Crude Enzyme Extract Research Articles

Influence of feeding habits of fish on the characteristics of their visceral alkaline proteases with special reference to detergent compatibility

Technical characteristics and detergent compatibility of visceral alkaline proteases of three freshwater fish, namely Labeo rohita, Pangasianodon hypophthalmus and Cyprinus carpio of different feeding habits, were studied. Crude enzyme extract was partially purified by (NH4)2SO4 precipitation and dialysis. The molecular weight was in the range of 20–63 kDa. The enzyme purification folds post-dialysis were found to be 1.55, 1.81 and 2.17 in case of Rohu, Pangas and Common carp respectively. The alkaline protease from Rohu, Pangas and Common carp exhibited maximum activity at pH 10.0, 9.0 and 11.0 respectively. The enzyme temperature optima observed were 60°C (Rohu and Pangas) and 70°C (Common carp). SBTI and EDTA inhibited more than 90% of the activity at conc. of 50 mM. Exposure of the proteases to non-ionic surfactants like Tween 20–80 retained about 92%–100% and 76%–100% of their activity at conc. (v/v) of 1% and 5% respectively. Proteases were found less stable in the presence of SDS. There was moderate to lesser influence of H2O2 and sodium perborate on the proteolytic activity. The alkaline protease from omnivorous fish was found superior compared to the herbivore and carnivore in respect of pH and temperature optima and stability with detergents and oxidizing agents.

Screening of in vitro nutrient digestibility coefficients of selected insect meals in broiler chickens, black-meat chickens and quails.

The objective of this study is to identify potential insect species comparing with commonly used protein sources based on efficiency of the in vitro digestibility on dry matter (DMd), organic matter (OMd) and crude protein (CPd) in broiler chickens, black-meat chickens (Native breed) and quails. Each of gastric mucosa, pancreas and duodenal mucosa were obtained from proventriculus, pancreas and duodenum, respectively. Crude digestive enzyme extracts (CTE) were extracted from these organs to perform in vitro digestibility. Eighteen insect samples and six commonly used protein sources were served as substrates which were evaluated on DMd, OMd and CPd in triplicate for each substrate. The CTE from gastric mucosa was used to simulate proventriculus, whereas small intestine was simulation by adding the CTE from pancreas and duodenum. The large variation of chemical composition between insect meals was presented. For commonly used protein sources, animal proteins were higher on digestibility than plant proteins (p<.001). Quails represented a great potential to digest insect meals comparing other animals. Based on CPd results, there were potential insect species for broiler chickens (Achroia grisella: AG, Tenebrio molitor: TM and Musca domestica), black-meat chickens (Patanga succincta, TM and AG) and quails (Hermetia illucens, Acheta domesticus and Locusta migratoria; p<.001). The evidences from this study suggest that these insect species contain a great potential to use as alternative protein sources promoting an animal production with sustainability. However, the in vivo experimentation must be performed to confirm in further study.

Characterization of 2,3,7,8-tetrachlorodibenzo-p-dioxin biodegradation by extracellular lignin-modifying enzymes from ligninolytic fungus

Ligninolytic fungi secrete extracellular lignin-modifying enzymes (LME) that degrade plant polymers for fungal nutrition but that are, because of their broad substrate specificity, also applicable for the degradation of many hazardous pollutants. Laccase is one of the most well characterized LME and is involved in the removal and degradation of recalcitrant aromatic compounds with or without the assistance of laccase-mediators. The Ligninolytic fungus Rigidoporus sp. FMD21 can degrade 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) with a half-life of 6.2 days. Using Rigidoporus sp. FMD21 crude extracellular enzyme extract (ExE) that mainly consisted of laccase, 77.4% of 2,3,7,8-TCDD was degraded within 36 days. The degradation rate did not depend on the 2,3,7,8-TCDD concentration in the tested range between 0.005 and 0.5 pgTEQ/μL. 2,3,7,8-TCDD was analysed by DR-CALUX® bioassay and the degradation was confirmed by GC-HRMS. In this study, we found evidence for cleavage of the diaryl ether bond in the 2,3,7,8-TCDD molecule and here we propose a new degradation mechanism in which 3,4-dichlorophenol is the main metabolite of 2,3,7,8-TCDD degradation by FMD21’s ExE. Six laccase-mediators were tested. Three of them 1-hydroxybenzotriazole (HBT), syringaldehyde (Syr) and violuric acid (Vio) showed an equipotent added effect on 2,3,7,8-TCDD degradation by ExE, however only in case of Vio a level of significance was reached. The others showed no effect or negatively impacted degradation. In conclusion, we have shown that Rigidoporus sp. FMD21 produces extracellular enzymes, mainly laccases that apparently are able to degrade the highly recalcitrant and most toxic 2,3,7,8-congener of TCDD via diaryl bond cleavage into 3,4-dichlorophenol.

