The demand for synthetic flavor ester is high, especially in the food, beverage, and cosmetic and pharmaceutical industries. It is derived from the reaction between a short-chain fatty acid and alcohol. Lipases from Antarctic bacteria have gained huge interest in the industry due to its ability react at low temperatures. The use of immobilization enzymes is one of the methods that can improve the stability of the enzyme. The current work encompasses the low temperature enzymatic synthesis of ethyl hexanoate by direct esterification of ethanol with hexanoic acid in a toluene and solvent-free system. The effects of various reaction parameters such as the organic solvent, temperature, time, substrate, substrate ratio and concentration, enzyme concentration on ethyl hexanoate synthesis were tested. Several matrices were used for immobilization and comparisons of the efficiency of immobilized enzyme with free enzyme in the synthesis of flavor ester were conducted. Ester production was optimally synthesized at 20 °C in both systems— immobilized and free enzyme. A 69% ester conversion rate was achieved after a two-hour incubation in toluene, compared to 47% in a solvent-free system for free enzyme. Immobilized AMS8 lipase showed a higher conversion of ester in toluene with respect to free-solvents, from 80% to 59%, respectively. Immobilized enzymes showed enhancement to the stability of the enzyme in the presence of the organic solvent. The development of AMS8 lipase as an immobilized biocatalyst demonstrates great potential as a cost-effective enzyme for biocatalysis and biotransformation in the food industry.

The role of phosphotriesterase as an enzyme which is able to hydrolyze organophosphate compounds cannot be disputed. Contamination by organophosphate (OP) compounds in the environment is alarming, and even more worrying is the toxicity of this compound, which affects the nervous system. Thus, it is important to find a safer way to detoxify, detect and recuperate from the toxicity effects of this compound. Phosphotriesterases (PTEs) are mostly isolated from soil bacteria and are classified as metalloenzymes or metal-dependent enzymes that contain bimetals at the active site. There are three separate pockets to accommodate the substrate into the active site of each PTE. This enzyme generally shows a high catalytic activity towards phosphotriesters. These microbial enzymes are robust and easy to manipulate. Currently, PTEs are widely studied for the detection, detoxification, and enzyme therapies for OP compound poisoning incidents. The discovery and understanding of PTEs would pave ways for greener approaches in biotechnological applications and to solve environmental issues relating to OP contamination.

Lipase plays an important role in industrial and biotechnological applications. Lipases have been subject to modification at the N and C terminals, allowing better understanding of lipase stability and the discovery of novel properties. A thermotolerant lipase has been isolated from Antarctic Pseudomonas sp. The purified Antarctic AMS3 lipase (native) was found to be stable across a broad range of temperatures and pH levels. The lipase has a partial Glutathione-S-transferase type C (GST-C) domain at the N-terminal not found in other lipases. To understand the influence of N-terminal GST-C domain on the biochemical and structural features of the native lipase, the deletion of the GST-C domain was carried out. The truncated protein was successfully expressed in E. coli BL21(DE3). The molecular weight of truncated AMS3 lipase was approximately ~45 kDa. The number of truncated AMS3 lipase purification folds was higher than native lipase. Various mono and divalent metal ions increased the activity of the AMS3 lipase. The truncated AMS3 lipase demonstrated a similarly broad temperature range, with the pH profile exhibiting higher activity under alkaline conditions. The purified lipase showed a substrate preference for a long carbon chain substrate. In addition, the enzyme activity in organic solvents was enhanced, especially for toluene, Dimethylsulfoxide (DMSO), chloroform and xylene. Molecular simulation revealed that the truncated lipase had increased structural compactness and rigidity as compared to native lipase. Removal of the N terminal GST-C generally improved the lipase biochemical characteristics. This enzyme may be utilized for industrial purposes.

Enzymatic detection of organophosphate (OP) compounds can be tailored using highly sensitive and selective enzymes in the development of biosensors. Previously, mutant (YT) phosphotriesterase (PTE) was reported to efficiently hydrolyze Sp and Rp enantiomers of phosphotriester. This study reports the use of phosphotriesterase mutant YT (YT-PTE) immobilized onto reduced graphene oxide (rGO) and fabricated onto a screen-printed carbon electrode (SPCE) for electrochemical detection of OP compounds. Immobilization of YT-PTE onto rGO was secured using N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) cross-linker, and the resulting immobilized enzyme was able to retain up to 90 of its activity. Electrochemical analysis of the SPCE/rGO/YT-PTE showed detection of paraoxon in a linear range of 1mM0.005μM with its limit of detection as low as 0.11μM. SPCE/rGO/YT-PTE exhibited high selectivity towards paraoxon and parathion and have good reproducibility. Furthermore, detection of paraoxon was also possible in a real water sample with only minor interferences.

A multi-domain oxidoreductase, carboxylic acid reductase (CAR), can catalyze the one-step reduction of carboxylic acid to aldehyde. This study aimed to immobilize bacterial CAR from a moderate thermophile Mycobacterium phlei (MpCAR). It was the first work reported on immobilizing bacterial CAR onto a polymeric support, Seplite LX120, via simple adsorption. Immobilization time and protein load were optimized for MpCAR immobilization. The immobilized MpCAR showed optimal activity at 60 °C and pH 9. It was stable over a wide range of temperatures (10 to 100 °C) and pHs (4–11), retaining more than 50% of its activity. The immobilized MpCAR also showed stability in polar solvents. The adsorption of MpCAR onto the support was confirmed by Scanning Electron Microscopy (SEM), Fourier-Transform Infrared (FTIR) spectroscopy, and Brunauer–Emmett–Teller (BET) analysis. The immobilized MpCAR could be stored for up to 6 weeks at 4 °C and 3 weeks at 25 °C. Immobilized MpCAR showed great operational stability, as 59.68% of its activity was preserved after 10 assay cycles. The immobilized MpCAR could also convert approximately 2.6 mM of benzoic acid to benzaldehyde at 60 °C. The successfully immobilized MpCAR on Seplite LX120 exhibited improved properties that benefit green industrial processes.

Immobilization is a method for making an enzyme more robust in the environment, especially in terms of its stability and reusability. A mutant phosphotriesterase (YT PTE) isolated from Pseudomonas dimunita has been reported to have high proficiency in hydrolyzing the S_p and R_p-enantiomers of organophosphate chromophoric analogs and therefore has great potential as a decontamination agent and biosensor. This work aims to investigate the feasibility of using Fuller’s earth (FE) as a YT PTE immobilization support and characterize its biochemical features after immobilization. The immobilized YT PTE was found to show improvement in thermal stability with a half-life of 24 h compared to that of the free enzyme, which was only 8 h. The stability of the immobilized YT PTE allowed storage for up to 4 months and reuse for up to 6 times. The immobilized YT PTE showed high tolerance against all tested metal ions, Tween 40 and 80 surfactants and inorganic solvents. These findings showed that the immobilized YT PTE became more robust for use especially with regards to its stability and reusability. These features would enhance the future applicability of this enzyme as a decontamination agent and its use in other suitable industrial applications.