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Enzyme Preparations (enzyme + preparation)
Kinds of Enzyme Preparations Selected AbstractsHeterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloidsFEBS JOURNAL, Issue 8 2002Irina Gerasimenko Strictosidine glucosidase (SG) is an enzyme that catalyses the second step in the biosynthesis of various classes of monoterpenoid indole alkaloids. Based on the comparison of cDNA sequences of SG from Catharanthus roseus and raucaffricine glucosidase (RG) from Rauvolfia serpentina, primers for RT-PCR were designed and the cDNA encoding SG was cloned from R. serpentina cell suspension cultures. The active enzyme was expressed in Escherichia coli and purified to homogeneity. Analysis of its deduced amino-acid sequence assigned the SG from R. serpentina to family 1 of glycosyl hydrolases. In contrast to the SG from C. roseus, the enzyme from R. serpentina is predicted to lack an uncleavable N-terminal signal sequence, which is believed to direct proteins to the endoplasmic reticulum. The temperature and pH optimum, enzyme kinetic parameters and substrate specificity of the heterologously expressed SG were studied and compared to those of the C. roseus enzyme, revealing some differences between the two glucosidases. In vitro deglucosylation of strictosidine by R. serpentina SG proceeds by the same mechanism as has been shown for the C. roseus enzyme preparation. The reaction gives rise to the end product cathenamine and involves 4,21-dehydrocorynantheine aldehyde as an intermediate. The enzymatic hydrolysis of dolichantoside (N,-methylstrictosidine) leads to several products. One of them was identified as a new compound, 3-isocorreantine A. From the data it can be concluded that the divergence of the biosynthetic pathways leading to different classes of indole alkaloids formed in R. serpentina and C. roseus cell suspension cultures occurs at a later stage than strictosidine deglucosylation. [source] Orange peel degradation and enzyme recovery in the enzymatic peeling processINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 2 2006Axel Pagán Summary A detailed study was carried out on enzymatic peeling of oranges in a reactor using an enzyme preparation. This work was focused on determining the changes that happen in the peel albedo of Navelina oranges. Thus, the conditions of temperature and concentration of the enzymatic preparation were optimized in order to produce the maximum weight loss, which indicates good peeling efficiency. Experiments to study the efficiency of the reactor effluents by reusing them for successive enzymatic peelings were also carried out with the aim of diminishing the cost of the process. Finally, the recovery of the enzymes after use in multiple enzymatic peelings by means of ultrafiltration was tested and an increment in their enzymatic activity was observed. [source] The effect of hydration time and ethanol concentration on the rate of hydrolysis of extracted vanilla beans by commercial cellulase preparationsINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 9 2005Sandy L. Ovando Summary The rate of producing reducing sugars from extracted vanilla beans by using cellulolytic enzymes was studied. The hydration of cellulose improved the efficiency of hydrolysis. The treatment using Crystalzyme PML-MX at a concentration of 2.64 International Filter Paper Units (IFPU) g,1 of bean was the most successful and the enzyme stable for up to 5:100 (weight:volume) of ethanol. After 48 h of prehydration and 26 h of enzymatic hydrolysis with this enzyme preparation, 196.6 mg g,1 reducing sugars containing 15.9 mg g,1 glucose were liberated. Less active enzyme products, Zymafilt L-300 and Novozym, had greater cellulolytic activity when 10 or 15:100 (weight:volume) of ethanol were added respectively. Because of their stability with ethanol, the enzyme products could be used in the pretreatment of botanical material that are rich in flavour compounds and in this manner improve the final extraction of valuable flavours. [source] L-methioninase production by Aspergillus flavipes under solid-state fermentationJOURNAL OF BASIC MICROBIOLOGY, Issue 4 2009Ashraf S. A. El-Sayed Abstract Solid-state fermentation was carried out for the production of extra-cellular L-methioninase by Aspergillus flavipes (Bain and Sart.) using nine agro-industrial residues, namely wheat bran, rice bran, wheat flour, coconut seeds, cotton seeds, ground nut cake, lentil hulls, soya beans and chicken feathers. Chicken feathers were selected as solid substrate for L-methioninase production by A. flavipes. The maximum L-methioninase productivity (71.0 U/mg protein) and growth (11 mg protein/ml) of A. flavipes was obtained using alkali pretreated chicken feathers of 50% initial moisture content as substrate supplemented with D-glucose (1.0% w/v) and L-methionine (0.2% w/v). External supplementation of the fermentation medium with various vitamin sources has no overinductive effect on L-methioninase biosynthesis. The partially purified A. flavipes L-methioninase preparation showed highest activity (181 U/ml) at pH 8.0 with stability over a pH range (pH 6,8) for 2 h. L-methioninase activity was increased by preincubation of the enzyme for 2 h with Co2+, Mn2+, Cu2+ and Mg2+ and strongly inhibited by the presence of EDTA, NaN3, Li2+, Cd2+, DMSO and 2-mercaptoethanol. The enzyme preparation has a broad substrate spectrum showing a higher affinity to deaminate L-glycine, N -acetylglucosamine and glutamic acid, in addition to their proteolytic activity against bovine serum albumin, casein, gelatin and keratin. The partially purified enzyme was found to be glyco-metalloproteinic in nature as concluded from the analytical and spectroscopic profiles of the enzyme preparation. The demethiolating activity of the enzyme was also visualized chromogenially. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Stabilization of penicillin V acylase from Streptomyces lavendulae by covalent immobilizationJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 5 2001Jesús Torres-Bacete Abstract Penicillin,V acylase from the actinomycete Streptomyces lavendulae ATCC 13664 has been immobilized to epoxy-activated acrylic beads (Eupergit C®) by covalent binding. Further linkage of bovine serum albumin after enzyme immobilization was carried out in order to remove the remaining oxirane groups of the support. The obtained immobilized biocatalyst displayed double exponential deactivation kinetics at temperatures below 55,°C, while the native enzyme followed single exponential decay at the same temperatures. We concluded that soluble penicillin acylase was deactivated in one step, whereas the immobilized enzyme showed an enzymatic intermediate state which is highly thermostable. As a consequence of the immobilization process, the enzyme displayed a 10-fold increase in its half-life at 40,°C. At this temperature, the enzymatic intermediate state was progressively destabilized as the pH of the medium was increased. Thus, the optimum pH range for the immobilized enzyme preparation was established as being from 7.0 to 8.0. Higher pH values led to quicker enzyme deactivation. © 2001 Society of Chemical Industry [source] A Novel Process for the Recovery of Polyphenols from Grape (Vitis vinifera L.) PomaceJOURNAL OF FOOD SCIENCE, Issue 2 2005Dietmar Kammerer ABSTRACT: A novel process for enzyme-assisted extraction of polyphenols from winery by-products was established on a pilot-plant scale. Optimization of enzymatic hydrolysis of grape skins, that is, selection of pectinolytic and cellulolytic enzymes, enzyme-substrate ratio, and time-temperature regime of enzymatic treatment, was conducted on a laboratory scale. Enzyme activities were monitored by viscosity measurement of resuspended grape pomace and by quantification of oligomeric pectin and cellulose degradation products released from cell wall material. Optimal conditions were obtained with 5000 ppm (based on dry matter) of a pectinolytic and 2500 ppm of a cellulolytic enzyme preparation, respectively, at 50°C, which were also applied in pilot-plant scale experiments. Concomitant determination of individual polyphenolics demonstrated a significantly improved yield for most compounds when compared with experiments without enzyme addition. Recovery rates were comparable to those obtained when grape pomace was extracted using sulfite. Pre-extraction of the pomace with hot water followed by treatment with cell wall degrading enzymes even increased yields of phenolic compounds. Only some quercetin glycosides and malvidin coumaroylglucoside were partly hydrolyzed due to enzyme side activities. This new process may provide a valuable alternative to the application of sulfite, which is considered crucial in food processing. [source] The first enzymatic method for C,18F bond formation: the synthesis of 5,-[18F]-fluoro-5,-deoxyadenosine for imaging with PETJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 13 2003Laurent Martarello Abstract The use of the key enzyme involved in carbon,fluorine bond formation in Streptomyces cattleya catalysing the formation of 5,-fluoro-5,-deoxyadenosine (5,-FDA) from fluoride ion and S -adenosyl-l-methionine (SAM) was explored for its potential application in fluorine-18 labelling of the adenosine derivative. Enzymatic radiolabelling of [18F]-5,-FDA was successfully carried out starting from SAM and [18F]HF when the concentration of the enzyme preparation was increased from sub-mg/ml values to mg/ml values. The purity of the enzyme had no measurable effect on the radiochemical yield of the reaction and the radiochemical purity of [18F]-5,-FDA. Copyright © 2003 John Wiley & Sons, Ltd. [source] Trichoderma enzymes promote Fibrobacter succinogenes S85 adhesion to, and degradation of, complex substrates but not pure cellulose,JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 10 2004Diego P Morgavi Abstract The effects of an enzyme preparation from Trichoderma longibrachiatum (TE) on adhesion and growth of the fibrolytic rumen bacterium Fibrobacter succinogenes S85 was studied to gain a better understanding of the action of feed enzyme additives on fibre digestion by ruminants. Adhesion experiments were performed on crystalline cellulose, corn silage and alfalfa hay. Adhesion of F succinogenes to cellulose was negatively related to the concentration of TE (p < 0.05). At the highest concentration used, TE reduced adhesion to cellulose from 65 to 39%. For corn silage and alfalfa hay, TE stimulated adhesion at low levels (p < 0.05) but this effect was lost at higher levels. Culture experiments were performed on crystalline cellulose and corn silage. The presence of TE in media containing cellulose failed to increase substrate disappearance or gas production although it increased numbers of non-adherent bacteria (p < 0.05). When corn silage was used, the addition of TE increased NDF disappearance (p < 0.05) at 24 and 48 h (33 and 52% in controls versus 53 and 65% in TE treatments). Growth rate and gas production were also stimulated (p < 0.05). We conclude that, for cellulose, the hydrolytic enzymes in TE obstructed available binding sites decreasing bacterial adherence. Fibrobacter succinogenes digested cellulose efficiently and addition of exogenous cellulases did not further increase substrate disappearance. However, for complex plant substrates, low concentration of TE increased bacterial adhesion and plant (corn) fiber degradation. For the Department of Agriculture and Agri-Food, Government of Canada, © Minister of Public Works and Government Services Canada 2004. Published for SCI by John Wiley & Sons, Ltd. [source] Standard assays do not predict the efficiency of commercial cellulase preparations towards plant materialsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006Mirjam A. Kabel Abstract Commercial