Hemicellulase Activity

Application and Principle This procedure is used to determine hemicellulase activity of preparations derived from A.J-pergillus niger var. The test is based on the enzymatic hydrolysis of the interior glucosidic bonds of a defined locust (carob) bean gum substrate at 40° and pH 4.5. Determine the corresponding reduction in substrate viscosity with a calibrated viscometer. 

Amyloglucosidase Activity), 798 Glucose Isomerase Activity, 796 Glucose Oxidase Activity, 798 P-D-Glucosidase Activity, 798 Hemicellulase Activity, 799 Invertase Activity, 800 Lactase (Acid) (P-Galactosidase) Activity, 802... 

Aspergillus niger Mould for commercial enzyme production (pectinase, hemicellulase) Active cinnamoyl esterase releasing free hydroxycinnamic acids in juices Dugelay et al. (1993)... 

The commercial liquefaction enzyme typically used (Rhoapect 7016) contained predominantly cellulase and hemicellulase activity but with some pectinase activity. The use of more pectinase and less liquefaction enzyme represented a more favorable treatment economically and was actually more effective in reducing viscosity (Table II). Also, as predicted, the steady state flux of the membranes was higher when pectinase predominated. 

Enzymes most frequently proposed to fruit juices producers are pectinases coming from Aspergillus. Pectinases are exocellular enzymes and are the main activities produced among numerous side activities type hemicellulases, glycosidases. The Table 1 gives the spectrum of enzymatic activities contained in three commercial preparations A, B and C. 

For apple maceration, the main activity is the endopectinlyase (PL) which decreases the viscosity due to the colloidal pectin by decreasing tlie molecular weight of this substrate methylated at about 90%. In association to this PL, exo B and endoarabanase (ARAs), pectinacetylesterase (PAE), rhamnogalacturonase (RG) and other hemicellulases allow to hydrolyse side chains of the pectin, decrease the steric hindrance and make the access of pectinases to the rhamnogalacturonic backbone more easy. 

Figure 1. Enzyme activities in crude extracts made from cultures of different ages detected with substrates selected for cellulases, hemicellulases, amylases, and pectinases. Cultures were grown at 22 C. Black bars indicate activity for 105-day-old cultures given a 12-day-long cold shock at 5 C starting on day 90. (Reproduced with permission from ref. 8. Copyright 1985 American Society for Microbiology.)...

However, we have observed that values obtained with crude extracts were only qualitative. Often, they did not accurately estimate the quantities of the individual enzymes present. Inhibitors were typically present that caused the underestimation of certain enzymes ie.g., ligninases Table II) and that could potentially mask less dominant enzymes. Also, certain polysaccharidases e.g., hemicellulases) were often overestimated due to the action of non-specific or synergistic enzymes e.g., other hemicellulases or cellulases) (9,14), This artifact resulted in low apparent recovery of a given activity and only moderate increases in specific activity upon purification of the major corresponding enzyme present, in spite of the fact that SDS polyacrylamide gels indicated good recovery and substantial removal of contaminants (14),... 

The complex nature and interconnectivity of plant cell wall polymers preclude straightforward enzymatic digestion. There are dozens of enzyme families involved in plant cell wall hydrolysis, including cellulases, hemicellu-lases, pectinases, and lignin-modifying enzymes. The Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) has classified cellulases and hemicellulases, like all enzymes, into different classes based on activity. Table 33.2 and Table 33.3, compiled from the IUBMB enzyme nomenclature database (http //www.chem.qmul.ac.uk/iubmb/ enzyme/), list the IUBMB enzyme classifications for cellulases and hemicellulases.153... 

In contrast to cellulases, the hemicellulases encompass a much broader suite of activities. In addition to analogue versions of endo-, exo-, and glycosidase cellulase activities, multiple debranching activities are needed to handle the high complexity of the heterogeneous hemicelluloses (Table 33.3). The varied backbone composition of hemicelluloses also adds complexity. Xylans, xyloglucans, man-nans, and numerous other minor polysaccharide chains form the backbone for different hemicelluloses. 

Carbohydrase [(Trichoderma longibrachiatum var.) (formerly reesei)] Produced as an off white to tan, amorphous powder or as a liquid by controlled fermentation using Trichoderma longibrachiatum var. Soluble in water (the solution is usually tan to brown), but practically insoluble in alcohol, in chloroform, and in ether. Major active principles (1) cellulose, (2) /3-glucanase, (3) /3-D-glucosidase, (4) hemicellulase, and (5) pentosanase. Typical applications used in the preparation of fruit juices, wine, vegetable oils, beer, and baked goods. 

Calculation One hemicellulase unit (HCU) is defined as that activity that will produce a relative fluidity change of 1 over a period of 5 min in a locust bean gum substrate under the conditions specified. Calculate the relative fluidities (FR) and Ln values (see definition below) for each of the four efflux times (T-y) and reaction times (7 R) as follows ... 

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