Study on the potential of cold-active lipases from psychrotrophic fungi for detergent formulation

Lipases from psychrotrophic fungal isolates BPF4 and BPF6 identified as Penicilium canesense and Pseudogymnoascus roseus respectively were characterized for their compatibility towards laundry detergent. BPF4 and BPF6 lipases showed maximum activity at pH 11 and 9 respectively and at 40?°C. The residual activities at 20?°C and 4?°C of BPF4 lipase were 35% and 20% and of BPF6 lipase were 70% and 20?°C respectively. Both the enzymes were stable at 4?°C, 20?°C and 40?°C for 2?h losing at the most 20% of activities. Both the enzymes were metalloenzymes with activity enhancement by nearly threefold by Ca²⁺. Contrary to BPF6 lipase, BPF4 enzyme was not stimulated by EDTA nor inhibited, rather stimulated by SDS and Triton X-100 by 125% and 330% respectively. Both the lipases showed minor to moderate inhibition by NaClO₃ and H₂O₂, and exhibited nearly 90% residual activity after 1?h of incubation in selected detergent brands thus indicating potential for their inclusion in detergent formulation thereby facilitating cold-washing as a step towards mitigation of climate change.     

The crystal structure of 2-deoxy-β-d-arabino-hexopyranose at −150°

2-Deoxy-β-d-arabino-hexopyranose, C₆H₁₂O₅, is orthorhombic, P2₁2₁2₁, with cell dimensions at ?150° [20°], a = 6.484(2) [6.510(3)], b = 10.364(2) [10.427(4)], c = 11.134(3) [11.153(5)] Å, V = 748.2 [757.1] ų, Z = 4, D_x = 1.457 [1.440], and D_m = [1.455] g.cm^?3. The intensities of 1269 reflections were measured by using MoKα radiation. The structure was solved by direct methods, and refined by full-matrix least-squares, with anisotropic, thermal parameters for the carbon and oxygen atoms, and isotropic parameters for the hydrogen atoms. The pyranose has the ⁴C₁(d) conformation, with puckering parameters Q = 0.563 Å, θ = 3.9°, and ? = 350.3°. The departure from ideality is very small, and less than that in β-d-glucopyranose, Q = 0.584 Å and θ = 6.9°. The β-glycosidic, CO bond is short, 1.383(4) Å, and the OCOH torsion angle is ?87°, consistent with the anomeric effect. The hydrogen-bonding scheme consists of infinite chains, with side chains terminating at a ring-oxygen atom.     

Biochemical properties of the alkaline lipase of Bacillus flexus XJU-1 and its detergent compatibility

The possibility of using Bacillus flexus XJU-1 lipase in detergent preparations was studied. The enzyme was monomeric protein as confirmed by liquid chromatography-mass spectrometry and its molecular weight was 15.95 kDa. The lipase showed optimum activity at pH 10.0 and was 100% stable for 24 h at pH 10.0 and 11.0. It exhibited maximum activity at 70°C and retained more than 70% of the initial activity at 60, 70 and 80°C for 24 h. The activity was stimulated by Ca²⁺, Ba²⁺, Mg²⁺ and Co²⁺, whereas 50% of the initial activity was lost with Fe³⁺ and Hg²⁺. The activity was inhibited by 10 mM N-bromosuccinimide and tosyl-L-lysylchloromethylketone, while N-ethylmaleimide, phenylmethylsulphonylfluoride and urea did not show any effect. The enzyme significantly hydrolysed olive, cottonseed, sunflower, groundnut, and gingelly oils. With p-nitrophenyl palmitate, V_max and K_m were 62.5 U/mL and 2.25 mM, respectively. The lipase maintained its stability in Tween-80, Triton-100 and H₂O₂ at 1%, but an activation of 10% and a reduction of 15% in relative activity were observed with NaClO and sodium dodecyl sulphate, respectively. The enzyme retained maximum storage stability for 20 days at ?20, 4 and 30°C. In the presence of 0.7% (w/v) Ariel, Henko, Super wheel, Tide plus and Rin, a retention of more than 84.90% initial activity was recorded after 24 h at 60°C. The supplementation of the lipase to the detergents improved the olive oil stain removal. These properties suggested the present enzyme as a potential additive for detergent preparations.     

