Cold Acetone Precipitation Research Articles

Biophysical Properties of Thermostable Amidase Produced by Aspergillus fumigatus in Submerged Fermentation

Amidases, also known as amidohydrolases (EC 3.5.1.4), are enzymes within the nitrilase superfamily or amidase signature family. They hydrolyze amides and nitriles into their corresponding acids while releasing ammonia. These enzymes are widely used in biotechnology and industry. However, their production is limited to a few organisms, which cannot meet industrial demand. Therefore, exploring new amidase sources is crucial. This study focuses on purifying and characterizing amidase from Aspergillus fumigatus using cold acetone precipitation and column chromatography with Sephadex G-100. The effects of temperature, pH and metal ions, on the enzyme activity were examined for both crude and purified amidase. The thermal and pH stability of the enzyme alongside the enzyme kinetics were also assessed. The purified amidase exhibited optimal activity of 13.1 U/mL at 70°C, with 65% residual activity after 5 hours at this temperature. The pH studies showed optimal activity at pH 5.0 for the purified enzyme (7.8 U/mL) and at pH 4.0 for the crude enzyme (5.5 U/mL). Metal ion effects indicated that Zn²⁺, Mg²⁺, and EDTA enhanced, while Cu²⁺ inhibited, the activities of both crude and purified amidase. Kinetic analysis showed that the enzyme followed Michaelis-Menten kinetics, with Km and Vmax values of 0.104 mM and 1.884 U/mL for crude amidase, and 0.017 mM and 1.872 U/mL for purified amidase. These findings suggest that amidase from Aspergillus fumigatus holds potential for industrial applications where amidases are essential.

Physicochemical Properties, Functionalities, and Antioxidant Activity of Protein Extracts from New Zealand Wild Sea Cucumbers (Australostichopus mollis).

This study investigated the physicochemical properties, functionalities, and antioxidant capacities of protein extracts from wild sea cucumber Australostichopus mollis collected from four distinct locations in New Zealand. Protein was extracted from sea cucumber body walls using trypsin enzymatic extraction, followed by cold acetone precipitation. The amino acid analysis revealed high glycine (189.08 mg/g), glutamic acid (119.45 mg/g), and aspartic acid (91.91 mg/g) concentrations in all samples. The essential amino acid indexes of the protein extracts (62.96, average) were higher than the WHO/FAO standard references, indicating the excellent protein quality of A. mollis. Furthermore, protein extracts from A. mollis demonstrated superior emulsifying activity (202.3-349.5 m2/g average) compared to commercial soy and whey protein isolates under all tested pH conditions, and enhanced foaming capacity (109.9-126.4%) and stability (52.7-72%) in neutral and acidic conditions. The extracts also exhibited good solubility, exceeding 70% across pH 3-11. Antioxidant capacities (ABTS and DPPH free radical scavenging activity and ferric reducing antioxidant power) were identified in A. mollis protein extracts for the first time, with clear variations observed among different locations. These findings elucidate the advantageous functional properties of protein extracts from wild New Zealand A. mollis and highlight their potential application as high-quality antioxidant food ingredients.

Effect of Glycolipoprotein Biosurfactant from Enterococcus faecium on the viability of Breast cancer cell lines.

Objective : This study , was research for cytotoxicity of bio-surfactant created by Enterococcus faecium isolated from feces of Iraqi healthy breast-fed infants with age < 6 months.
 Methods : Cold acetone precipitation was used to the extraction of extracellular Glycolipoprotein biosurfactant and partially purify it. Biosurfactant was then evaluated against two the cell lines, a Breast cancer MCF-7 cell line and a human normal fibroblast cell line NHF), specifically for cell survival and proliferation. 
 Results : At all concentrations with varying percentage, The viability of the MCF-7 cancer cell line was shown to be reduced with the addition of biosurfactant.; maximum inhibition percentage was 74.2% at a 100 µg/ml concentration, which is lesser than 45.5% cytotoxicity Of NHF healthy fibroblasts cell line.
 Conclusions : The findings of this study are highly encouraging in terms of the potential of Glycolipoprotein biosurfactants to treat cancer and encourage additional research with different cell lines.

