Thermal stability is one of the most desirable characteristics in the search for novel lipases. The search for thermophilic microorganisms for synthesising functional enzyme biocatalysts with the ability to withstand high temperature, and capacity to maintain their native state in extreme conditions opens up new opportunities for their biotechnological applications. Thermophilic organisms are one of the most favoured organisms, whose distinctive characteristics are extremely related to their cellular constituent particularly biologically active proteins. Modifications on the enzyme structure are critical in optimizing the stability of enzyme to thermophilic conditions. Thermostable lipases are one of the most favourable enzymes used in food industries, pharmaceutical field, and actively been studied as potential biocatalyst in biodiesel production and other biotechnology application. Particularly, there is a trade-off between the use of enzymes in high concentration of organic solvents and product generation. Enhancement of the enzyme stability needs to be achieved for them to maintain their enzymatic activity regardless the environment. Various approaches on protein modification applied since decades ago conveyed a better understanding on how to improve the enzymatic properties in thermophilic bacteria. In fact, preliminary approach using advanced computational analysis is practically conducted before any modification is being performed experimentally. Apart from that, isolation of novel extremozymes from various microorganisms are offering great frontier in explaining the crucial native interaction within the molecules which could help in protein engineering. In this review, the thermostability prospect of lipases and the utility of protein engineering insights into achieving functional industrial usefulness at their high temperature habitat are highlighted. Similarly, the underlying thermodynamic and structural basis that defines the forces that stabilize these thermostable lipase is discussed. KEY POINTS: • The dynamics of lipases contributes to their non-covalent interactions and structural stability. • Thermostability can be enhanced by well-established genetic tools for improved kinetic efficiency. • Molecular dynamics greatly provides structure-function insights on thermodynamics of lipase.
A thermostable lipase from Geobacillus zalihae strain T1 was chemically modified using propionaldehyde via reductive alkylation. The targeted alkylation sites were lysines, in which T1 lipase possessed 11 residues. Far-UV circular dichroism (CD) spectra of both native and alkylated enzyme showed a similar broad minimum between 208 and 222 nm, thus suggesting a substantial amount of secondary structures in modified enzyme, as compared with the corresponding native enzyme. The hydrolytic activity of the modified enzymes dropped drastically by nearly 15-fold upon chemical modification, despite both the native and modified form showed distinctive α-helical bands at 208 and 222 nm in CD spectra, leading us to the hypothesis of formation of a molten globule (MG)-like structure. As cooperative unfolding transitions were observed, the modified lipase was distinguished from the native state, in which the former possessed a denaturation temperature (T(m)) in lower temperature range at 61 °C while the latter at 68 °C. This was further supported by 8-anilino-1-naphthalenesulfonic acid (ANS) probed fluorescence which indicated higher exposure of hydrophobic residues, consequential of chemical modification. Based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, a small number of lysine residues were confirmed to be alkylated.
Liquid Biphasic Flotation (LBF) is an advanced recovery method that has been effectively applied for biomolecules extraction. The objective of this investigation is to incorporate the fermentation and extraction process of lipase from Burkholderia cepacia using flotation system. Initial study was conducted to compare the performance of bacteria growth and lipase production using flotation and shaker system. From the results obtained, bacteria shows quicker growth and high lipase yield via flotation system. Integration process for lipase separation was investigated and the result showed high efficiency reaching 92.29% and yield of 95.73%. Upscaling of the flotation system exhibited consistent result with the lab-scale which are 89.53% efficiency and 93.82% yield. The combination of upstream and downstream processes in a single system enables the acceleration of product formation, improves the product yield and facilitates downstream processing. This integration system demonstrated its potential for biomolecules fermentation and separation that possibly open new opportunities for industrial production.
