Biocatalysts Entrapped By Prepolymer Methods

As described above, various kinds of prepolymers having different physicochemical properties have been developed for the entrapment of different kinds of bio-catalysts. Examples of enzymes entrapped with prepolymers are listed in Table 3. Some enzymes listed in Table 3 have been employed for development or appraisal of new prepolymer methods. Not many reports are available concerning the applications of enzymes entrapped by prepolymer methods. Lipases entrapped with hydrophobic...

Modification Of Triglycerides A Modifying the Fatty Acid Composition

Chemical interesterification is one of the major modification techniques and basically involves a random rearrangement of the sn-1, sn-2, and sn-3 fatty acids over the triglycerides present in the mixture (Fig. 1). A similar result can be obtained when using a random lipase such as that from Candida rugosa* (38, 67), Pseudomonas fluorescens (68), or Pseudomonas cepacia (69). Using a 1,3-specific lipase, only the fatty acids on the outer positions of the triglyceride molecules will be...

Effect of the Thermodynamic Water Activity

Apart from being a substrate, water also has a vital function in maintaining the three-dimensional conformation of the enzyme and hence its intrinsic activity (24, 25). This effect is most conveniently expressed in terms of the thermodynamic water activity maintained in the reaction medium. It allows comparison of results of enzymatic reactions in various organic solvents, even though absolute water concentrations are significantly different (26). It has been shown that the form of this...

Alcohol Oxidases and Dehydrogenases

In general, aldehydes are more potent flavor compounds than their alcoholic counterparts. Hence, alcohol oxidases are interesting enzymes for the in vitro production of flavoring preparations which, among others, can be applied in beverages. Typical examples include the use of methanol oxidase from Pichia, Hansenula, and Candida (17) for the production of natural acetaldehyde from ethanol. This enzyme is induced during growth on methanol. At the end of the logarithmic growth phase cells are...

Properties As Enzyme

The main function of TGase is formation of e-(y-gluta-myl) lysine isopeptide crosslinks between naturally occurring proteins or peptides, resulting in polymerization (Figs. 1, 3). Amines such as hydroxylamine, mono-dansylcadaverine, cadaverine, spermine, spermidine, and putrescine can be incorporated into the protein substrate by the TGase reaction to bind covalently to glu-tamine residues resulting in the inhibition of the polymerization of proteins (Fig. 2). Thus, amines are competitive...

References

Carbohydrate active enzymes. At Url http afmb.cnrs-mrs.fr pedro CAZY db.html. 1999. 2. L Dijkhuizen ,D Penninga, HJ Rozeboom, B Strokopytov, BW Dijkstra. Protein engineering of cyclodextrin glycosyltransferase from Bacillus circu-lans strain 251. In SB Petersen, B Svensson, S Pedersen, eds. Carbohydrate Bioengineering. Amsterdam Elsevier Science, 1995, pp 165-174. 3. L Dijkhuizen, BA van der Veen, JCM Uitdehaag, BW Dijkstra. Engineering of cyclodextrin...

Substrate Binding in CGTases

The first important step in enzyme catalysis is binding of the substrate. In several starch-degrading enzymes a separate domain responsible for adsorption onto raw starch has been found. Evidence for a starch-binding site in CGTases separate from the active site was presented (55), and fusion of the E-domain of the Bacillus macerans CGTase to -galactosidase demonstrated that it can indeed function as a starch-binding domain (56). The function of the E-domain has been studied in more detail with...

Fatty Acid Synthase

The molecular organization of the plant fatty acid synthase is composed of dissociable enzyme activities, similar to E. coli and termed type II FAS, has been recognized since the early 1980s (Fig. 2). The initial reactions of FAS are catalyzed by transacylases which transfer the acetyl and malonyl units from their CoA esters to the corresponding ACP derivatives (18). The initial condensation reaction in plant fatty acid biosynthesis takes place between acetyl (C2) ACP and malonyl (C3) ACP,...

