NUTRIENT ACQUISITION : DIGESTION & TRANSPORT

Group 1

DISACCHARIDES

Examples: Sucrose, maltose and cellobiose

May be transported into fungus:

Intact or

Hydrolyzed before being transported.

• E.g., In S. cerevisiae, sucrose was converted

to glucose and fructose at the cell surface and

the monosaccharides were transported.

• Isolated cell walls of S. cerevisiae contained

most of the invertase.

• Invertase was solubilized by treated with snail

digestive enzyme in mannitol.

• Protoplast were unable to ferment sucrose, but

could ferment glucose

• Maltose was not located in wall, but retained in

the protoplast.

• Yeast cannot ferment maltose unless the have

been grown on maltose.

CELLULOSE

Structure : microfibrillar substance of linear molecules

packed into crystalline arrays interspersed with

amorphous regions.

•Non- ordered structure•Helps in β-linkage to adapt to microenvironment

Native cellulose : Insoluble : Comminuted to produce fine particulate

suspension

Modified, soluble cellulose derivatives

Carboxymethyl cellulose ( CMC) and Hydroxyethylcellulose ( HEC )

- Thickener in Food

Umbelliferyl cellodextrins• Chromogenic substances• Enzyme activities measured based on the colored

products

Cellobiohydrolase Endoglucanases

Digest

ONLY Amorphous Region Crystalline Arrays and Amorphous Region

• CBHI and EGI have greater than 50% nucleotide

sequence similarity and about 45% amino acid sequence

similarity.

• CBHII and EGIII were unrelated to each other or the first

pair.

Two reasons for the expression of the genes for the

enzymes in Saccharomyces cerevisiae:

• Since S. cerevisiae has no known exocellular

cellulases, expression of the genes individually resulted in

single-enzyme activities with no cross contamination.

• Since cellulose substrates are highly variable, conversion

of cellulose to glucose may not be optimal with the native

mix from Trichoderma reesei.

• Expression of the four cloned cellulase genes of T. ressei in S. cerevisiae

succeeded by using cDNA clones from the mRNAs to eliminate the introns

that were not correctly spliced by yeast, and by providing suitable yeast

promoters.

• The recombinant enzymes from S. cerevisiae were active toward the

natural substrates, barley β-glucan and lichenin, and several artificial

substrates.

• The specific activity and binding of the recombinant CBHII were reduced

in comparison with the natural enzyme, suggesting that the

hyperglycosylation affected activity.