Ammonium Sulfate Fractionation Research Articles

Comparative proteome analysis of matured dry and germinating Moringa oleifera seeds provides insights into protease activity during germination

This study is the first to apply label-free based proteomics to investigate the proteome changes during Moringa oleifera seed germination. In total, 1267 proteins were identified, with proteins within the molecular weight range of 10–60 kDa being most abundant. Among the 174 differentially expressed proteins (DEPs), 42 were upregulated and 26 downregulated, whereas 45 and 61 were uniquely expressed in the matured dry and germinating seeds, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the main DEPs had significant molecular functions related to catalytic activity and were involved in metabolism, degradation and biosynthesis, with 49 enzymes highly expressed in germination, including 9 proteases with hydrolytic activity, of which a peptidase and three types of endopeptidases were associated with milk-clotting activity. We further demonstrated that proteolytic activity (PA) and milk-clotting specific activity (MCSA) of protease extracts from M. oleifera seeds were increased during germination, particularly for samples from the ammonium sulfate (AS) fractionation that were statistically significance (p value < 0.05). Overall, proteases derived from germinating M. oleifera seeds can be used in the food industry, especially for potential application in the production of bioactive peptides and cheese processing.

Purification of Keratinase from Bacillus sp. MD24 using Ammonium Sulfate Fractionation

A keratin degrading bacterium, Bacillus sp MD24, was isolated from soil. The crude keratinase produced by the bacterium has been reported to dehair goat skin. However, the dehairing process took 72 hours. In order to shorten the dehairing time it is necessary to increase the keratinase concentration. This could be done by optimizing keratinase production either finding the best fermentation media or optimizing fermentation condition. Another way to increase the concentration could be done by partially purifying the enzyme. Keratinase from Bacillus sp MD24 had been produced under submerged fermentation, however, it produced a relatively low amount of enzyme. Although an effort to increase enzyme production had been reported by solid state fermentation, the enzyme concentration was not enough for industrial purposes. This work aimed to increase enzyme concentration by partial purification through enzyme precipitation using ammonium sulphate. The research was conducted in three stages: (1) regeneration the bacterium, (2) production of keratinase, and (3) purification of keratinase with ammonium sulfate fractionation. Keratinase activity was measured by Anson method and protein concentration was measured by Lowry method. Enzyme purity was clarified using a combination of specific activity, purity level, and SDS-PAGE analysis. Based on the analysis result, ammonium sulphate did not act as a good precipitation agent for the keratinase. Two major bands were suggested as keratinase with an estimated molecular weight of 25 and 66 kDa as monomer and dimer form, respectively.

Evaluation of The Stability and In Vitro Anti-inflammatory Activity of Partially Purified Bromelain Nanoemulsion

Bromelain is an enzyme belonging to cysteine protease. It is known for topical use as debridement for inflammation treatment. Nanoemulsion of bromelain (NEB) aims to solve this problem and also improve its stability. NEB was made by high emulsification method using homogenizer and sonicator. Nanoemulsion physicochemical stabilities were evaluated. The bromelain as an active compound was isolated from the core of pineapple (Ananas comosus [L.] Merr) which was purified by ammonium sulphate fractionation and then dialysis followed. Nanoemulsion formula 2 was found to be the most suitable formula for encapsulating bromelain with good physicochemical stability after 30 days of storage. This formula showed clear visual appearance with globule diameter of 37.42 nm with polydispersity index of 0.40. The result showed that the fraction of bromelain obtained from each purification step showed an increase in specific activity. The specific activities of fractionation using 20-50% ammonium sulphate and dialysis were 152.05 and 266.58 U/mg, respectively. The purest bromelain fraction was encapsulated into nanoemulsion formula 2. The bromelain fraction’s anti-inflammatory activity was tested based on HRBC membrane stabilization method. The result showed that bromelain dialysis fraction shows the ability as anti-inflammatory agent with stability percentage of 79.76%.

Protein Fractionation Methods Applied to the Delineation of Bioactive Proteins from the Zebra Mussel, Dreissena polymorpha.

