Engineering of 3 methylaspartate ammonia-lyases for biobased adipic acid production
Examensarbete för masterexamen
Bioteknik 300 hp (civilingenjör)
Van Havere, Robin
The current traditional adipic acid production is a highly polluting process. Researchers have been studying bio‐based pathways to produce adipic acid in an environmentally sustainable way. One of these bioprocesses could involve the use of a metabolic pathway to produce adipic acid from lysine. The pathway first enzymatic reaction is the deamination of lysine into 6‐aminohex‐2‐enoic acid. No enzyme has been discovered yet to catalyse this reaction. The study of an enzyme able to do this conversion was the main aim of this thesis. Several ammonia lyases were considered as interesting enzymes to study since these enzymes are able to do a deamination on aminoacids, though different than than lysine. The enzyme studied in this research project is 3‐methylaspartate ammonia lyase (MAL) which catalyses the deamination reaction on 3methylaspartic acid. The 3‐methylaspartic acid has a comparable structure to lysine and makes the MAL enzyme an appropriate candidate to study the conversion. However, the MAL enzyme did not show any activity towards lysine. Preliminary results obtained from computational structural biology experiments of MAL in the presence of lysine suggested some mutations that could be done on the enzyme to gain activity on lysine. Therefore, the suggested mutations were implemented in the MAL enzyme, aiming at increasing the size of the binding pocket of MAL to give lysine more space to fit in the active site and make the reaction more feasible. The mutations were successfully introduced in the MAL gene with a site‐directed mutagenic PCR protocol. The mutated MAL genes were then expressed in E.coli BL21 (DE3) and the protein purified. The protein purification was done with two methods that used the same basic principle: an Immobilized Metal‐ion Affinity Chromatography. After the purification, the activity of the mutated enzymes was monitored by various activity assays. The assays provided the kinetic constants of the conversion of the natural substrate, 3‐methyl aspartate, by the mutated enzymes and their plausible activity on lysine by detecting the theoretical conversion products: 6‐aminohex‐2‐enoic acid and ammonia. No activity on lysine was detected, but still valuable information was retrieved from the activity assays. Some of the mutated enzymes like L384A, C361A and C361S showed a tremendous impairment in the activity, which led me to conclude that those residues that were altered are important for the enzyme catalysis.
Industriell bioteknik , Industrial Biotechnology