Polysaccharide- Degrading enzymes from humicola lanuginosa (griffon and maublanc): isolation and properties of xylanase and p- glucosidase

Abstract

The thermophilic fungus Humicola lanuginosa (Griffon and Maublanc) Bunce: Isolation and Properties of Xylanase and Glucosidase, submitted by Lalitha Anand for the Degree of Doctor of Philosophy, Indian Institute of Science, Bangalore. This thesis deals with the isolation of xylanase and glucosidase produced by the thermophilic fungus Humicola lanuginosa (Griffon and Maublanc) Bunce, and a study of their mode of action on xylans from oat spelt, larchwood, and sugarcane bagasse. The major biopolymer constituents of plant cell walls are cellulose, hemicellulose, pectin, and lignin. Polysaccharidedegrading enzymes with welldefined specificities serve as powerful tools for understanding the structures of these polymers and their organization within the plant cell wall. In this study, a xylanase and a glucosidase were obtained in pure form, and their modes of action on three xylans were examined. Xylanase (EC 3.2.1.8) is primarily responsible for hydrolyzing the 1,4 linkages between xylose residues in the hemicellulose xylan. This enzyme has been isolated from several mesophilic organisms and well characterized. However, limited information is available regarding xylanases from thermophilic fungi, both in terms of their protein properties and mode of action. Such information would greatly aid our understanding of xylanase structure-function relationships and the structural features of xylan. The thesis contains an introductory chapter reviewing literature on plant cellwall polysaccharides and the enzymes that degrade them, followed by four chapters describing the experimental work. Screening of several soil samples for thermophilic fungi capable of producing cellulases and xylanases led to the isolation of Humicola lanuginosa, which produced high levels of extracellular xylanase. Culture conditions were standardized for optimal enzyme production. A purification procedure involving anionexchange chromatography and gel filtration was developed to isolate xylanase and glucosidase from culture filtrates of H. lanuginosa. Xylanase was purified fourfold, and glucosidase was purified 82fold, with yields of 8% and 43%, respectively. The aminoacid compositions of both enzymes resembled those of fungal xylanases and glucosidases, with high levels of acidic amino acids and low levels of sulfurcontaining amino acids. The molecular weight of xylanase was 22,500 by SDSPAGE and 29,000 by gel filtration; its sedimentation coefficient was 2.76 × 10¹³ sec. For glucosidase, the molecular weight was 110,000 by SDSPAGE and 134,900 by gel filtration, with a sedimentation coefficient of 10.5 × 10¹³ sec. Catalytic properties such as Km and temperature optima were also determined. The xylanase was highly stable when stored at room temperature or even at 50°C, either dry or in solution, for at least 24 hours. However, storage at -20°C in the dry state for more than two months resulted in loss of activity-an observation not previously reported for any xylanase. Chromatographic techniques revealed:

a minor charged variant of the native enzyme retaining activity, a large, enzymatically inactive, highmolecularweight species.

Ultracentrifugation and electrophoresis suggested that this inactive species was an aggregate of the native protein, likely formed during storage at -20°C. A comparative analysis of enzyme action on larchwood xylan, oatspelt xylan, and sugarcanebagasse xylan was carried out. Sugarcanebagasse xylan was extracted, purified, and characterized as an arabinoglucuronoxylan. All three xylans were hydrolyzed to varying degrees. Paper chromatography of products indicated that xylanase exhibited endoaction on xylan. The susceptibility of the 1,3 linkage between arabinose and xylose to acid hydrolysis, and its resistance to xylanase, was exploited to prepare:

xylooligomers free of arabinose arabinoxylooligomers retaining side chains.

Studies on xylanase and glucosidase action on xylooligosaccharides (xylobiose to xylopentaose) showed:

Xylanase hydrolysis rate increased with chain length. Glucosidase acted slowly on all oligomers. Xylanase exhibited predominant endoaction, attacking the chain randomly from the reducing end. Glucosidase exhibited exoaction, releasing xylose from the nonreducing end.

The direction of attack was determined using reduced xylotriose. On arabinoxylooligosaccharides, interesting differences were observed:

Xylanase showed predominant exoaction on arabinoxylotetraose (AX) and arabinoxylopentaose (AX), unlike its endoaction on unsubstituted oligomers. Even after extensive hydrolysis, significant amounts of parent arabinosugars remained unhydrolyzed, whereas the corresponding xylooligomers were completely hydrolyzed.

These results indicated that arabinose substitution creates linkages resistant to both enzymes. The smallest resistant sugar was arabinoxylobiose (AX), which likely contains the resistant linkage pattern. Based on the hydrolysis patterns of arabinoxylooligomers, structures of AX, AX, and AX were proposed, demonstrating the usefulness of hydrolytic enzymes in structural elucidation. In conclusion, xylanolytic enzymes from Humicola lanuginosa were purified to homogeneity and characterized. Although the enzyme system does not completely hydrolyze xylan due to the absence of sidechaincleaving enzymes, it provides valuable insights into xylan structure. The physicochemical characterization contributes to understanding enzyme structure and function.