cellulase preparations are potentially effective for processing biomass feedstocks in order to obtain bioethanol. In plant cell walls, cellulose fibrils occur in close association with xylans (monocotyls) or xyloglucans (dicotyls). The enzymatic conversion of cellulose/xylans is a complex process involving the concerted action of exo/endocellulases and cellobiases yielding glucose and xylanases yielding xylooligomers and xylose. An overview of commonly measured cellulase-, cellobiase-, and xylanase-activity, using respectively filter paper, cellobiose, and AZCL-dyed xylan as a substrate of 14 commercially available enzyme preparations from several suppliers is presented. In addition to these standardized tests, the enzyme-efficiency of degrading native substrates was studied. Grass and wheat bran were fractionated into a water unsoluble fraction (WUS), which was free of oligosaccharides and starch. Additionally, cellulose- and xylan-rich fractions were prepared by alkaline extraction of the WUS and were enzymatically digested. Hereby, the capability of cellulose and xylan conversion of the commercial enzyme preparations tested was measured. The results obtained showed that there was a large difference in the performance of the fourteen enzyme samples. Comparing all results, it was concluded that the choice of an enzyme preparation is more dependent on the characteristics of the substrate rather than on standard enzyme-activities measured. © 2005 Wiley Periodicals, Inc. [source] Evaluation of Minimal Trichoderma reesei Cellulase Mixtures on Differently Pretreated Barley Straw SubstratesBIOTECHNOLOGY PROGRESS, Issue 6 2007Lisa Rosgaard The commercial cellulase product Celluclast 1.5, derived from Trichoderma reesei (Novozymes A/S, Bagsværd, Denmark), is widely employed for hydrolysis of lignocellulosic biomass feedstocks. This enzyme preparation contains a broad spectrum of cellulolytic enzyme activities, most notably cellobiohydrolases (CBHs) and endo-1,4-,-glucanases (EGs). Since the original T. reesei strain was isolated from decaying canvas, the T. reesei CBH and EG activities might be present in suboptimal ratios for hydrolysis of pretreated lignocellulosic substrates. We employed statistically designed combinations of the four main activities of Celluclast 1.5, CBHI, CBHII, EGI, and EGII, to identify the optimal glucose-releasing combination of these four enzymes to degrade barley straw substrates subjected to three different pretreatments. The data signified that EGII activity is not required for efficient lignocellulose hydrolysis when addition of this activity occurs at the expense of the remaining three activities. The optimal ratios of the remaining three enzymes were similar for the two pretreated barley samples that had been subjeced to different hot water pretreatments, but the relative levels of EGI and CBHII activities required in the enzyme mixture for optimal hydrolysis of the acid-impregnated, steam-exploded barley straw substrate were somewhat different from those required for the other two substrates. The optimal ratios of the cellulolytic activities in all cases differed from that of the cellulases secreted by T. reesei. Hence, the data indicate the feasibility of designing minimal enzyme mixtures for pretreated lignocellulosic biomass by careful combination of monocomponent enzymes. This strategy can promote both a more efficient enzymatic hydrolysis of (ligno)cellulose and a more rational utilization of enzymes. [source] Optimization of Reaction Conditions for Enzymatic Viscosity Reduction and Hydrolysis of Wheat Arabinoxylan in an Industrial Ethanol Fermentation ResidueBIOTECHNOLOGY PROGRESS, Issue 2 2006Hanne R. Sørensen This study examined enzyme-catalyzed viscosity reduction and evaluated the effects of substrate dry matter concentration on enzymatic degradation of arabinoxylan in a fermentation residue, "vinasse", resulting from industrial ethanol manufacture on wheat. Enzymatic catalysis was accomplished with a 50:50 mixture of an enzyme preparation from Humicola insolens, Ultraflo L, and a cellulolytic enzyme preparation from Trichoderma reesei, Celluclast 1.5 L. This enzyme mixture was previously shown to exhibit a synergistic action on arabinoxylan degradation. The viscosity of vinasse decreased with increased enzyme dosage and treatment time at pH 5, 50 °C, 5 wt % vinasse dry matter. After 24 h of enzymatic treatment, 76,84%, 75,80%, and 43,47%, respectively, of the theoretically maximal arabinose, xylose, and glucose releases were achieved, indicating that the viscosity decrease was a result of enzyme-catalyzed hydrolysis of arabinoxylan, ,-glucan, and cellulose. In designed response surface experiments, the optimal enzyme reaction conditions with respect to pH and temperature of the vinasse, the vinasse supernatant (mainly soluble material), and the vinasse sediment (mainly insoluble substances) varied from pH 5.2,6.4 and 41,49 °C for arabinose release and from pH 4.9,5.3 and 42,46 °C for xylose release. Even though only limited hydrolysis of the arabinoxylan in the vinasse sediment fraction was obtained, the results indicated that the same enzyme activities acted on the arabinoxylan in the different vinasse fractions irrespective of the state of solubility of the substrate material. The levels of liberated arabinose and xylose increased with increased dry matter concentration during enzymatic hydrolysis in the vinasse and the vinasse supernatant, but at the same time, increased substrate dry matter concentrations gave corresponding linear decreases in the hydrolytic efficiency as evaluated from levels of monosaccharide release per weight unit dry matter. The study thus documents that enzymatic arabinoxylan hydrolysis of the vinasse significantly decreases the vinasse viscosity and that a compromise in the dry matter must be found if enzymatic efficiency must be balanced with monosaccharide yields. [source] Composite