T1 Lipase Research Articles

Facile modulation of enantioselectivity of thermophilic Geobacillus zalihae lipase by regulating hydrophobicity of its Q114 oxyanion.

Site-directed mutagenesis of the oxyanion-containing amino acid Q114 in the recombinant thermophilic T1 lipase previously isolated from Geobacillus zalihae was performed to elucidate its role in the enzyme's enantioselectivity and reactivity. Substitution of Q114 with a hydrophobic methionine to yield mutant Q114M increased enantioselectivity (3.2-fold) and marginally improved reactivity (1.4-fold) of the lipase in catalysing esterification of ibuprofen with oleyl alcohol. The improved catalytic efficiency of Q114L was concomitant with reduced flexibility in the active site while the decreased enantioselectivity of Q114L could be directly attributed to diminished electrostatic repulsion of the substrate carboxylate ion that rendered partial loss in steric hindrance and thus enantioselectivity. The highest E-values for both Q114L (E-value 14.6) and Q114M (E-value 48.5) mutant lipases were attained at 50°C, after 12-16h, with a molar ratio of oleyl alcohol to ibuprofen of 1.5:1 and at 2.0% (w/v) enzyme load without addition of molecular sieves. Pertinently, site-directed mutagenesis on the Q114 oxyanion of T1 resulted in improved enantioselectivity and such approach may be applicable to other lipases of the same family. We demonstrated that electrostatic repulsion phenomena could affect flexibility/rigidity of the enzyme-substrate complex, aspects vital for enzyme activity and enantioselectivity of T1.

Development of a catalytically stable and efficient lipase through an increase in hydrophobicity of the oxyanion residue

In-silico and empirical site-directed substitutions of oxyanion Q114 of the wildtype T1 lipase with that of hydrophobic Leu and Met residues were carried out to afford the Q114L and Q114M lipases, respectively. Using the esterification production of menthyl butyrate as a reaction model, evaluation on the catalytic efficiency of the three lipases was performed. It was found that Leu evidently improved the catalytic activity of the Q114L lipase, achieving the highest conversions of menthyl butyrate under varying experimental conditions that may be attributable to its lower total energy when compared with both the T1 and Q114M lipases. The diminishing catalytic activity of T1 lipase observable following substitution with Met (Q114M lipase) may be due to formation of three additional cavities within the vicinity of the mutation, lesser density of the protein core and susceptibility to high temperature, particularly under prolonged reaction time. Therefore, it can be concluded that the substitution of Leu into the oxyanion-114 had rendered favorable structural changes that enhanced the catalytic activity of the T1 lipase, envisaging that such approach may also be of applied value for improving the catalytic activity of other bacterial lipases within the I.5 family.

A Comparative Analysis of Microgravity and Earth Grown Thermostable T1 Lipase Crystals Using HDPCG Apparatus

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.

A newly isolated yeast as an expression host for recombinant lipase.

Pichia guilliermondii strain SO isolated from spoiled orange was developed for use as an alternative expression host by using Pichia pastoris as the model of the experiment. This is the first study to report on the capability of P. guilliermondii SO as a host to express thermostable T1 lipase from Geobacillus zalihae. Alcohol oxidase and formaldehyde dehydrogenase promoters were present in the yeast genome. Interestingly, the recombinant yeast [SO/pPICZαB/T1-2 (SO2)] took only 30 h to reach optimal production with minimal methanol induction [1.5% (v/v)] in YPTM medium, as compared to P. pastoris, which took longer to reach its optimal condition. The purification yield of the His-tagged fusion lipase was 68.58%, with specific activity of 194.58 U/mg. The optimum temperature was 65°C at pH 9 in glycine-NaOH buffer, and it was stable up to 70°C in a wide pH range from pH 5 to 12. In conclusion, a newly isolated yeast from spoiled orange has been proven suitable for use as an expression host.

