Crude Enzyme Extract Research Articles

“Biosynthesis of volatile compounds by hydroperoxide lyase enzymatic activity during virgin olive oil extraction process”

The approach of this research was to describe the biochemical transformations of minor components of virgin olive oil with high impact on its sensory quality, specifically “volatile compounds” during its extraction process. For this purpose `Picual´, `Arbequina´ and `Hojiblanca´ cultivars were processed at three different harvesting times. Their volatile profiles and levels of enzymatic activity of “hydroperoxide lyase”, responsible for the biosynthesis of C6 aldehydes related to “green and fruity notes”, were monitored and identified in mesocarp, paste before kneading and paste after kneading based on the catalytic action of crude enzyme extracts. Both of them, volatiles and HPL, were analyzed by incubating with their corresponding substrates and reaction products formed were analyzed by Solid Phase Microextraction-Gas Chromatography and Mass Spectrometry. The results obtained in the present research can be useful in order to develop new markers biochemical whereby improved aroma quality in olive breeding programs or in the design of new protocol of VOO extraction.

In vitro evaluation of lignocellulolytic activity of thermophilic bacteria isolated from different composts and soils of Iran

Abstract Microorganisms with cellulolytic and ligninolytic activities have important roles in the composting process but they may not tolerate thermophilic phase. Using thermophilic microbes possessing lignocellulolytic activity could facilitate the composting process. In this study, soil and compost samples were collected from different regions of Iran. The sample dilutions were cultured on nutrient agar at 58 ± 2 °C for five days for isolating thermophiles. They were then screened according to their qualitative ligninolytic and cellulolytic activities at 25 °C and 58 °C. The superior isolates were then quantitatively assayed for cellulase, ligninase, amylase, β-glucosidase, xylanase and mannanase activities in crude enzyme extracts. The efficient isolates were identified by molecular methods. Among 327 thermophilic isolates, 118 and 137 isolates were lacking ligninase and cellulase activity, respectively. The isolates 1 vermicompost (VC), 12 compost (C), 47 Yazd soils (YZ) and 13 compost (C) had the highest ligninase activity and the highest cellulase activity was observed in isolates 194 Tabriz soils (TB), 104C and 12C. With increasing temperature from 25 °C to 58 °C, both enzymatic activities increased in all thermophilic isolates. The isolates 1VC, 12C, 13C, 47YZ, 151VC and 104 C were thermophilic bacteria possessing both the highest ligninase and cellulase activities. They were identified as Paenibacillus validus, Paenibacillus koreensis, Paenibacillus thailandensis, Paenibacillus cellulositrophicus, Paenibacillus lautus, Bacillus nealsonii, respectively.

Identification of novel enzymes to enhance the ruminal digestion of barley straw

Crude enzyme extracts typically contain a broad spectrum of enzyme activities, most of which are redundant to those naturally produced by the rumen microbiome. Identification of enzyme activities that are synergistic to those produced by the rumen microbiome could enable formulation of enzyme cocktails that improve fiber digestion in ruminants. Compared to untreated barley straw, Viscozyme® increased gas production, dry matter digestion (P < 0.01) and volatile fatty acid production (P < 0.001) in ruminal batch cultures. Fractionation of Viscozyme® by Blue Native PAGE and analyses using a microassay and mass-spectrometry revealed a GH74 endoglucanase, GH71 α-1,3-glucanase, GH5 mannanase, GH7 cellobiohydrolase, GH28 pectinase, and esterases from Viscozyme® contributed to enhanced saccharification of barley straw by rumen mix enzymes. Grouping of these identified activities with their carbohydrate active enzymes (CAZy) counterparts enabled selection of similar CAZymes for downstream production and screening. Mining of these specific activities from other biological systems could lead to high value enzyme formulations for ruminants.