Particles of Novozyme,435 and Silicone: Advancing Technical Applicability of Macroporous Enzyme CarriersCHEMCATCHEM, Issue 4 2009Abstract The mechanical and leaching stability of enzymes adsorbed on macroporous carriers is an important issue for the technical applicability of such biocatalysts. Both can considerably benefit from the deposition of silicone coating on the carrier surface. The coating of the immobilized lipase Novozyme,435 (NZ435), as a model enzyme preparation, with different silicone loadings was studied in detail by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), as well as by energy-dispersive X-ray spectroscopy (EDX) and BET isotherms, and offers explanations and prerequisites for its stabilizing effects. The deposition of silicone on the poly(methyl methacrylate) (PMMA) carrier was found to form an interpenetrating network composite rather than the anticipated core-shell structure. The silicone precursors homogeneously wet the carrier surface including all inner pores and gradually fill the complete carrier. In parallel, the surface area of NZ435 decreases from an initial value of 89,m2g,1to 0.2,m2g,1after silicone loading. A visible layer of silicone on the outer surface of the carrier was only observed at a silicone concentration of 54,%,w/w and more. Maximum leaching stability corresponds to the formation of this layer. The mechanical stability increases with the amount of deposited silicone. It can be expected that stabilization against leaching and/or mechanical stress by formation of silicone composites can easily be transferred to a whole range of alternative biocatalytic systems. This should considerably advance their general technical applicability and overall implementation of biocatalysts in chemical synthesis. [source] The value of debridement and Vacuum-Assisted Closure (V.A.C.) Therapy in diabetic foot ulcersDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue S1 2008Magnus Eneroth Abstract Background Treatment of diabetic foot ulcers includes a number of different regimes such as glycaemic control, re-vascularization, surgical, local wound treatment, offloading and other non-surgical treatments. Although considered the standard of care, the scientific evidence behind the various debridements used is scarce. This presentation will focus on debridement and V.A.C. Therapy, two treatments widely used in patients with diabetes and foot ulcers. Methods A review of existing literature on these treatments in diabetic foot ulcers, with focus on description of the various types of debridements used, the principles behind negative pressure wound therapy (NPWT) using the V.A.C. Therapy system and level of evidence. Results Five randomized controlled trials (RCT) of debridement were identified; three assessed the effectiveness of a hydrogel as a debridement method, one evaluated surgical debridement and one evaluated larval therapy. Pooling the three hydrogel RCTs suggested that hydrogels are significantly more effective than gauze or standard care in healing diabetic foot ulcers. Surgical debridement and larval therapy showed no significant benefit. Other debridement methods such as enzyme preparations or polysaccharide beads have not been evaluated in RCTs of people with diabetes. More than 300 articles have been published on negative pressure wound therapy, including several small RCTs and a larger multi-centre RCT of diabetic foot ulcers. Negative pressure wound therapy seems to be a safe and effective treatment for complex diabetic foot wounds, and could lead to a higher proportion of healed wounds, faster healing rates, and potentially fewer re-amputations than standard care. Conclusions Although debridement of the ulcer is considered a prerequisite for healing of diabetic foot ulcers, the grade of evidence is quite low. This may be due to a lack of studies rather than lack of effect. Negative pressure wound therapy seems to be safe and effective in the treatment of some diabetic foot ulcers, although there is still only one well-performed trial that evaluates the effect. Copyright © 2008 John Wiley & Sons, Ltd. [source] Comparison of two glutaraldehyde immobilization techniques for solid-phase tryptic peptide mapping of human hemoglobin by capillary zone electrophoresis and mass spectrometryELECTROPHORESIS, Issue 9 2004Isabelle Migneault Abstract Stabilization of proteolytic enzymes, especially by immobilization, is of considerable interest because of their potential applications in medicine and the chemical and pharmaceutical industries. We report here a detailed comparison of two procedures for trypsin immobilization using the same homobifunctional agent, glutaraldehyde, for the purpose of peptide mapping. These methods include covalent coupling either to controlled pore glass (solid support) or via a cross-linking reaction (without any solid support). The immobilized trypsin preparations were characterized by the determination of immobilization efficiency, which ranged from 68 to > 95%, and measurement of apparent kinetic parameters toward a synthetic peptide-like substrate. Batch digestions of whole denaturated human normal adult hemoglobin (HbA) were performed to obtain peptide maps by capillary zone electrophoresis (CZE). Migration time reproducibility of the CZE maps was excellent, with a mean relative standard deviation of 1.5%. Moreover, the two immobilized enzyme preparations showed excellent reproducibility for repeated digestions. Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry was also used for peptide mass mapping of denaturated HbA digested using the two immobilized trypsin preparations. Even though the two immobilized trypsin preparations do not behave identically, similar sequence coverages of 57% and 61% (for the two HbA chains merged) were achieved for the support-based and cross-linked trypsin preparations, respectively. [source] Effects of different maceration enzymes on yield, clarity and anthocyanin and other polyphenol contents in blackberry juiceINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 12 2009Wei-Dong Wang Summary Enzymatic maceration of blackberries was conducted with eight different pectinolytic enzyme preparations. Juice yields were increased greatly when macerated blackberries were treated with enzyme preparations (P < 0.05), but no significant difference in yield was found among different enzymes (P > 0.05). The amounts of anthocyanins and polyphenols in the juices as well as clarity of the juices were greatly varied because of different enzyme treatment. Juice prepared with Klerzyme 150 showed better clarity and greater amount of anthocyanins than the juices prepared with other enzyme preparations (P < 0.05). Studies on the uniform design for Klerzyme 150 enzyme revealed that the optimum conditions were 0.063% (v/w), 44 °C and 110 min for enzyme dosage, reaction temperature and reaction time, respectively. [source] Partial purification of proteases that are generated by processing of the Northern shrimp Pandalus borealis and which can tenderize beefINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 5 2004Hitoshi Aoki Summary The crude extracts obtained from the heads of Northern shrimps, Pandalus borealis, (adapted to cold), showed considerable collagenolytic activities. When tested for beef tenderization, resulted in an overdegradation of meat proteins, which was detected organoleptically. Subsequently, four fractions with proteolytic activity were partially purified from the crude extracts by hydroxyapatite followed by MonoQ or Superdex 200 column chromatography. Warner-Bratzler shear force values of steaks treated with three protease fractions (Q, S2, S3) at 10 °C were significantly lower (P < 0.01) than that of the control and the enzyme preparations were completely inactivated after mild heat treatment. These results suggest that the potential for Northern shrimp enzymes to be used in industrial processes, particularly in the food industry, is quite large, where working at lower temperatures to prevent undesirable chemical reactions is necessary. [source] NBT-PABA test to assess efficiency and kinetics of substituted proteolytic enzyme action in pancreatic duct ligated minipigs,JOURNAL OF ANIMAL PHYSIOLOGY AND NUTRITION, Issue 3 2008A. Mößeler Summary The NBT-PABA test is an established method for diagnosis of pancreatic exocrine insufficiency. In the present study the NBT-PABA test was used to test and compare the efficacy of two multienzyme preparations (product A and B) differing in galenic preparation in minipigs in which pancreatic exocrine insufficiency (PEI) was induced by pancreatic duct ligation. Without enzyme substitution no distinct increase in PABA was found in blood after oral administration of NBT-PABA. Administration of both enzyme preparations led to a clear dose dependent rise in PABA-concentrations in blood. Interestingly, the two preparations showed different time curves of serum PABA concentration, indicating differences in the kinetic of proteolytic enzyme action. It is concluded that the NBT-PABA test can be a very useful test for indirectly evaluating proteolytic enzyme efficacy in vivo, and also gives information about the kinetics of enzyme action, not only the end-result of enzyme action (like digestibility trials which were used traditionally). A single test is performed in a few hours and there is no need for fistulated animals. [source] OPTIMIZATION OF PERMEABILIZATION PROCESS FOR LACTOSE HYDROLYSIS IN WHEY USING RESPONSE SURFACE METHODOLOGYJOURNAL OF FOOD PROCESS ENGINEERING, Issue 3 2009GURPREET KAUR ABSTRACT To overcome the permeability barrier and prepare whole cell biocatalysts with high activities, permeabilization of Kluyveromyces marxianus var. lactis NCIM 3566 in relation to, -galactosidase activity was optimized using cetyltrimethylammonium bromide (CTAB) as permeabilizing agent. Permeabilized whole cells can be advantageous over pure enzyme preparations in terms of cost-effectiveness and increased stability maintained by the intracellular environment. Response surface methodology (RSM) was applied to optimize concentration of CTAB, temperature and the treatment time for maximum permeabilization of yeast cells. The optimum operating conditions for permeabilization process to achieve maximum enzyme activity obtained by RSM were 0.06% (w/v) CTAB concentration, 28C temperature and process duration of 14 min. At these conditions of process variables, the maximum value of enzyme activity was found to be 1,334 IU/g. The permeabilized yeast cells were highly effective and resulted in 90.5% lactose hydrolysis in whey. PRACTICAL APPLICATION , -Galactosidase is one of the most promising enzymes, which has several applications in the food, fermentation and dairy industry. However, the industrial applications of , -galactosidase have been hampered by the costs involved in downstream processing. The present investigation was focused on developing the low-cost technology for lactose hydrolysis based on permeabilization process. Disposal of lactose in whey and whey permeates is one of the most significant problems with regard to economics and environmental impact faced by the dairy industries. Keeping this in view, lactose hydrolysis in whey has been successfully performed using permeabilized Kluyveromyces marxianus cells. Hydrolysis of lactose using , -galactosidase converts whey into a potentially very useful food ingredient, which has immense applications in food industries. Its use has increased significantly in recent years, mainly in the dairy products and in digestive preparations. Lactose hydrolysis causes several potential changes in the manufacture and marketing of dairy products, including increased solubility, sweetness and broader fermentation possibilities. [source] Effect of Enzyme Treatments and Drying Temperatures on Methylpyrazine Content in