Other Uses of Cellulases

Clearly the largest industrial use of cellulases is outside the food and feed sector in textile processing of natural textile fibers composed of cellulose (85, 98). Cellulases have also found potential applications in the pulp and paper industry (99). The textile and pulp fibers must retain their strength after cellulase treatment. Thus, novel commercial products with altered cellulase profiles have been developed. The detergent industry is the other major user of enzymes. The main enzymes used...

Catalytic Properties And Mechanisms

The kinetics of l-LDH are explained by an ordered bi-bi mechanism, in which coenzyme NADH (or NAD+) is bound first, and subsequently the substrate, pyruvate (or L-lactate), is bound to the enzyme active site. In the l-LDH catalysis, the imidazole group of the His195 (the numbering is based on the vertebrate enzyme according to Eventof et al. 15 ) residue acts as an essential acid base catalyst, which mediates proton transfer between the substrate and the solvent. The catalytic reaction occurs...

Nitrogenfixing Organisms And Crop Plants

Only prokaryotes, i.e., those living things without an organized nucleus (Eubacteria, Archaea, and actino- mycetes), can perform biological nitrogen fixation. The ability to fix N2 is widely spread among bacterial genera (3, 4) and, despite a number of claims, no eukar-yote has been clearly established to fix N2. Although farmers recognized the benefits of crop rotations centuries ago, the source of much of that benefit was unknown to them. The first report of nitrogen fixation in 1838 involved...

Reaction Mechanism of HLADH

The enzyme binds coenzyme and substrate to form a ternary complex which, in general, can be assumed to be ordered with coenzyme binding preceding substrate binding and product release preceding coenzyme release. Aldehyde formation during the catalytic action of the enzyme requires a net removal of two hydrogen atoms from an alcohol substrate. This dehydrogena-tion process is known to proceed by a mechanism of combined proton and hydride ion transfer. It is well established that transfer of the...

Process Modeling

Many models have been developed to describe lipase-catalyzed reactions. The simplest models generally involve Michaelis-Menten kinetics or similar type of rate expressions describing, e.g., substrate or product inhibition. These types of models are often used to describe single reactions such as hydrolysis and ester-ification (59). Acidolysis (transesterification) and interesterifica-tion of triglycerides are much more complex. Being multistep and multisubstrate reactions a full-kinetic...

Molecular Structure

A large number of amino acid sequences of catalases are known, primarily deduced from the gene sequences. These include (based on entries listed in Swiss-Prot) Aspergillus fumigatus (Sartorya fumi-gata), A. niger, Arabidopsis thaliana (mouse ear cress), Bacillus subtilis, B. firmus, B. stearothermophi-lus, Bacteroides fragilis, Bordetella pertussis, Bos taurus (bovine), Botrytis cinerea (Botryotinia fuckeliana), Brucella abortus, Caenor habditis elegans, Campylobacter jejuni, Candida albicans,...

Utilization Of Laccase In Food

Besides its use as a bleaching agent in the wood pulp and paper industry, laccase has been used in dechlorination and for removal of phenolic compounds from wastewater. In relation to the food industry, laccase has been used in assays for phenols in natural juices (23). It has also been used as a biosensor for determination of polyphenols catechols in tea (24). Immobilized laccase has been used to remove phenols from apple juice (25) and in must and wine (26). Lante et al. (27) also reported...

Determination of HRP Activity Using ABTS 42

The reaction catalyzed is shown in equation (2). HRP ABTS has an absorption maximum at 340 nm (pH 4.4) and e340 3.97 x 104 M-1 cm-1. It is oxidized by HRP to a radical cation that has an adsorption at pH 5.0 with e414 3.6 x 104 M-1 cm-1. Table 4 Kinetic Parameters of HRP with Different Substrates Table 4 Kinetic Parameters of HRP with Different Substrates

Lipase Specificity

The types of specificity that can be ascribed to lipases include 1. Substrate specificity The enzyme shows a different rate of lipolysis of various acylglycerols, tri-, di-, and monoacylglycerols, or various types of fatty acids. 2. Positional specificity The enzyme catalyzes the release of fatty acids preferentially at the primary ester, secondary ester, or nonspecific at all esters. 3. Stereospecificity The enzyme hydrolyzes the two primary esters (sn-1 or sn-3) at different rates (91)....