Diacylglycerol (DAG) and monoacylglycerol (MAG) are two natural occurring minor components found in most edible fats and oils. These compounds have gained increasing market demand owing to their unique physicochemical properties. Enzymatic glycerolysis in solvent-free system might be a promising approach in producing DAG and MAG-enriched oil. Understanding on glycerolysis mechanism is therefore of great importance for process simulation and optimization. In this study, a commercial immobilized lipase (Lipozyme TL IM) was used to catalyze the glycerolysis reaction. The kinetics of enzymatic glycerolysis reaction between triacylglycerol (TAG) and glycerol (G) were modeled using rate equation with unsteady-state assumption. Ternary complex, ping-pong bi-bi and complex ping-pong bi-bi models were proposed and compared in this study. The reaction rate constants were determined using non-linear regression and sum of square errors (SSE) were minimized. Present work revealed satisfactory agreement between experimental data and the result generated by complex ping-pong bi-bi model as compared to other models. The proposed kinetic model would facilitate understanding on enzymatic glycerolysis for DAG and MAG production and design optimization of a pilot-scale reactor.
Psychrophilic microorganisms are cold-adapted with distinct properties from other thermal classes thriving in cold conditions in large areas of the earth's cold environment. Maintenance of functional membranes, evolving cold-adapted enzymes and synthesizing a range of structural features are basic adaptive strategies of psychrophiles. Among the cold-evolved enzymes are the cold-active lipases, a group of microbial lipases with inherent stability-activity-flexibility property that have engaged the interest of researchers over the years. Current knowledge regarding these cold-evolved enzymes in psychrophilic bacteria proves a display of high catalytic efficiency with low thermal stability, which is a differentiating feature with that of their mesophilic and thermophilic counterparts. Improvement strategies of their adaptive structural features have significantly benefited the enzyme industry. Based on their homogeneity and purity, molecular characterizations of these enzymes have been successful and their properties make them unique biocatalysts for various industrial and biotechnological applications. Although, strong association of lipopolysaccharides from Antarctic microorganisms with lipid hydrolases pose a challenge in their purification, heterologous expression of the cold-adapted lipases with affinity tags simplifies purification with higher yield. The review discusses these cold-evolved lipases from bacteria and their peculiar properties, in addition to their potential biotechnological and industrial applications.
Candida rugosa lipase was modified via reductive alkylation to increase its hydrophobicity to work better in organic solvents. The free amino group of lysines was alkylated using propionaldehyde with different degrees of modification obtained (49 and 86%). Far-ultraviolet circular dichroism (CD) spectroscopy of the lipase in aqueous solvent showed that such chemical modifications at the enzyme surface caused a loss in secondary and tertiary structure that is attributed to the enzyme unfolding. Using molecular modeling, we propose that in an aqueous environment the loss in protein structure of the modified lipase is owing to disruption of stabilizing salt bridges, particularly of surface lysines. Indeed, molecular modeling and simulation of a salt bridge formed by Lys-75 to Asp-79, in a nonpolar environment, suggests the adoption of a more flexible alkylated lysine that may explain higher lipase activity in organic solvents on alkylation.
5M mutant lipase was derived through cumulative mutagenesis of amino acid residues (D43E/T118N/E226D/E250L/N304E) of T1 lipase from Geobacillus zalihae. A previous study revealed that cumulative mutations in 5M mutant lipase resulted in decreased thermostability compared to wild-type T1 lipase. Multiple amino acids substitution might cause structural destabilization due to negative cooperation. Hence, the three-dimensional structure of 5M mutant lipase was elucidated to determine the evolution in structural elements caused by amino acids substitution. A suitable crystal for X-ray diffraction was obtained from an optimized formulation containing 0.5 M sodium cacodylate trihydrate, 0.4 M sodium citrate tribasic pH 6.4 and 0.2 M sodium chloride with 2.5 mg/mL protein concentration. The three-dimensional structure of 5M mutant lipase was solved at 2.64 Å with two molecules per asymmetric unit. The detailed analysis of the structure revealed that there was a decrease in the number of molecular interactions, including hydrogen bonds and ion interactions, which are important in maintaining the stability of lipase. This study facilitates understanding of and highlights the importance of hydrogen bonds and ion interactions towards protein stability. Substrate specificity and docking analysis on the open structure of 5M mutant lipase revealed changes in substrate preference. The molecular dynamics simulation of 5M-substrates complexes validated the substrate preference of 5M lipase towards long-chain p-nitrophenyl-esters.