Dreissena polymorpha is an invasive species that has infested the Great Lakes, portions of the Mississippi River, and roughly 250 lakes in Wisconsin. To date, there is no real ability to manage zebra mussels; efforts are limited to preventing the Zebra mussels from spreading further into Wisconsin. However, some groups in Europe have turned to the zebra mussel as a source for livestock feeds (McLaughlin, 2014.) In a tangential line of research, there is much interest in the study of marine shelled organisms, including abalone and crab, as sources of bioactive proteins. We have methods commonly used to understand the proteome of the shells of marine organisms to understand the protein content of the shells of zebra mussels. This poster reports on efforts to fractionate proteins and peptides as the first step towards understanding the antibiotic potential of zebra mussel shell extracts. Ammonium sulfate fractionation methods, as well as ion exchange chromatography and lectin chromatography, were investigated.This work may lead to the discovery a useful protein that creates a need for a bivalve that is otherwise an invasive pest in the United States.

Purification and characterization of thermostable α-amylase from germinating Sword bean (Canavalia gladiata (Jacq.) DC.) seeds.

The thermostable α-amylase from germinating sword bean (Canavalia gladiata (Jacq.) DC.) seeds (CgAmy) was successfully purified by a combination of ammonium sulphate fractionation and Epoxy-activated Sepharose 6B affinity chromatography. The purified α-amylase showed 507.8 fold with a specific activity of 750.0 U/mg. SDS-PAGE of the purified enzyme revealed a single protein band of 50.0 kDa. Purified enzyme was confirmed as α-amylase type by LC-MS/MS analysis and activity on specific substrate of ethylidene-pNP-G7. The CgAmy revealed extreme activity at a high temperature of 50.0-70.0°C with optimum activity at 70.0°C. The optimal pH of enzyme activity was observed at 6.0. The CgAmy exhibited stability in pH range of 5.0-8.0 and highly thermostable with a temperature of 40.0-60.0°C. The kinetic parameters K m for hydrolysis of starch were found to be 3.12 mg/ml. The α-amylase activity was enhanced in the presence of Co2+ and β-mercaptoethanol. While, Na2+, K2+, Ca2+, Mg2+, Zn2+, Ba2+, Fe2+ and Cd2+ slightly inhibited α-amylase activity. Interestingly, the CgAmy displayed stability towards some organic solvents and detergents. Stability at high temperature and some metal ions, organic solvents and detergents indicated that this enzyme has potential for various applications.

A Mesohaline Thraustochytrid Produces Extremely Halophilic Alpha-Amylases.

Halophilic bacteria are well known to produce highly salt-tolerant enzymes that have unusual applications in biotechnology. Production of halophilic proteins is generally not expected in mesohaline microorganisms. Ulkenia sp. AH-2, a mesohaline, marine straminipilan thraustochytrid isolated from waters of a mangrove ecosystem, produces halophilic alpha-amylases. Four enzyme fractions, viz.., A, B, C, and D, were obtained upon ammonium sulfate fractionation and gel filtration. These had a broad salinity tolerance ranging from 0 to 4M NaCl, with an optimum at 3M NaCl. Pools A, C, and D each resolved as a single band on PAGE and zymogram analysis, and the purified proteins were designated Amy a, Amy c, and Amy h. The major activity resided in "pool B," consisting of several amylases which could not be further resolved into pure fractions. Together, these had an optimum at 2M NaCl. All the enzymes were stable to storage for 2 to 24h at 4°C in a range of salt concentrations and even showed enhanced activity following such incubations. True to halophilic enzymes, the complex of "pool B" amylases showed improved activity in the presence of a wide range of organic solvents at 20% concentration. These enzymes are of particular interest by virtue of their constitutive nature as well as production under culture conditions that do not require salinity beyond that of seawater.