Biological activities of purified prolidase from pathogenic E.coli

Prolidase is a ubiquitous enzyme that plays a chief role in the metabolism of proline-rich proteins. The goal of this study extraction and purify prolidase from E. coli and evaluate some applications such as anti-biofilm and anticancer. Hundred stool Samples were collected from infants with breastfeeding, non-vomiting, and non-diarrhea to isolate E.coli bacteria. A 16S rRNA gene (585 bp) was found in all isolates of E. coli via PCR identification. Depending on the qualitative method on prolidase agar, only 40 (80%) isolates could produce prolidase from 50 isolates that were considered non-pathogens, then only 32 isolates revealed different levels in prolidase production with specific activity equal to (2.1U/mg) of E.coli. MS12. Sucrose, casein, and 40Co were the chosen isolate's best conditions for producing prolidase. Cold acetone precipitation and dialysis were used to extract the enzyme, and DEAE-cellulose and the Sephadex G-150 column were used in purification with specific activity (2 U/ml) and (6.6 U/mg) protein. Prolidase showed the highest effect on biofilm at 500 μg/ml concentration against P. aeruginosa, then E. coli, 65% and 60.3% respectively. Brain Tumor Cell Line (A127), Colorectal Adenocarcinoma cells (CaCo-2), and Normal embryonic liver cell line (WRL-68) were used to test the prolidase effect on these cell lines. An assay of MTT was used to detect the inhibiter concentration (IC50) values and cytotoxic effect of purified prolidase. Keywords: E.coli, Prolidase, purification, antibiofilm, anticancer activity

A combination of semi-purified L-methioninase with tamoxifen citrate to ameliorate breast cancer in athymic nude mice.

Chemotherapy nonspecifically targets both tumor and healthy proliferating cells. Methionine deprivation using L-methioninase along with chemotherapy appears promising towards cancer management. The present study is an attempt to use a new combination of L-methioninase with Tamoxifen (TAM) to treat breast cancer in mice. L-Methioninase from Methylobacterium sp. was partially purified (SPMet's) by cold acetone precipitation and lyophilized. Its cytotoxicity effect, alone and in combination with Tamoxifen, was evaluated in vitro (MCF-7) cells and in vivo (athymic nude mice) conditions. SPMet's was found to inhibit the growth of MCF-7 cells with an IC50 value of 47.05µg/ml, while the combination of SPMet's and TAM had an IC50 of 6.4µg/ml. Athymic nude mice were grouped into: Group-I - Tumor control; Group-II - TAM; Group-III - SPMet's; Group-IV - SPMet's + TAM. Tumor growth inhibition (TGI) was maximum in Group-IV with 84.65% followed by Group-II with 65.12%. Hematological and Biochemical parameters in Group-II, III, and IV were restored to normal levels. Tumor histopathology showed increased apoptosis and necrosis in Group-IV. Caspases 3 & 8 gene upregulation was significantly higher in Group-IV than other treated groups, indicating higher efficacy of the combination approach. This is the first study report about a combination of SPMet's and TAM on in vivo breast cancer model, with significantly higher anticancer activity and without noticeable side effects. The findings of this study have several important implications for future clinical studies.

Partial Purification of Phytocystatin from Moringa oleifera lam

Background: Phytocystatins are plants cysteine protease inhibitors (CPIs) that are known for their numerous uses in medicine and biotechnology. Methods: Different extraction media which include, Sodium Hydroxide, Hydrochloric acid, Sodium Chloride, Sodium phosphate buffer and distilled Water were used to evaluate flower, leave, root, latex, and stem bark of Moringa oleifera for inhibitory activity against cysteine protease (Papain enzyme). The phosphate buffer extract with higher protease inhibitory activity was concentrated by cold acetone precipitation and further subjected to partial purification using ammonium sulphate fractionation and Hydrophobic chromatography on Phenyl Sepharose column. The molecular weight of partially purified protein was estimated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Results: The extracts of Sodium phosphate buffer from the seeds of Moringa oleifera showed higher CPI activity of 11.23 units/mg protein. Cold acetone precipitated extract gave high yield of 94.6% protein with 60% inhibition of protease. The partially purified protein showed a fifty percent inhibitory concentration (IC50) of 1.22±0.3 mg/ml protein against the enzyme with 63% inhibition. An estimated molecular weight of 13.5kDa was obtained from electrophoretic analysis of partially purified protein. Conclusion: Moringa oleifera seeds could be a good source of low molecular weight protein Inhibitor of Cysteine protease and might be useful in biotechnology of traditional medicinal plants, in transgenic crops to arrest the negative pathogenic over expressions of cysteine proteases

Developing a cloud point extraction process for lipopeptide recovery from cell-free broth of Bacillus sp. GY19