Coordination chemistry of 7,9-disubstituted 6-oxopurine metal compounds. 4. Platinum(II) coordination at N(1). Molecular and crystal structure of [(ethylenediamine)bis(7,9-dimethylhypoxanthine)platinum(II)] hexafluorophosphate

The preparation and molecular and crystal structure of the complex [(ethylenediamine)bis(7,9,-dimethylhypoxanthine)platinum(II)] hexafluorophosphate, [Pt(C₂H₈N₂)(C₇H₈N₄O)₂] (PF₆)₂, are reported. The complex crystallizes in the monoclinic system, space group C2/c, with a = 12.334(2)Å, b = 10.256(2)Å, c = 22.339(3)Å, β = 101.31(1)°, V = 2771.0ų, Z = 4, D_measd = 2.087(3) g cm^?3, D_calc = 2.094 g cm^?3. Intensities for 3992 symmetry-averaged reflections were collected in the θ-2o scan mode on an automated diffractometer employing graphite-monochromatized MoKα radiation. The structure was solved by standard heavy-atom Patterson and Fourier methods. Full matrix least-squares refinement led to a final R value of 0.051. Both the ethylenediamine chelate and the PF₆^? anion are disordered. The primary coordination sphere about the Pt(II) center is approximately square planar with the bidentate ethylenediamine ligand and the N(1) atoms [Pt(II) ? N(1) = 2.020(5)Å] of two 7,9-dimethylhypoxanthine bases (related by a crystallographic twofold axis of symmetry) occupying the four coordination sites. The exocyclic O(6) carbonyl oxygen atoms of the two 7,9-dimethylhypoxanthine ligands participate in intracomplex hydrogen bonding with the amino groups of the ethylenediamine chelate [N(ethylenediamine) ? O(6) = 2.89( )Å]. The observed Pt ? O(6) intramolecular distances of 3.074(6)Å are similar to those found in other Pt(II) N(1)-bound 6-oxopurine complexes and in several Pt(II) N(3)-bound cytosine systems.     

Functional and structural studies of a novel cold-adapted esterase from an Arctic intertidal metagenomic library

A novel cold-adapted lipolytic enzyme gene, est97, was identified from a high Arctic intertidal zone sediment metagenomic library. The deduced amino acid sequence of Est97 showed low similarity with other lipolytic enzymes, the maximum being 30 % identity with a putative lipase from Vibrio caribbenthicus. Common features of lipolytic enzymes, such as the GXSXG sequence motif, were detected. The gene product was over-expressed in Escherichia coli and purified. The recombinant Est97 (rEst97) hydrolysed various ρ-nitrophenyl esters with the best substrate being ρ-nitrophenyl hexanoate (K _m and k _cat of 39 μM and 25.8 s^?1, respectively). This esterase activity of rEst97 was optimal at 35 °C and pH 7.5 and the enzyme was unstable at temperatures above 25 °C. The apparent melting temperature, as determined by differential scanning calorimetry was 39 °C, substantiating Est97 as a cold-adapted esterase. The crystal structure of rEst97 was determined by the single wavelength anomalous dispersion method to 1.6 Å resolution. The protein was found to have a typical α/β-hydrolase fold with Ser144-His226-Asp197 as the catalytic triad. A suggested, relatively short lid domain of rEst97 is composed of residues 80–114, which form an α-helix and a disordered loop. The cold adaptation features seem primarily related to a high number of methionine and glycine residues and flexible loops in the high-resolution structures.     