Selection of a versatile Lactobacillus plantarum for wine production and identification and preliminary characterisation of a novel histamine‐degrading enzyme

SummaryIn this study, a desirable and versatile Lactobacillus plantarum strain possessing great ability of L‐malic acid consumption, biogenic amine degradation and resistance to wine harsh environment was obtained through successive screenings. Malolactic fermentation (MLF) in contaminated grape wine and cherry wine (supplemented with histamine, tyramine and cadaverine) conducted by this strain was finished within 24 and 18 days, respectively, and the concentration of histamine, tyramine and cadaverine was decreased by over 57% following MLF. The enzyme from L. plantarum responsible for the amine degradation was purified to homogeneity by four steps including cell disruption, ammonium sulphate fractionation, an anion chromatography and a gel filtration chromatography. Such enzyme was identified as glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) after N‐terminal sequence analysis and protein Blast, a tetramer enzyme with a subunit molecular weight of 36 kDa. The optimum values of pH and temperature of this enzyme were at pH 7.5 and 40 °C, and stable between pH 5.5–8.5 and 30–50 °C.

Dissolution profile of partially purified bromelain from pineapple core encapsulated in alginate-guar gum with in vitro study of antiplatelet activity

The aim of this study is to maintain the antiplatelet activity of pineapple core bromelain from degradation in gastric fluid by encapsulating the enzyme in alginate-guar gum crosslinked with glutaraldehyde. Bromelain isolation was followed by purification process such as ammonium sulfate precipitation and dialysis. The specific activity obtained showed an enhancement started for crude enzyme (20.11 U/mg), ammonium sulfate fraction (190.64 U/mg), and dialysis fraction (229.77 U/mg). Dialysis fraction was encapsulated by in situ loading method in alginate-guar gum hydrogel with 1.25% (v/v) glutaraldehyde concentration. The swelling ratio of the hydrogel is 234.37 % in acidity of 1.2; and 1375.91 % in acidity of 7.4; while the encapsulation efficiency amount was 60.42%. I n addition, maximum concentration of bromelain released in dissolution test was higher in artificial intestinal environment (1.79 mg/L) than in artificial gastric fluid (0.13 mg/L), with maximum proteolytic activity of 1.2 U/mL and 0.11 U/mL, respectively. In vitro study of antiplatelet activity showed a good inhibition for dialysis fraction (50.6 %) and dissolution fraction (34.86 %), indicating that the hydrogel is good enough to maintain the activity of bromelain.

Determination of the effect of EDTA and PCMB on purified bromelain activity from pineapple core and in vitro antiplatelet activity

Bromelain is an enzyme belongs to the cysteine protease. In this study, bromelain isolated from pineapple core (Ananascomosus [L.] Merr) was purified by fractionation using ammonium sulfate followed by dialysis and then ion exchange chromatography. The fraction of bromelain obtained from each purification step showed an increase in specific activity. The highest specific activity of protease was found in 20-50% ammonium sulfate fraction of 104.018 U/mg with a purity level 3.2-fold compared to crude extract. Further purification by ion exchange chromatography using DEAE-Cellulose, the fraction of bromelain showed an increase in specific activity to 278.333 U/mg with a purity level 8.8-fold compared to crude extract. The determination of kinetics parameter of purified bromelain using Lineweaver-Burk plot gives Km value of 0.15 % (w/v) and Vmax of 0.056 U/min. This bromelain can be strongly inhibited by EDTA and PCMB. The addition of EDTA and PCMB at a concentration of 0.5 mM can decrease the activity of the enzyme up to 88.50% by showing the competitive and mix-inhibition types of inhibition, respectively. The antiplatelet activity of the bromelain fraction was tested in-vitro based on the Born method, by using plasma (PRP), acetosal as a positive control and ADP as an aggregator. The purified bromelain showed the ability of an antiplatelet agent with percentage of aggregation 29.51% and percentage of inhibition 68.91%.