Relationships In Cellulases

A two-domain structure consisting of a catalytic domain and a separate substrate-binding domain is typical for cellulose-degrading enzymes from both bacteria and fungi. The existence of substrate-binding domains is also common in other enzymes which degrade solid substrates. In fungal enzymes the cellulose-binding domains (CBDs) are typically linked to the catalytic domain (catalytic core) by linkers which clearly separate the two domains (Fig. 3). The CBDs can be found in either the C or N...

Isoforms of Pectate and Pectin Lyases

The advances in molecular-biology techniques and tools in the past decade have allowed the rapid accumulation of nucleotide sequences of genes encoding pectate and pectin lyases. Comparison of the number of pectate lyase sequences and pectin lyase sequences present in databases shows that the pectate lyases strongly outnumber the pectin lyases. This is exemplified by an organism like E. chrysanthemi 3937. For this organism so far only one pectin lyase and nine pectate lyases have been...

Extremophilic Enzymes A Search of Enzymes from Exotic Environments

A majority of the currently known 4000 enzymes have been isolated from mesophilic organisms. In terms of microbial diversity, < 1 of the world microbial species have been identified and cultured in laboratories. These enzymes have evolved to function under a narrow range of pH, temperature, ionic strength, pressure, and chemical concentration, and are not optimized for performance under industrial conditions. Considerable efforts have been devoted to harvest organisms, mostly in the...

Purification of Papain

Papain might be purified by the method of Stepanov and Rudenskaya (66) on a bacitracin-Sepharose column, but this has not been proved since crude papain contains papain, chymopapain, and an aminopepti-dase, which may not be separated by this method. The Kimmel and Smith method (71) described here is an improved method of that of Balls and Lineweaver (72) and gives crystalline papain. They started with hard particles of commercial latex that had to be ground to a fine powder to permit...

Enzymic Degradation Of Rhamnogalacturonans

Rhamnogalacturonan (RG, also referred to as RG-I) is the second heterogalacturonan of which the enzymic degradation is described in this chapter. This polysaccharide can have a backbone composed of as many as 100 repeats of the disaccharide 2)-a-L-Rhap-(1 4)-a-D-GalpA-(1 (5). The rhamnosyl residues can be substituted at O-4 with single unit -D-Galp- (1 4) or longer neutral sidechains. The latter are often referred to as the hairs, and are mainly composed of galactosyl and or arabinosyl...

Introduction

Xyloglucans are plant cell wall polysaccharides, which belong to the hemicellulose class i.e., they can bind to cellulose. They are the most abundant hemicellulose in the walls of many nongraminaceous species. Xyloglucans can function both as a structural and as a reserve polysaccharide. In the primary cell wall, xyloglucans can crosslink cellulose fibers, yielding a network that determines to a large extent the strength of the wall. This complex usually constitutes approximately half of the...

Phosphatidylserine and Phosphatidylethanolamine

In yeast PS is synthesized from CDP-diacylglycerol and serine, a reaction catalyzed by the enzyme CDP-diacylglycerol L-serine O-phosphatidyltransferase (PS synthase, EC 2.7.8.8). Plant PS synthase activity was originally reported in spinach leaf microsomes (10) and has since been detected in many other plant tissues (2). The recent characterization of a wheat cDNA encoding a PS synthase provides definitive evidence that plants are capable of producing PS directly from CDP-diacylglycerol (60)....

Endotransglycosylase And Related Enzymes

A separate section has been devoted to xyloglucan endotransglycosylase (XET), mainly because it is a very interesting enzyme from an enzymological point of view. The enzyme occurs throughout the plant kingdom, although some plants (for instance tomato) seem a richer source than others (38). XET or XET-related (referred to as XTR) proteins probably play a role in a number of important plant processes (a) anchoring newly (synthesized and) deposited xyloglucan into the plant cell wall (b) ripening...

Other Proteinases

The presence of other minor proteolytic enzymes in milk, including thrombin and a lysine aminopeptidase, has been reported (32). In addition to cathepsin D, other proteolytic enzymes from somatic cells are probably present in milk. Verdi and Barbano (33), who studied the degradation of caseins in milk by somatic cells or plasmin, found that somatic cell proteinases and plasmin produced distinctly different peptides, and that the plasmin inhibitor 6-aminohexanoic acid was suitable for studying...