Purification and Characterization of Amyloglucosidase Enzyme from the Thermophilic &lt;i&gt;Endomycopsis fibuligera&lt;/i&gt; Using Sago Starch as a Substrate

An investigation on purification and characterization of amyloglucosidase enzyme from Endomycopsis fibuligera by fermentation of sago starch has been carried out. This enzyme is inductive and can be produced by fermenting sago starch in a medium containing E. fibuligera. Crude enzyme was obtained by centrifuging the medium cultures containing E. fibuligera at 6,000 rpm for 20 min and then adding with 0.15 M acetate buffer (pH 5.0). Enzyme activity was determined using Somogyi-Nelson method by quantifying the released glucose from amyloglucosidase catalysis of starch (0.2%) as substrate. Prepurification process was conducted by ammonium sulphate fractionation and it showed that the ammonium sulphate fractionation with the degree of saturation of 40-60% produced a maximum activity of enzyme. Purification by DEAE-Cellulose and Sephadex G-75 column chromatography produced three and one fractions with purifity 17.4 and 22.5 times, respectively, compared to the crude extract enzyme. Characterization of this enzyme showed the optimum condition at pH 5.0 and 55 °C with 0.2% starch as substrate. The amyloglucosidase activities was strongly increased by addition of Co2+ and Mn2+ ions, whereas the activities were weakly decreased by addition of K+, Mg2+, and Fe3+ ions.

Saccharification of Sugarcane Bagasse Using an Enzymatic Extract Produced by Aspergillus fumigatus

This study investigates the efficiency of a crude enzymatic extract produced by Aspergillus fumigatus SCBM6 by solid state fermentation (SSF) in the hydrolysis of alkali pretreated sugarcane bagasse (PTB). After SSF using in ... | Find, read and cite all the research you need on Tech Science Press

Enantioselective biotransformation of sterically hindered amine substrates by the fungus Stemphylium lycopersici.

To screen 20 micro-organisms for ω-transaminase (ω-TA) activity by the kinetic resolution of rac-1-phenylethylamine, followed by testing rac-amines of pharmaceutical interest with bulky substituents and to conduct the asymmetric synthesis of a chiral amine. Stemphylium lycopersici was selected as the best biocatalyst. By the central composite rotatable design (CCRD), it was found that, at lower pH (5·5 and 6·5), the lyophilized micro-organism biocatalysed the kinetic resolution of rac-1-phenylethylamine with 99% enantiomeric excess (e.e.) ((R)-enantiomer) with acetophenone conversions ranged from 41 to 45%. Interestingly, the lyophilized crude enzymatic extract lead to better results at pH from 7·0 to 9·0, with conversions up to 47% and about 99% e.e. We also attested that as much as higher is the pyruvate (amino acceptor) concentration, higher is the acetophenone conversion, corroborating the presence of ω-TA-type enzymes. Among different sterically hindered racemic amines tested, rac-1,2,3,4-tetrahydro-1-naphthylamine and rac-phenylbutylamine were satisfactorily kinetically resolved in up to 91% e.e. (R). The results for the asymmetric synthesis showed excellent conversion (>85%) for the S-1-phenylethylamine, indicating (S)-stereopreference. Stemphylium lycopersici showed to be an important tool for broader substrate scope transaminases and a relevant player on the development of new biocatalysts with ability in asymmetric synthesis reactions. Here in, we contribute to the improvement of the biocatalytic toolbox for chiral amines synthesis. Interestingly, we have found that the crude enzymatic extract of the endophytic fungus S. lycopersici could accept bulky substrates with reasonable activity, compared to the wild-type transaminase already published over literature, and with high enantioselectivity.