DISSOLUTION KINETICS OF PARTIALLY PURIFIED BROMELAIN FROM PINEAPPLE CORES (ANANAS COMOSUS [L.] MERR.) ENCAPSULATED IN GLUTARALDEHYDE-CROSSLINKED ALGINATE-GUAR GUM

Objective: Bromelain is susceptible to low pH and thus must be encapsulated in glutaraldehyde-crosslinked alginate-guar gum (Alg-GG) hydrogels to avoid bromelain activity degradation in the stomach.&#x0D; Methods: Isolated crude bromelain was purified through ammonium sulfate precipitation, sodium benzoate precipitation, and dialysis. Bromelain-loaded Alg-GG was dissolved in artificial gastric fluid and intestinal environment.&#x0D; Results: The bromelain fractions showed a higher specific activity (U/mg) than the crude enzyme (51.32), as follows ammonium sulfate fraction (267.70±4.67), sodium benzoate fraction (115.63±3.35), and dialysis fraction (332.22). The dialysis fraction was encapsulated in Alg-GG hydrogel containing 0.75% (v/v) glutaraldehyde through the post-loading method. The swelling ratios of the hydrogel are 188.43% at pH 1.2 and 563.83% at pH 7.4. The highest encapsulation efficiency is 72.2%. The maximum bromelain concentration released during dissolution is higher in artificial intestinal environment (1.97 mg/L) than in artificial gastric fluid (0.18 mg/L), and the maximum proteolytic activities are 1.3 and 0.15 U/mL, respectively. Data were incorporated into the zero-order, first-order, Higuchi, and Korsmeyer–Peppas models to determine the kinetics and mechanism of bromelain dissolution. All bromelain concentrations (70, 140, and 210 ppm) follow the Korsmeyer–Peppas model. The dissolution mechanism is a combination of diffusion and erosion. Both the dialysis fraction (56.59%) and the dissolution product (47.45%) showed a good in vitro antiplatelet activity.&#x0D; Conclusion: The present data show the promise of Alg-GG encapsulation as a vehicle for orally administered therapeutic enzymes.

Carbonyl Reductase 1 Plays a Significant Role in Converting Doxorubicin to Cardiotoxic Doxorubicinol in Mouse Liver, but the Majority of the Doxorubicinol-Forming Activity Remains Unidentified.

Doxorubicin is a widely used cancer therapeutic, but its effectiveness is limited by cardiotoxic side effects. Evidence suggests cardiotoxicity is due not to doxorubicin, but rather its metabolite, doxorubicinol. Identification of the enzymes responsible for doxorubicinol formation is important in developing strategies to prevent cardiotoxicity. In this study, the contributions of three murine candidate enzymes to doxorubicinol formation were evaluated: carbonyl reductase (Cbr) 1, Cbr3, and thioredoxin reductase 1 (Tr1). Analyses with purified proteins revealed that all three enzymes catalyzed doxorubicin-dependent NADPH oxidation, but only Cbr1 and Cbr3 catalyzed doxorubicinol formation. Doxorubicin-dependent NADPH oxidation by Tr1 was likely due to redox cycling. Subcellular fractionation results showed that doxorubicin-dependent redox cycling activity was primarily microsomal, whereas doxorubicinol-forming activity was exclusively cytosolic, as were all three enzymes. An immunoclearing approach was used to assess the contributions of the three enzymes to doxorubicinol formation in the complex milieu of the cytosol. Immunoclearing Cbr1 eliminated 25% of the total doxorubicinol-forming activity in cytosol, but immunoclearing Cbr3 had no effect, even in Tr1 null livers that overexpressed Cbr3. The immunoclearing results constituted strong evidence that Cbr1 contributed to doxorubicinol formation in mouse liver but that enzymes other than Cbr1 also played a role, a conclusion supported by ammonium sulfate fractionation results, which showed that doxorubicinol-forming activity was found in fractions that contained little Cbr1. In conclusion, the results show that Cbr1 accounts for 25% of the doxorubicinol-forming activity in mouse liver cytosol but that the majority of the doxorubicinol-forming activity remains unidentified. SIGNIFICANCE STATEMENT: Earlier studies suggested carbonyl reductase (Cbr) 1 plays a dominant role in converting chemotherapeutic doxorubicin to cardiotoxic doxorubicinol, but a new immunoclearing approach described herein shows that Cbr1 accounts for only 25% of the doxorubicinol-forming activity in mouse liver cytosol, that two other candidate enzymes-Cbr3 and thioredoxin reductase 1-play no role, and that the majority of the activity remains unidentified. Thus, targeting Cbr1 is necessary but not sufficient to eliminate doxorubicinol-associated cardiotoxicity; identification of the additional doxorubicinol-forming activity is an important next challenge.

