Lecture 5 - Biologicaly degradation of lignocellulose Flashcards

Question

What bacteria can degrade lignin and how do they do this?

Answer 1

* *Sphinobium* sp. SYK-6 * certain actinobacteria, a-proteobacteria and y-proteobacteria * needs large number of enzymes to trim down the aromatics and catalyse the ring opening * evidence for anaerobic bacteria that can degrade and grown on lignin

Answer 2

**Hardwood** * hemicellulose mainly xylan * backbone of b-1,4-linked xylose with decorations **Softwood** * hemicellulose main galactoglucan * b-1,4- linked D-glucose and D-galactose

Answer 3

The gemicellulose targeted is that of the biofuel grass miscanthus

Answer 4

*Caldanaerobius polysaccharolyticus* * gram +ve thermophillium * from garbag dump in illinois * characterised a range of its hemicellulose degrading enzymes following a partial genome sequence * take a biochemical and structural approach to characterise components of a xylan utilisation cluster

Answer 5

1. used simple BlastP with known endo-xylanases 2. found one in caldanerobius polysaccharolyticus 3. took sequence and examined it in software (InterPro, Pfam) revealing it had mutliple domains 4. Found that it contains: 1. a GH10 flanked by 2 family 22 CBMs on the N-term 2. 2 family 9 CMBs on the C-term 3. 3 surface later homology (SLH) domains also at the c-terminus (attach to the surface of the cell) 4. Clear N-terminal signal peptide 5. Cloned by PCR into an E.coli expression vecetor to make recombinant protein

Answer 6

* Ran protein on gel, identified a soluble protein at 170 kDa as expected * First functional assay: (prior to: had to clone protein and his-tag for affinity chromotography. Cleaned up protein by breaking open E.coli heat cell lystae to ~50degrees C, E.coli proteins denature but not protein from the thermophile) -\> incubate the protein with birchwood xylan (BWX) overnight and measure the increase in reducing sugar ends. This was shown, demonstrating xylanase activity * Also followed the reaction using TLC (thin layer chromotography plate) and can resolve the different forms of the xylan, although bands don't match to the standards exactly * spot products of the reaction onto thin layer chromotography plates and this separates the molecules * products stop at X3 - are they modified in some way? * Identified that their protein (Xy10A) produced mainly xylo-oligomers

Answer 7

1. Xy10A produced mainly xlo-oligomers 2. must be a region on the genome that encodes genes for further breakdown and use of the xylo-oligomers 3. found a large 18kb cluser with: 1. ABC transporter 2. genes for pentose phosphate pathway 3. xylanase was not in this cluster 4. Looked further at the xylosidase (Xyl3A), the glucuronidase (Agu67A) and the ABC transporter

Answer 8

* expressed and purified the Xyl3A (xylosidase) in e.coli in the same way as the xylanase * protein at the expected size of 86kDa (SDS-PAGE) * size exclusion chromatography: protein eluted at a position that was consistent with it being a dimer in solution (157kDa) Tested for b-xylosidase activity of the Xyl3A * screened for activity using a range of pNP-linked substrates * most active on pNP-D-xylopyranoside (pNPX) * then tested against a range of real xylo-oligomers and saw efficient cleavage down to xylose (monomer) * therefore clearly a xylosidase * has weak activity against cellulo-oligosaccharides (has a weak glucosidase activity)

Answer 9

Expression of Agu67A * expressed and purified the Agu67A in E.coli the same was as the xylanase * See a protein of ~ expected size (below 75kDa when should be 79kDa) * size exclusion chromotography: protein is a dimer * Identified some GH domains (N, M, C) * Expected to be a debranching enzyme to remove the 4-O-methylglucuronic acid groups attached to the xylose backbone Activity of Agu67A * looked at whether it produced undecorated xylo-oligomers from branched substrate (aldobiouronic, aldotriouronic and aldotetrauronic) * then look at the release of glucuronic acid equivelents in the reaction * used HPLC - to look at the substrates that were put in with and without the enzyme. With the enzyme other molecular weights begin to appear * Works best on alduronic acids

Answer 10

* mixed three enzymes together to show that the combination of the 3 could convert more of the BWX xylan into free xylose * the addition of the xylosidase to the xylanase gave the largest increase * the extra addition of the a-glucuronisdase made a small further improvement (helping the xylanase) Odd experiment to do as would expect all these enzymes to be in diff places in the cell. With all 3 proteins get more single sugar products than separate

Answer 11

* whether the ABC-T is specific for xylo-oligosaccharides * as the xylanase is extracellular and the xylosidase and glucuronidase are intracellular substrates must be transported in as branched xylo-oligomers * Expressed the SBP from the candidate ABC transporters in teh cluster and examined ligand binding by isothermal titration chromotrography * found that it doesn't bind xylose but binds tightly to xylo -biose, -triose and -tetraose with the highest affinity for xylotriose, then xylobiose, then xylotetraose.

Answer 12

1. looks at enzyme-substrate interactions 2. when proteins bind to a substrate, small amounts of energy are released 3. compare to a reference cell (kept at constant temperature) and measure small temperature changes 4. measure the amount of energy need to be added/taken out in order to match test cell to reference cell 5. allows the calculation of the affinity and how well substrate binds Steepest = best binding

Answer 13

* solved the structure of the SBP * to learn about how it bound the xylo-oligomers * structure resembles other monosaccharide transporters from other thermophiles * clearly binds the xylo-oligosaccharide * analysis of the binding site suggests it is suited to binding the triose and nothing longer (confirming ITC data)

Answer 14

1. enzyme binds to cell surface and breads down hemicellulose to alduronic acid (Xylanase - Xyn10A) 2. This is transported into the cell (ABC transporter) 3. Processes inside the cell to xylase monomers which are ran through the cells metabolism to generate energy (Xylosidase - Xyl3A, glucuronidase - Agu67A)