Lipases of Endophytic Fungi Stemphylium lycopersici and Sordaria sp.: Application in the synthesis of solketal derived Monoacylglycerols

Monoacylglycerols (MAGs) are amphiphilic compounds with wide range of applications such as emulsifiers, solubility agents, and chiral building blocks. These compounds are currently produced by chemical approaches involving alkaline glycerolysis or esterification under high temperatures and pressure, resulting in low yields and with by-products. Lipase-catalyzed processes have been alternative tools to provide more ecological approaches since MAGs can be obtained under milder reaction conditions and with higher selectivity. However, just a few papers have been explored the potential of endophytic fungi as lipase sources. In this work we summarized the screening of lipolytic activity of endophytic fungus S. lycopersici and Sordaria spp isolated from vegetal species collected in Jurubatiba Sandbank National Park, RJ, Brazil, as well as its applications as biocatalysts on the lipase-catalyzed synthesis of solketal 1-MAG derivatives. As a result, the crude enzymatic extract of S. lycopersici showed 98 U/mL and 110 U/mL of hydrolytic activity after 72 h and 96 h, respectively, against 74 U/mL (96 h) and, 86 U/mL (120 h) expressed by enzymatic extract of Sordaria spp.. Concerning the esterification activity, both crude enzymatic extracts and lyophilized fungi showed about 80 % conversion into ethyl oleate, in 100 min. On solketal derived 1-MAG synthesis, S. lycopersici both lyophilized and immobilized in polyurethane (PU) forms showed more than 75 % of conversion in the presence and absence of organic solvents. On MAG recycle assays, the PU biocatalyst could be reused after five reaction cycles while for the ethyl oleate synthesis, PU biocatalyst could be reused after six reaction cycles. Both microorganisms, immobilized in polyurethane, were successfully applied as biocatalysts in esterification reactions for solketal 1-MAG derivative production, in a solvent-free media.

Electrochemical analysis of specific catalase activity during development of Aspergillus flavus and its correlation with aflatoxin B1 production

Aflatoxin B1 (AFB1) contamination causes huge economic losses. To explore the correlation between catalase (CAT) and AFB1 production during fungal development, we fabricated an electrochemical CAT-activity sensor by measuring residual H2O2 after enzymatic degradation. The sensor made by palladium nanoparticles/carbonized bacterial cellulose nanocomposites exhibits a linear range over 0.5–3.5 U/mL and a detection limit of 0.434 U/mL. Both dry weight and CAT activity of mycelia continuously increase. But, the latter shows a greater increase than the former after three days. Specific CAT activity in crude enzyme extract of A. flavus was quantified. It maintains at ~25.00 U/mg for 3 days and enhances to 28.91 and 45.30 U/mg, respectively, on days 4 and 5. AFB1 production follows the same trend. On days 4 and 5, AFB1 concentration reaches 201.35 and 767.9 ng/mL, respectively. The positive correlation between specific CAT activity and AFB1 production suggests that CAT is involved in AFB1 biosynthesis.

Thermal inactivation kinetics of peroxidase and polyphenol oxidase from pomegranate arils (Punica granatum L. cv. Wonderful).