Enzymatic production of a solvent-free menthyl butyrate via response surface methodology catalyzed by a novel thermostable lipase from Geobacillus zalihae

Most substrate for esterification has the inherent problem of low miscibility which requires addition of solvents into the reaction media. In this contribution, we would like to present an alternative and feasible option for an efficient solvent-free synthesis of menthyl butyrate using a novel thermostable crude T1 lipase. We investigated the effects of incubation time, temperature, enzyme loading and substrate molar ratio and determined the optimum conditions. The high conversion of menthyl butyrate catalyzed by crude T1 lipase in a solvent-free system is greatly affected by temperature and time of the reaction media. The highest yield of menthyl butyrate was 99.3% under optimized conditions of 60 °C, incubation time of 13.15 h, 2.53 mg, 0.43% (w/w) enzyme to substrate ratio and at molar ratio of butyric anhydride/menthol 2.7:1. Hence, the investigation revealed that the thermostable crude T1 lipase successfully catalyzed the high-yield production of menthyl butyrate in a solvent-free system. The finding suggests that the crude T1 lipase was a promising alternative to overcome shortcomings associated with solvent-assisted enzymatic reactions.

Pichia pastoris as a host to overexpress the thermostable T1 lipase from Geobacillus zalihae

Pichia pastoris was known to be a good expression system in producing various heterologous proteins. The gene encoding thermostable T1 lipase from Geobacillus zalihae was cloned into pPICZαB and expressed in P. pastoris strains (GS115, X-33 and KM71H) under regulation of alcohol oxidase promoter. The expression of the gene in Escherichia coli system showed low expression level. Therefore, this study would highlight on the overexpression of the T1 lipase in yeast extracellularly. Recombinant X-33/pPICZαB/T1-2 (XPB2), GS115/pPICZαB/T1-5 (GPB5) and KM71H/pPICZαB/T1-7 (KPB7) were chosen for optimization in shake flask. Optimization strategies showed that these recombinants preferred YPTM medium with initial induction OD600nm = 7 cell biomass and 2% (v/v) methanol to provide optimal expression conditions. Hyper-resistant transformants at 3000 μg/mL zeocin gave better expression than 100 μg/mL zeocin selection. Time course study by using different inocula age showed that OD600nm = 7 expressed lipase at the highest level. The highest expression level was attained with GPB5 (88 U/ml), XPB2 (81 U/ml) and KPB7 (51U/ml). Western Blot analysis confirmed that the molecular mass of recombinant T1 lipase was 45 kDa. In conclusion, thermostable T1 lipase was successfully overexpressed by using secretory P. pastoris system with two-fold higher than E. coli system.

Crystallographic Analysis of Ground and Space Thermostable T1 Lipase Crystal Obtained via Counter Diffusion Method Approach

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.

Fatty acid specificity ofT1lipase and its potential in acylglycerol synthesis

T1 lipase has received considerable attention due to its thermostability. Fatty acid specificity of T1 lipase (crude and purified) was investigated, and its potential in the synthesis of acylglycerols was also evaluated. Fatty acid specificity of T1 lipase (crude and purified) was investigated in the esterification of fatty acids (C6:0 to C18:3), suggesting that crude and purified T1 lipase had the lowest preference for C18:0 [specificity constant (1/α) = 0.08] followed by C18:1 (1/α = 0.12) and showed the highest preference for C8:0 (1/α = 1). A structural model was constructed to briefly explore interactions between the lipase and its substrate. Furthermore, crude T1 lipase-catalysed synthesis of diacylglycerols (DAGs) and monoacylglycerols (MAGs) by esterification of glycerol with C18:1 was studied for evaluating its potential in acylglycerols synthesis. The optimal conditions were glycerol/oleic acid molar ratio 5:1, the lipase concentration 9.7 U g(-1) of substrates, water content 50 g kg(-1) of substrates and temperature 50 °C, which yielded 42.25% DAGs, 26.34% MAGs and 9.18% triacylglycerols at 2 h. DAGs and MAGs were synthesised in good yields although C18:1 (a much poorer substrate) was used. Our work demonstrates that T1 lipase, which was discovered to show 1,3-regio-selectivity, is a promising biocatalyst for lipids modification.

Typoselectivity of CrudeGeobacillussp. T1 Lipase Fused with a Cellulose‐Binding Domain and Its Use in the Synthesis of Structured Lipids