ABSTRACT Biosurfactants can be potentially used in food, cosmetics, pharmaceuticals, and environmental remediation because of their biodegradability and low toxicity. However, it is very challenging to recover biosurfactants from fermented broth as they are temperature-sensitive and present at dilute concentrations. This study aims to develop an alternative process that uses cloud point extraction (CPE) to recover lipopeptides biosurfactant from cell-free broth of Bacillus sp. GY19. The nonionic surfactant, Triton X-100, was selected as the aqueous-based solvent for CPE. Under the optimal conditions lipopeptides concentration in Triton X-100 was 23,000 mg/L, which is 14.8-folds higher than that in cell-free broth. Lipopeptides and Triton X-100 exerted a positive synergistic effect in reducing the critical micelle concentration of mixed micelles with a β value of −1.9. This could be considered a co-benefit of using CPE for biosurfactant recovery. Two techniques for back extraction of lipopeptides after the CPE process were proposed. Silica gel 60 selectively adsorbed Triton X-100 with 44% removal, while maintaining lipopeptides recovery at 86%. Cold acetone precipitation could remove 99% of Triton X-100 by precipitating lipopeptides. However, a 1%(w/v) SDS solution had to be used to re-solubilize the dehydrated lipopeptide

Síntesis de dextrano por microorganismos nativos de caña de azúcar

The sugarcane agri-food industry boosts the Mexican economy in producing regions. Basing its relevance in sugar production yields. However, by-products are not widely exploited leaving an opportunity for diversification. In this study, three microorganism isolates (A, B, and C) were obtained from sugarcane kefir; the morphology of isolates B and C corresponded to the lactic acid bacterial genus Leuconostoc. Thus, we examined the potential for these isolates to produce EPSs, like dextran, a molecule with applications in pharmaceuticals, industrials, and foods. The experiment was performed adjusting the active culture concentration to 1 x 106 colony-forming units (CFU)/ml, the culture was maintained at 37°C in agitation at 150 rpm. The obtained EPSs were purified by ethanol and cold acetone precipitation. The results showed that B and C bacterial isolates had the capacity to produce EPSs (14 g/L for isolate B and 32 g/L for isolate C) after 24 h. Fourier-transform infrared spectroscopy (FT-IR) characterization indicated that the EPS was dextran. Further, the produced biopolymer had high solubility in water, avoided freezing at -4°C, and boiled at 85°C.

Cysteine Protease Inhibitors from the Methanol Extract of the Root Bark of Securidaca longepedunculata with Antimalarial Potentials in Chloroquine-Resistant P. berghei Parasite

Malaria parasite resistance against Artemisinin-based Combination Therapy (ACT) in some parts of the world necessitates the search for antimalarial compounds or plant extracts with novel mode of action and active against the chloroquine-resistant strain of the parasite to serve as alternative to ACT. Cysteine protease inhibitors' fraction of the methanol extract of the root bark of Securidaca longepedunculata (CPI) was investigated for antiplasmodial activity against chloroquine-resistant P. berghei-infected mice and its inhibitory effect on papain, P. berghei cysteine proteases and heme biocystallization were also evaluated. The methanol extract of the root bark was obtained by soxhlet extraction with 1,000 mL of 70% (v/v) methanol for 48 hours and concentrated to dryness at 45∘C. CPI was obtained using PBS (pH 7) extraction followed by cold acetone precipitation. Peter's four-day suppressive and Rane's four-day curative test was employed to assess the antimalarial potentials of CPI. Data was analysed with one way ANOVA followed by Dunnett's post hoc test, differences were considered significant at p≤0.05. The suppressive effect of CPI was significant (p≤0.05) at 34 and 23 mg/kg doses. Doses of 34, 23 and 11 mg/kg produced significant (p≤0.05) dose-dependent curative effect. CPI inhibited the proteolytic activity of papain enzyme and P. berghei cysteine proteases in vitro with IC50 values of 20.1 and 5.6 μg/mL respectively. The present study showed that cysteine protease inhibitors fraction of the methanol extract of the root bark of Securidaca longepedunculata is a potential source of novel antimalarial agents that could target malaria parasite cysteine proteases.