Purification and Biochemical Characterization of a Lipase Produced by Aspergillus Niger: Interfacial Binding, Activity of Lipase at the Lipid-Water Interface Using Monomolecular Film Techniques

Aspergillus Niger lipase (ANL) was purified from a culture medium. Pure ANL was obtained after ammonium sulfate fractionation, Sephacryl S-200 gel filtration and cation exchange chromatography (Mono-Q). The pure lipase, which is not a glycoprotein, was presented as a monomer having a molecular mass of about 99 kDa. The lipase activity was maximal at pH 8 and at 39°C. ANL hydrolyses the long chains of triacylglycerols more efficiently than the short ones. A specific activity of 3779 U/mg was measured on olive oil as substrate at 39°C and at pH 8 in the presence of 4 mM NaTDC. ANL was inactivated when the enzyme was incubated at 75°C or at pH less than 4. Natural detergent (NaTDC), pH dependence of the catalytic activity of ANL was at pH 8, which confirm that ANL present one pH profile.

Production of extracellular amylase by Aspergillus niger under submerged fermentation using jack fruit rag as the carbon source

Production of extracellular amylase by Aspergillus niger was studied under submerged fermentation using jackfruit rag as the carbon source. Different parameters, such as incubation period, pH of the culture broth and level of substrate were changed to optimise the conditions for amylase production. Maximum enzyme production ~ 8400 units/g was obtained in 5 days old cultures, grown at pH, 6.5 and 30°C with substrate level 20 gL -1 . As nitrogen sources NH 4 Cl, KNO 3 casein, peptone and beef extract were tested. Except NH 4 Cl all other sources enhanced the amylase production. Study on the kinetics of extracellular and intracellular amylase production revealed that extracellular amylase production was always higher than that in intracellular. Crude amylase obtained from culture broth was partially purified by ammonium sulphate fractionation followed by DEAE Cellulose chromatography. Partially purified enzyme exhibited optimum pH and incubation temperature at pH 6 and 60 ° C respectively and higher thermal and pH stability at 50-60°C and pH 5-7 respectively and enhanced activity with Ca 2+ . These unique features of the enzyme indicates its suitability for various industrial applications. Shorter incubation period and lower substrate cost offer the potential for inexpensive production of amylase, making the process industrially and economically feasible.

Characterization of ammonium sulphate fraction of pepsin from fish stomach

Pepsin is one of the digestive enzymes often used in various fields, especially for industry. Pepsin can be isolated from fish viscera. This study was aimed to obtain the source of pepsin having high activity and the characteristics of the pepsin. Fish used in this study including tuna, Hemigaleus balfouri, skipjack, and cob. Pepsin was purified using ammonium sulfate. The results showed that pepsin from tuna and H. balfouri had the highest activity. The activity of the purified pepsin as 101,061 AU/mg. The pepsin worked in a temperature range of 40-60 °C and pH 2 having activity up to 9,000 AU mL, and 9,800 AU/mL, respectively.

Characteristics of an Acidic Phytase from Aspergillus aculeatus APF1 for Dephytinization of Biofortified Wheat Genotypes.