Partial characterization, quantification and activity of pancreatic lipase in the gastrointestinal tract of Totoaba Macdonaldi

Lipids are one of the main macronutrients that constitute balanced feeds used in aquaculture. Adequate utilization of dietary lipid is influenced by the activity of pancreatic lipase, one of the enzymes that promotes digestion of dietary lipids in the gastrointestinal tract of fish. The culture of Totoaba macdonaldi is quite recent; its nutritional requirements have been partially established. Knowing the characteristics of pancreatic lipase for this species could help optimize the dietary lipids included in balanced feeds for its culture. Therefore, the aim of this work is to partially characterize and evaluate the enzymatic activity of pancreatic lipase for T. macdonaldi. Biological indices showed that experimental organisms had a good nutritional status. Pancreatic lipase molecular weight was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and its activity was evaluated in crude enzymatic extracts from different gastrointestinal tract regions. The molecular weight of lipase was estimated to be 70.4 kDa; the highest lipolytic activity was observed at 45?C and at a pH optimum of 8.0 in the anterior intestine and pyloric caeca, where the concentration and activity of the enzyme was significantly higher (P=0.004) compared to the distal parts of the intestine. Biochemical characteristics observed for the pancreatic lipase of T. macdonaldi are quite similar to other lipases of fish cultured worldwide; results provided in this study will help understand the role this lipolytic enzyme plays in the digestive process of this species.

EFFECT OF EXOGENOUSE LOCAL LIGNINOLYTIC CRUDE ENZYMES ON IN VITRO DIGESTABILITY AND LIGNIN CONTENT OF ROUGHAGES

In current study use local ligninolytic crude extract enzyme (HAMU 8%) to improve IVDMD and reducing lignin content. The result suggest that treated wheat straw (WS), Corncobs (Cc), alfalfa hay (AH), by HAMU increased significantly (p &lt;0.05) in vitro dry matter digestibility (IVDMD) and reduction in lignin content, However, extra enzyme improvement is shown with Corncobs treated with HAMU (average improvement, 19.84%) and lower improvement was shown in alfalfa hay treated with HAMU (average improvement, 10.07%). The higher reduction (P&lt;0.01) in lignin content was shown in Cc treated with HAMU from 9.8 to 7.901%, with average reduction 1.899%; when the lower reduction was observed significantly (P&lt;0.05) with AH treated from 8.26 to 7.77%, average reduction was 0.49%.It could be recommended, treated low quality roughages with HAMU improved the nutritional value of roughages through the digestion of Structural carbohydrates.

Ultrasound effects on the activity of Aspergillus niger lipases in their application in dairy wastewater treatment

AbstractWe evaluated the effect of ultrasound radiation on lipase activity. The experiments were conducted using a low‐cost crude enzyme extract. We then used this lipase to treat dairy effluent. The results of our study presented an increase of approximately 430% in the concentration of free fatty acids.

Bacterial Biofilm Removal Using Solid-State-Produced Enzymes

The objective of this study was to evaluate the removal capacity of bacterial film formed by Pseudomonas sp., using commercial enzymes (pectinase, cellulase, and amylase) and the enzymatic extract obtained by solid-state cultivation (SSC) from Aspergillus niger ATCC 9642. The bacterial biofilm removal (Pseudomonas sp.) was evaluated using the commercial enzyme microplate technique and the crude enzyme extract (SSC). The effects of orange peel (9.28–22.72 g), corn steep liquor (0.32–3.68 g), and wheat bran (0–4 g) concentrations were evaluated on pectinase production in solid-state cultivation. The enzyme extract (SSC) satisfactorily removed 77 % of biofilm after 10 min contact, taking into account that there was no enzymatic extract purification treatment. The results indicated a promising and economically viable alternative to enzymatic removal of biofilms in the food industry.

In situ purification of periplasmatic L‐asparaginase by aqueous two phase systems with ionic liquids (ILs) as adjuvants

