Question

You have been hired at Genentech to reengineer trypsin to become a nylonase, to be used for bioremediation of environmental contamination by nylon. Based on what you have learned about directed evolution and design, in no more than 300 words, describe the approach you would take to perform this reengineering. Justify why this is approach is optimal.

Answer 1

• Nylon comprises a family of man-made substances that were first manufactured in 1935. Nylonases are biological enzymes that can break down nylon oligomers.
• The most prominent nylonases are within the family of enzymes classified as 6-aminohexanoate hydrolases, some enzymes not formally classified as 6-aminohexanoate hydrolases also have the ability to breakdown nylons, and so can also be classified as nylonases.
• 6-aminohexanoate hydrolases, proteases like trypsin and certain lipases have been experimentally demonstrated to have nylonase activity.
• The most biological nylonases can only degrade short nylon polymers. Bonds joining the nylon-6 monomer units are similar to the peptide bonds joining amino acids in a protein.
• Trypsin is a very common protein-cleaving enzyme that appears in vertebrate eukaryotes and various prokaryotes. Unfortunately, Ebata and Morita did not specify what the organismal source was of the trypsin they used in their experiments.
• The eukaryotic serine protease, rat anionic trypsin, and various mutants created by site-directed mutagenesis have been heterologously expressed in Escherichia coli.
• The bacterial alkaline phosphatase (phoA) promoter was used to control the expression of the enzymes in an induced or constitutive fashion. The DNA coding for the eukaryotic signal peptide of pretrypsinogen was replaced with DNA coding for the phoA signal peptide.
• The phoA signal peptide successfully directs the secretion of the mammalian trypsinogen to the periplasmic space of E. coli. Active trypsin was expressed in the periplasm of E. coli by deleting the DNA coding for the activation hexapeptide of the zymogen.
• The activity of trypsin in the periplasm suggests that the enzyme is correctly activated and has folded such that the 12 cysteine residues involved in the six disulfide bonds of rat anionic trypsin have paired correctly. A transcription terminator increased the level of expression by a factor of two.
• However, increasing the copy number of the plasmid decreased the levels of expression. Localization of the active enzyme in the periplasm allows rapid screening of modified trypsin activities and facilitates the purification of protein to homogeneity and subsequently to crystallinity.