Lysozyme Ec 32117

Lysozyme (muramidase, mucopeptide N-acetyl-mura-myl hydrolase) is a widely distributed enzyme which lyses certain bacteria by hydrolyzing the P(1-4) linkage between muramic acid and N-acetylglucosamine of mucopolysaccharides in the bacterial cell wall. Lysozyme activity is normally assayed by the lysis of cultures of Micrococcus lysodeikticus measured by a decrease in turbidity. Lysozyme was isolated from human milk by Jolles and Jolles (69), who believed that bovine milk was devoid of...

Unsaturated and Polyunsaturated Fatty Acids

In many oleaceous species the products of fatty acid synthesis in the plastid are palmitic (C16 0), stearic (C18 0), and oleic (C18 1) acids (see above). These are made available to the endoplasmic reticulum for triacylglycerol assembly (see below) and the formation of other fatty acids, particularly polyunsaturated fatty acids. Unsaturated fatty acids, therefore, are synthesized by the sequential insertion of double bonds into a more saturated precursor. The first double bond in the production...

Purification

Details of the method used for purification of a pull-ulanase limit dextrinase depend on the source of the enzyme and the response of the enzyme to pH and temperature. B. acidopullulyticus pullulanase, for example, can be purified from Promozyme (Novozymes North America, Inc., Franklinton, NC, U.S.A.), a commercially available source of the enzyme (10). After dialysis of Promozyme overnight against acetate buffer (50 mM, pH 5.0), the pullulanase can be precipitated by addition of cold acetone (...

Purification of Carboxypeptidases A and B

Earlier sections of this chapter have not included car-boxypeptidase B. Carboxypeptidase B is a metal-containing exopeptidase similar to carboxypeptidase A, but it is specific only for arginine and lysine residues at the carboxy-terminal end of proteins and peptides. Carboxypeptidase A can remove C-terminal amino acids other than arginine or lysine residues. These two exopeptidases are found in the pancreas of animals. Figure 24 and Table 5 show the chromatography of activated proenzymes...

Immobilized Enzyme Processes That Have Been Commercialized In The Food Industry

Production of High-Fructose Corn Syrup Using Immobilized Glucose Isomerase This industry has grown from the initial commercial production in 1970 by Clinton Corn Pocessing to become the largest industrial use of an immobilized enzyme process in the world (16, 17). Most recent data indicate that the global annual production of high fructose corn syrup is 10 million metric tons dry substance(dsb) which is produced with 1500 metric tons of immobilized enzyme (J. Shetty, personal communication,...

Potential Adverse Effects Of Enzyme Preparations

Certain side activities of fungal enzyme preparations can be detrimental to fruit juice and wine quality owing to the formation of off-flavors and the decolor-ization. Hydroxycinnamic acids, such as p-coumaric and feru-lic acids, occur mainly in bound forms in fruits. Quinic acid and or glucosidic esters of these hydroxycinnamic acids have been detected in numerous fruits apple, pear, apricot, peach, orange, grapefruit, strawberry, raspberry (167), while in grapes they are esterified with...

Xanthine Oxidase from Cows Milk

An abbreviated description of the method of Avis et al. (20) for the purification of xanthine oxidase from cow's milk, published in 1955, is given below. This method is chosen for several reasons 1. Although XO had been partially purified by other researchers, Avis et al. (20) were the first to obtain a homogeneous XO preparation and to crystallize it. 2. Researchers have shown this method to be the best one, even after 45 years. Some modifications of the method have been published, but all...

Glucoamylase In Foods

GA is produced by some eubacteria, a few archaea, a number of yeasts, and many filamentous fungi. Although there are reports of animal and plant GAs, these appear to be fundamentally different enzymes with kinetic properties that overlap those of true GAs. GA is therefore not a significant factor in most unprocessed foods. GA plays a major role in food processing. In Asia, filamentous fungi from Aspergillus and Rhizopus and filamentous yeast such as Saccharomycopsis fibuligera that secrete GA...

