Enzymes, cell-wall hydrolysis with

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. 

Hen egg-white lysozyme catalyzes the hydrolysis of various oligosaccharides, especially those of bacterial cell walls. The elucidation of the X-ray structure of this enzyme by David Phillips and co-workers (Ref. 1) provided the first glimpse of the structure of an enzyme-active site. The determination of the structure of this enzyme with trisaccharide competitive inhibitors and biochemical studies led to a detailed model for lysozyme and its hexa N-acetyl glucoseamine (hexa-NAG) substrate (Fig. 6.1). These studies identified the C-O bond between the D and E residues of the substrate as the bond which is being specifically cleaved by the enzyme and located the residues Glu 37 and Asp 52 as the major catalytic residues. The initial structural studies led to various proposals of how catalysis might take place. Here we consider these proposals and show how to examine their validity by computer modeling approaches. 

In soil, the chances that any enzyme will retain its activity are very slim indeed, because inactivation can occur by denaturation, microbial degradation, and sorption (61,62), although it is possible that sorption may protect an enzyme from microbial degradation or chemical hydrolysis and retain its activity. The nature of most enzymes, particularly size and charge characteristics, is such that they would have very low mobility in soils, so that if a secreted enzyme is to have any effect, it must operate close to the point of secretion and its substrate must be able to diffuse to the enzyme. Secretory acid phosphatase was found to be produced in response to P-deficiency stress by epidermal cells of the main tap roots of white lupin and in the cell walls and intercellular spaces of lateral roots (63). Such apoplastic phosphatase is safe from soil but can be effective only when presented with soluble organophosphates, which are often present in the soil. solution (64). However, because the phosphatase activity in the rhizo-sphere originates from a number of sources (65), mostly microbial, and is much higher in the rhizosphere than in bulk soil (66), it seems curious that plants would have a need to secrete phosphatase at all. 

In one case, a small peptide with enzyme-like capability has been claimed. On the basis of model building and conformation studies, the peptide Glu-Phe-Ala-Ala-Glu-Glu-Phe-Ala-Ser-Phe was synthesized in the hope that the carboxyl groups in the center of the model would act like the carboxyl groups in lysozyme 17). The kinetic data in this article come from assays of cell wall lysis of M. lysodeikticus, chitin hydrolysis, and dextran hydrolysis. All of these assays are turbidimetric. Although details of the assay procedures were not given, the final equilibrium positions are apparently different for the reaction catalyzed by lysozyme and the reaction catalyzed by the decapeptide. Similar peptide models for proteases were made on the basis of empirical rules for predicting polypeptide conformations. These materials had no amidase activity and esterase activity only slightly better than that of histidine 59, 60). 

Synergism between a-arabinosidase, xylanase and j8-xylosidase has been demonstrated in the hydrolysis of wheat straw arabinoxylan with purified enzymes of T, reesei (71). > en only xylanase and )8-xylosidase were used in the hydrolysis, the xylose yield was only 66% of that produced by the whole culture filtrate at the same activity levels of these two enzymes, and no arabinose was produced. Addition of a-arabinosidase increased the yields of both xylose and arabinose. Enhanced hydrolytic action of hemicellulolytic or pectinolytic enzymes in the hydrolysis of alfalfa cell wall polymers by addition of Ruminococcus albus a-arabinosidase has also been reported (37). 

The maximum rate is directly related to the rate at which the enzyme processes or permits conversion of the reactant molecule(s). The number of moles of reactants processed per mole of enzyme per second is called the turnover number. Turnover numbers vary widely. Some are high, such as for the scavenging of harmful free radicals by catalase, with a turnover number of about 40 million. Others are small, such as the hydrolysis of bacterial cell walls by the enzyme lysozyme, with a turnover number of about one half. 

Poly(ribitol phosphate) synthetase has been found in particulate fractions from Staphylococcus aureus H, and Lactobacillus plantatrum.lt ll-m The bulk of the activity in Lactobacillus plantarum was in crude, cell-wall preparations, and the enzyme is apparently located in the membrane, although intimate association with the wall itself has been suggested. Unlike the natural teichoic acid, the enzymically synthesized ribitol phosphate polymer was readily extracted with phenol hydrolysis by acid and by alkali gave the expected products, and oxidation with periodate indicated a chain length of 5-9 units, a value which compares well with that of 8 units for the natural polymer in the walls of this organism. 

The current observations confirm previous studies on beechwood and sprucewood holocellulose (7,10,19). The attack of the hemicellulose proceeds from the primary wall/Si as well as from the tertiary wall into S2 the pit chambers constitute preferred paths of enzyme diffusion into the walls. Also, substances of the middle lamella, especially in the cell corners, are removed by the xylanase and the mannanase treatments. Parallel to the removal of hemicelluloses, the fibrillar structure of the cellulose and its lamellar arrangement in transections of cell walls became obvious. In samples treated with cellulases, the cellulose fibrils were often completely hydrolyzed in the Si layer, occasionally accompanied by complete dissolution of cell-wall portions. This is also in conformity with the previous conclusion that the cellulases hydrolyze highly ordered zones of cellulose and remove hemicelluloses by hydrolysis or by detachment. 

---