Sucrose hydrolysis, enzymic

Enzymes. Invertase (P-fmctofuranosidase) is commercially produced from S. cerevisiae or S. uvarum. The enzyme, a glycoproteia, is not excreted but transported to the cell wall. It is, therefore, isolated by subjecting the cells to autolysis followed by filtration and precipitation with either ethanol or isopropanol. The commercial product is available dry or ia the form of a solutioa containing 50% glycerol as a stabilizer. The maia uses are ia sucrose hydrolysis ia high-test molasses and ia the productioa of cream-ceatered candies. 

FIG. 7-2 Linear analysis of catalytic rate equations, a), (h) Sucrose hydrolysis with an enzyme, r = 1curve-fitted with a fourth-degree polynomial and differentiated for r — (—dC/dt). Integrated equation,... 

Sucrose hydrolysis is catalyzed by an enzyme. Time- concentration data are given in the first two columns. The applicability of the M-M equation, r = kC/(M+C), is to be checked. 

After sucrose hydrolysis by the invertase enzyme in solution, perform all measurements at a constant applied potential of +0.60 Y in order to oxidase the hydrogen peroxide produced by the enzyme reaction of glucose oxidase biosensors (Scheme 20.1). 

Commercial yeast invertase (Bioinvert ) was immobilized by adsorption on anion-exchange resins, collectively named Dowex (1x8 50-400,1x4 50-400, and 1x2 100-400). Optimal binding was obtained at pH 5.5 and 32°C. Among different polystyrene beads, the complex Dowex-1x4-200/invertase showed a yield coupling and an immobilization coefficient equal to 100%. The thermodynamic and kinetic parameters for sucrose hydrolysis for both soluble and insoluble enzyme were evaluated. The complex Dowex/inver-tase was stable without any desorption of enzyme from the support during the reaction, and it had thermodynamic parameters equal to the soluble form. The stability against pH presented by the soluble invertase was between 4.0 and 5.0, whereas for insoluble enzyme it was between 5.0 and 6.0. In both cases, the optimal pH values were found in the range of the stability interval. The Km and Vmax for the immobilized invertase were 38.2 mM and 0.0489 U/mL, and for the soluble enzyme were 40.3 mM and 0.0320 U/mL. 

Table 1 shows that some Dowex/invertase complexes, in which the resins used were 1x2-200, 1x4-50, 1x4-100, 1x4-200, 1x8-50, and 1x8-200, retained 100% of protein molecules during sucrose hydrolysis. The absence of enzyme desorption from the support enhances the half-life of the immobilized complex when employed in repeated-batch or continuous processes (18). 

Both fructooligosaccharide synthesis and sucrose hydrolysis are catalyzed by most of the fructosyltransferases and 3-fmctofuranosidases (invertases) in the presence of sucrose. The transferase hydrolase raho, which determines the maximum yield of fructooligosaccharide, depends basically on two parameters the concentra-hon of sucrose and the intrinsic enzyme properties, that is its ability to bind the nucleophile (to which a fructose is transferred) and to exclude H2O from the acceptor binding site [11]. 

Stefuca et al. (1990) proposed an ET method offering a rapid, convenient, and general approach to determine kinetic constants of immobilized biocatalysts. Here, a differential reactor (DR) was used for the measurement of the initial reaction rate of sucrose hydrolysis (Vallat et al. 1986). The enzyme column of the ET has been considered as a differential packed-bed reactor, and with a mathematical model, intrinsic kinetic constants of immobilized invertase were calculated from experimental DR and ET data. 