This review is focused on the production of microbial lipases by high cell density fermentation. Lipases are among the most widely used of the enzyme catalysts. Although lipases are produced by animals and plants, industrial lipases are sourced almost exclusively from microorganisms. Many of the commercial lipases are produced using recombinant species. Microbial lipases are mostly produced by batch and fed-batch fermentation. Lipases are generally secreted by the cell into the extracellular environment. Thus, a crude preparation of lipases can be obtained by removing the microbial cells from the fermentation broth. This crude cell-free broth may be further concentrated and used as is, or lipases may be purified from it to various levels. For many large volume applications, lipases must be produced at extremely low cost. High cell density fermentation is a promising method for low-cost production: it allows a high concentration of the biomass and the enzyme to be attained rapidly and this eases the downstream recovery of the enzyme. High density fermentation enhances enzyme productivity compared with the traditional submerged culture batch fermentation. In production of enzymes, a high cell density is generally achieved through fed-batch operation, not through perfusion culture which is cumbersome. The feeding strategies used in fed-batch fermentations for producing lipases and the implications of these strategies are discussed. Most lipase-producing microbial fermentations require oxygen. Oxygen transfer in such fermentations is discussed.
Lipase-catalyzed production of palm esters by alcoholysis of palm oil with oleyl alcohol in n-hexane was performed in 2L stirred-tank reactor (STR). Investigation on the performance of reactor operation was carried out in batch mode STR with single impeller mounted on the centrally located shaft. Rushton turbine (RT) impellers provide the highest reaction yield (95.8%) at lower agitation speed as compared to AL-hydrofoil (AL-H) and 2-bladed elephant ear (EE) impellers. Homogenous enzyme particles suspension was obtained at 250 rpm by using RT impeller. At higher impeller speed, the shear effect on the enzyme particles caused by agitation has decreased the reaction performance. Palm esters reaction mixture in STR follows Newtons' law due to the linear relation between the shear stress (tau) and shear rate (dupsilon/dy). High stability of Lipozyme RM IM was observed as shown by its ability to be repeatedly used to give high percentage yield (79%) of palm esters even after 15 cycles of reaction. The process was successfully scale-up to 75 L STR (50 L working volume) based on a constant impeller tip speed approach, which gave the yield of 97.2% after 5h reaction time.
The digestion behavior of epoxy triglyceride, the main cytotoxic product of deep-frying oil, remains unknown, which may affect its biosafety. In this study, epoxy triglyceride (EGT) and triglyceride (GT) were used to reveal the effect of epoxy group on digestion. Digestibility rate analysis showed that the free fatty acids release rate of EGT was slower. To clarify this phenomenon, binding ability with salt ions in digestive juice and particle size were also been studied. Cluster size analysis indicated that epoxy group increased triglyceride particle size, resulting in smaller contact area between EGT and lipase. Interface behaviors displayed EGT decreased binding ability with salt ions in digestive juice. Spectroscopic analysis showed EGT caused the red shift of lipase peak, indicating that epoxy group changed lipase structure. Molecular dynamics simulation suggested EGT leads to loosen lipase structure. In conclusion, this study highlights that epoxy group could weaken the triglyceride digestion.