Catalytic characteristics of a sn-1(3) regioselective lipase from Cordyceps militaris

A total of 39 agricultural products were screened for natural sources of lipases with distinctive positional specificity. Based on this, Cordyceps militaris lipase (CML) was selected and subsequently purified by sequential chromatography involving anion-exchange, hydrophobic-interaction, and gel-permeation columns. As a result of the overall purification procedure, a remarkable increase in the specific activity of the CML (4.733 U/mg protein) was achieved, with a yield of 2.47% (purification fold of 94.54). The purified CML has a monomeric structure with a molecular mass of approximately 62 kDa. It was further identified as a putative extracellular lipase from C. militaris by the partial sequence analysis using ESI-Q-TOF MS. In a kinetic study of the CML-catalyzed hydrolysis, the values of V_max, K_m, and k_cat were determined to be 4.86 μmol·min⁻¹·mg⁻¹, 0.07 mM, and 0.29 min⁻¹, respectively. In particular, the relatively low K_m value indicated that CML has a high affinity for its substrate. With regard to positional specificity, CML selectively cleaved triolein at the sn-1 or 3 positions of glycerol backbone, releasing 1,2(2,3)-diolein as the major products. Therefore, CML can be considered a distinctive biocatalyst with sn-1(3) regioselectivity. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2744, 2019.     

Immobilisation of Candida rugosa lipase on polyhydroxybutyrate via a combination of adsorption and cross-linking agents to enhance acylglycerol production

In the present study, a combination of immobilisation processes was utilised to prepare robust biocatalysts. First, lipase from Candida rugosa was adsorbed on polyhydroxybutyrate (PHB) particles, followed by cross-linking with glutaraldehyde. Conditions for creating immobilised lipase involved the addition of 0.6 M glutaraldehyde and 45 U mL⁻¹ lipase while mixing at 150 rpm (4 °C) for 30 min. These conditions produced the highest yield of immobilised lipase (92 %) and the highest levels of activity (1.94 mg g⁻¹ support). At 40 °C and pH 9 the immobilised enzyme was optimally active with a K_m and V_maxat 1.2 mM and 2.5 × 10^-3 mmol min⁻¹, respectively. The use of immobilised lipase improved thermal stability, storage stability, and reusability.The immobilised lipase retained 80 % of its activity after incubation at 30–60 °C for 2 h and 4 °C for 30 d in 0.2 M sodium phosphate buffer (pH 7.0). Moreover, the immobilised enzyme retained 50 % of its activity after more than 14 cycles under optimal conditions. The immobilised lipase was used to produce monoacylglycerol MAG. The existence of a carbonyl group at 1,743 and 1,744 cm⁻¹ was identified using attenuated total reflectance (ATR)-Fourier transformed infrared spectroscopy. Results showed that 48 % MAG was produced.     

Topography of cyclodextrin inclusion complexes : Part II. The iodine-cyclohexa-amylose tetrahydrate complex; its molecular geometry and cage-type crystal structure

Iodine-cyclohexa-amylose tetrahydrate [(C₆H₁₀O₅)₆ ·I₂·d4H₂O] crystallizes in the orthorhombic space-group P2₁2₁2₁, a  14.240 Å, b  36.014 Å, c  9.558 Å. The structure was solved by heavy-atom techniques and refined by least-squares methods to a conventional discrepancy index R  0.148 for the 2872 observed data. The six d-glucose residues are in the C1 chair conformation; the conformational angles vary in magnitude from 45 to 66°, the angles O(5)-C(5)-C(6)-O(6) are close to · 70°, and the six O(4) atoms are almost coplanar (r.m. s. displacement 0.13 Å). Only four of the six O(2) ?O(3) intramolecular hydrogen bonds have formed, which renders the molecule less symmetrical and more conical-shaped than in the previously determined α-cyclodextrin-potassium acetate complex. The iodine molecule is coaxial with the cyclohexa-amylose molecule. The I-I distance is a conventional 2.677 Å. Close interactions between the iodine atoms and the host molecule comprise carbon atoms C(5) and C(6) and oxygen atoms O(4), with interatomic distances all equal to or greater than van der Waals contacts. Intermolecular, almost-linear, short contacts O ? I-I?O with I?O distances of 3.22 and 3.07 Å indicate attractive interaction.The molecules are arranged in herring-bone “cage-type” fashion, with the four water molecules as space-filling mediators; the structure is held together by an intricate network of hydrogen bonds.     