Phytases are the special class of enzymes which have excellent application potential for enhancing the quality of food by decreasing its inherent anti-nutrient components. In current study, a protease-resistant, acidic phytase from Aspergillus aculeatus APF1 was partially purified by ammonium sulfate fractionation followed by chromatography techniques. The molecular weight of partially purified phytase was in range of 25-35kDa. The purified APF1 phytase was biochemically characterized and found catalytically active at pH3.0 and 50°C. The Km and Vmax values of APF1 phytase for calcium phytate were 3.21mM and 3.78U/mg protein, respectively. Variable activity was observed with metal ions and among inhibitors, chaotropic agents and organic solvents; phenyl glyoxal, potassium iodide, and butanol inhibited enzyme activity, respectively, while the enzyme activity was not majorly influenced by EDTA, urea, ethanol, and hexane. APF1 phytase treatment was found effective in dephytinization of flour biofortified wheat genotypes. Maximum decrease in phytic acid content was noticed in genotype MB-16-1-4 (89.98%) followed by PRH3-30-3 (82.32%) and PRH3-43-1 (81.47%). Overall, the study revealed that phytase from Aspergillus aculeatus APF1 could be effectively used in food and feed processing industry for enhancing nutritional value of food.

Detection and Cryoprotective Activity of Dehydrin Proteins from Rice Bran and Soybean Whey.

Dehydrin proteins, group2 LEA proteins in rice bran and soybean whey were analyzed by two-dimensional (2D) electrophoresis, and the cryoprotective activity on freeze/thaw inactivation of lactate dehydrogenase was examined as a criterion of its function. Dehydrins in rice bran were detected by immunoblotting using antibody raised against a conserved lysine-rich motif sequence. In the water-soluble fraction of rice bran, 10 spots of 44 kDa and 23 kDa dehydrin-like polypeptides were detected on the immunoblotted membrane. Isoelectric points of the polypeptides were between 6.6 and 7.4. The 23 kDa dehydrin polypeptide was partially purified by ammonium sulfate fractionation and ion exchange column chromatography. CP50 value, protein amount necessary to keep 50% of enzyme activity, of the 23 kDa dehydrin was 0.78 μM (15.6 μg/mL), slightly lower than that of bovine serum albumin. Heat-soluble soybean whey proteins were analyzed by SDS-PAGE and 2D-electrophoresis. Dehydrin appeared to be the most abundant protein in the fraction. CP50 value for heat-soluble whey protein was estimated to be 15.8 μg/mL, while that of total whey was 355 μg/mL. The result indicated that simple heat fractionation is efficient to concentrate cryoprotective protein from soybean whey.

Anti-dengue potential of bioactive protein from exophytic bacteria that are symbiotic with brown algae Sargassum sp.

Symbiont bacteria of algae are bioactive metabolite sources with potential as medicinal raw materials. This study aims to find out the anti-dengue potential of a protein fraction isolated from Enterobacter agglomerans SB 5(1) as the symbiont of brown algae Sargassum binderi collected from Lae-Lae island, South Sulawesi. These extracellular and intracellular fractions were isolated by ammonium sulphate fractionation at saturation levels of 0-20 %, 20-40 %, 40-60 %, and 60-80 %. The protein was purified by dialysis method using cellophane bag. Toxicity was tested by BSLT method using shrimp larvae of Artemia salina, Leach. Cytotoxicity test against vero cells infected with dengue virus DEVN-2 was performed by MTT method. Study findings indicate that intracellular protein fraction from E. agglomerans SB 5(1), a symbiont of brown algae Sargassum binderi, showed the presence of bioactive protein having strong toxicity with LC50 of 48.67 µg/mL. Anti-dengue activity toward vero cells indicates inhibition percentage and CC50 value of 70% and 260.37 µg./mL, respectively, therefore it had no potential as anti-virus dengue agent. In future studies, it is recommended to perform hydrolysis of protein compound from symbiont bacteria of Sargassum sp. to explore other peptide compounds with more potential as anti-dengue agents.

