from the blood of diabetic patients using gel, filtration, dialysis, and sephadex G100 gel, the single beak was obtained in the fourth part of purification and the degree of purification reached 16.1 with an enzymatic outcome of 108.2% and specific activity was 0.189ng/mg. The kinetic study of the purified enzyme appeared an optimum concentration of the substrate of 10 ng / mg,the Km was 5.55 ng , maximum acivity Vmax, was 0.98ng / ml,the optimum temp. was (37C°) and the opt. pH was 7.5.

Distribution of Important Probiotic Genes and Identification of the Biogenic Amines Produced by Lactobacillus acidophilus PNW3.

The genome of Lactobacillus acidophilus PNW3 was assessed for probiotic and safety potentials. The genome was completely sequenced, assembled using SPAdes, and thereafter annotated with NCBI prokaryotic genome annotation pipeline (PGAP) and rapid annotation using subsystem technology (RAST). Further downstream assessment was determined using appropriate bioinformatics tools. The production of biogenic amines was confirmed through HPLC analysis and the evolutionary trend of the strain was determined through the Codon Tree pipeline. The strain was predicted as a non-human pathogen. A plethora of encoding genes for lactic acids and bioactive peptides production, adhesion molecules, resistance to the harsh gut environmental conditions, and improvement of the host metabolism, which are putative for important probiotic functionalities, were located at different loci within the genome. A bacteriocin predicted to be helveticin J was identified as one of the secondary metabolites. The maximum zone of inhibition exhibited by the crude bacteriocin against STEC E. coli O177 was 21.7 ± 0.58 mm and 24.3 ± 1.15 mm after partial purification (250 µg/mL). Three coding sequences were identified for insertion sequences and one for the CRISPR-Cas fragment. The protein-encoding sequence for Ornithine decarboxylase was found within the genome. L. acidophilus PNW3 presents important features categorizing it as a viable and safe probiotic candidate, though further safety investigations are necessary. The application of probiotics in livestock-keeping would ensure improved public health and food security.

Capacity of ethanol adjunct-treated interface of ionic liquid aqueous two phase system in simultaneous extraction and purification of sorghum leaf sheath polysaccharides

ABSTRACT Employing response surface methodology, partially purified polysaccharides from Sorghum bicolor L. leaf sheath were extracted using synergized ethanol adjunct-treated ionic liquid aqueous two phase system (1-octyl-3-methylimidazolium chloride, [C8mim]Cl and K2CO3) and dual frequency ultrasound-assisted extraction. Under ultrasound conditions of 35 °C, 20&60 KHz, 25 mins an experimental yield of 16% PPS was achieved. Dual frequency ultrasound-assisted dialysis effectively reduced salt content of extracted PPS solution in a liquid membrane. The polysaccharides collected after dialysis maintained primary structures. The introduction of ethanol in the ionic liquid aqueous two phase system therefore ensured an excellent simultaneous extraction and partial purification.

Microwave-assisted extraction, partial purification and biological activity in vitro of polysaccharides from bladder-wrack (Fucus vesiculosus) by using deep eutectic solvents

Based on natural deep eutectic solvents (DESs), microwave-assisted green extraction was applied for production of polysaccharides from bladder-wrack (Fucus vesiculosus). In this study, nine different combinations of deep eutectic solvents were evaluated for extraction of polysaccharides from bladder-wrack, and the results showed that DES system composed of choline chloride and 1,4-butanediol with molar ratio of 1:5 possessed the optimal extraction efficiency for polysaccharides. A three-level and four-variable Box-Behnken design (BBD), a specific design of response surface methodology (RSM), was used to determine the optimum extraction conditions for the maximum yields of polysaccharides from bladder-wrack by using DESs. The maximum yields of polysaccharides attained 116.33 mg/g within DES water content of 32%, extraction time of 35 min, extraction temperature of 168 °C and solid–liquid ratio of 39 mg/mL. After partial separation and purification of the polysaccharides extracted by DESs, antioxidant activity and anticancer activity in vitro were evaluated. The results revealed that polysaccharides extracted from bladder-wrack exhibited excellent antioxidant activities in vitro including DPPH and ABTS radical-scavenging activity. For human cervical cancer HeLa cells, the polysaccharides possessed a strong inhibition effect on the growth rate. So these results indicate that deep eutectic solvent, as an environmentally-friendly solvent, can be applied to extract polysaccharides from multifarious plant materials.