AbstractBACKGROUNDL‐asparaginase (ASNase) is an important biopharmaceutical used to treat the acute lymphoblastic leukemia (ALL) and lymphosarcoma. Considering its main use in cancer therapy, the most important request for ASNase production is the need for a highly pure biopharmaceutical obtained in the final of the downstream process, which is considered as the crucial step in its production.RESULTSThis work proposes the use of polymer–salt aqueous two‐phase systems (ATPS) based on polyethylene glycol and citrate buffer, with ionic liquids (ILs) as adjuvants, combined with the permeabilization of cell membrane using n‐dodecane and glycine for the in situ purification of periplasmatic ASNase from Escherichia coli cells. The process proposed was optimized (polymer molecular weight, pH, tie‐line length/mixture point, presence, nature and concentration of the adjuvant). The results show that ASNase partitions mostly to the PEG‐rich phase, due to hydrophobic interactions between both PEG and enzyme. Remarkably, the addition of 5 wt% of 1‐butyl‐3‐methylimidazolium methanesulfonate [C4mim][CH3SO3] as adjuvant lead to high recoveries [87.94 ± 0.03 (%)], purification factors (20.09 ± 0.35), and a final specific activity SA = 3.61 ± 0.38 U mg‐1 protein, from a crude enzyme extract with a SA = 0.18 ± 0.05 U mg‐1 protein. Moreover, better results were achieved when a prepurification step consisting of an ammonium sulfate precipitation was combined with the optimized ATPS, achieving an increased SA = 22.01 ± 1.36 U mg‐1 protein and PF = 173.8.CONCLUSIONSA novel integrated downstream process was successfully implemented for the in situ purification of ASNase from fermentation broth. © 2017 Society of Chemical Industry

The non-edible parts of fennel (Fœniculum vulgare) as a new milk-clotting protease source

Although fennel stems and leaves represent an abundant biomass, they remain poorly exploited in terms of extraction of biomolecules. This work aimed at the valorization of the non-edible parts of fennel by the extraction and the characterization of a new milk-clotting protease. Both, enzymatic extraction and milk-clotting conditions were optimized in the context of developing new aspects of cheese. Fennel stems and leaves were sorted and coarsely grounded. The extraction consists in an aqueous maceration and extracts were used to coagulate raw cow's milk. Maximum milk-clotting activity was obtained under a temperature of 20°C, extraction time of 12h and solid-to-solvent ratio of 150:100gmL−1. Besides, the impact of extract concentration, milk pH, calcium content and temperature on clotting-time were studied. The statistical analysis of the results confirmed significant effects of the four studied parameters. The use of specific inhibitors suggested the presence of a serine protease in the crude enzymatic extracts. The findings of this research confirmed that the use of fennel protease could be a new alternative for the commercial proteases in dairy products.

Toxic effects and mechanism of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) on Lemna minor

To investigate the toxic effect and mechanism of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) in aquatic plants, in vivo and in vitro exposure to BDE-47 were conducted. After 14-d exposure to 5–20 μg/L BDE-47, the growth of Lemna minor plants was significantly suppressed, and the chlorophyll and soluble protein contents in fronds markedly decreased. Accordingly, the photosynthetic efficiency (Fv/Fm, PI) decreased. When the thylakoid membranes isolated from healthy fronds was exposed to 5–20 mg/L BDE-47 directly in vitro for 1 h, the photosynthetic efficiency also decreased significantly. In both the in vitro (5–20 μg/L) and in vivo (5–20 mg/L) experiments, BDE-47 led to an increased plasma membrane permeability. Hence, we concluded that BDE-47 had a direct toxicity to photosynthetic membranes and plasma membranes. However, direct effects on the activities of peroxidase (POD), malate dehydrogenase (MDH) and nitroreductase (NR) were not observed by adding 5–20 mg/L BDE-47 into crude enzyme extracts. The malondialdehyde (MDA) and superoxide anion radical (O2−) contents in the BDE-47 treated fronds were higher than those in the control fronds, suggesting that L. minor can not effectively relieve reactive oxygen species (ROS). The data above indicates that BDE-47 is toxic to L. minor through acting directly on biomembranes, which induces the production of ROS and thus causes remarkable oxidative damage to cells.

