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As outlined in the original proposal, our studies over the last 12 months have focused on evaluating the impact of circular permutation on the structure and function of lipase B from Candida antarctica. To evaluate the structural dynamics of the termini regions in cp283 by time-resolved fluorescence anisotropy. Our initial studies of several BODIPY-labeled variants of cp283 with cys-tags in the new N and C-terminal regions were tainted by strong interactions of the fluorophor with the protein. A second set of experiments, using fluorescein as a tag instead, resolved the issue and revealed clear differences in the termini dynamics between wild type CALB and cp283 (Fig. 1). A manuscript is in preparation for submission.


 To explore the functional significance of the new termini regions by NMR spectroscopy. Given the results from previous truncation and time-resolved fluorescence anisotropy experiments, we are pursuing a third strategy to explore the structural properties of the C-terminal region in cp283. The originally proposed idea to label the entire protein with N15,C13-enriched alanine (taking advantage of the high density of this particular amino acid in the new termini region) was abandoned due to the central role of alanine in cellular metabolism. Instead, we have adapted a new strategy, using native chemical ligation to fuse a synthetic, isotopically labeled 17-amino acid peptide of the C-terminal sequence onto cp283D17. Based on previous data, we know that the truncated cp283 expresses and folds correctly, as well as shows catalytic activity. Our initial experiments are attempting the ligation of the protein with the labeled peptide moiety via the IMPACT system (New England Biolabs, Beverly, MA). Re-design the active site cleft of cp283D7. As a consequence of circular permutation, the ternary and quaternary structures of CALB have been altered significantly. At the ternary level, the termini relocation has transformed the active site binding pocket into an wide-open binding cleft. On the quaternary structure level, dimerization has created a massive groove at the protein interface which renders itself to redesign of a new catalytic site based on the symmetric arrangement of two aspartic acids in the active site of aspartyl-proteases. Based on the crystal structure for cp283D7 and computational design studies with Rosetta software, the introduction of a single mutation at position R202 to a glutamic or aspartic acid could position the amino acid side chain in close proximity to its counterpart in the other subunit (due to the C2-symmetry of the cp283D7 dimer) and mimic the active site topology of aspartyl proteases. While such a ‘primitive' active site is unlikely to perform at the level of native proteases, the presence of even residual peptide hydrolysis activity would be of interest from a perspective of protein evolution. We are currently optimizing the conditions for protein overexpression and will perform initial activity assays with fluorescent casein as substrate. Alternative fuel production with engineered CALB & changes in the reaction pathway. We have investigated the potential practical use of our engineered lipase in the production of biodiesel fuel from waste products such as spent cooking oil and animal fats. The transesterification reaction currently uses sodium hydroxide as a catalyst which creates substantial problems in the down-stream processing. Enzyme-based catalysts can circumvent these problems, yet have a significant economical disadvantage due to the high costs for the biocatalyst. Our improvements in catalytic performance of CALB could translate into lower catalyst loads and therefore directly benefit the competitiveness of the ‘green alternative'. Consistent with our previous findings, the engineered lipase cp283 consistently outperforms the wild-type enzyme for the transesterification of pure triglycerides, as well as clean vegetable oils and spent cooking oil. Besides demonstrating the engineered enzymes' usefulness for practical application, the study also provided interesting new insight into changes in the enzyme's reaction pathway. While substrate turnover in the native CALB is limited by the rate of hydrolysis of the enzyme-acyl intermediate, our kinetic data for the circular permutated CALB and several triglyceride substrates are consistent with a shift in the rate-determining step to the first reaction step, the release of the alcohol moiety. Further experiments, using additional substrates and pre-steady state kinetics to investigate the phenomenon, are ongoing. A manuscript describing the above findings has recently been accepted for publication in the journal “Biotechnology & Bioengineering” (DOI: 10.1002/bit.22471). Additional experiments to further explore the use of protein engineering for the improvement of biocatalysts in alternative fuel production are under way.

Figure 1: Summary of time-resolved fluorescence anisotropy measurements with tagged wild type CALB and cp283. A.) The fluorescence measurements capture tumbling of the entire protein (φslow; >1 nsec) and domain flexibility (φfast <1 nsec) while the fluorophor dynamics (φfaster) lies beyond detection. B.) In the case of our engineering project, cysteine mutations were introduced at positions 286, 279, and 272 of wild type CALB and cp283 to subsequently attach the fluorescein tag. The three corresponding fluorescence decays for CALB (blue) and cp283 (green) are shown. The slower decay of cp283 at position 286 indicates a more well-defined orientation of this region, an observation consistent with observations in the x-ray structure of cp283D7. In contrast, the fast signal drop of the fluorophor at residue 279 suggests increased flexibility. Interestingly, the two decays for position 279 are identical, suggesting very similar degree of flexibility for this region of the protein. These last findings disagree with the results from the x-ray structures, yet could (together with our previous truncation data) support the idea of a flexible region with a defined (helical) secondary structure.