Isolation and identification of a novel algicidal peptide from mackerel muscle hydrolysate

To help remedy damage from harmful algal blooms, an attempt was made to isolate an algicidal substance previously observed to be present in mackerel muscle hydrolysate. Crude extract was obtained by cold acetone precipitation, and it dissolved best in water. Through molecular weight cut-off determination and tricine-SDS PAGE, the algicidal substance was determined to be a peptide of <1 kDa. Based on this result, purification was first performed using size exclusion chromatography and preparative reverse phase high-performance liquid chromatography. Then, the active algicidal fraction was applied to an ultra-performance liquid chromatography-electrospray ionization-mass spectrometry system, followed by MS/MS analysis. The algicidal peptide had linear structure consisting of amino acids with sequence NH-KMNF-COOH. Its calculated properties were: molecular weight 538.66 g/mol; isoelectric point 9.91; net charge +1 at pH 7.0; and 50% hydrophobicity. Algicidal ability of the identified peptide was confirmed using synthesized peptide. The LC50 values toward four harmful algal blooming species were 0.69, 0.83, 0.85 and 1.24 mg/ml for Alexandrium fundyense, A. catenella, Heterocapsa triquetra, and Prorocentrum minimum, respectively. There was no coincidence in the sequence of the identified peptide with those of known metabolites in the APD, Norine, CAMP, UniProt and METLIN databases. Consequently, this algicidal substance originating from mackerel protein was deduced to be a novel peptide that can usefully be applied to relieve harmful algal blooms.

A SIMPLIFIED METHOD FOR THE EXTRACTION OF RECOMBINANT TAQ DNA POLYMERASE FROM ESCHERICHIA COLI

DNA polymerase (Taq) enzyme isolated from Thermus aquaticus, a thermostable gram-negative bacterium, is a basic component of PCR, widely used in life sciences. The extraction and purification of this enzyme involves time-consuming and expensive steps such as precipitation of proteins with PEI and/or ammonium sulfate, column chromatography techniques, and removal of salts or other small molecular weight contaminants by dialysis. In this work, a novel and simplified method for extraction and purification of the recombinant Taq polymerase from Escherichia coli was employed, which used cold acetone instead of PEI or ammonium sulfate to precipitate the enzyme. The enzyme was efficiently recovered as active form from both the crude cell lysate and column fractions with cold acetone precipitation. This simplified method enabled us to obtain high quality Taq DNA polymerase in a much shorter time and at a lower cost.

Electrokinetic Removal of Dodecyl Sulfate Micelles from Digested Protein Samples Prior to Electrospray-Ionization Mass Spectrometry.

In proteomics, dodecyl sulfate (DS-) as sodium salt is commonly used in protein solubilization prior to tryptic digestion, but the presence of the DS- hampers the electrospray ionization mass spectrometric (ESI-MS) analysis. The development of DS- depletion techniques is therefore important especially when dealing with small samples where there could be poor sensitivity due to sample loss or dilution during sample preparation. Here, we present a simple and fast electrokinetic removal method of DS- from small volumes of peptide and digested protein samples prior to ESI-MS. The selective removal was accomplished using an acidic extraction solution (ES) containing acetonitrile (ACN) inside a fused-silica capillary that was dipped into the sample. The use of acidic ES suppressed the electroosmotic flow; allowing the electrokinetic movement of DS- monomers and micelles into the capillary. The high amount of ACN present at the tip of the capillary served to collapse the micelles migrating into the capillary, thereby releasing the peptides that were bound to these micelles, facilitating peptide retention in the sample and efficient DS- removal. Increased % MS signal intensity (SI) restoration of the peptide was observed, while DS- removal was unaffected when the amount of ACN in the ES was increased. This is because of the micelle to solvent stacking mechanism (effective electrophoretic mobility reversal) working at high concentration of ACN for the improved recovery of the peptides. % MS SI restoration for the Z-Gly-Gly-Val and bradykinin peptides were 75-83% while % MS SI reduction of DS- was up to 99% under optimal conditions, that is, 40% ACN in the ES. Higher % peptide recoveries from digested protein samples were obtained using the proposed method compared to the conventional cold acetone precipitation method.

Sample preparation for detection of low abundance proteins in human plasma using ultra-high performance liquid chromatography coupled with highly accurate mass spectrometry.

Proteomics is a valuable approach to discover biomarkers in human plasma for early diagnosis. However, detection of biomarkers in the plasma is still challenging because of its large protein content. In our study, we established albumin/IgG depletion methods for identification of low abundance proteins using two commercial kits with additional buffer conditions and various concentrations of cold acetone. Trypsin digestion, desalting, and data-dependent acquisition were also optimized. More than 80% depletion of albumin/IgG was achieved with two commercial kits and 98% depletion of albumin was obtained with 70% cold acetone. Recovery of four reference proteins, BNP (47-76), insulin, cytochrome c, and ubiquitin was obtained in all optimized methods. The best recovery of reference proteins was obtained using the ProteoExtract albumin/IgG removal kit with buffer A (61%-106%). After cold acetone precipitation, three reference proteins were recovered more than 48% except ubiquitin (12%). The number of identified proteins by Mascot was 28, 35, 17, and 34 for ProteoExtract, ProteoPrep, 70%, and 50% cold acetone, respectively. Furthermore, optimized methods detected MS/MS fragmentation patterns of elevated BNP in patient samples with cardiac disease. Our study provides the conditions for efficient biomarker discovery by minimal removing of high abundant proteins.