In this work, porous glass beads grafted with polyethylene glycol (PEG) were used as an adsorbent to purify lipase from Burkholderia metallica in column chromatography. The purification parameters viz. salt stability, types and concentrations of PEG and salt, pH of the binding solution, and flow rate were studied to determine the performance of the purification system in an XK16/20 column. The crude lipase was mixed with different types and concentrations of salts 1-5% (w/w) (sodium citrate, potassium citrate, and sodium acetate) and subjected to the column containing the polymeric glass bead. One-variable-at-a-time experimentation revealed that 20% (w/w) PEG 6000 g/mol impregnated glass beads with a binding solution of 5% sodium citrate at pH 7.7, a flow rate of 1.0 mL/min and extraction time of 10 min resulted in the highest purification factor and recovery yield at 3.67 and 88%, respectively. The purified lipase has 55 ∼ 60 kDa molecular mass. The outcome of the study showed PEG could be applied to modify the inert glass beads into polymeric form, providing a biocompatible and mild separation condition for lipase. Thus, PEG could be successfully applied for the purification of lipase from B. metallica fermentation broth using column chromatography.
Purified thermostable recombinant L2 lipase from Bacillus sp. L2 was crystallized by the counter-diffusion method using 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl as precipitant. X-ray diffraction data were collected to 2.7 A resolution using an in-house Bruker X8 PROTEUM single-crystal diffractometer system. The crystal belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 A. The asymmetric unit contained one single molecule of protein, with a Matthews coefficient (V(M)) of 2.85 A(3) Da(-1) and a solvent content of 57%.
In recent years, studies on psychrophilic lipases have been an emerging area of research in the field of enzymology. This study focuses on bacterial strains isolated from anthropogenically-influenced soil samples collected around Signy Island Research Station (South Orkney Islands, maritime Antarctic). Limited information on lipase activities from bacteria isolated from Signy station is currently available. The presence of lipase genes was determined using real time quantification PCR (qPCR) in samples obtained from three different locations on Signy Island. Twenty strains from the location with highest lipase gene detection were screened for lipolytic activities at a temperature of 4 °C, and from this one strain was selected for further examination based on the highest enzymatic activities obtained. Analysis of 16S rRNA sequence data of this strain showed the highest level of sequence similarity (98%) to a Pseudomonas sp. strain also isolated from Antarctica. In order to increase lipase production of this psychrophilic strain, optimisation of different parameters of physical and nutritional factors were investigated. Optimal production was obtained at 10 °C and pH 7.0, at 150 rev/min shaking rate over 36 h incubation.
Geobacillus zalihae sp. nov., which produces a putative thermostable lipase, represents a novel species, with type strain T1. The characterisation of this intrinsically thermostable T1 lipase either physicochemically or structurally is an important task. The crystallisation of T1lipase in space was carried out using a High-Density Protein Crystal Growth (HDPCG) apparatus with the vapour diffusion method, and X-ray diffraction data were collected. The microgravity environment has improved the size and quality of the crystals as compared to earth grown crystal. The effect of microgravity on the crystallisation of T1 lipase was clearly evidenced by the finer atomic details at 1.35 A resolution. Better electron densities were observed overall compared with the Earth-grown crystals, and comparison shows the subtle but distinct conformations around Na(+) ion binding site stabilized via cation-π interactions. This approach could be useful for solving structure and function of lipases towards exploiting its potentials to various industrial applications.
Three-dimensional structure of thermostable lipase is much sought after nowadays as it is important for industrial application mainly found in the food, detergent, and pharmaceutical sectors. Crystallization utilizing the counter diffusion method in space was performed with the aim to obtain high resolution diffracting crystals with better internal order to improve the accuracy of the structure. Thermostable T1 lipase enzyme has been crystallized in laboratory on earth and also under microgravity condition aboard Progress spacecraft to the ISS in collaboration with JAXA (Japanese Aerospace Exploration Agency). This study is conducted with the aims of improving crystal packing and structure resolution. The diffraction data set for ground grown crystal was collected to 1.3 Å resolution and belonged to monoclinic C2 space group with unit cell parameters a = 117.40 Å, b = 80.95 Å, and c = 99.81 Å, whereas the diffraction data set for space grown crystal was collected to 1.1 Å resolution and belonged to monoclinic C2 space group with unit cell parameters a = 117.31 Å, b = 80.85 Å, and c = 99.81 Å. The major difference between the two crystal growth systems is the lack of convection and sedimentation in microgravity environment resulted in the growth of much higher quality crystals of T1 lipase.