AbstractTyposelectivity of crude CBD‐T1 lipase (Geobacillussp. T1 lipase fused with a cellulose binding domain) was investigated. Multi‐competitive reaction mixtures including a set ofn‐chain fatty acids (C8:0, C10:0, C12:0, C14:0, C18:1 n‐9, C18:2 n‐6 and C18:3 n‐3) and tripalmitin‐enriched triacylglycerols were studied in hexane. The crude CBD‐T1 lipase discriminated strongly against C18:1 n‐9 [competitive factor (α) = 0.23] and showed the highest preference for C8:0 (α= 1). Utilizing the catalytic properties of crude CBD‐T1 lipase, acidolysis of soybean oil with C8:0 was selected as a model reaction to investigate the ability of the lipase to produce MLM‐type (medium‐long‐medium) structured lipids. Several reaction parameters (added water amount, reaction temperature, substrate molar ratio and reaction time) examined for incorporating C8:0 into soybean oil, the optimum conditions were: 1:3 (soybean oil/C8:0) of molar ratio, 3 mL of hexane, 50 °C of temperature, 48 h of reaction time, 20 % of crude CBD‐T1 lipase (w/w total substrates), and 7.5 % of water (w/w enzyme). Under these conditions, the incorporation of C8:0 was 29.6 mol%. The results suggest that crude CBD‐T1 lipase, which showed different fatty acid specificity profiles, is a potential biocatalyst for the modification of fats and oils.

Enzymatic hydrolysis of palm stearin to produce diacylglycerol with a highly thermostable lipase

AbstractThis paper for the first time presents the production of diacylglycerol (DAG) by hydrolysis of high‐melting oil by a highly thermostable lipase (T1 lipase). T1 lipase was expressed in recombinant Escherichia coli, and used to produce DAG by catalyzing the hydrolysis of palm stearin. The optimal conditions were obtained by a water content of 30% w/w (with respect to oil mass), T1 lipase at a concentration of 7.5 U/g (U/w, with respect to oil mass) and at 60°C. Scale‐up reaction was attempted based on the optimized conditions and 28.1% of DAG was achieved. Molecular distillation was employed for purification of DAG from the reaction mixture and the final DAG product yielded 42.5% of DAG (27.8% for 1,3‐DAG and 14.7% for 1,2‐DAG) and 57.5% of triacylglycerol (TAG). During hydrolysis, T1 lipase was discovered to show 1,3‐regiospecific activity toward TAGs. The results indicated that T1 lipase is a prospective enzyme which can be used in the modification of high‐melting oils and fats.Practical applications: Enzymatic modification of high‐melting oils is a challenging task. Here DAG production by T1 lipase‐catalyzed hydrolysis of palm stearin (PS) is reported for the first time.

Formulation and Evaluation of an Automatic Dishwashing Detergent Containing T1 Lipase

AbstractThe use of enzymes in detergent formulations is becoming popular due to the concerns about the environment. T1 lipase (E.C. 3.1.1.3) was evaluated for its stability and performance in dishwashing along with other common components of an automatic dishwashing detergent. Therefore, the process of formulating the detergent would depend on the stability of T1 lipase, which may also reflect the performance during the washing. T1 lipase was mostly stable in nonionic surfactants, especially those that were made of polyhydric alcohols. T1 lipase was also stable in a mixture of sodium carbonate and glycine. However, sodium carbonate alone destabilized T1 lipase possibly due to the interaction between carbonates and Ca2+. These results indicated that polyhydric alcohols and glycine had stabilizing effects on T1 lipase. The dishwashing performance was evaluated in term of percent soil removed. The dishwashing performance of the formulated detergent was positively affected by the increase in temperature but negatively affected by the presence of hard water, specifically Ca2+ and Mg2+. However, T1 lipase was not negatively affected by the presence of hard water, and this enzyme was enhanced by the presence of polyacrylates. The presence of Ca2+ improved the structural integrity of T1 lipase. It is generally known that most enzymes that depend on Ca2+ for their structural integrity would be greatly destabilized in the presence of metal chelators; thus, stabilizing strategies such as adding glycine would be essential to maintain enzyme activity during the wash.

Combination of Oxyanion Gln114 Mutation and Medium Engineering to Influence the Enantioselectivity of Thermophilic Lipase from Geobacillus zalihae

The substitution of the oxyanion Q114 with Met and Leu was carried out to investigate the role of Q114 in imparting enantioselectivity on T1 lipase. The mutation improved enantioselectivity in Q114M over the wild-type, while enantioselectivity in Q114L was reduced. The enantioselectivity of the thermophilic lipases, T1, Q114L and Q114M correlated better with log p as compared to the dielectric constant and dipole moment of the solvents. Enzyme activity was good in solvents with log p < 3.5, with the exception of hexane which deviated substantially. Isooctane was found to be the best solvent for the esterification of (R,S)-ibuprofen with oleyl alcohol for lipases Q114M and Q114L, to afford E values of 53.7 and 12.2, respectively. Selectivity of T1 was highest in tetradecane with E value 49.2. Solvents with low log p reduced overall lipase activity and dimethyl sulfoxide (DMSO) completely inhibited the lipases. Ester conversions, however, were still low. Molecular sieves employed as desiccant were found to adversely affect catalysis in the lipase variants, particularly in Q114M. The higher desiccant loading also increased viscosity in the reaction and further reduced the efficiency of the lipase-catalyzed esterifications.