Extraction, Partial Purification, Characteristics, and Antimicrobial Activity of Plant Protease From Moringa Oleifera Leaves

Introduction: Proteases are hydrolyzing enzymes and are considered to be one of the most important groups of enzymes for industry and are used in leather, pharmaceutical, and food industry along with detergents, and bioremediation processes. The main objectives of this study were (i) to extract, partially purify, and characterize the protease enzyme from Moringa (Moringa oleifera) leaves; and (ii) to investigate the effect of such an enzyme against some pathogenic bacteria.Materials and Methods: This enzyme was extracted in a 0.1 M phosphate buffer pH 7. It was left for 24 hours in a refrigerator and was then filtered using filter paper Whatman no. 41. The aqueous filtrate was used to estimate the proteolytic activity.Results: Purification by ammonium sulfate gave the best results at 50%-70% concentration which had the highest specific activity, highest purification fold with the percentage yield of 45.3%. Gel filtration by Sephadex G-100 gave the best specific activity and the best purification fold with the yield from fraction of 34%-43%. The protease enzyme has optimum pH 7 and temperature 50 oC. The enzyme was thermally stable at 40-70 oC for 20-30 minutes. Some metal ions were activator on the enzyme-like Mn2+ (highest), Ba2+, Ca2+, and Na+. The efficacy of protease enzyme was improved when integration with antibiotic against certain bacterial including Bacillus cereus (S3), Staphylococcus aureus, Salmonella typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes. Also, E. coli O157:H7, L. monocytogenes, and Yersinia enterocolitica did not show any growth at pH 10.Conclusions: To conclude, it can be stated that protease enzyme can be considered as a promising agent, cheap, and safe source which is suitable for using in various industries.

Euthynnus affinis viscera-an alternative source for protease and lipase enzymes: Characteristic and potential application as destainer agent

Abstract. Mardina V, Harmawan T, Fitriani, Sufriadi E, Febriani, Yusof F. 2020. Euthynnus affinis viscera-an alternative source for protease and lipase enzymes: Characteristic and potential application as destainer agent. Biodiversitas 21: 5858-5864. This study described the characterization of protease and lipase, extracted from the viscera of Euthynnus affinis and their applications as stains removal. Enzyme increased a few folds (3.99 folds for protease and 2.45 folds for lipase) after the submission to partial purification processes by ammonium sulfate precipitation and dialysis. Molecular mass assessment by SDS-PAGE showed that the enzyme solution contains three protein bands, estimated at 70, 42, and 24 kDa for protease and 32 kDa for lipase. Zymography confirmed the presence of protease and lipase in the sample. The protease and lipase exhibited optimum activities at pH 7.0 and pH 8.0 respectively, active at temperatures from 35 to 75oC with optimal activities at 65oC for both. The enzymes were stable at alkaline pH after 90 minutes (mins) of incubation and were also stable up to 65oC, showing the remaining activities of more than 70%. Samples containing protease and lipase as de-staining agents were tested by their ability to remove blood and palm oil stains, with results proving they were effective. Overall, the study showed that crude fish visceral extract contains protease and lipase and is useful in removing household stains, and could substitute for commercial detergents.

Evidence for a Novel Key Regulatory Step in the Triglyceride Synthetic Pathway of Human Adipose Tissue

The triglyceride (TG) synthetic pathway is believed to be regulated at the level of the rate-limiting enzyme acyl-CoA:1,2-diacylglycerol O-acyltransferase (DGAT). Recent reports using rat hepatic tissue suggest a kinase-dependent mechanism for the regulation of DGAT activity. To examine this process further, the present study investigates the regulatory mechanisms involved in the modulation of DGAT in human adipocytes. Adipocytes were fractionated into a microsomal fraction containing DGAT and a cytosolic fraction containing a putative regulatory kinase. DGAT activity was determined bymeasuring the incorporation of 14C-oleoyl-CoA into TG with exogenously supplied 1,2-dioleoyl-sn-glycerol. Kinase activity was assayed by addition of the cytosolic fraction in the presence of Mg2+ and ATP. The results indicate a significant inhibition of human adipose tissue DGAT activity by as much as 43% (avg: 17.5% ± 10.4%, p < 0.01) via a mechanism consistent with a phosphorylation event. Partial purification of the putative cytosolic kinase was achieved by multidimensional chromatography. This study thus provides evidence for a novel and key regulatory step in the human TG biosynthetic pathway. Further research is necessary to determine whether the model outlined here is a physiologic conduit through which extracellular hormones exert a regulatory influence on TG synthesis.