The psychrophilic enzyme is an interesting subject to study due to its special ability to adapt to extreme temperatures, unlike typical enzymes. Utilizing computer-aided software, the predicted structure and function of the enzyme lipase AMS8 (LipAMS8) (isolated from the psychrophilic Pseudomonas sp., obtained from the Antarctic soil) are studied. The enzyme shows significant sequence similarities with lipases from Pseudomonas sp. MIS38 and Serratia marcescens. These similarities aid in the prediction of the 3D molecular structure of the enzyme. In this study, 12 ns MD simulation is performed at different temperatures for structural flexibility and stability analysis. The results show that the enzyme is most stable at 0°C and 5°C. In terms of stability and flexibility, the catalytic domain (N-terminus) maintained its stability more than the noncatalytic domain (C-terminus), but the non-catalytic domain showed higher flexibility than the catalytic domain. The analysis of the structure and function of LipAMS8 provides new insights into the structural adaptation of this protein at low temperatures. The information obtained could be a useful tool for low temperature industrial applications and molecular engineering purposes, in the near future.
In order to characterize enzyme activity and stability corresponding to temperature effects, thermodynamic studies on commercial immobilized lipase have been carried out via enzymatic transesterification. An optimum temperature of 40 degrees C was obtained in the reaction. The decreasing reaction rates beyond the optimum temperature indicated the occurrence of reversible enzyme deactivation. Thermodynamic studies on lipase denaturation exhibited a first-order kinetics pattern, with considerable stability through time shown by the lipase as well. The activation and deactivation energies were 22.15 kJ mol(-1) and 45.18 kJ mol(-1), respectively, implying more energy was required for the irreversible denaturation of the enzyme to occur. At water content of 0.42%, the initial reaction rate and FAME yield displayed optimum values of 3.317 g/L min and 98%, respectively.
Acidolysis to incorporate oleic acid into refined, bleached and deodorized (RBD) palm olein (IV 56) using various lipases (enzymes) as catalysts to increase the oleic content of the oil was investigated. Immobilised lipases (lipase PLG, Lipozyme TL IM, Lipozyme RM IM and Novozym 435) and non-immobilised lipase (lipase PL) were used in this study to compare the effectiveness of the selected lipases in catalyzing the reaction to produce a high oleic oil. The results showed that the TAG of OLO/OOL content was increased at least 4 fold and OOO content was increased at least 3 fold when a 5% enzyme load was used. Lipase PL showed the greatest increase in tri-unsaturated triacylglycerols (TAGs) content. A pilot scale experiment conducted using TL IM enzyme, followed by recovery of the oil and fractionation allows the production of oils with varying oleic contents. A high oleic content of 56% was achievable.
Less sedimentation and convection in a microgravity environment has become a well-suited condition for growing high quality protein crystals. Thermostable T1 lipase derived from bacterium Geobacilluszalihae has been crystallized using the counter diffusion method under space and earth conditions. Preliminary study using YASARA molecular modeling structure program for both structures showed differences in number of hydrogen bond, ionic interaction, and conformation. The space-grown crystal structure contains more hydrogen bonds as compared with the earth-grown crystal structure. A molecular dynamics simulation study was used to provide insight on the fluctuations and conformational changes of both T1 lipase structures. The analysis of root mean square deviation (RMSD), radius of gyration, and root mean square fluctuation (RMSF) showed that space-grown structure is more stable than the earth-grown structure. Space-structure also showed more hydrogen bonds and ion interactions compared to the earth-grown structure. Further analysis also revealed that the space-grown structure has long-lived interactions, hence it is considered as the more stable structure. This study provides the conformational dynamics of T1 lipase crystal structure grown in space and earth condition.