Endo-action

The endo-action of the K. marxianus PG was demonstrated by a extremely rapid attack on plant tissue. This activity appears to be at least equivalent to that of several commercial preparations used for separating plant cells for protoplast preparation (RMC, unpublished data). Most of the endo-PGs produced by plant pathogens and saprophytes have so far been reported to possess macerating activity. PG secreted by K. marxianus CCT 3172 also had a strong activity in reducing the viscosity of cocoa pulp. Cocoa pulp generally contains 1 - 1.5% (w/w) of pectin consisting of 68% esterification and 11.6% methoxyl content [18]. 

Johnson (55) has proposed that cell wall extension in yeasts takes place by the concerted action of a nucleoside diphosphate glucose glucosyl transferase and a glucanase or glucanases, such that the hydrolytic enzyme(s), presumably of an endo-action pattern, produces breaks in the existing glucan network into which new glucosyl units are incorporated. 

The fi-glucan exo- and endo-hydrolases are discussed with reference to newer techniques for the investigation of their specificity and action pattern. Those exo-hydrolases which have been well characterized are described individually. The endo-hydrolases are examined from the point of view of their linkage specificity, action on substituted glucans and their specificity for various monomer units. The significance of more random and less random endo-action patterns is considered in relation to single or multiple attack mechanisms. Certain features of p-glucan endo-hydrolase catalyzed reactions are discussed in relation to current views on the three-dimensional structure and mechanism of action of lysozyme. 

Phadebas-starch, obtained by reaction of starch with a triazine dye (Cibacron Blue F8GA), continues to be used as a substrate for the assay of a-amylase. Comparison of assays using Phadebas-starch with saccharogenic and amyloclastic procedures showed that the method is reliable and specific for a-amylase, since Phadebas-starch is not hydrolysed by glucoamylase. Further work on Phadebas-starch showed that groups substituted on the helices inhibit the exo-action of jS-amylase, but function only as accidental barriers to the endo-action of a-amylase. ... 

Figure 13.19 Chromatograms obtained by on-line SPE-GC-MS(SIM) of (a) 10 ml of tap water spiked with pesticides at levels of 0.1 ng 1 (b) 10 ml of a sample of unspiked tap water. Peak identification foi (a) is as follows 1, molinate 2, a-HCH 3, dimethoate 4, simazine 5, ati azine 6, y-HCH 7, S-HCH 8, heptachloi 9, ametiyn 10, prometiyn 11, fen-itrothion 12, aldrin 13, malatliion 14, endo-heptachlor 15, a-endosulfan 16, teti achlor-vinphos 17, dieldrin. Reprinted from Journal of Chromatography, A 818, E. Pocumll et al., On-line coupling of solid-phase exti action to gas cliromatography with mass specti ometiic detection to determine pesticides in water , pp. 85-93, copyright 1998, with permission from Elsevier Science.

Endo-polygalacturonases I and II (PGI and PGII) isolated from recombinant A. niger were characterized with respect to pH optimum, activity on polygalacturonic acid (pga), mode of action and kinetics on oligogalacturonates. 

Substrate specificity and mode of action. Previous information, which we had obtained from FORL crude culture filtrates, showed that the pectin lyase (characterized by an isoelectric point of 9.2) had a predominantly "endo" way of action. This fact has been confirmed with the purified protein it decreased the viscosity of reaction mixtures with pectin, but no increase in absorbance was detected in standard conditions. Moreover, the enzyme showed a great specificity for the substrate, as no activity was detected when the decrease in viscosity of pectate was tried. So, properties of the purified enzyme were studied by using pectin as substrate and following the decrease in viscosity of the reaction mixtures. 

The more abundant lyase produced by FORL has been purified to homogeneity as it is shown by analytical isoelectric focusing (figure 5). The data in Table 1 show that a 32.48-fold increase in specific activity is achieved with a recovery of approximately 2.36%. The enzyme showed an "endo" type of action and a great specificity for pectin. 

A viscometric assay and identification of hydrolysis products were used to determine the mechanism of action of PG. An endo-PG is characterized by a strong reduction in viscosity (e.g. 50%) with a concomitantly low (e.g. 1-3%) release of reducing groups [9]. The time required for 50% decrease in viscosity of a 3.0% (w/v) sodium polypectate solution at 25°C was approximately 10 min, at which time about 1.5% of the total galacturonide bonds had been hydrolysed (data not shown). These results reveal a random mechanism of hydrolysis of sodium polypectate and the enzyme was a poly oc(l,4)-D-galacturonide glycanohydrolase (EC 3.2.1.15) or endo-PG. 

The decreased denaturating action of the precursor and procedure enables one to immobilize reduced amounts of biomaterial. It was demonstrated in Ref. [55] that biocatalysts prepared by entrapping endo-l,3-P-D-glucanase and a-D-galactosidasc in amounts comparable to that in living cells had a reasonable level of activity. When the TEOS is applied, the enzyme content in silica matrix can be up to 20-30 wt.% to counterbalance losses due to denaturation [50]. 

Deshayes described the cydoaddition of 1-amino-1,3-butadienes 63 with ethyl acrylate (34) under the action of microwave irradiation in a monomode reactor with temperature control [59]. Irradiation for 30 min at 70 °C in the absence of solvent afforded a 60 40 ratio of an inseparable mixture of the endo and exo isomers in 90% yield. Classical heating under the same conditions did not affect the selectivity but the yield was lower (Scheme 9.17). 

The action of t-butyllithium on 5-methylene-8-nonenyl iodide (206) leads to the lithium compound 207, which undergoes a tandem cyclization to yield eventually 84% of 2-methylspiro[4.4]nonane (208) (equation 102). An analogous reaction of the iodide 209 (equation 103) results in the [4.3.3]propellane 210 (81%) as a mixture of endo- and excMSomers1. ... 

The characterization of the end products of the degradation of high-molecular substrates, as well as experiments in which the effect of enzymes on oligomeric substrates was investigated, have contributed significantly to the elucidation of the mode of action of endo-D-galacturonanases. 

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