Foodprocessing Properties Of Glycosidases

This section will examine the properties of enzymes, focusing on the specific conditions encountered in the processing of fruit juice and derived beverages. In particular, three important parameters influencing flavor release will be discussed the effect of pH aglycone and sugar specificity of glycosidases and inhibition of activity by sugar and sugar analogues. A summary of the properties of -glucosidase from some plants and microorganisms is given in Table 2. The optimum pH activity of plant...

Mechanism of Nitrogenase Action

During catalysis, the Fe protein delivers electrons, one at a time, to the MoFe protein in a process that couples MgATP binding and hydrolysis to the association and dissociation of the two component proteins and concomitant electron transfer (33, 60). Both component proteins are required for MgATP hydrolysis, and neither component protein alone will reduce substrate. Based on a substantial amount of kinetic data, a numerical model has been developed to describe the process by which electrons...

Endogenous And Exogenous Pectic Enzymes In Fruit And Vegetable Processing

Pectic enzymes occur naturally in many fruits and vegetables (endogenous enzymes), but they are also added as processing aids (exogenous enzymes). The latter are mostly derived from food-grade fungi, e.g., Aspergillus niger, A. aculeatus. In higher plants, particularly pectin methyl esterase and polygalacturonase, both endo- and exoacting, are present. Pectin acetyl esterase has been found in citrus fruit and more recently also pectate lyase and rhamnogalacturonan hydrolase have been found in...

Maturation Of Nitrogenase

The primary translation products of the nitrogenase structural genes are not active. Many other nif-specific genes are required to activate these immature structural components. The function of the products of these nif-specific maturation genes is to catalyze the formation and then insertion of the individual metalloclusters into apo-forms of the Fe-protein and MoFe-protein. In the case of the Fe-protein, only the nifM gene product is specifically required for its maturation (86). The nifM...

Removal of Oxygen

During storage of food, oxygen can have a detrimental effect on quality. As an example, oxidation of unsatu-rated fatty acids can lead to rancidity of vegetable oils. Oxidation of colored components will change the color of beverages or wine. In addition, oxygen influences the taste of beer in a negative way during storage. The majority of foods contain certain quantitites of glucose. In closed systems, the quantity of oxygen to be removed, in order to prevent oxidation, is generally rather...

USE OF bGlucosidases In Food Processing And Quality Enhancement

The importance of -glycosidases to food quality and processing is ably reviewed and discussed by Giinata (see Chapter 21) with emphasis on flavor enhancement in fruit juices and various beverages derived from them. Therefore, the reader is referred to Chapter 21 for more information on this aspect. There are several hundred different y8-glucosidic flavor precursors identified from plants whose aglycones are products of mevalonate or shikimate pathways. Obviously, there are y8-glucosidases in...

Reducing Group Methods

Reducing-group measurements are the best methods for general cellulase activity determinations. One of the greatest difficulties with this type of method is the nonuniform response between oligosaccharides with different degrees of polymerization (DP). Another difficulty is the incompatibility of some methods with certain substrates. Among the three reducing- group methods described here for measurement of cel-lulase activity, the disodium 2,2 '-bicinchoninate (BCA) method gives the greatest...

Thermophilic Enzymes

As mentioned earlier, it is generally advantageous to run industrial processes at elevated temperatures, as long as sensitive components in the reaction are not damaged under such conditions. It is therefore not surprising that many of the biotechnological processes involving enzymes are carried out at relatively high temperatures, and most of the enzymes used are quite thermostable, despite them being usually of mesophilic origin (148). A useful industrial enzyme must have several specific...

Reaction Mechanism for YADH

The main features of the reaction mechanisms for YADH are in all probability essentially the same as in HLADH (because of structural similarities of the catalytic domain of the two enzymes). Finer details of the reaction mechanism, however, are different between the two enzymes. There are differences in substrate specificity, in the rate of catalysis, and in the requirements for ordered events during the catalytic mechanism which may be random or ordered depending on substrate (31). The step of...