Partial Purification of Superoxide Dismutase and Antigenicity Nature in Setaria digitata, A Filarial Parasite

Partial Purification of Superoxide Dismutase and Antigenicity Nature in Setaria digitata, A Filarial Parasite

Partial purification and biochemical characterization of a new highly acidic NYSO laccase from Alcaligenes faecalis

BackgroundDue to the multitude industrial applications of ligninolytic enzymes, their demands are increasing. Partial purification and intensive characterization of contemporary highly acidic laccase enzyme produced by an Egyptian local isolate designated Alcaligenes faecalis NYSO were studied in the present investigation. ResultsAlcaligenes faecalis NYSO laccase has been partially purified and intensively biochemically characterized. It was noticed that 40–60% ammonium sulfate saturation showed maximum activity. A protein band with an apparent molecular mass of ~ 50 kDa related to NYSO laccase was identified through SDS-PAGE and zymography. The partially purified enzyme exhibited maximum activity at 55 °C and pH suboptimal (2.5–5.0). Remarkable activation for enzyme activity was recognized after 10-min exposure to temperatures (T) 50, 60, and 70 °C; time elongation caused inactivation, where ~ 50% of activity was lost after a 7-h exposure to 60 °C.Some metal ions Cu2+, Zn2+, Co2+, Ni2+, Mn2+, Cd2+, Cr2+, and Mg2+ caused strong stimulation for enzyme activity, but Fe2+ and Hg2+ reduced the activity. One millimolar of chelating agents [ethylenediamine tetraacetic acid (EDTA), sodium citrate, and sodium oxalate] caused strong activation for enzyme activity. Sodium dodecyl sulfate (SDS), cysteine-HCl, dithiothreitol (DTT), β-mercaptoethanol, thioglycolic acid, and sodium azide caused strong inhibition for NYSO laccase activity even at low concentration. One millimolar of urea, imidazole, kojic acid, phenylmethylsulfonyl fluoride (PMSF), H2O2, and Triton X-100 caused activation. The partially purified NYSO laccase had decolorization activity towards different dyes such as congo red, crystal violet, methylene blue, fast green, basic fuchsin, bromophenol blue, malachite green, bromocresol purple eriochrome black T, and Coomassie Brilliant Blue R-250 with various degree of degradation. Also, it had a vast range of substrate specificity including lignin, but with high affinity towards p-anisidine. ConclusionThe promising properties of the newly studied laccase enzyme from Alcaligenes faecalis NYSO strain would support several industries such as textile, food, and paper and open the possibility for commercial use in water treatment. It will also open the door to new applications due to its ligninolytic properties in the near future.

A newly-isolated Bacillus subtilis BSC35 produces bacteriocin-like inhibitory substance with high potential to control Clostridium perfringens in food

Previous studies on Bacillus subtilis-producing bacteriocins (or bacteriocin-like inhibitory substance, BLIS) against Clostridium perfringens provided with limited information to show their potentials. In this study, B. subtilis BSC35 was newly-isolated and its BLIS-containing cell-free supernatant exhibited a strong antimicrobial activity (maximum 24 mm of lysis zone in standard well diffusion assay) against all the tested C. perfringens strains (22 farm isolates and 2 references). The culture condition was optimized (30 °C, 240 rpm, TSB at pH 7.5) to maximize BLIS BSC35 production and stability of BLIS BSC35 was tested. After zymogram study following partial purification, BLIS BSC35 was measured approximately 4 kDa in size. Finally, when cell-free-supernatant or B. subtilis BSC35 culture was used to control C. perfringens in liquid medium and a food model (Cheonggukjang), as low as 0.1% and 104 CFU/g addition, respectively, was enough to strongly inhibit the growth of C. perfringens with a rapid bactericidal mode of action, implying high anti-C. perfringens potentials and wide range of applicabilities.