Characterisation of extracellular polysaccharides from bacteria isolated from a full-scale desalination plant

Bacterial extracellular polymeric substances (EPS), particularly slime consisting of high-molecular weight soluble exopolysaccharides contribute significantly to seawater reverse osmosis (SWRO) membrane fouling. In this study, we characterized the soluble exopolysaccharides of 25 model bacterial strains isolated from different locations of a full-scale desalination plant in Western Australia. Biofilms of individual strains were initially quantified using crystal violet assay; significant biofilm production was detected in 21/25 isolates. Enzyme-linked lectin sorbent assay using lectins, Conacavalin A (ConA) and Ulex identified glucose and/or mannose in 11/25 isolates, and fucose in 24/25 isolates to significant levels. Exopolysaccharides were extracted and purified from bacterial cultures using centrifugation, TCA precipitation, cold acetone precipitation, dialysis and vacuum drying. Yield of exopolysaccharides varied between 90mg to 480mg/L of broth culture. Purified exopolysaccharides of 14 strains were analysed by High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) and ATR-FTIR. Fucose, rhamnose, glucuronic and galacturonic acids were present in majority of the isolates. These sugars are common to polysaccharides of glycosphingolipid-producing bacteria, a predominant subset of RO membrane biofilm community of the full-scale plant. They are known to impart better physical integrity to EPS and form strong, sticky recalcitrant biofilms. Mannose, glucose, galactose, xylose and ribose were also present.

A novel multicopper oxidase (laccase) from cyanobacteria: Purification, characterization with potential in the decolorization of anthraquinonic dye.

A novel extracellular laccase enzyme produced from Spirulina platensis CFTRI was purified by ultrafiltration, cold acetone precipitation, anion exchange and size exclusion chromatography with 51.5% recovery and 5.8 purification fold. The purified laccase was a monomeric protein with molecular mass of ~66 kDa that was confirmed by zymogram analysis and peptide mass fingerprinting. The optimum pH and temperature of the enzyme activity was found at 3.0 and 30°C using ABTS as substrate but the enzyme was quite stable at high temperature and alkaline pH. The laccase activity was enhanced by Cu+2, Zn+2 and Mn+2. In addition, the dye decolorization potential of purified laccase was much higher in terms of extent as well as time. The purified laccase decolorized (96%) of anthraquinonic dye Reactive blue- 4 within 4 h and its biodegradation studies was monitored by UV visible spectra, FTIR and HPLC which concluded that cyanobacterial laccase can be efficiently used to decolorize synthetic dye and help in waste water treatment.

Screening and optimization of laccase from cyanobacteria with its potential in decolorization of anthraquinonic dye Remazol Brilliant Blue R

Abstract In the present study 35 heterocystous and non heterocystous strains of cyanobacteria has been investigated for laccase production. Among all selected strain 29 strains showed the positive results and Spirulina platensis CFTRI gave the highest laccase activity. The laccase production was found to be maximum on 10 th day under normal growth condition. The average time of laccase production was decreased to 4 th day in presence of inducer guaiacol. The combination of (2,5-Xylidine + guaicol) act as best inducer that enhanced the laccase production 36.2 folds (582.92 Uml −1 ) due to synergistic effect. The optimum condition for laccase production was 30 °C & pH 9, glucose as carbon source and sodium nitrate as nitrogen source. The enzyme preparation was done by cold acetone precipitation and further characterized through SDS-PAGE and activity staining using ABTS as substrate. The molecular weight of protein was ≈66KDa. The crude laccase showed 46.74% decolorization of anthaquinonic dye RBBR within 48 h, The rate of decolorization was increased to 63.61% & 89.90% in presence of redox mediator (1-hydroxybenzotriazole & syringaldehyde) which suggested that biological methods using laccase are generally considered environmentally friendly as they can lead to complete degradation of organic pollutants at low cost.

Amylase Zymography.