Isolation and Cleaning of Lactate Dehydrogenase from Pea (Pisum sativum L.) Roots by Hypoxia and the Study of Its Regulatory Properties

The placement of pea plants (Pisum sativum L.) under flooding conditions led to an increase in lactate dehydrogenase (EC 1.1.1.27) activity in the roots. The enzyme was purified to an electrophoretic homogeneous state by a multistage purification method including ammonium sulfate fractionation, ion exchange chromatography on DEAE-Sephacel, and gel chromatography on Sephadex G-200. The degree of purification was 43.4, the yield was 2.5%, and the specific activity was 80.5 U/mg protein. Its physicochemical properties were studied: the molecular weight of the native lactate dehydrogenase molecule was 138 kDa. The molecular weight of the subunits was determined by PAGE by electrophoresis in the presence of DDS-Na. Its value was 34 kDa, which indicates that the enzyme is a homotetramer. The kinetic and regulatory properties of the enzyme and the values ​​of the Michaelis constants were established. he effect of the concentration of hydrogen ions and temperature on direct and reverse reactions catalyzed by it was obtained. It was determined that lactate dehydrogenase inhibited ATP.

Fractionation and activity profiling of fruit bromelain from pineapples of Phuket variety growing in Thailand.

Bromelain is a type of protease found in both fruits and stems of pineapples. Stem bromelain has been extensively studied and is commercially available for applications in various industries. In contrast, studies of fruit bromelain are quite limited since most of pineapples have been consumed freshly, canned or juiced. Nowadays, the consumption of canned fruits, including canned pineapples has decreased greatly. Fruit bromelain could be a new growth point for pineapple industry. In this study, fruit bromelain was extracted from the pineapple juice of Phuket variety and some of its properties were studied. The enzyme was purified by precipitation using ammonium sulfate fractionation followed by ion-exchange and gel filtration chromatography. Consequently, the protease purification level was increased by 95.2 fold. The final specific activity was getting to 448,590 U/mg on average, dominated by cysteine proteases, with optimal activity at 45°C and pH ranging from 6 to 8. The study facilitates the molecular and application research of fruit bromelain. PRACTICAL APPLICATIONS: The research has been carried out at Funong Food Technology Co., Ltd., Guangdong, China, which produces primarily pineapple chunks and juice. As plenty of by-products, like peels and cores of pineapples, are produced, the techniques are employed to extract bromelain from the by-products. The techniques reported in this work are not new or advanced, however, they are applicable during the manufacturing process and the related equipment is easy to set up and operate. What's more, the practical application of the techniques is cost-effective for the manufactories. Take Funong Food Technology Co., Ltd. as an example, they was using 80% saturation ammonium sulfate to precipitate protein from pineapple juice and obtained a bromelain with activity of approximately 8,000 U/mg and yield of 1.7kg per ton of juice. With the application of the techniques reported in this work, bromelain was first extracted by ammonium sulfate gradient precipitation, and then purified through ion-exchange and gel filtration chromatography. Each step of precipitation and purification generates a different level of activity and recovery of bromelain, ranging from around 2,506 to 448,590 U/mg, which allows for the production of bromelain according to the requirement of the market and brings more profits.

Purification and characterization of a glycoside hydrolase family 5 endoglucanase from Tricholoma matsutake grown on barley based solid-state medium

An endoglucanase was isolated from solid-state culture of the ectomycorrhizal fungus Tricholoma matsutake (TmEgl5A) grown on rolled barley and vermiculite. The enzyme was purified by ammonium sulfate fractionation, ion-exchange, hydrophobic, and gel filtration. TmEgl5A showed a molecular mass of approximately 40kDa as determined by SDS-PAGE. The single band of the protein was analyzed by peptide-mass-finger-printing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the trypsin-digested peptide sequences were matched to a putative endoglucanase sequence (protein ID1465229) in the JGI T. matsutake 945 v3.0 genome database. Based on the sequence information, the gene encoding TmEgl was cloned and expressed in Pichia pastoris KM71H. The deduced amino acid sequence was similar to GH5 family endoglucanases from Basidiomycetes. The enzyme acts on barley β-glucan, lichenan, and CMC-Na. The hydrolyzation products from these substrates were detected by thin-layer chromatography as oligosaccharides with minimal disaccharides. These results suggested that T. matsutake produces a typical endoglucanase in solid-state culture, and the fungus has the potential to degrade β-linkage polysaccharides.