Bacillus subtilis levansucrase (sucrose 2,6-/8-D-fructan 6-)8-D-fructosyltrans-ferase, EC 2.4.1.10) catalyzes fructosyl transfer from sucrose to levan (154). In the absence of a fructosyl acceptor, the primary reaction is sucrose hydrolysis, although a limited amount of self-initiated levan synthesis occurs as well (155). As with sucrose phosphorylase, acceptor specificity is broad a number of saccharides and other nucleophiles are suitable fructosyl acceptors (154-158). The complete amino acid sequence of the approximately SO-kDa enzyme has been determined by both protein (159) and gene (160) sequence analyses. The three-dimensional structure at 3.8 A reveals a rod- or ellipsoid-shaped protein with a length some four times the diameter (161). 

There is no direct evidence on the form of the glycosyl-enzyme intermediate. Nonetheless, stabilization of a noncovalent oxocarbonium for the period between product fructose release and acceptor binding may not be realistic for slow reactions, given the extremely short lifetime of a glycosyl oxocarbonium ion (9). Levansucrase and particularly GTase-S are quite slow enzymes, with sucrose hydrolysis Heat of 48 sec 122) and 9.1 see" 213), respectively. Thus, the carbonium ion may well collapse to a more stable covalent complex or develop an equilibrium between the two forms. Nucleophilic catalysis is consistent with... 

Mild alkaline hydrolysis of S. sobrinus GTase-S glucosyl-enzyme releases glucose exclusively as the /3-anomer. This contrasts with a-D-glucose liberated during sucrose hydrolysis by native enzyme. The difference can be attributed to the character of acylals, which can hydrolyze at the acetal carbon or the ester carbon with respective anomeric retention or inversion (229, 230). The native enzyme hydrolyzes sucrose as an acetal so that released a-o-glucose translates to an active site carboxyl linked as the )3-anomer or ion paired to a carbonium ion from the re face. The denatured covalent complex, without the active site structure to cleave at the acetal carbon, hydrolyzes the /3-linked D-glucose at the ester carbon, with release of the retained jS-anomer. 

Fig. 2-3. Rate of sucrose hydrolysis by yeast sacchar-ase as a function of substrate concentration. Adapted from R. Kuhn in J. B. S. Haldane, "Enzymes," Longmans Greene and Co., London, 1930. Reprinted by permission of the Longman Group Limited.

Mandenius CF, Billow L, Danielsson B et al. (1985) Monitoring and control of enzymic sucrose hydrolysis using on-line biosensors. Appl Microbiol Biotechnol 21(3 ) 135-142 Marsden WL, Gray PP (1986) Enzymatic hydrolysis of ceUulase in lignocellulosic materials. CRC Critic Rev Biotechnol 3 235-274... 

Fructose is the sweetest common sugar, being about twice as sweet as sucrose consequently, invert sugar is sweeter than sucrose. The enzyme invertase, which bees use in making honey, accomplishes the same chemical result as does the acid-catalyzed hydrolysis of sucrose. 

A second Gram positive system that has been studied in some detail is levansucrase synthesis in B. subtilis. This extracellular enzyme catalyses transfructorylation from sucrose to various acceptors which results in levan synthesis and sucrose hydrolysis. 

For a-glucosidase, 7—18 isoenzymes are known. In a wide pH optimum between 5.8—6.5 the enzyme hydrolyzes maltose and other a-glucosides. The Km with sucrose as substrate is 0.030 mol/1. It also possesses transglucosylase activity. During the first stage of sucrose hydrolysis the trisaccharide erlose (a-maltosyl-P-D-fmctofiiranoside)... 

A simple technique for identification of raffinose and sucrose involves enzymic hydrolysis on paper chromatograms (255). 

White (87), investigating the sucrose inversion, confirmed the formation of four compounds but could not detect the tetrasaccharide found by White and Secor. Similar inversion effects have been observed during the action of mold invertase on sucrose. Bealing and Bacon (9) noticed that, during incomplete hydrolysis, enzyme preparations from molds formed a number of nonreducing substances with R values lower than that of sucrose (in addition to the expected monosaccharides), which apparently were similar but not identical with those produced by yeast invertase. They later investigated this effect more extensively and were able to show that at least four intermediate compounds could be produced in the breakdown of sucrose. 

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