Antibacterial Bioactivity of Some Lactic Acid Bacteria Isolated from Various Egyptian Products

The current research aimed to isolate and screening of antibacterial bioactive lactic acid bacteria (LAB) from different foods such as raw milk, dairy products and fermented meat and evaluate their potential for production of antibacterial substances, namely bacteriocins. Ten samples were collected from different market foods, examined and used for LAB isolation on MRS medium. A total of 71 bacterial isolates were selected based on some variation properties, such as color, shape, size and oxygen requirements. These isolates were confirmed as lactic acid bacteria based on their microscopic observation and chemical properties, i.e. catalase negative and acid producing from glucose fermentation. All selected isolates were grown on MRS medium at 37oC for 48 h, and compared to identified LAB strains, i.e. Lactococcus lactis, Streptococcus thermophilus, Befidobacterium sp, Lactobacillus casi, Lb rhamnosus, Lb. acidophilus, Lb. helveticus and Lb. plantarum. Isolates and strains were surveyed for bacteriocin production as inhibitory activity test. Partial purification of bacteriocin was carried out by salt precipitation method; this method was done by ammonium sulphate 40-60% saturation and centrifuged at 9500 rpm for 20 minutes at 4oC. Some of microbial indicator strains found to test by inhibitory biological activity substance (partial purification of bacteriocin) produced by lactic acid cultures. Indicator strains such as staphylococcus aureus, Listeria monocytogenes and others were grown by nutrient agar at 37o C for 24h. An inhibitory activity test was executed by paper diffusion method. The inhibition effect was examined by form clear inhibition zone around indicator strains. Twenty-eight isolates and the 8 LAB strains showed bioactivities against the tested pathogens. Gram positive indicator bacteria were found to be the most sensitive to inhibitory substances produced by LAB culture as compared to gram negative indicator strains. Staphylococcus aureus was exhibited the highest sensitivity to it. The highest bacteriocins activities were produced by strain Lactobacillus rhamnosus while R5 and S6 from LAB isolates. They were shown broad spectrum against gram positive and gram-negative pathogenic bacteria. Diameters of inhibition zone refer to degree of sensitive between indicator strains and bacteriocin activity

Identification of Genetic Variants via Bacterial Respiration Gas Analysis.

Indole is a signal molecule derived from the conversion of tryptophan, and it is present in bacterial respiratory gas. Besides influencing bacterial growth, indole exhibits effects on human health, including a positive effect on inflammation and protection against pathogens. However, a high fecal indole concentration (FIC) can suggest an unbalanced gut flora or the presence of certain pathogens. To analyze the indole produced by bacteria, its collection and detection is required. Traditional methods usually require centrifugation of liquid bacterial culture medium and subsequent extraction of indole from the medium or partial purification of indole from fecal samples (e.g., by distillation or extraction). In this study, we demonstrate the possibility of identifying gas contents directly from bacteria, and we distinguish the difference in species and their genetics without the need to centrifuge or extract. Using an absorbent sheet placed above a liquid culture, we were able to collect gas content directly from bacteria. Gas chromatography-mass spectrometry (GC-MS) was used for the analysis. The GC-MS results showed a clear peak attributed to indole for wild-type Escherichia coli cells (MG1655 and MC4100 strains), whereas the indole peak was absent in the chromatograms of cells where proteins, part of the indole production pathway from tryptophan (TnaA and TnaB), were not expressed (by using tnaAB-deleted cells). The indole observed was measured to be present in a low nmol-range. This method can distinguish whether the bacterial genome contains the tnaAB gene or not and can be used to collect gas compounds from bacterial cultures quickly and easily. This method is useful for other goals and future research, such as for measurements in restrooms, for food-handling facilities, and for various applications in medical settings.

