Lignocellulosic biomass characteristics

Tuan-hua David Ho, Ph.D (賀端華 )

Institute of Plant & Microbial Biology

Academia Sinica

02-27891709

tho@sinica.edu.tw

Carbohydrate and cell wallsOutline

• Basic sugar chemistry

• Sucrose metabolism

• Starch metabolism

• Cell walls--structure and function

• Cell wall degrading enzymes

Basic sugar biochemistry

Polysaccharides can get complicated such as mixed polymers with branches.

Glucose-P

ADPG

AmyloseAmylose

Amylopection

Starch biosynthesis

ADPG pyrophosphorylase

Branching enzymes

Soluble starch synthase

Bound starch synthase

Chloroplast

Other plastids

Winkled pea mutant deficient in a starch branching enzyme

Waxy mutant in maize: amylopectin replaces amylose due to deficient in granule bound starch synthase

Wild type potatoTransgenic potato with a bacterial ADPG pyrophosphorylase not subject to feedback inhibition

Iodine stained potato slices

Shrunken maize mutant: deficient in sucrose synthase

Sucrose

Glucose + FructoseInvertase

Sucrose synthase Fructose + UDPG

UTP

Synthesis of starch or cellulose

Sucrose-P synthase

Starch degradation during cereal grain germination

Cell walls determine the shape and size of a cell.

Protoplasts are always spherical, but walled cells are different in shape and size.

Involvement of cell walls in cell differentiation

• Shape and size of cells• Aging and senescence• Vascular system formation• Fiber formation• Abscission formation• Fruit ripening• Defense against pathogens and predators• Sensing changes in the environment

Layers of walls: middle lamella, primary walls and secondary walls

A new wall begins in the “phragomosome”/”phragmoplast” in a dividing cell.

From sugar to polysaccharides:

Glucose (sugar) -------> glucan (such as (1-->4)glucan, cellulose)Mannose -------> mannanGlactose -------> galactanXylose -------> xylanXylose and glucose -------> xyloglucan (hemicellulose)Glactose ----> oxidized to glacturonic acid ------> galacturonan

Techniques used to determine the structure of complexpolysaccharides: 1. Gas-liquid chromatography (GLC)--composition2. Mass spectrometry--structure3. Nuclear magnetic resonance---interacting groups4. Electrone microscopy---”seeing is believing”

Cellulose fibrils in the primary cell wall are oriented perpendicular to the main axis of the cell.

H-bondings between the cellulose fibrils provide the strength of the wall.

Cell wall proteins

Dicots and some monocots Most of the monocots

Cell wall biosynthesis requires ER, Golgi, and plasma membrane.Components remain soluble until they can be cross-linked at the cell surface.

From starch and other sources

Polysaccharides

Cellulose microfibrils are assembled at the surface of plasms memberane.

Cellulose synthase “rosettes” in action!

Cellulose synthase

• First isolated from certain bacteria (Acetobactor xylinum and Agrobacterium tumefaciens) mutants--CesA genes

• Apparent homologs of CesA have been isolated from cotton, Arabidopsis, etc.

Thickness of walls remains the same when a cell expands

Cell

Expands

XET (xyloglucan endotransglycosylase) cuts and rejoins the tethering hemicellulose molecules,

“ Expansins” loosen the interactions between cellulose fibrils and the tethering hemicellulose molecules

Lignin is an important component in cell walls

Pre-treatment

Cellulose hydrolysis

Sugars

Fermentation

Ethanol

Plant Materials(rice straws, wood chips, etc)

Search for enzymes/microbes forlignocellulosic bio-ethanol formation

Hemi-cellulasesLigninasesCutinasesProteases (?)

Cellulases X 3

Cel

l Wal

ls Liganins

Cuticle

Biological sources of cell wall degrading enzymes

• Rice straw composts • Guts of herbivorous animals• Guts of wood/grass consuming insects• Guts of grass consuming fishes and aquatic

organisms

Integrative Processes

Gene Cloning

Microbes IsolationComposts

Microbes from ATCC etc.

Data Bases

Recombinant ProteinCharacterization

Transformation into energy cropsGene SelectionProtein Production

Environments

Metagenomic Libraries

Proteomics

Protein Engineering

Proteinisolation,

purification

Genomic libraries

Enzymatic treatmentsby adding to feedstock

In planta deconstruction &bioconversion

SDS-PAGE zymogram of endoglucanse activities from Geobaccillus spp.

Substrate: 0.1% CMC

Cellulases: endoglucanases, exoglucanases (such as CBH), -glucosidases

2-D gel zymogram detection for endoglucanse activities

Substrate: 0.4% CMC

Cloning of endoglucanase genes via activity staining

Geobaccillus spp. Geobaccillus thermodentrificans

Endoglucanase activities in gut tissues of Taiwanese grasshopper

SDS-PAGE zymogram of xylanase from Geobaccillus spp.

Substrate: Birch wood xylan

Xalanase activities in gut tissues of Taiwanese grasshopper

Laccase activities in Basdiomyctous fungi

1,4 benzenediol + O2 ------> quinone + H2O (Laccase is a ligninase)

Detection of laccase activity on SDS-PAGE

Source: Pycnoporus cinnabarinus

Take home messages

• Lignocellulosic materials are virtually the cell walls of plants

• Plant cell walls are complicated interlocking polymers of cellulose, hemicellulose, lignins, pectins and proteins

• A combination of enzymes are needed to “deconstruct” the cell wall complex

• Much effort is needed to search for novel cell wall degrading enzymes/microbes