The use of immobilized enzymes as biocatalysts has great potential to improve the efficiency and environmental sustainability of many industrial processes. Here, we report a novel approach that allows for the direct production of a highly active immobilized lipase within the bacterium Bacillus thuringiensis. Cry3Aa-lipA crystals were generated by genetically fusing Bacillus subtilis lipase A to Cry3Aa, a protein that naturally forms crystals in the bacteria. The crystal framework significantly stabilized the lipase against denaturation in organic solvents and high temperatures, resulting in a highly efficient fusion crystal that could catalyze the conversion of triacylglycerols to fatty acid methyl ester biodiesel to near-completion over 10 cycles. The simplicity and robustness of the Cry-fusion crystal (CFC) immobilization system could make it an appealing platform for generating industrial biocatalysts for multiple bioprocesses.

Cry3Aa is a protein that forms crystals naturally in the bacterium Bacillus thuringiensis. Here we report that coexpression of Cry3Aa and a Proteus mirabilis lipase without recombinant fusion results in the efficient passive entrapment of the lipase within the nanoporous channels of the resulting crystals. This Cry3Aa crystal-mediated entrapment provides multiple benefits to the lipase including a high enzyme loading, significantly improved thermostability, increased proteolytic resistance, and the ability to be utilized as a recyclable biodiesel catalyst. These characteristics, along with its greatly simplified method of isolation, highlight the potential of Cry3Aa crystal-mediated enzyme entrapment for use in biocatalysis and other biotechnological applications.N anoporous crystalline materials are invaluable frameworks for gas storage, 1,2 molecular separations, 3,4 catalysis, 5−7 and medical applications, 8−10 among others. 11−13 Such materials can be synthesized from metal−organic frameworks, 14,15 activated carbon, 16,17 zeolites, 18 aerogels, 19,20 and even biological components such as DNA 21,22 and proteins. 12,23,24 Each of these systems has its advantages, but the attraction of using protein-based frameworks is their ability to be modified by genetic manipulation with spatial precision, their biodegradability, and their diversity and tunability given the plethora of canonical and noncanonical amino acids 25,26 available.Recently, there has been growing interest in using nanoporous crystalline materials to entrap enzymes for the fabrication of stable and recyclable catalysts. 27−29 In these frameworks it is essential that the scaffold contains nanopores large enough to accommodate protein biomolecules. 30

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Background We have recently developed a one-step, genetically encoded immobilization approach based on fusion of a target enzyme to the self-crystallizing protein Cry3Aa, followed by direct production and isolation of the fusion crystals from Bacillus thuringiensis . Using this approach, Bacillus subtilis lipase A was genetically fused to Cry3Aa to produce a Cry3Aa–lipA catalyst capable of the facile conversion of coconut oil into biodiesel over 10 reaction cycles. Here, we investigate the fusion of another lipase to Cry3Aa with the goal of producing a catalyst suitable for the conversion of waste cooking oil into biodiesel. Results Genetic fusion of the Proteus mirabilis lipase (PML) to Cry3Aa allowed for the production of immobilized lipase crystals (Cry3Aa–PML) directly in bacterial cells. The fusion resulted in the loss of PML activity, however, and so taking advantage of its genetically encoded immobilization, directed evolution was performed on Cry3Aa–PML directly in its immobilized state in vivo. This novel strategy allowed for the selection of an immobilized PML mutant with 4.3-fold higher catalytic efficiency and improved stability. The resulting improved Cry3Aa–PML catalyst could be used to catalyze the conversion of waste cooking oil into biodiesel for at least 15 cycles with minimal loss in conversion efficiency. Conclusions The genetically encoded nature of our Cry3Aa-fusion immobilization platform makes it possible to perform both directed evolution and screening of immobilized enzymes directly in vivo. This work is the first example of the use of directed evolution to optimize an enzyme in its immobilized state allowing for identification of a mutant that would unlikely have been identified from screening of its soluble form. We demonstrate that the resulting Cry3Aa–PML catalyst is suitable for the recyclable conversion of waste cooking oil into biodiesel. Electronic supplementary material The online version of this article (10.1186/s13068-019-1509-5) contains supplementary material, which is available to authorized users.

The N-terminal domain of Cry3Aa can be used to generate sub-micron particles Genetic fusion of functional peptides to protein particles targets them to A549 cells Myoglobin is an effective oxygen carrier for delivery of O 2 into hypoxic cancer cellsTargeted myoglobin delivery to hypoxic cancer cells increased their radiosensitivity