Thermal inactivation of peroxidase (POD) and polyphenol oxidase (PPO) in both pomegranate arils crude enzyme extract and fresh juice was investigated. The optimum conditions for reactions were studied using a mixture of guaiacol and H2O2 as substrate for POD and pyrogallol for PPO. The experimental work indicated that optimum pH for both enzymes was 7.0; meanwhile optimum temperature was 30°C for POD and 25°C for PPO. Michaelis-Menten constant (Km) values for POD were 8.33 and 8.00mM for guaiacol and H2O2, respectively. The Km value was 5.88mM pyrogallol for PPO. Thermal inactivation results revealed that the inactivation kinetics followed a monophasic first-order model. Activation energies (Ea) were 74.68 and 112.97kJ/mol for POD and PPO, respectively. Therefore, POD was more heat-stable than PPO. This result is very useful to optimize pomegranate processing (canning or freezing), which represents the most important food industries in many tropical and subtropical regions. PRACTICAL APPLICATIONS: Pomegranate has been attracted more interest due to its antioxidant and nutritional values. For industrial applications, there is a growing interest to obtain pomegranate products such as juice, jams, food supplements, etc. It is well established that the residual activities of endogenous enzymes in either raw materials or processed products may cause loss of quality during storage. Inhibition of enzymatic browning is an important factor to maintain pomegranate quality. This work offers a better insight of characterization of browning enzymes and their thermosatbility, which will be useful to control the possible browning of pomegranate arils during processing and storage. In addition, the results of this study is very important in pomegranate processing where it showed a higher temperature with shortened time can be applied.

Manufacture of fresh cheese using east African Calotropis procera leaves extract crude enzyme as milk coagulant.

The present work was aimed to develop fresh cheese using the crude extract from calotropis procera leaves and thereby performed evaluating milk‐clotting activity and acute toxicity of crude extract. The extraction of coagulant was performed at 30, 45, and 60℃ using 2, 6, and 10 g of dried calotropis procera leaves, respectively. The highest yield of crude enzyme extract was acquired with 2 g and 30℃, and results were varied significantly (p < .05). The highest cheese yield and fastest clotting time were 17.89 kg cheese/100kg milk and 14:50 min acquired with 10 g C. procera powder at 60℃ extraction temperature, respectively. E. coli, total bacterial count, yeast, and mold microbial load detection were observed below the limit established by codex alimentarius. This study recommends that use of C. procera crude enzyme leaves extract can be used as alternate milk coagulant for production of fresh cheeses. Soft cheeses manufactured with calotropin enzyme as milk coagulant revealed no adverse effects of toxicity tested on albino rates to the highest dose.

Digestive proteases and in vitro protein digestibility in bagrid catfish Mystus nemurus (Cuvier and Valenciennes 1840)

Digestive proteases in the stomach and intestine of bagrid catfish Mystus nemurus juveniles and in vitro protein digestibility of fishmeal (FM), soya bean meal (SBM) and squid meal (SM) were determined. Postprandial data demonstrated highest protease activity 2 and 5 hr in the stomach and intestine, respectively, after feeding. Acidic-like protease in the stomach exhibited an optimum activity at pH 2, while alkaline proteases dominated the intestine, with optimum activities between pH 9 and 11. Investigations using specific commercial inhibitors confirmed the presence of pepsin in the stomach. Chymotrypsin, trypsin and metalloproteases were present in the intestine; however, the results indicated a possible involvement of other serine proteases. The highest inhibition of intestinal protease activity of bagrid catfish occurred during incubation with SM, followed by SBM and FM. Fishmeal had the highest relative protein digestibility (RPD), compared with SBM and SM, regardless of enzyme system used. Crude enzyme extract and the 4E system resulted in the highest RPD followed by 3E and 1E systems. The current results suggest that bagrid catfish adopt the typical protein digestion pathway of a carnivorous fish of endoprotease and exoprotease activities from the stomach to the intestine. Pepsin acts in the stomach, while trypsin, chymotrypsin, metalloproteases and other serine proteases are involved in the intestine. This study also recommends the use of crude intestinal enzyme extract for digestibiliy studies, instead of commercial enzyme systems, to better identify the suitable protein sources for bagrid catfish.