Cocoa (Theobroma Cacao L.) Powder ExtractJOURNAL OF FOOD SCIENCE, Issue 9 2006Leila Moulay ABSTRACT:, The effects of combining enzyme treatments and heating protocols on pyrazine formation in cocoa powder extracts have been studied. Five commercial enzyme preparations containing protease and carbohydrase activities were initially assessed for their ability to release amino acids and reducing sugars, both of which are substrates for Maillard reactions. The enzyme preparation Flavourzyme was subsequently selected for further study given its ability to liberate both types of substrate. Cocoa powder solutions were treated with 2 doses of Flavourzyme and processed at 2 drying temperatures. The combination of the higher dose of the enzyme (12% w/w enzyme/substrate) and the higher drying temperature (150 °C) resulted in a synergistic increase in the concentration of methyl- and dimethylpyrazines. A statistically significant increase in the concentration of tetramethylpyrazine occurred in samples treated at 150 °C that was independent of the enzyme dose assayed. No significant changes in trimethylpyrazine content were detected in treated compared to untreated samples. [source] Effect of endo -xylanase-containing enzyme preparations and laccase on the solubility of rye bran arabinoxylanJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 7 2003Roger Andersson Abstract Three commercial enzyme preparations with endo -xylanase activity, namely Bio-Feed Wheat, Bio-Feed Plus and Grindamyl H 640, and laccase have been tested for their effects on the solubilisation of arabinoxylan (AX) in rye bran or autoclaved rye bran. Autoclaving efficiently increased the availability of AX for enzymatic degradation. Both Bio-Feed Wheat (a monocomponent enzyme) and Bio-Feed Plus (a multicomponent preparation with different enzymatic activities) efficiently degraded the autoclaved rye bran AX into lower-molecular-weight fragments. As much as 70% of the xylose residues and 58% of the arabinose residues in the autoclaved bran were soluble after treatment with Bio-Feed Plus; the weight,average molecular weight of the detectable portion of these soluble polymers was 104,000,Da. Grindamyl H 640 solubilised only a small fraction of the AX in autoclaved rye bran; the molecular weight of these soluble fragments was higher than that of those released by the Bio-Feed xylanases. Addition of laccase during treatment with Bio-Feed Wheat or Grindamyl H 640 decreased the yield of water-soluble AX. © 2003 Society of Chemical Industry [source] In vitro comparative studies on pancreatic enzyme preparationsALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 1 2010J.-M. LÖhr No abstract is available for this article. [source] Preclinical pharmacology of robenacoxib: a novel selective inhibitor of cyclooxygenase-2JOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 1 2009J. N. KING This manuscript reports the results of preclinical studies in the rat with robenacoxib, a novel selective cyclooxygenase (COX)-2 inhibitor. Robenacoxib selectively inhibited COX-2 in vitro as evidenced from COX-1:COX-2 IC50 ratios of 27:1 in purified enzyme preparations and >967:1 in isolated cell assays. Binding to COX-1 was rapid and readily reversible (dissociation t1/2 << 1 min), whilst COX-2 binding was slowly reversible (t1/2 = 25 min). In vivo, robenacoxib inhibited PGE2 production (an index of COX-2 inhibition) in lipopolysaccharide (LPS)-stimulated air pouches (ID50 0.3 mg/kg) and for at least 24 h in zymosan-induced inflammatory exudate (at 2 mg/kg). Robenacoxib was COX-1 sparing, as it inhibited serum TxB2 synthesis ex vivo (an index of COX-1 inhibition) only at very high doses (100 mg/kg but not at 2,30 mg/kg). Robenacoxib inhibited carrageenan-induced paw oedema (ID50 0.40,0.48 mg/kg), LPS-induced fever (ID50 1.1 mg/kg) and Randall,Selitto pain (10 mg/kg). Robenacoxib was highly bound to plasma protein (99.9% at 50 ng/mL in vitro). After intravenous dosing, clearance was 2.4 mL/min/kg and volume of distribution at steady-state was 306 mL/kg. Robenacoxib was preferentially distributed into inflammatory exudate; the AUC for exudate was 2.9 times higher than for blood and the MRT in exudate (15.9 h) was three times longer than in blood (5.3 h). Robenacoxib produced significantly less gastric ulceration and intestinal permeability as compared with the reference nonsteroidal anti-inflammatory drug (NSAID), diclofenac, and did not inhibit PGE2 or 6-keto PGF1, concentrations in the stomach and ileum at 30 mg/kg. Robenacoxib also had no relevant effects on kidney function at 30 mg/kg. In summary, results of preclinical studies in rats studies suggest that robenacoxib has an attractive pharmacological profile for potential use in the intended target species, cats and dogs. [source] In vitro comparative study of three pancreatic enzyme preparations: dissolution profiles, active enzyme release and acid stabilityALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 3 2008A. ALOULOU Summary Background, Various pancreatic enzyme preparations are used for the treatment of pancreatic insufficiency but their bioequivalence is often unknown. Aim, To determine in vitro the pH-dependent release and acid resistance of enzymes from three commercially available pancreatin capsules, two containing enteric-coated (Creon 25000; Eurobiol 25000) and one uncoated (Eurobiol 12500) microspheres. Methods, Dissolution experiments were performed at pH values ranging from 4.0 to 5.8. Lipase, chymotrypsin and amylase activities were measured in the solution as a function of time. Results, Eurobiol 25000 started to release its enzymes significantly at pH 5.0 (t1/2 = 71 min), whereas the enzymes from Creon 25000 were only released at higher pH value (5.4; t1/2 = 49.2 min). Unlike chymotrypsin, lipase and amylase were highly sensitive to acidic conditions at the lowest pH values tested. Both enzymes were also found to be sensitive to