Chapter 24 : Cellulases, Hemicellulases, and Pectinases

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The study of cellulases is important from the standpoint of microbial conversion of biomass to feeds and chemical feed stock. The complex structure of hemicelluloses has dictated a correspondingly diverse array of hemicellulases. The concentration (or actually dilution) of enzyme preparation required to effect this level of depolymerization is converted, through a somewhat indirect procedure, to the cellulase activity in filter paper units (FPU) per milliliter. Rather than being an exact representation of the saccharification process that occurs in simultaneous saccharification and fermentation (SSF), diafiltration saccharification assay (DSA) data were useful for comparison with SSF data in efforts to identify the influences of factors other than product inhibition on the performance of cellulases in SSF. Cellulases may also be detected in slab gels using either Western blotting or enzyme-linked immunosorbent assay, as reported for enzymes from . When preparing enzyme-treated substrates, care must be taken to employ phenolic acid esterase-free cellulases. Hemicellulose-depolymerizing enzymes are divided into three classes; the endoacting, exoacting, and oligomer-hydrolyzing. The process of detecting and verifying exoglucanases (cellobiohydrolases [CBHs] in context of the fungal cellulose systems) has long been controversial. If purified proteins are available, careful comparisons of reducing-sugar yields and fluidity values from carboxymethylcellulose (CMC) hydrolysis as a function of enzyme concentration can be used to judge whether an enzyme is more endoglucanase-like or CBH-like. Recent reviews by Kashyap and Naidu and Panda outline the pectinase enzymes in detail. Ruthenium red staining in plates and zymograms has also been used for assay of pectinase enzymes.

Citation: Himmel M, Baker J, Adney W, Decker S. 2007. Cellulases, Hemicellulases, and Pectinases, p 596-610. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch24
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Image of FIGURE 1

Action of the major cellulase enzymes on cellulose. Endoglucanases cleave random internal β-1→4 bonds. The major exoglucanases cleave cellobiose units from the reducing chain ends in a processive manner, whereas minor exoglucanases may cleave glucose units from the nonreducing end of the chain (not shown). β-Glucosidases cleave cellobiose to two glucose units.

Citation: Himmel M, Baker J, Adney W, Decker S. 2007. Cellulases, Hemicellulases, and Pectinases, p 596-610. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch24
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Image of FIGURE 2

Action of major enzymes involved in the depolymerization of generic xylan (A) and glucomannan (B) chains. Additional debranching enzymes are required for the numerous variants found in the different hemicelluloses.

Citation: Himmel M, Baker J, Adney W, Decker S. 2007. Cellulases, Hemicellulases, and Pectinases, p 596-610. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch24
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Image of FIGURE 3

More specific action of debranching enzymes involved in the deconstruction of arabinoxylan. The structure is a generalized diagram of arabinoglucuronoxylan. Enzyme activities: 1, endoxylanase; 2, acetyl xylan esterase; 3, α-L-arabinofuranosidase; 4, α-D-glucuronidase; 5, ferulic acid esterase; 6, acetyl esterase; and 7, β-xylosidase.

Citation: Himmel M, Baker J, Adney W, Decker S. 2007. Cellulases, Hemicellulases, and Pectinases, p 596-610. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch24
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Image of FIGURE 4

DSA progress curve of commercial cellulase preparation (Spezyme from Genencor International) at a loading of 20 FPU/g of cellulose acting on Sigmacell 20 at 38°C. After the actual experimental measurements, empirical fits to the data were done. The expression “time-to-target kinetics” is considered more descriptive of the approach than is the classical “integrated-rate kinetics.”

Citation: Himmel M, Baker J, Adney W, Decker S. 2007. Cellulases, Hemicellulases, and Pectinases, p 596-610. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch24
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Image of FIGURE 5

AZCL-polysaccharide hydrolysis in a petri plate (top) and a microtiter plate. The dark particulates are the AZCL-β-glucan (top) and AZCL-galactan (bottom). Soluble blue dye is released upon hydrolysis.

Citation: Himmel M, Baker J, Adney W, Decker S. 2007. Cellulases, Hemicellulases, and Pectinases, p 596-610. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch24
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