The Application of Enzymological Techniques for Determination of the Inhibitor Activity of Bio Enzym Extracted from Seawater Bacteria Pseudomonas aerogenosa to Fight Biocorrosion of Carbon Steel

The paper presents a studies for the development of new corrosion inhibitors by biotechnological way applicable to many areas of petroleum industry including, characterization of protective films on carbon steel and biocorrosion inhibition. Our research is oriented towards the isolation of new strains of bacteria from the sea waters of the Mediterranean region that have power (inhibitory and/or bactericidal that blocks the growth of sulfate-reducing bacteria responsible for microbiologically influenced by production of Pyocyanin (1 hydroxyphenazin) or 5-methylphenazin-1-one. Our first objective was to isolate the population of Pseudomonas Aerugenosa on a specific medium, characterization gram, then fermentation in a nutrient broth. The second objective of this study is evaluation of corrosion rate by weight loss method with injecting different dose of the crude enzyme extract CEE containing the methylphenazine (Pyocyanin). An efficacy test was performed on test kit vials containing the specific culture medium with the SRB contaminated with industrial injection water from SONAHESS region. The total disappearance of the SRB is 40% of CEE by blocking their metabolism in the growth phase confirms the efficiency of treatment.

Development of a controlled bioconversion process for the recovery of chitosan from blue crab (Portunus segnis) exoskeleton

The present work aimed to extract chitin and chitosan from blue crab (Portunus segnis) shells, via an optimized procedure keeping as much as possible its native structure. Thus, several microbial, visceral and commercial proteases were tested for their deproteinization efficiency. High levels of protein removal of about 82% and 85% were recorded using Bacillus safensis proteases and crude enzyme extract of P. segnis, respectively. Further, 0.5 M HCl baths based system was adopted for minerals removal. Blue crab chitin had the following characteristics: acetylation degree of 97%, deproteinization and demineralization rates of 92% and 100%, respectively. Subsequently, blue crab chitosan (BCC) was prepared by chitin N-deacetylation, leading to an acetylation degree of 10%. After characterization by FTIR spectroscopy and X-ray diffraction, the functional properties and antioxidant and antimicrobial activities of BCC were investigated. Interestingly, BCC exhibited notable in vitro antioxidant activity including DPPH and hydroxyl radicals scavenging, β-carotene bleaching inhibition and metal chelating effects (92%, 89%, 72%, and 93%, at 5 mg/ml, respectively). Moreover, chitosan markedly inhibited growth and adhesion of all microorganisms tested, and was efficient in the disruption of pre-formed biofilms.

Application of crude proteolytic enzyme extract from fresh rhizome ginger to produce coconut oil

Application of crude proteolytic enzyme extract from fresh rhizome ginger to produce coconut oil

Lignocellulose Degrading Capabilities of Sphaerobolus stellatus in Creeping Bentgrass

Thatch collapse, caused by Sphaerobolus stellatus (Tode) Persoon, is a new disease of finely managed turfgrasses. Symptoms include dark‐green, circular patches of turf atop degraded organic matter resulting in an indentation of the playing surface. Within a 6‐wk period, S. stellatus reduces organic matter and thatch depth by 21 and 25%, respectively, but the mechanism of thatch reduction has not been defined. To characterize the underlying biochemical processes of thatch collapse, lignin degradation was assessed, and the activities of associated lignocellulose enzymes within the organic matter of turfgrass thatch were quantified. One isolate of S. stellatus was inoculated on creeping bentgrass (Agrostis stolonifera L.) plugs for 6 wk. Using tetramethylammonium hydroxide (TMAH) thermochemolysis to analyze the fate of lignin from golf course thatch, we found a significant depolymerization of S. stellatus‐colonized lignin. To understand the broader mechanism responsible for organic matter degradation, we used crude enzyme extracts from one S. stellatus isolate from turf and mulch along with one isolate of a Panaeolina spp. at four time points in 3 mo. Extracts were assayed for Mn‐independent and Mn‐dependent peroxidase, laccase, xylanase, and cellulase activity. Elevated levels of laccase were found in the S. stellatus isolate from turf, and both S. stellatus isolates produced levels of Mn‐independent peroxidases. Both S. stellatus isolates and the Panaeolina spp. isolate exhibited xylanase activity. Cellulase was not verifiably detected. The presence of laccase in S. stellatus from turfgrass, Mn‐independent peroxidase in both S. stellatus isolates, and xylanase in all fungal extracts illustrates a mechanism of organic matter reduction in thatch collapse.