proteolytic inactivation at the highest pH values tested. Overall, Eurobiol 25000 released higher amounts of active amylase and lipase than Creon 25000 at the pH values usually found in duodenal contents. The uncoated Eurobiol 12500 preparation was, however, the only one that could immediately release rather high levels of active chymotrypsin and lipase at low pH (4.5). Conclusion, These findings suggest that pH-sensitive enteric-coated pancreatin products containing similar amounts of enzymes might not be bioequivalent depending on the pH of duodenal contents. [source] A dimeric 5- enol -pyruvyl-shikimate-3-phosphate synthase from the cyanobacterium Spirulina platensisNEW PHYTOLOGIST, Issue 2 2001Giuseppe Forlani Summary ,,Isolation and biochemical characterization is reported here of 5- enol -pyruvyl-shikimate-3-phosphate (EPSP) synthase, the enzyme that catalyses the sixth step in the common prechorismate pathway of aromatic amino acid biosynthesis and the target of the widely used herbicide glyphosate, from the cyanobacterium Spirulina platensis. ,,Homogeneous enzyme preparations were obtained by ammonium sulphate fractionation, anion-exchange and substrate-elution chromatography, and chromatofocusing. Protein characterization was carried out by conventional kinetic analysis, PAGE and gel permeation. ,,A 2800-fold purification was achieved, with a recovery of 20% of initial activity. Unusually low apparent affinities for both substrates, phosphoenolpyruvate and shikimate-3-phosphate, did not correspond to decreased glyphosate sensitivity. During SDS-PAGE, the protein migrated as a single band corresponding to a molecular mass of c. 49 kDa. The behaviour of the protein upon gel permeation chromatography under nondenaturing conditions was, however, consistent with a mass of c. 91 kDa. ,,The native enzyme appears to be homodimeric, a remarkable feature that has not been previously reported for EPSP synthases from either cyanobacteria or higher plants. The presence of mono- and dimeric EPSP synthases could represent an important tool for cyanobacterial classification. [source] Bifunctional indole-3-acetyl transferase catalyses synthesis and hydrolysis of indole-3-acetyl- myo -inositol in immature endosperm of Zea maysPHYSIOLOGIA PLANTARUM, Issue 2 2003Stanislaw Kowalczyk 1- O -(indole-3-acetyl)- , - d -glucose: myo -inositol indoleacetyl transferase (IA- myo -inositol synthase) is an important enzyme in IAA metabolism. This enzyme catalyses the transfer of the indole acetyl (IA) moiety from 1- O -(indole-3-acetyl)- , - d -glucose to myo -inositol to form IA- myo- inositol and glucose. IA- myo -inositol synthase was purified to an electrophoretically homogenous state from maize liquid endosperm by fractionation with ammonium sulphate, anion-exchange, adsorption on hydroxylapatite, affinity chromatography on ConA-Sepharose, preparative PAGE and isoelectric focusing. We thus obtained two enzyme preparations which differ in their Rf on 8% polyacrylamide gel. The preparation of Rf 0.36 contained a single 56.4 kDa polypeptide, whereas the preparation of Rf 0.39 consisted of two polypeptides of 56.4 and 53.5 kDa. Both purified preparations of IAInos synthase also exhibited the activity of an IAInos hydrolase, showing that the dual activity was associated with a single protein. Results of gel filtration and analytical SDS-PAGE suggest that the native enzyme exists as both a monomeric (65 kDa) and homo- or heterodimeric form (110,130 kDa). Analysis of peptide maps and amino acid sequences of two 21 amino-acid peptides showed that polypeptides of 56.4 and 53.5 kDa have the same primary structure and that the 3 kDa difference in molecular mass is probably caused by different glycosylation levels. Comparison of this partial and internal amino acid sequence with sequences of other plant acyltransferases indicated similarity to several proteins which belonged to the serine carboxypeptidase-like (SCPL) acyltransferase family. [source] Temperature dependence of nitrate reductase in the psychrophilic unicellular alga Koliella antarctica and the mesophilic alga Chlorella sorokinianaPLANT CELL & ENVIRONMENT, Issue 7 2006VITTORIA DI MARTINO RIGANO ABSTRACT Temperature responses of nitrate reductase (NR) were studied in the psychrophilic unicellular alga, Koliella antarctica, and in the mesophilic species, Chlorella sorokiniana. Enzymes from both species were purified to near homogeneity by Blue Sepharose (Pharmacia, Uppsala, Sweden) affinity chromatography and high-resolution anion-exchange chromatography (MonoQ; Pharmacia; Uppsala, Sweden). Both enzymes have a subunit molecular mass of 100 kDa, and K. antarctica NR has a native molecular mass of 367 kDa. NR from K. antarctica used both NADPH and NADH, whereas NR from C. sorokiniana used NADH only. Both NRs used reduced methyl viologen (MVH) or benzyl viologen (BVH). In crude extracts, maximal NADH and MVH-dependent activities of cryophilic NR were found at 15 and 35 °C, respectively, and retained 77 and 62% of maximal activity, respectively, at 10 °C. Maximal NADH and MVH-dependent activities of mesophilic NR, however, were found at 25 and 45 °C, respectively, with only 33 and 23% of maximal activities being retained at 10 °C. In presence of 2 µm flavin adenine dinucleotide (FAD), activities of cryophilic NADH:NR and mesophilic NADH:NR were stable up to 25 and 35 °C, respectively. Arrhenius plots constructed with cryophilic and mesophilic MVH:NR rate constants, in both presence or absence of FAD, showed break points at 15 and 25 °C, respectively. Essentially, similar results were obtained for purified enzymes and for activities measured in crude extracts. Factors by which the rate increases by raising temperature 10 °C (Q10) and apparent activation energy (Ea) values for NADH and MVH activities measured in enzyme preparations without added FAD differed slightly from those measured with FAD. Overall thermal features of