Widening the Active Site of Bacillus circulans Xylanase by Amino Acid Insertion Open Access

Tran, Hai (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/5712m686z?locale=en
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Abstract

Bacillus circulans xylanase (Bcx) is an enzyme of the glycoside hydrolase family which natively binds and degrades xylan, a polysaccharide abundant in plant cell walls. This characteristic makes it a highly desirable enzyme for industrial purposes particularly in papermaking, animal feed, and more recently the biofuel industry. Bcx adopts a β-jellyroll structure resembling a human right hand whereby the active site is located in the resulting cleft. Bridging the cleft, a loop region holds the finger and thumb regions in place to stabilize the protein. I hypothesize that the cleft width is an important determinant of the rate of hydrolysis and substrate specificity for glycoside hydrolase family members. By inserting 1-4 amino acids in the peptide bridge, the Bcx finger and thumb domains will spread open which could translate into new and broader substrate specificity and also an increased rate of catalysis. Here, we have created five Bcx mutants with glycine and proline residues inserted into the bridge to compare and contrast the effects of flexible and rigid residues. Our results suggest that out of the six variants tested, WT-Bcx has the highest activity for xylan substrate. Although the activity level for cellulose was low, G1-Bcx exhibited approximately a 35% increase in activity over wild-type. Circular dichroism data suggests that inserting flexible glycine residues decrease thermostability while inserting rigid proline residues maintain or improve it.

Table of Contents

Table of Contents
1. Introduction...1

a. Biofuels...1
b. Glycoside Hydrolases...2
c. Protein Engineering...6

2. Materials and Methods...9

a. Materials...9
b. Mutagenesis...9
c. Cloning and Sequencing...10
d. Protein Expression...11
e. Protein Purification...11
f. Enzyme Activity Experiment...13
g. Circular Dichroism Analysis...14

3. Results...15

a. Gene Construction...15
b. Protein Expression...15
c. Protein Purification...16
d. Enzyme Activity...18
e. Circular Dichroism...19

4. Discussion...22
5. References...24

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