Phosphatidylglycerol and Diphosphatidylglycerol

PG in plants has been found in a number of plant organelles but is mainly associated with chloroplast thylakoids (16), whereas diphosphatidylglycerol (DPG), also known as cardiolipin, is restricted to the inner mitochondrial membrane (2, 16). In yeast the synthesis of PG is catalyzed by two enzymes, PGP synthase (glycerophosphate CDP diacylglycerol phos-phatidyltransferase, EC 2.7.8.5) and PGP phosphatase (phosphatidylglycerolphosphate phosphohydrolase, EC 3.1.3.27). The first catalyzes the...

Purification of Subtilisin

Chandrasekaran and Dhar (69) found that subtilisin can be purified in a single step by affinity chromato-graphy. The affinity matrix was activated CH-Sepharose 4B coupled to the dye 4-(4-aminopheny-lazo)-phenylarsonic acid. The coupling conditions are given in the paper. For chromatography, purified crystalline subtilisin (5 mg mL) was dialyzed overnight against 20 mM acetate buffer, pH 5.9, containing 5 mM CaCl2. The dia-lyzed solution (4.8 mL) was loaded to the column (1 x 51 cm). The column...

Use Of Glycosidases For Aroma Enrichment

The use of glycosidases to release flavor compounds from glycosidic precursors, was initially examined in wines. Two major reasons for this are (i) important flavor compounds in wines of Vitis vinifera cultivars are accumulated in grapes as flavorless glycoconju-gates, and especially (ii) the glucose inhibition of ft-glucosidase in available enzyme preparations, limits the use of glycosidases to media, like wine, containing trace levels of glucose (< 1 g L). Nevertheless, because of the...

Phosphatidylethanolamine And Phosphatidylcholine From Diacylglycerol

Flux modeling experiments in tobacco support the idea that the flux of ethanolamine into PE is split equally between PS decarboxylation (or base exchange see below) and incorporation of phosphoethanolamine via CDP-ethanolamine and diacylglycerol (63). Phosphoethanolamine is probably derived from free or phosphorylated serine by decarboxylation (73, 74) although no enzyme operating on anything other than phosphatidylserine has been convincingly demonstrated. In contrast, it is apparent that...

Developments Since 1940

Even the development of citric acid by fermentation, (Pfizer) and penicillin (Beecham, Glaxo, Merck, Pfizer, Squibb, Bristol-Myers) in the 1940s did not really trigger a scale-up of industrial applications of enzymes. We must go forward to around 1955 before the development of enzyme production was gaining speed by growing sales of bacterial amylase and protease. It began in a very modest fashion. As an illustration, the turnover of the enzyme division of Novo Industri (now Novozymes), the...

Purification Of Laccases A Methods

There is no best method for the purification of lac-case because of the varied number of isoforms and different characteristics. Laccases have been purified most often from extracellular fluids from fungal and plant cell cultures. In general, fungal laccase purifications employ some method for removal of cells (filtration, centrifugation) followed by concentration of the extracellular fluid using ultrafiltration or precipitation methods (ethanol, ammonium sulfate). The dialyzed and concentrated...

Qualitative And Quantitative Determination Of Activity

The enzyme activity is usually assayed based on pyr-uvate reduction, in which the catalytic efficiency of most LDHs together with the equilibrium are much more favorable than those of lactate oxidation. The rate of decrease of the NADH is quantitatively determined by monitoring absorbance at 340 nm e of NADH 6,220 M cm 1 at 338 nm, pH 7.5 . Pyruvate concentrations of and 0.3 mM are usually appropriate for the A4 and B4 isozymes of mammals, respectively, at pH 7.2 and 25 C. In general, however,...

T T t T T

Figure 1 Distribution of linkages in barley 1 3,1 4 - -glucans. G -glucosyl residues 3 1 3 -linkages 4 1 4 -linkages red reducing end arrows sites of hydrolysis by 1 3,1 4 - -glucan endohydrolases EC 3.2.1.73 . From Ref. 10. barley 1 3, 1 4 - -glucans have weight average molecular weights in the range 200,000-300,000 12, 13 . These values correspond to polysaccharides containing 1200-1800 glucosyl residues. The high viscosities of cereal 1 3, 1 4 - -glu-can solutions can be attributed not only...