Unlocking the mystery behind the activation phenomenon of T1 lipase: a molecular dynamics simulations approach.

The activation of lipases has been postulated to proceed by interfacial activation, temperature switch activation, or aqueous activation. Recently, based on molecular dynamics (MD) simulation experiments, the T1 lipase activation mechanism was proposed to involve aqueous activation in addition to a double-flap mechanism. Because the open conformation structure is still unavailable, it is difficult to validate the proposed theory unambiguously to understand the behavior of the enzyme. In this study, we try to validate the previous reports and uncover the mystery behind the activation process using structural analysis and MD simulations. To investigate the effects of temperature and environmental conditions on the activation process, MD simulations in different solvent environments (water and water-octane interface) and temperatures (20, 50, 70, 80, and 100°C) were performed. Based on the structural analysis of the lipases in the same family of T1 lipase (I.5 lipase family), we proposed that the lid domain comprises α6 and α7 helices connected by a loop, thus forming a helix-loop-helix motif involved in interfacial activation. Throughout the MD simulations experiments, lid displacements were only observed in the water-octane interface, not in the aqueous environment with respect to the temperature effect, suggesting that the activation process is governed by interfacial activation coupled with temperature switch activation. Examining the activation process in detail revealed that the large structural rearrangement of the lid domain was caused by the interaction between the hydrophobic residues of the lid with octane, a nonpolar solvent, and this conformation was found to be thermodynamically favorable.

Manipulation of the Conformation and Enzymatic Properties of T1 Lipase by Site-Directed Mutagenesis of the Protein Core

In silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues, Val, Met, Leu, Ile, Trp, and Phe into Gln 114 of T1 lipase. The in silico investigations accurately predicted the enzymatic characteristics of the mutants in the experimental studies and provided rationalization for some of the experimental observations. Substitution with Leu successfully improved the conformational stability and enzymatic characteristics of T1 lipase. However, replacement of Gln114 with Trp negatively affected T1 lipase and resulted in the largest disruption of protein stability, diminished lipase activity and inferior enzymatic characteristics. These results suggested that the substitution of a larger residue in a densely packed area of the protein core can have considerable effects on the structure and function of an enzyme. This is especially true when the residue is next to the catalytic serine as demonstrated with the Phe and Trp mutation.

Improvement of Thermal Stability via Outer-Loop Ion Pair Interaction of Mutated T1 Lipase from Geobacillus zalihae Strain T1

Mutant D311E and K344R were constructed using site-directed mutagenesis to introduce an additional ion pair at the inter-loop and the intra-loop, respectively, to determine the effect of ion pairs on the stability of T1 lipase isolated from Geobacillus zalihae. A series of purification steps was applied, and the pure lipases of T1, D311E and K344R were obtained. The wild-type and mutant lipases were analyzed using circular dichroism. The Tm for T1 lipase, D311E lipase and K344R lipase were approximately 68.52 °C, 70.59 °C and 68.54 °C, respectively. Mutation at D311 increases the stability of T1 lipase and exhibited higher Tm as compared to the wild-type and K344R. Based on the above, D311E lipase was chosen for further study. D311E lipase was successfully crystallized using the sitting drop vapor diffusion method. The crystal was diffracted at 2.1 Å using an in-house X-ray beam and belonged to the monoclinic space group C2 with the unit cell parameters a = 117.32 Å, b = 81.16 Å and c = 100.14 Å. Structural analysis showed the existence of an additional ion pair around E311 in the structure of D311E. The additional ion pair in D311E may regulate the stability of this mutant lipase at high temperatures as predicted in silico and spectroscopically.