Homologous expression,purification and characterization of a novel high-alkaline and thermal stable lipase from Burkholderia cepacia ATCC 25416

The lipase secreted by Burkholderia cepacia ATCC 25416 was particularly attractive in detergent and leather industry due to its specific characteristics of high alkaline and thermal stability. The lipase gene (lipA), lipase chaperone gene (lipB), and native promoter upstream of lipA were cloned. The lipA was composed of 1095 bp, corresponding to 364 amino acid residues. The lipB located immediately downstream of lipA was composed of 1035 bp, corresponding to 344 amino acid residues. The lipase operon was inserted into broad host vector pBBRMCS1 and electroporated into original strain. The homologous expression of recombinant strain showed a significant increase in the lipase activity. LipA was purified by three-step procedure of ammonium sulfate precipitation, phenyl-sepharose FF and DEAE-sepharose FF. SDS-PAGE showed the molecular mass of the lipase was 33 kDa. The enzyme optimal temperature and pH were 60 °C and 11.0, respectively. The enzyme was stable at 30–70 °C. After incubated in 70 °C for 1 h, enzyme remained 72% of its maximal activity. The enzyme exhibited a good stability at pH 9.0–11.5. The lipase preferentially hydrolyzed medium-chain fatty acid esters. The enzyme was strongly activated by Mg²⁺, Ca²⁺, Cu²⁺, Zn²⁺, Co²⁺, and apparently inhibited by PMSF, EDTA and also DTT with SDS. The enzyme was compatible with various ionic and non-ionic surfactants as well as oxidant H₂O₂. The enzyme had good stability in the low- and non-polar solvents.     

UPregulated single-stranded DNA-binding protein 1 induces cell chemoresistance to cisplatin in lung cancer cell lines

In order to understand the molecular basis of cold adaptation, we have used directed evolution to transform a thermophilic lipase LipR1 into its psychrophilic counterpart. A single round of random mutagenesis followed by screening for improved variants yielded a mutant with single-point mutation LipR1M1 (S130T), with optimum activity at 20?°C. Its activity at 50?°C is only 20% as compared to wild type (100%). It showed catalytic rate constant (k _cat) 3 times higher and a catalytic efficiency (k _cat/K _m) 4 times that of wild type. Circular dichroism and fluorescence studies also supported our observation of mutant structural flexibility. Structure analysis using homology models showed that Threonine 130 is exposed to solvent and has lost H-bond interaction with neighboring amino acid, thereby increasing flexibility of this lipase structure.     

EXAFS studies on the PN and POX states of the P-clusters in nitrogenase

The MoFe protein of nitrogenase is an α₂β₂ tetramer that contains two each of two different types of metal centers, the FeMo-cofactor and the P-clusters. The function of the P-clusters is believed to be to accept electrons from the Fe protein of nitrogenase and to donate them to the FeMo-cofactor. We have studied the P-clusters of Azotobacter vinelandii nitrogenase in both the P^N and P^OX states utilizing Fe K-edge X-ray absorption spectroscopy. Since the MoFe holoprotein contains the seven iron FeMo-cofactor centers in addition to P-clusters, we have utilized a FeMo-cofactor-deficient MoFe protein synthesized by the Δnif H strain DJ54. That MoFe protein is an α₂β₂ tetramer that contains P-clusters by the criteria of metal analysis, CD spectroscopy, cluster extrusion, and electrochemical reduction of the P^OX state. Several important results have emerged from our XAS studies. The first shell Fe-S coordination shows the same average Fe-S distance (2.26?Å) in both states. The second coordination shells could only be well fit using two different Fe-Fe contributions. In both states, short Fe-Fe components with distances of 2.57?Å and 2.42?Å for the P^N and P^OX states, respectively, were required to complement longer 2.75?Å and 2.70?Å distances. Understanding of the P-cluster structure is essential if we are to make advances in understanding the role of the P-clusters and their participation in electron transfer through the nitrogenase system.     