Production of cellulases by Aureobasidium pullulans LB83: optimization, characterization, and hydrolytic potential for the production of cellulosic sugars

Aureobasidium pullulans LB83 was evaluated for cellulase production under submerged fermentation conditions. Different process variables such as carbon sources (corn cob, sugarcane bagasse, and sugarcane straw), synthetic (urea, ammonium sulfate, and peptone), and non-synthetic (soybean meal, rice, and corn meal) nitrogen sources and inoculum size were evaluated by one parameter at-a-time strategy. Aureobasidium pullulans LB83 showed maximum cellulase activity (FPase, 2.27 U/mL; CMCase, 7.42 U/mL) on sugarcane bagasse. Among the nitrogen sources, soybean meal as a non-synthetic nitrogen sources showed a maximum cellulase activity (FPase 2.45 U/mL; CMCase, 6.86 U/mL) after 60 hr. The inoculum size of 1.6 × 106 CFU/mL had the maximum FPase and CMCase activities of 3.14 and 8.74 U/mL, respectively. For the enzymatic hydrolysis, both the commercial cellulase (10 FPU/g of Cellic CTec 2 (#A) and 10 FPU/g of crude enzyme extract (CEE) (#B), and varying ratio of CTec 2 and CEE in combination #C (5 FPU/g of CTec 2 + 5 FPU/g CEE), combination #D (2.5 FPU/g of CTec 2 + 7.5 FPU/g CEE), and combination #E (7.5 FPU/g of CTec 2 + 2.5 FPU/g CEE) were assessed for enzymatic hydrolysis of delignified sugarcane bagasse. Enzyme combination #C showed maximum hydrolysis yield of 92.40%. The study shows the hydrolytic potential of cellulolytic enzymes from A. pullulans LB83 for lignocellulosic sugars production from delignified sugarcane bagasse.

Determination of the activity and kinetic parameters of polyphenol oxidase enzyme in crude extracts of some Jordanian plants

The activity and kinetic parameters of the enzyme polyphenol oxidase (PPO) in crude extracts of the fruits of apple (Pyrus malus L.), cucumber (Cucumis sativus L.), squash (Cucurbita pepo L.), pomegranate rind and seeds (Punica granatum L.), and the leaf of cactus (Opuntia ficus-indica (L.) were investigated. The highest browning intensity was found in apple, followed by pomegranate seeds and cucumber fruits. The optimum activity of the enzyme was shown at pH 7.0 for apple and cucurbita, but it was found at pH 6.0 for the rind and seeds of the pomegranate. The PPO of cactus leaf and cucumber fruits showed two peaks at pH 4.0 and pH 7.0. On the other hand, the optimal temperature found for PPO activity in these plants was around 40oC. The relative activity of PPO was ranging from 45% in cactus leaf to 72% in cucumber fruit at 30oC. A strong correlation with value (R) = 0.9485 between the browning intensity and PPO activity in all studied samples was obtained. These results indicated that the studied activity of PPO on different plants could serve as a guideline for predicting the conduct of enzymes to effectively prevent the browning of plants during grading and storage.

Extraction of pectinase from Candida isolated from textile mill effluent and its application in bio-scouring of cotton

Pectinase is an important enzyme in industrial biotechnology and is widely utilized in industries producing food and beverages. In textile chemical processing, pectinase is utilized for a pretreatment of cotton called bio-scouring where it helps to increase the absorbency of cotton fabric and makes it suitable for further coloration. Various products based on pectinase are available in the market; however, the extraction of pectinase from a novel source which can meet the requirements of bio-scouring remains the quest of research. The present work deals with the isolation of fungus (Candida) from textile mill effluent for the extraction of extracellular thermostable pectinase. Optimization of temperature and pH of pectinase activity was studied. The crude enzyme extract showed efficient activity under the broad range of temperature and pH. The bio-scoured cotton showed an acceptable level of absorbency (absorbency < 3 s). The colour values of the dyed fabrics, scoured using conventional and bio-scouring, were comparable. The effluent load was lower for a bio-scouring process as compared to that of traditional scouring.