the NADH and MVH activities of the cryophilic NR, including optimal temperatures, heat inactivation (with/without added FAD) and break-point temperature in Arrhenius plots, are all shifted by about 10 °C towards lower temperatures than those of the mesophilic enzyme. Transfer of electrons from NADH to nitrate occurs via all three redox centres within NR molecule, whereas transfer from MVH requires Mo-pterin prosthetic group only; therefore, our results strongly suggest that structural modification(s) for cold adaptation affect thermodynamic properties of each of the functional domains within NR holoenzyme in equal measure. [source] Optimization of enzyme complexes for lignocellulose hydrolysisBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2007Alex Berlin Abstract The ability of a commercial Trichoderma reesei cellulase preparation (Celluclast 1.5L), to hydrolyze the cellulose and xylan components of pretreated corn stover (PCS) was significantly improved by supplementation with three types of crude commercial enzyme preparations nominally enriched in xylanase, pectinase, and ,-glucosidase activity. Although the well-documented relief of product inhibition by ,-glucosidase contributed to the observed improvement in cellulase performance, significant benefits could also be attributed to enzymes components that hydrolyze non-cellulosic polysaccharides. It is suggested that so-called "accessory" enzymes such as xylanase and pectinase stimulate cellulose hydrolysis by removing non-cellulosic polysaccharides that coat cellulose fibers. A high-throughput microassay, in combination with response surface methodology, enabled production of an optimally supplemented enzyme mixture. This mixture allowed for a ,twofold reduction in the total protein required to reach glucan to glucose and xylan to xylose hydrolysis targets (99% and 88% conversion, respectively), thereby validating this approach towards enzyme improvement and process cost reduction for lignocellulose hydrolysis. Biotechnol. Bioeng. 2007;97: 287,296. © 2006 Wiley Periodicals, Inc. [source] Methodological analysis for determination of enzymatic digestibility of cellulosic materialsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2007Y.-H. Percival Zhang Abstract Accurate measurement of enzymatic cellulose digestibility (X) is important in evaluating the efficiency of lignocellulose pretreatment technologies, assessing the performance of reconstituted cellulase mixtures, and conducting economic analysis for biorefinery processes. We analyzed the effect of sugars contained in enzymes solutions, usually added as a preservative, and random measurement errors on the accuracy of X calculated by various methods. The analysis suggests that exogenous sugars at levels measured in several commercial enzyme preparations significantly bias the results and that this error should be minimized by accounting for these sugars in the calculation of X. Additionally, a method of calculating X equating the ratio of the soluble glucose equivalent in the liquid phase after hydrolysis to the sum of the soluble glucose equivalent in the liquid phase and the insoluble glucose equivalent in the residual solid after hydrolysis was found to be the most accurate, particularly at high conversion levels (>ca. 50%). Biotechnol. Bioeng. 2007;96: 188,194. © 2006 Wiley Periodicals, Inc. [source] Standard assays do not predict the efficiency of commercial cellulase preparations towards plant materialsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006Mirjam A. Kabel Abstract Commercial cellulase preparations are potentially effective for processing biomass feedstocks in order to obtain bioethanol. In plant cell walls, cellulose fibrils occur in close association with xylans (monocotyls) or xyloglucans (dicotyls). The enzymatic conversion of cellulose/xylans is a complex process involving the concerted action of exo/endocellulases and cellobiases yielding glucose and xylanases yielding xylooligomers and xylose. An overview of commonly measured cellulase-, cellobiase-, and xylanase-activity, using respectively filter paper, cellobiose, and AZCL-dyed xylan as a substrate of 14 commercially available enzyme preparations from several suppliers is presented. In addition to these standardized tests, the enzyme-efficiency of degrading native substrates was studied. Grass and wheat bran were fractionated into a water unsoluble fraction (WUS), which was free of oligosaccharides and starch. Additionally, cellulose- and xylan-rich fractions were prepared by alkaline extraction of the WUS and were enzymatically digested. Hereby, the capability of cellulose and xylan conversion of the commercial enzyme preparations tested was measured. The results obtained showed that there was a large difference in the performance of the fourteen enzyme samples. Comparing all results, it was concluded that the choice of an enzyme preparation is more dependent on the characteristics of the substrate rather than on standard enzyme-activities measured. © 2005 Wiley Periodicals, Inc. [source] Enzymatic Treatment of Mechanical Pulp Fibers for Improving Papermaking PropertiesBIOTECHNOLOGY PROGRESS, Issue 6 2000Ken K. Y. Wong Three enzyme preparations (crude cellulase, laccase, and proteinase) were evaluated for their potential to improve the papermaking properties of mechanical pulp. After treating a long fibre-rich fraction of the pulp with enzyme, the fibres were recombined with untreated fines for handsheet making and testing. None of the enzymes altered the retention of fines or the consolidation of the furnish mix during handsheet formation. All three enzymes increased tensile stiffness index, which is a measure of the initial resistance of the handsheets to strain. Only the laccase preparation, an enzyme that modifies pulp lignin, consistently increased fibre bonding to enhance other strength properties of the handsheets. [source] | ||||||||||||||||