Title: Bridges to Stability: Engineering Disulfide Bonds Towards Enhanced Lipase Biodiesel Synthesis Gihaz S, Bash Y, Rush I, Shahar A, Pazy Y, Fishman A Ref: ChemCatChem, 12:181, 2020 : PubMed
Computational design of disulfide bonds was performed for lipase from Geobacillus stearothermophilus T6 (LipT6) for enhanced methanol stability and improved biodiesel production. Thirteen double mutants comprising new cysteine pairs were screened and evaluated for their stability in 70% methanol. Superior stability was found with variant E251C/ G332C (M13) having a 5.5-fold higher hydrolysis activity and enhanced unfolding temperature (Tm) of +7.9 C in methanol compared with wild-type. Moreover, M13 converted nearly 80% waste chicken oil to biodiesel, representing a 2.4-fold improvement relative to the WT. Structural studies using X-ray crystallography confirmed the existence of the engineered disulfide bonds shedding light on the link between the bond location and backbone architecture with its stabilization impact. Rational integration of disulfide bonds is suggested to be a feasible method to promote elevated stability in organic solvents for various industrial applications such as biodiesel synthesis
Title: Filling the Void: Introducing Aromatic Interactions into Solvent Tunnels To Enhance Lipase Stability in Methanol Gihaz S, Kanteev M, Pazy Y, Fishman A Ref: Applied Environmental Microbiology, 84:, 2018 : PubMed
An enhanced stability of enzymes in organic solvents is desirable under industrial conditions. The potential of lipases as biocatalysts is mainly limited by their denaturation in polar alcohols. In this study, we focused on selected solvent tunnels in lipase from Geobacillus stearothermophilus T6 to improve its stability in methanol during biodiesel synthesis. Using rational mutagenesis, bulky aromatic residues were incorporated to occupy solvent channels and induce aromatic interactions leading to a better inner core packing. The chemical and structural characteristics of each solvent tunnel were systematically analyzed. Selected residues were replaced with Phe, Tyr, or Trp. Overall, 16 mutants were generated and screened in 60% methanol, from which 3 variants showed an enhanced stability up to 81-fold compared with that of the wild type. All stabilizing mutations were found in the longest tunnel detected in the "closed-lid" X-ray structure. The combination of Phe substitutions in an A187F/L360F double mutant resulted in an increase in unfolding temperature (Tm ) of 7 degrees C in methanol and a 3-fold increase in biodiesel synthesis yield from waste chicken oil. A kinetic analysis with p-nitrophenyl laurate revealed that all mutants displayed lower hydrolysis rates (k cat), though their stability properties mostly determined the transesterification capability. Seven crystal structures of different variants were solved, disclosing new pi-pi or CH/pi intramolecular interactions and emphasizing the significance of aromatic interactions for improved solvent stability. This rational approach could be implemented for the stabilization of other enzymes in organic solvents.IMPORTANCE Enzymatic synthesis in organic solvents holds increasing industrial opportunities in many fields; however, one major obstacle is the limited stability of biocatalysts in such a denaturing environment. Aromatic interactions play a major role in protein folding and stability, and we were inspired by this to redesign enzyme voids. The rational protein engineering of solvent tunnels of lipase from Geobacillus stearothermophilus is presented here, offering a promising approach to introduce new aromatic interactions within the enzyme core. We discovered that longer tunnels leading from the surface to the enzyme active site were more beneficial targets for mutagenesis for improving lipase stability in methanol during biodiesel biosynthesis. A structural analysis of the variants confirmed the generation of new interactions involving aromatic residues. This work provides insights into stability-driven enzyme design by targeting the solvent channel void.
Two ternary sol-gel matrices, an octyltriethoxysilane-based aliphatic matrix and a phenyltriethoxysilane (PTEOS)-based aromatic matrix, were used to immobilize a methanol-stable variant of lipase from Geobacillus stearothermophilus T6 for the synthesis of biodiesel from waste oil. Superior thermal stability of the mutant versus the wildtype in methanol was confirmed by intrinsic protein fluorescence measurements. The influence of skim milk and soluble E. coli lysate proteins as bulking and stabilizing agents in conjunction with sol-gel entrapment were investigated. E. coli lysate proteins were better stabilizing agents of the purified lipase mutant than skim milk, as evidenced by reverse engineering of the aromatic-based system. This was also shown for commercial Candida antarctica lipase B (CaLB) and Thermomyces lanuginosus lipase (TLL). Uniform, dense, and nonaggregated particles imaged by scanning electron microscopy and a small particle size of 13 mum pertaining to the system comprising PTEOS and E. coli lysate proteins correlated well with high esterification activity. Combining protein and immobilization engineering resulted in a durable biocatalyst with efficient recycling ability and high biodiesel conversion rates.
Title: Structural insights into methanol-stable variants of lipase T6 from Geobacillus stearothermophilus Dror A, Kanteev M, Kagan I, Gihaz S, Shahar A, Fishman A Ref: Applied Microbiology & Biotechnology, 99:9449, 2015 : PubMed
Enzymatic production of biodiesel by transesterification of triglycerides and alcohol, catalyzed by lipases, offers an environmentally friendly and efficient alternative to the chemically catalyzed process while using low-grade feedstocks. Methanol is utilized frequently as the alcohol in the reaction due to its reactivity and low cost. However, one of the major drawbacks of the enzymatic system is the presence of high methanol concentrations which leads to methanol-induced unfolding and inactivation of the biocatalyst. Therefore, a methanol-stable lipase is of great interest for the biodiesel industry. In this study, protein engineering was applied to substitute charged surface residues with hydrophobic ones to enhance the stability in methanol of a lipase from Geobacillus stearothermophilus T6. We identified a methanol-stable variant, R374W, and combined it with a variant found previously, H86Y/A269T. The triple mutant, H86Y/A269T/R374W, had a half-life value at 70 % methanol of 324 min which reflects an 87-fold enhanced stability compared to the wild type together with elevated thermostability in buffer and in 50 % methanol. This variant also exhibited an improved biodiesel yield from waste chicken oil compared to commercial Lipolase 100L(R) and Novozyme(R) CALB. Crystal structures of the wild type and the methanol-stable variants provided insights regarding structure-stability correlations. The most prominent features were the extensive formation of new hydrogen bonds between surface residues directly or mediated by structural water molecules and the stabilization of Zn and Ca binding sites. Mutation sites were also characterized by lower B-factor values calculated from the X-ray structures indicating improved rigidity.