Genome sequencing and functional characterization of a Dictyopanus pusillus fungal enzymatic extract offers a promising alternative for lignocellulose pretreatment of oil palm residues.

The pretreatment of biomass remains a critical requirement for bio-renewable fuel production from lignocellulose. Although current processes primarily involve chemical and physical approaches, the biological breakdown of lignin using enzymes and microorganisms is quickly becoming an interesting eco-friendly alternative to classical processes. As a result, bioprospection of wild fungi from naturally occurring lignin-rich sources remains a suitable method to uncover and isolate new species exhibiting ligninolytic activity. In this study, wild species of white rot fungi were collected from Colombian forests based on their natural wood decay ability and high capacity to secrete oxidoreductases with high affinity for phenolic polymers such as lignin. Based on high activity obtained from solid-state fermentation using a lignocellulose source from oil palm as matrix, we describe the isolation and whole-genome sequencing of Dictyopanus pusillus, a wild basidiomycete fungus exhibiting ABTS oxidation as an indication of laccase activity. Functional characterization of a crude enzymatic extract identified laccase activity as the main enzymatic contributor to fungal extracts, an observation supported by the identification of 13 putative genes encoding for homologous laccases in the genome. To the best of our knowledge, this represents the first report of an enzymatic extract exhibiting laccase activity in the Dictyopanus genera, offering means to exploit this species and its enzymes for the delignification process of lignocellulosic by-products from oil palm.

Biochemical characterization of endoglucanases produced by Myceliophthora thermophila M.7.7 in solid-state culture

Cellulases have been investigated for their potential application in lignocellulosic residues, used to produce second-generation ethanol (2 GE), as these enzymes can hydrolyze cellulose and release glucose for alcoholic fermentation by yeasts. The aim of this project was to characterize the endoglucanases produced by the thermophilic fungus Myceliophthora thermophila M.7.7 in solid-state cultivation. Tests were carried out with the crude enzyme extract (CEE) to assess the effects of pH, temperature, and various chemicals including cations and phenolic compounds on endoglucanolytic activity. The molecular weight estimation was performed by polyacrylamide gel electrophoresis (SDS-PAGE), and it was possible to observe seven endoglucanases detected by zymography, with estimated Mw in the range between 26 and 82 kDa. The optimum pH for the CEE was 5.5 and the optimum incubation temperature was 70 °C. Concerning endoglucanase stability, the highest activities were achieved in the pH range of 6.0–6.5, and between temperatures of 30 and 40 °C up to 120 min. Among the tested reagents, Triton, dithiotreitol (DTT), and Isopropanol increased the enzymatic activity on the crude extract by about 10%. Among cations, only Sr2+ significantly increased the endoglucanases’ activity. Of the phenolic compounds, although all induced a decrease of the endoglucanolytic activity, none could nullify it after a pre-incubation for 24 h. The apparent kinetic parameters were determined using carboxymethylcellulose (CMC), being Vmax = 51.7 ± 2.7 μmol min−1 and Km = 0.99 ± 0.15 g% CMC (9.9 mg mL−1).

Optimization of culture conditions for enhanced decolorization of Amido black by Calocybe indica (cbe 1515) spent mushroom substrate

The objective of this study was to exploit the decolorization potential of Spent Mushroom Substrate (SMS) of Calocybe indica (Strain-CBE 1515) for the biodegradation of reactive textile dye Amido black. Initial studies have been made on screening of mycelia and crude enzyme extract of spent compost of Milky mushroom (Calocybe indica) strains for their dyes (Amido black, Congo red and RBBR) decolorization potential. It was observed that strains of C. indica were found to be hyper specific to laccase production and significant decolorization potential was showed by CBE 1515 for Amido black. Various process parameters like composition of basal nutrient medium, pH, temperature, additional carbon and nitrogen sources, and initial dyestuff concentration were optimized to develop an economic decolorization process. The optimum dye decolorization was achieved in LME medium containing dextrose and yeast extract as carbon and nitrogen sources respectively and adjusting the pH to7.5 and incubated at 30 °C.