Amylase zymography was carried out for the detection of amylases produced by a Geobacillus stearothermophilus strain isolated from the Thermal Center "Las Trincheras" in Venezuela. Zymography is an electrophoretic technique used to study hydrolases by means of thin gels containing copolymerized-specific substrates, under nonreducing conditions. In this study, 0.1% starch was incorporated into the gel as substrate. The formation of clear zones against a dark background in the gel stained with iodine indicated the presence of amylolytic activity. The thermophilic bacteria released several extracellular amylases to a selective growth medium supplemented with 1% soluble starch at 55 °C after 40 h incubation. The amylolytic enzymes showed an optimum temperature of 60 °C and an optimum pH at 6.0. The amylases were partially purified by cold acetone precipitation followed by two chromatographic techniques. These purified amylases showed different molecular masses which were determined by sodium dodecyl sulfate gel electrophoresis and confirmed by zymography.

Production of a Health-Beneficial Food Emulsifier by Enzymatic Partial Hydrolysis of Phospholipids Obtained from the Head of Autumn Chum Salmon.

Phospholipids and their partial hydrolysates, namely lysophospholipids (LPLs), have been widely used in food, pharmaceutical, and cosmetic products as highly efficient emulsifiers. This study was conducted to produce docosahexaenoic acid (DHA)-esterified LPLs by enzymatic modification of phospholipids obtained from the head of autumn chum salmon (Oncorhynchus keta). The emulsifying properties of the obtained LPLs were also evaluated. Two different types of substrates of salmon head phospholipids were prepared via silica gel and cold acetone precipitation. Enzymatic partial hydrolysis was carried out using immobilized phospholipase A1 (PLA1) and Lipozyme RM IM. Results showed that the increase in DHA in the LPLs was much higher in the silica-separated phospholipids than in the acetone-precipitated phospholipids. When silica-separated phospholipids were used as the substrate, the DHA content of the LPLs increased from 23.1% to 40.6% and 42.6% after 8 h of partial hydrolysis with Lipozyme RM IM and immobilized PLA1, respectively. The yield of the LPLs was comparatively higher in the Lipozyme RM IM than in the immobilized PLA1 hydrolysis reaction. The critical micelle concentration values of the LPLs and purified lysophosphatidylcholine (LPC) were 100 mg/L and 5 mg/L, respectively. The surface tension values of the LPLs and LPC were reduced to 30.0 mN/m and 30.5 mN/m, respectively. The hydrophilic-lipophilic balance of the LPLs and LPC were 6.0 and 9.4, respectively. Based on the emulsifying properties observed, we conclude that LPLs derived from the phospholipids of salmon head lipids could be used as a health-beneficial emulsifier in the food industry.

Purification and identification of β-casein phosphopeptide (1-25).

The β-casein phosphopeptide 1-25 (βCPP) is involved in calcium binding, cellular transduction, and dental remineralization. The objective of this work was to improve upon the original protocol commonly used for isolation of this phosphopeptide from β-casein. This method exploits the isoelectric point of β-casein fragments to selectively precipitate βCPP. The highest βCPP extraction yield reported to date with this protocol is 14.4±0.5% of theoretically available βCPP. The present work optimizes 2 steps in this procedure, namely the length of trypsin digestion and incorporation of cold acetone precipitation, to increase the yield to 32.3±5.4%. Reverse-phase HPLC indicated high purity of the isolate, whereas mass spectrometry confirmed 2 forms of the phosphopeptide: fragments 1-25 (87%) and 2-25 (13%). The adaptation of the existing protocol represents a significant improvement in extraction yield and facilitates preparation of larger amounts of high-purity βCPP for subsequent analysis and use in functional foods and other applications.

Improvement of shelf life of soymilk using immobilized protease of Oerskovia xanthineolytica NCIM 2839.

Protease enzyme has lot of commercial applications, so the cost-effective production of protease using sunflower oil seed waste was carried out from Oerskovia xanthineolyitca NCIM 2839. The maximum protease production was after 24 h of incubation with 2.5 % oil seed waste concentration. O. xanthineolytica was found to produce two proteases—P1 and P2. The proteases were purified using 60 % cold acetone precipitation and DEAE-cellulose ion exchange chromatography. SDS-PAGE revealed molecular weight of P1 and P2 was 36 and 24 kDa, respectively. P1 and P2 were optimally active at pH 7.0 and pH 7.5 at temperature 35 and 40 °C, respectively. Analysis of hydrolyzed product of P1 and P2 by HPLC reveals that the P1 has endoprotease and P2 has exoprotease activity. The treated soy milk with immobilized proteases showed increased shelf life and removal of off flavor.