Cloning and upscale production of monoamine oxidase N (MAO-N D5) by Pichia pastoris.

To clone monoamine oxidase N, that catalyses the selective oxidative deamination or deracemisation of amines into imines, in Pichia pastoris and prove the importance of choosing the proper expression system for its recombinant production. Monoamine oxidase, originating from Aspergillus niger and subjected to directed evolution (MAO-N D5), was cloned and expressed in Pichia pastoris CBS7435 MutS strain for the first time. Various transformants were screened at microscale level. The production of the clone expressing the most active enzyme was scaled-up to a 1.5l fermenter and preparation of MAO-N D5 as a crude enzyme extract was optimised. The obstacles in the production of the enzyme in both expression systems, Escherichia coli and P. pastoris, are discussed and demonstrated. There was an improvement in specific productivity, which was 83 times higher in P. pastoris, clearly proving the importance of choosing the right expression host system for the specific enzymes.

Synergistic action of an Aspergillus (hemi-)cellulolytic consortium on sugarcane bagasse saccharification

This study evaluated the synergistic efficiency of crude enzymatic extracts (CEE) produced by fungal strains Aspergillus fumigatus SCBM6 (AFE) and Aspergillus niger SCBM1 (ANE) in the hydrolysis of alkali pretreated sugarcane bagasse (2% NaOH (w v−1), ∼100 °C, 4 h and liquid-to-solid ratio of 25 (v w−1)). CEEs were produced by solid-state fermentation (SSF) and presented high concentration of β-glucosidase (AFE = 78.4 U g−1 and ANE = 43.02 U g−1), β-xylosidase (ANE = 78.0 U g−1) and xylanase (AFE = 1774.5 U g−1 and ANE = 3289.6 U g−1). A 24 full design of experiments was used to optimize the hydrolysis of pretreated bagasse as a function of time, temperature, amount of pretreated bagasse and AFE-to-ANE ratio. After hydrolysis optimization, the maximum concentration of total reducing sugars (TRS) was 21.68 g L−1. It was also noticed a significant production of xylose (135.59 mg) and arabinose (58.10 mg) per gram of hemicelluloses and a slight production of glucose (20.18 mg) per gram of cellulose. As fungi consortium were excellent producers of hemicellulases, it is suggested that they could be used for pentoses saccharification and production of ethanol via C-5 fermentation, xylitol, biopolymers, among other bioproducts.

Production, characterization and dye decolorization ability of a high level laccase from Marasmiellus palmivorus

Production of phenol oxidases by Marasmiellus palmivorus was evaluated in submerged cultures at different pH conditions: uncontrolled (free), fixed at 5.0, 6.0 and 7.0 and controlled after reaching pH 5.0, 6.0 and 7.0. pH (3–8) and temperature (20–45 °C) were optimized for laccase production and the thermal stability (30–60 °C) of laccases was evaluated. In addition, laccase activity in concentrated crude enzyme extract was evaluated. Laccase activity was greatest in production conditions of free pH (1950.6 ± 42.7 U mL−1), free - pH 6 (1901 ± 85.5 U mL−1) and fixed - pH 7 (1851.8 ± 0.0 U mL−1). Laccase activity was highest when reaction conditions were pH 4.0–5.5 and 25–45 °C. By concentrating the crude enzyme extract it was possible to significantly increase laccase activity. The greater the enzymatic activity used in the decolorization of the dyes Reactive Blue 220 and Acid Green 28, the shorter the time required to remove 90% and 75% of the color respectively. Given the results, it is evident that M. palmivorus can secrete high concentrations of phenol oxidases with potential biotechnological applications.