Chemistry Reference
In-Depth Information
Endo-1,3:1,4-glucanase
Cellobiohydrolase
1,4
1,4
1,3
1,3
1,4
1,4
1,4
β
-Glucosidase
1,4
1,4
1,4
1,3
Endo-1,3-glucanase
Endo1,4-glucanase
Fig. 8.2
Enzymatic hydrolysis of
β
-glucan.
of complex enzymes and processes involved in its breakdown. In several studies,
5, 6
it was
shown how
Trichoderma viride
(which commonly occurs in nature) acts to naturally decom-
pose organic material. When
T. viride
was grown on denatured crude barley cell walls as sole
source of carbon and energy, it produced a range of enzymes. The first enzyme to be switched
on in substantial quantities was endoxylanase. This highlights the prior need to remove pen-
tosan before glucan. A carboxypeptidase was also measured early on in the study. This will act
to break protein structures in the barley cell wall and thereby help to release β-glucan. Endo-β
1,4-glucanase, arabinofuranosidase and general esterase enzymes (xyloacetylesterase, feru-
loyl esterase) were next in sequence to be detected. The arabinofuranosidase releases arabi-
nose from the xylan backbone, while it has been proposed that xyloacetylesterase and feruloyl
esterase release acetate and ferulate respectively from arabinoxylan. These enzymes therefore
increase the accessibility of the β-glucan to enzyme attack. Of particular significance is their
elaboration before the development of endo-barley-β-glucanase. The endo-β-glucanases act
to break down the β 1-3 and β 1-4 linkages in the barley β-glucan (Fig. 8.2). This enhances
the solubility of β-glucan and reduces potential downstream brewing difficulties that are
associated with under-modified or poorly malted grains.
Using whole barley kernels, it has been shown
7
that during malting, the order of synthesis
of enzyme development also supports the model proposed by the
T. viride
study;
5
namely
the early synthesis of xylanase and carboxypeptidase, followed next by β-glucanase and
arabinofuranosidase and lastly by α-amylase. This also supports the model of an arabinoxylan
sheath, covering a β-glucan core in the cell walls, which must first be hydrolyzed in order to
give glucanases better access to the cell wall core.
To further explain the proposed model
5
in Fig. 8.1: a layer of pentosan is located in the
outer regions of the wall, rendering it accessible to xylanolytic enzymes and ensuring that
the solubilization of β-glucan is restricted. The covering of β-glucan by pentosan, however,
is not complete. This allows glucanases to access their substrate, with the low molecular
weight digestion products readily leaking back through the pentosan cover. It allows for
calcoflour to stain β-glucan and for a certain amount of soluble β-glucan to be accessible
to solvating water in the absence of enzyme activity. Enzymes that remove the arabinosyl-,
acetyl- and feruloyl-plugs increase this accessibility still further. A further more substantial
layer of pentosan is hidden within the wall. This model gives a very good indication of the
complexity of the barley cell wall structure. It also serves to give a visual understanding of the
difficulties encountered when mashing with unmalted adjunct material and under-modified
malt. It provides a simple explanation of why commercial enzyme complexes that contain
arrays of glucanases, xylanases and cellulases are better to reduce brewhouse-processing
difficulties than, for example, a purified β-glucanase.
