Holistic engineering of Cal-A lipase chain-length selectivity identifies triglyceride binding hot-spot

Abstract

Through the application of a region-focused saturation mutagenesis and randomization approach, protein engineering of the Cal-A enzyme was undertaken with the goal of conferring new triglyceride selectivity. Little is known about the mode of triglyceride binding to Cal-A. Engineering Cal-A thus requires a systemic approach. Targeted and randomized Cal-A libraries were created, recombined using the Golden Gate approach and screened to detect variants able to discriminate between long-chain (olive oil) and short-chain (tributyrin) triglyceride substrates using a high-throughput in vivo method to visualize hydrolytic activity. Discriminative variants were analyzed using an in-house script to identify predominant substitutions. This approach allowed identification of variants that exhibit strong discrimination for the hydrolysis of short-chain triglycerides and others that discriminate towards hydrolysis of long-chain triglycerides. A clear pattern emerged from the discriminative variants, identifying the 217–245 helix-loop-helix motif as being a hot-spot for triglyceride recognition. This was the consequence of introducing the entire mutational load in selected regions, without putting a strain on distal parts of the protein. Our results improve our understanding of the Cal-A lipase mode of action and selectivity. This holistic perspective to protein engineering, where parts of the gene are individually mutated and the impact evaluated in the context of the whole protein, can be applied to any protein scaffold.

Enzymatic transesterification of urethane-bond containing ester

“…For the other tested lipases from Candida antarctica [49][50][51][52][53], Thermomyces lanuginosa [54][55][56][57], Rhizomucor Fig. 7 Biocatalyst effect: educt amounts and product yields after transesterification catalyzed by different immobilized lipases.…”

Methods for enzyme library creation: Which one will you choose?

“…We recently developed a strategy to provide flexibility in blending mutated and non-mutated, user-specified segments of a protein of interest. [26,102] This approach is based on the Golden-Gate assembly method, which makes use of Type IIs restriction enzymes that cut outside of their recognition site, allowing for seamless assembly of fragments. [110] Among its strengths (see details in Table 4), this method is quick, low-cost and allows for the introduction of highly mutated gene segments (or "parts") into the wild-type background.…”

“…They involve targeting specific residues or regions for mutation to one or many possibilities, often in a combinatorial manner, or they might imply the use of insertions and deletions in the gene sequence. [3,18,[25][26][27][28] Targeted mutagenesis is limited by the requirement for structural or biophysical knowledge of the enzyme. To address this limitation, it is common to perform exploratory rounds of randomization that inform subsequent rounds of targeted mutagenesis: Nobel laureate Frances Arnold once stated that bringing together rational design and directed evolution is the way forward to the generation of quality libraries containing a high number of sequences with the desired properties.…”

Enhancement of Lipase CAL‐A Selectivity by Protein Engineering for the Hydrolysis of Erucic Acid from Crambe Oil

“…[9][10][11] In this sense, due to the unique architecture of its substrate binding site, which forms a tunnel like cavity, Candida antarctica lipase A (CAL-A) has been proven to be an excellent candidate to undergo protein engineering approaches to tailor a desired property and several examples have been described in the literature. [12][13][14][15][16][17][18] For instance, in our previous work enrichment of erucic and gondoic fatty acids was accomplished from Crambe and Camelina seed oils. [17,18] These compounds are naturally present in high concentration in the oils of plants of the genera Cruciferae [19] and are relevant building blocks in industry for the production of surfactants, DOI: 10.1002/ejlt.201900115 polymers (e.g., erucamide), and lubricants.…”

“…Hence, the most appropriate biocatalyst could be chosen to directly fulfill the specific requirements of a determined biotransformation, or to serve as template for protein engineering to further optimize its features . In this sense, due to the unique architecture of its substrate binding site, which forms a tunnel like cavity, Candida antarctica lipase A (CAL‐A) has been proven to be an excellent candidate to undergo protein engineering approaches to tailor a desired property and several examples have been described in the literature . For instance, in our previous work enrichment of erucic and gondoic fatty acids was accomplished from Crambe and Camelina seed oils .…”