Rational engineering of low temperature activity in thermoalkalophilic Geobacillus thermocatenulatus lipase

While thermophilic enzymes have thermostability desired for broad industrial applications, they can lose activity at ambient temperatures far from their optimal. Engineering cold activity into thermophilic enzymes has the potential to broaden the range of temperatures resulting in significant activity (i.e., decreasing the temperature dependence of kcat). Even though it has been widely suggested that cold temperature enzyme activity results from active flexibility that is at odds with the rigidity necessary for thermostable enzymes; however, directed evolution experiments have shown us these properties are not mutually exclusive. In this study, rational protein engineering was used to introduce flexibility inducing mutations around the active sites of Geobacillus thermocatenulatus lipase (GTL). Two mutants were found to have enhanced specific activity compared to wild-type at temperatures between 283 K to 363 K with p-nitrophenol butyrate but not with larger substrates. Kinetics assay revealed both mutations resulted in psychrophilic traits, such as lower activation enthalpy and more negative entropy values compared to wild type in all substrates. Furthermore, the mutants had significantly improved thermostability compared to wild type enzyme, which proves that it is feasible to improve the cold activity without trade-off. Our study provides insight into the enzyme cold adaptation mechanism and design principles for engineering cold activity into thermostable enzymes.

Short Communication: Preliminary phylogenetic analysis of bacteria producing laccase isolated from Gunung Pancar, Bogor, Indonesia

Mar WW, Rohman A, Muwafiqi NH, Laras GA, Agustina D, Asmarani O, Puspaningsih NNT. 2020. Short Communication: Preliminary phylogenetic analysis of bacteria producing laccase isolated from Gunung Pancar, Bogor, Indonesia. Biodiversitas 21: 2113-2118. Interpretation of phylogenetic trees is essential in understanding relationships between organisms, as well as their characteristics, bionetwork, and even their genomic and developmental ecology. The aim of this research is to analyze the phylogenetic bacteria producing laccase isolated from Gunung Pancar Bogor, Indonesia. Phylogenetic analysis of Geobacillus kaustophilus TP-02 producing laccase was performed using alignments with all Bacillus clusters. A phylogenetic tree was constructed by the neighbor-joining (NJ) method, using the maximum likelihood parameter. Laccase was purified using ammonium sulfate precipitation to 2.67-fold. The activity of crude laccase was determined to be 93.39 U/ml using 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. Moreover, Geobacillus related to counting some Geobacillus genus such as Geobacillus thermocatenulatus, Geobacillus sp. MAS1, Geobacillus thermoleovorans, and Geobacillus zalihae, having similarity under 91% with comparison to the Geobacillus kaustophilius sequence in GenBank. Therefore, results show that G. kaustophilus TP-02 does not have any closeness with other Geobacillus genera, even though these species have the same ancestor.

Disulfide Engineered Lipase to Enhance the Catalytic Activity: A Structure-Based Approach on BTL2

Enhancement, control, and tuning of hydrolytic activity and specificity of lipases are major goals for the industry. Thermoalkaliphilic lipases from the I.5 family, with their native advantages such as high thermostability and tolerance to alkaline pHs, are a target for biotechnological applications. Although several strategies have been applied to increase lipases activity, the enhancement through protein engineering without compromising other capabilities is still elusive. Lipases from the I.5 family suffer a unique and delicate double lid restructuration to transition from a closed and inactive state to their open and enzymatically active conformation. In order to increase the activity of the wild type Geobacillus thermocatenulatus lipase 2 (BTL2) we rationally designed, based on its tridimensional structure, a mutant (ccBTL2) capable of forming a disulfide bond to lock the open state. ccBTL2 was generated replacing A191 and F206 to cysteine residues while both wild type C64 and C295 were mutated to serine. A covalently immobilized ccBTL2 showed a 3.5-fold increment in esterase activity with 0.1% Triton X-100 (2336 IU mg−1) and up to 6.0-fold higher with 0.01% CTAB (778 IU mg−1), both in the presence of oxidizing sulfhydryl agents, when compared to BTL2. The remarkable and industrially desired features of BTL2 such as optimal alkaliphilic pH and high thermal stability were not affected. The designed disulfide bond also conferred reversibility to the enhancement, as the increment on activity observed for ccBTL2 was controlled by redox pretreatments. MD simulations suggested that the most stable conformation for ccBTL2 (with the disulfide bond formed) was, as we predicted, similar to the open and active conformation of this lipase.

Ultra-Small Pd(0) Nanoparticles into a Designed Semisynthetic Lipase: An Efficient and Recyclable Heterogeneous Biohybrid Catalyst for the Heck Reaction under Mild Conditions

A novel heterogeneous enzyme-palladium (Pd) (0) nanoparticles (PdNPs) bionanohybrid has been synthesized by an efficient, green, and straightforward methodology. A designed Geobacillus thermocatenulatus lipase (GTL) variant genetically and then chemically modified by the introduction of a tailor-made cysteine-containing complementary peptide- was used as the stabilizing and reducing agent for the in situ formation of ultra-small PdNPs nanoparticles embedded on the protein structure. This bionanohybrid was an excellent catalyst in the synthesis of trans-ethyl cinnamate by Heck reaction at 65 °C. It showed the best catalytic performance in dimethylformamide (DMF) containing 10–25% of water as a solvent but was also able to catalyze the reaction in pure DMF or with a higher amount of water as co-solvent. The recyclability and stability were excellent, maintaining more than 90% of catalytic activity after five cycles of use.