Synthetic dyes biodegradation by fungal ligninolytic enzymes: Process optimization, metabolites evaluation and toxicity assessment

This work aimed to provide information that contributes to establishing environmental-friendly methods for synthetic dyes’ degradation. The potential decolorization capacity of the crude enzymatic extract produced by Phanerochaete chrysosporium CDBB 686 using corncob as a substrate was evaluated on seven different dyes. Critical variables affecting the in-vitro decolorization process were further evaluated and results were compared with an in-vivo decolorization system. Decolorization with enzymatic extracts presented advantages over the in-vivo system (higher or similar decolorization within a shorter period). Under improved in-vitro process conditions, the dyes with higher decolorization were: Congo red (41.84 %), Poly R-478 (56.86 %), Methyl green (69.79 %). Attempts were made to confirm the transformation of the dyes after the in-vitro process as well as to establish a molecular basis for interpreting changes in toxicity along with the degradation process. In-vitro degradation products of Methyl green presented a toxicity reduction compared with the original dye; however, increased toxicity was found for Congo red degradation products when compared with the original dyes. Thus, for future applications, it is crucial to evaluate the mechanisms of biodegradation of each target synthetic dye as well as the toxicity of the products obtained after enzymatic oxidation.

Fungal biosynthesis of lignin-modifying enzymes from pulp wash and Luffa cylindrica for azo dye RB5 biodecolorization using modeling by response surface methodology and artificial neural network

This study demonstrates the evaluation between the artificial neural network technique coupled to the genetic algorithm (ANN-GA) and the response surface methodology (RSM) for prediction of Reactive Black 5 (RB5) decolorization by crude enzyme from Pleurotus. sajor-caju. Fungal lignin-modifying enzymes (FLME) were synthesized using pulp wash (PW) as an inducing substrate, and L. cylindrica (L.C) for cell immobilization. When grown in PW, the fungus showed higher Lac activity (126.5 IU. mL−1), whereas when immobilized a higher MnP activity was achieved (22.79 IU. mL−1), but both methods were capable of decolorizing the dye in about 89.4 % and 75 %, respectively. This indicates applicability of PW as an alternative substrate for FLME induction and viability of immobilization for MnP synthesis. For RB5 decolorization, the action of the crude enzyme extract was considered as a function of pH, dye concentration, temperature, and reaction time. The models are well adjusted to predict the efficiency of biodecolorization, with no statistical difference between ANN-GA and RSM, which indicates potential for green enzymes prospecting application in bioprocess industry.

Production of Crude Xylanase From Trichoderma sp. Using Reutealis trisperma Exocarp Substrate in Solid State Fermentation

Reutealis trisperma exocarp is a waste containing hemicellulose, cellulose, and lignin which can be used to produce xylanase enzymes. Reutealis trisperma exocarp contains 44.48% of hemicellulose, a polymer of xylan. The xylanase is an enzyme that can be utilized for hydrolyzing hemicellulose (xylan) to xylose. Xylanase can be produced by solid-state fermentation using isolate Trichoderma sp and Reutealis trisperma exocarp as substrate. This research aims to determine the fermentation time and substrate concentration that produce the highest xylanase activity using Trichoderma sp. In this study, the crude extract enzyme was evaluated on xylanase activity, protein content and specific activity at various fermentation times of 12, 24, 36, 48, 60 h using substrate concentrations of 2%, 4%, 6%, and 8%. The highest xylanase activity was observed with the substrate concentration of 8% at the 60 hour fermentation time. In this process, 672.039 U/mL of xylanase activity, protein content around 0.590 mg / mL and 1137.638 U/mg of the specific activity were produced.