Engineering catalytic efficiency of thermophilic lipase from &amp;lt;i&amp;gt;Geobacillus zalihae&amp;lt;/i&amp;gt; by hydrophobic residue mutation near the catalytic pocket

In-silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues; Val, Met, Leu, Ile, Trp and Phe into the oxyanion Q114 of T1 lipase. We hypothesized that the oxyanion Q114, involved in substrate binding is also associated with modulation of conformational stability and in conferring specific enzyme attributes. The insilico investigations accurately predicted the quality of the protein packing in some of the variants. Our study found by altering the hydrophobicity of the oxyanion 114, remarkably altered enzyme conformational stability and catalytic attributes. Substitution with Leu resulted improvements in four out of the six tested characteristics. The hydrophobic Leu might have improved local structure folding and increased hydrophobic interactions with other residues in the vicinity of the mutation. The Met variant showed higher activity over the wild-type in hydrolyzing a wider range of natural oils. The bulky amino acids, Phe and Trp negatively affected T1 lipase and resulted in the largest disruption of protein stability and inferior enzyme characteristics. We have successfully illustrated that a single point residue changes at oxyanion 114 could result in a myriad of enzyme attributes, which implied there was some interplay between hydrophobicity and conformation for lipase catalytic functions.

2PT142 Theoretical evaluation of structural stability of the active site of T1 lipase : cation-π vs. water-π interactions(The 50th Annual Meeting of the Biophysical Society of Japan)

2PT142 Theoretical evaluation of structural stability of the active site of T1 lipase : cation-π vs. water-π interactions(The 50th Annual Meeting of the Biophysical Society of Japan)

Structural Instability of the Active Site of T1 Lipase Induced by Replacement of Na(+) with Water Complexed with the Phenylalanine Aromatic Ring.

The crystallographic analysis of T1 lipase suggested an interaction between Na(+) and the aromatic ring of Phe16 in the active site. However, experimental approaches could not dismiss the possible presence of water instead of Na(+). Our previous molecular dynamics (MD) simulations suggested that the significantly large enthalpy gain of the Na(+)-π interaction was required to preserve the catalytic core structure of T1 lipase. In this study, to examine the effects of water, we performed further MD simulations of T1 lipase involving the water-π interaction, instead of the Na(+)-π interaction, exploiting various force fields, such as ff99, ff02, and an accurate potential field to describe the water-π interaction, which was generated using our recently developed scheme (referred to as the grid-based energy representation). The analyses revealed that the water-π complex was unstable in the catalytic core of T1 lipase even when the accurate potential of the water-π complex represented by the grid-based energy function was employed in the MD simulations and led to the disruption of the coordinated structure. In contrast, the catalytic core structure of T1 lipase involving the Na(+)-π complex was significantly stable in the 10 ns MD simulation using the grid-based energy representation of the Na(+)-π interaction. Thus, the possible presence of water may be excluded, and our previous proposal concerning the functional role of the structural element involving the Na(+)-π interaction in the catalytic site of T1 lipase has unambiguously been confirmed. Further, the strong coordination of Na(+) and Nε of His358 was also shown to be substantial to preserve the core structure of the catalytic site.

Structural Instability of the Active Site of T1 Lipase where Na +-π Interaction is Replaced with Water-π Complex

The cation-π interaction is one of the strongest noncovalent forces. However, its biological role has been unknown, since few structures containing cation-π interaction have been determined in biological systems. Matsumura, et al. determined the crystal strcture of T1 lipase and found interactions between Na+ and the aromatic ring of Phe16 in the active site. However, this Na+-π interaction remained to be discussed whether it really exists or not. To investigate structural stability of Na+-Phe interactions, we performed molecular dynamics (MD) simulations of T1 lipase.It is well known that the current conventional force fields cannot estimate the cation-π interaction correctly, whereas ab initio calculations require huge computational costs for the MD simulations. Accordingly, we developed a novel scheme to calculate the interaction energy with a high accuracy compared with the CCSD(T) level, and with a low calculation cost compared with the force field calculations. The result of our calculations definitely showed that the large enthalpy gain of the Na+-π interaction was required to preserve the catalytic core structure. Since experimental approaches could not dismiss the possible presence of water instead of Na+ in the active site of T1 lipase, we also examined the effects of water by performing MD simulations. Our analyses revealed that the water-π complex was unstable and led to the collapse of the coordinated structure of the active site. Thus, we concluded that the possible presence of water may be excluded. Our novel scheme is currently the only way to perform long-time MD simulations involving cation-π interactions with reasonable computational cost and with high accuracy. Based on this analysis, we propose a novel structural and functional element indentified in the active site of T1 lipase.

Reductive Alkylation Causes the Formation of a Molten Globule-Like Intermediate Structure in Geobacillus zalihae Strain T1 Thermostable 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.