PURIFICACIÓN Y CARACTERIZACIÓN DE INHIBIDORES DE PAPAÍNA PROVENIENTES DE SEMILLAS DE AMARANTO (Amaranthus caudatus) Y FRIJOL (Phaseolus vulgaris)

Los inhibidores de proteasas vegetales con frecuencia son proteínas de baja masa molar, presentes en tejidos de almacenamiento y aéreos de las plantas. Su expresión se induce en respuesta al daño por insectos y microorganismos patógenos y el contenido de estos inhibidores puede ser alto en órganos de cereales y leguminosas. Bajo la premisa de que la combinación de técnicas de purificación de proteínas de baja y alta resolución permite incrementar la actividad específica de inhibidores de proteasas presentes en tejidos vegetales, el objetivo de este estudio fue aislar, purificar y caracterizar bioquímicamente inhibidores de papaína a partir de semillas de amaranto (Amaranthus caudatus) y frijol (Phaseolus vulgaris). El diseño experimental fue completamente al azar para seleccionar las técnicas de baja resolución que incrementan significativamente (p≤0.05) el grado de purificación de los extractos tratados. En todos los estudios se hicieron tres repeticiones y las mediciones se realizaron por triplicado. A partir de harinas de semillas de amaranto y frijol se obtuvieron extractos proteicos crudos que después se ultrafiltraron en membranas de 10 kDa. Las fracciones de baja masa molar se purificaron por cromatografía de afinidad en papaína-glioxil-sepharosa 6B-CL. El extracto crudo de amaranto presentó actividad inhibidora de 1.177 ± 0.067 mU·mL-1 y el de frijol 1.556 ± 0.542 mU·mL-1. Los extractos con purificación parcial de amaranto y frijol se purificaron por cromatografía de afinidad en papaína-glioxil-sepharosa 6B-CL, hasta 137.5 y 79.1 veces, respectivamente. La masa molar estimada fue 7.50 kDa para el inhibidor de amaranto y 8.20 kDa para el de frijol. El inhibidor de amaranto mostró inhibición competitiva con Ki de 0.872 µM, mientras que la del frijol fue no competitiva y con Ki de 0.058 µM. Esto evidencia la presencia de inhibidores de cisteíno proteasas en las semillas de amaranto y frijol.

Production, partial purification and characterization of alkaline phosphatase from a thermo-alkaliphile Geobacillus thermodenitrificans I2 isolate

Abstract Alkaline phosphatases producing microbes are wide spread in nature; ALP is a hydrolase enzyme functioning at alkaline pH, able to hydrolyze phosphates from many types of molecules; this initiating many applications. Experimental designs were applied to evaluate the culture conditions affecting ALP production by Geobacillus thermodenitrificans strain I2, where glucose, yeast-extract and agitation were the most significant variables according to Plackett-Burman design (PBD). The optimum levels of these variables were assessed through Box–Behnken design (BBD) to be (g/L): glucose 26.4, yeast-extract 29.72 and agitation 150rpm, with a predicted enzyme activity of 8.149U/ml. The measured activity upon application the optimized medium was 7.896U/ml. Enzyme was partially purified (42%; 12 fold) through precipitation by ammonium sulfate and anion column, with two protein bands (>70 & ~40kDa) in SDS-PAGE. A partial gene translated sequence proved that the natively expressed ALP by studied strain is related to the band with ~40kDa. The partially purified enzyme exhibited maximum activity at 60°C and pH9.0, with remarkable temperature stability up to 65°C. The results indicated an enhancement in enzyme activity with metals (Mg2+, Mn2+) and reducing agents (DTT & s-mercaptoethanol) whereas, other metals (Zn2+, Hg2+), and anionic surfactant (SDS); EDTA; and PMSF inhibited the enzyme activity. The dependence on enzyme and substrate concentration was represented in Lineweaver-Burk plot where, Km and Vmax were 40mM and 25U/mg protein, respectively. In conclusion, the promising traits of studied ALP would underpin its efficient exploitation in several industries to cope with demands of worldwide enzyme markets.

High Throughput Extraction, Partial Purification and Characterization of Antioxidant Enzyme Ascorbate Peroxidase (Apx) Present in Chilli (Capsicum Annuum, Var. Azad Mirch-1) Plant

High Throughput Extraction, Partial Purification and Characterization of Antioxidant Enzyme Ascorbate Peroxidase (Apx) Present in Chilli (Capsicum Annuum, Var. Azad Mirch-1) Plant