Enzymatic delignification of sugar cane bagasse and rice husks and its effect in saccharification

Sugarcane bagasse (SB) and rice husks (RH) are agroindustrial byproducts which can be hydrolyzed to release sugars for the production of valuable bioproducts. However, the exposure of their carbohydrates to the action of hydrolytic enzymes depends on the removal/modification of lignin by a pretreatment. Here we report a biological strategy for pretreatment of SB and RH using a crude enzyme extract rich in laccases to delignification and improvement of reducing sugars yield after saccharification. Enzyme extracts were produced by different species of fungi cultured in a medium with agroindustrial byproducts and other components in static semi-solid condition. The highest yields of laccase production were obtained by Trametes villosa (9467.8 U/L). SB and RH were pretreated with this crude enzyme extract in various conditions and the process optimization resulted in the choice of proper mediators for laccases oxidative action and an increase of more than 10-fold in reducing sugars concentration in the saccharified samples (291 mg/g of sugars were obtained from SB and 193 mg/g from RH) when compared to the control (non pre-treated). Results of MIR-ATR suggested that RH were less susceptible to enzymatic pretreatment than SB and partial removal/modification of lignin was sufficient to improve saccharification.

A novel fibrinolytic enzymes from the Korean traditional fermented food-Jotgal: Purification and characterization.

The fibrinolytic activity in Korean traditional fermented food, Jotgal (pickled fish) was identified. Though the fibrinolytic activity could vary in different kinds of Jotgal, this activity seems to be produced by microorganisms during the natural fermentation stage. From Gonjaengijot (pickled opossum shrimp), two novel fibrinolytic enzymes named by JP-I and JP-II, have been purified by ethanol precipitation, Bio-GEL P-100 gel filtration, and DEAE-cellulose ion-exchange chromatography. Compared to the crude enzyme extract, the specific activity of the JP-I and JP-II increased 258, 85-fold with the recovery of 22.1, 8.5%, respectively. The molecular weights of both enzymes were estimated as 36kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal condition for fibrinolytic activity of JP-I was at 50°C and pH 8.1, while that of JP-II was at 45°C and 9.9. Both enzymes were stable at a broad range of pH (5.0 to 10.5) and have metalloprotease nature. From these results, it concludes that these enzymes could be a novel potent thrombolytic agent. PRACTICAL APPLICATIONS: The fibrinolytic enzyme is one of the clinical agents for cardiovascular diseases which is the leading cause of morbidity and mortality worldwide with 17 million deaths every year. A variety of fibrinolytic enzymes are found and characterized from various sources such as plants, animals, and microorganisms, and new sources for fibrinolytic enzymes continue to be explored. Jotgal, widely used in Korean people's diet, is a traditional Korean seafood prepared from many different types of fishes, fish eggs, fish intestines, and shellfishes. Through an amount of research, some of fibrinolytic enzymes were found and purified from Jotgal, however, no studies have been done on fibrinolytic enzyme from opossum shrimp. In this study, the purification, enzymatic characteristics, and fibrinolytic activity of the proteases, originated from Korean traditional fermented food, Jotgal were reported. These enzymes could be novel potent thrombolytic agent.

Agave Leaves as a Substrate for the Production of Cellulases by Penicillium sp. and the Obtainment of Reducing Sugars

Lignocellulosic biomass can be used to obtain fermentable sugars by enzymatic hydrolysis, and also it serves as a carbon source to produce cellulases by solid-state fermentation. In this study, we propose the use of leaves of Agave salmiana as a carbon source to produce cellulases by the fungus Penicillium sp., isolated from the same plant. The crude enzymatic extract was used to obtain sugars from the hydrolysis of the parenchymal cells of the leaves. The enzymes produced were characterized (endoglucanase 14.4 U/g; exoglucanase 3.5 U/g; β-glucosidase 4.14 U/g). The enzymes showed activities at elevated temperatures: 50°C for endoglucanase and exoglucanase and 70°C for β-glucosidase. Furthermore, the crude enzymatic extract obtained was able to hydrolyze the parenchyma in 51.6% in 48 h. The evidence presented in this paper shows the potential of the agave leaves as a source of carbon in the production of enzymes by fermentation with the consequent production of reducing sugars. In addition, the enzymes produced by Penicillium sp. could be used in the production of bioethanol, since they work at high temperatures.