Molybdenum active centre of DMSO reductase from Rhodobacter capsulatus: crystal structure of the oxidised enzyme at 1.82-Å resolution and the dithionite-reduced enzyme at 2.8-Å resolution

The 1.82-Å X-ray crystal structure of the oxidised (Mo^(VI)) form of the enzyme dimethylsulfoxide reductase (DMSOR) isolated from Rhodobacter capsulatus is presented. The structure has been determined by building a partial model into a multiple isomorphous replacement map and fitting the crystal structure of DMSOR from Rhodobacter sphaeroides to the partial model. The enzyme structure has been refined, at 1.82-Å resolution, to an R factor of 14.8% (R _free?=?18.4%). The molybdenum is coordinated by seven ligands: four dithiolene sulfurs, Oγ of Ser147 and two oxo groups. The four sulfur ligands, at a metal-sulfur distance of 2.4?Å or 2.5?Å, are contributed by the two molybdopterin guanine dinucleotide (MGD) cofactors. The coordination sphere of the molybdenum is different from that in previously reported structures of DMSOR from R. sphaeroides and R. capsulatus. The 2.8-Å structure of DMSOR, reduced by addition of sodium dithionite, is also described and differs from the structure of the oxidised enzyme by the removal of a single oxo ligand from the molybdenum coordination sphere. A structure, at 2.5-Å resolution, has also been obtained from crystals soaked in mother liquor buffered at pH?7.0. No differences are observed in the structure at pH?7 when compared with the native crystal structure at pH?5.5.     

Synthesis of cinnamyl acetate catalysed by highly reusable cotton-immobilized Pseudomonas fluorescens lipase

Cinnamyl acetate as an important fragrance ingredient could be synthesized by lipase-catalysed transesterification in organic systems, but enzyme proteins tended to denature and inactivate for no water lubrication. To improve the non-aqueous stability of lipases, absorbent cotton was taken as an alternative “water” phase to stabilize enzyme proteins. In a mass ratio of 1:1, Pseudomonas fluorescens lipase was immobilized on cotton fibres by physical absorption in a column glass bottle, forming a facile cotton-lipase bioreactor in which the transesterification between cinnamyl alcohol and vinyl acetate processed efficiently. From the molar conversions after reaction for 2?h at 37?°C and 160?rpm, the ability of cotton-lipase to transform substrate was more than 5-folds of native lipase. And even in static state and at 4?°C, the conversion of reaction catalysed by cotton-PFL had 11-fold increase relative to native lipase after 8?h. Recycles showed that the cotton-lipase had an extra-long half-life of activity (t_1/2?=?693?h) and a negligible decay rate in the ability to transform substrate (D_r?=?0.08% h^?1). All these showed that this lipase had been effectively activated and stabilized by cotton fibres for the numerous hydroxyl groups and fluffy structure.     

A novel control of enzymatic enantioselectivity through the racemic temperature influenced by reaction media

The influence of reaction media on the racemic temperature (T_r) in the lipase-catalyzed resolution of ketoprofen vinyl ester was investigated. An effective approach to the control of the enzymatic enantioselectivity and the prediction of the increasing tendency was developed based on the T_r influenced by reaction media. The T_r for the resolution catalyzed by Candida rugosa lipase (CRL) was found at 29 °C in aqueous and S-ketoprofen was obtained predominantly at 40 °C. However, CRL showed R-selectivity at 40 °C in diisopropyl ether because the T_r was changed to 56 °C. CRL, lipase from AYS Amano^® and Mucor javanicus lipase were further applied for the investigation of the enzymatic enantioselectivity in dioxane, DIPE, isooctane and their mixed media with water. The effects of the reaction medium on T_r could be related to the solvent hydrophobicity, the lipase conformational flexibility and the interaction between the enantiomers and the lipase.     

Synthesis and crystal structure of bis(tetramethylammonium) aquotetra-fluorodioxouranate(VI) dihydrate, [(CI3)4 N]2[UO2 F4(H2 O)]·2H2O

The title compound has been synthesized and subjected to crystal structure analysis. M_r = 548.50, m.p. 108.1 °C (decom.), orthorhombic, Im2m,a = 7.006(2), b = 8.938(2), c = 13.619(2) Å V = 852.8(3) ų, Z = 2, D_x = 2.136, D_m, (flotation in CCl₄/CH₂I₂) = 2.128 g cm^?3, λ(Mo-Kα) = 0.71069 Å, μ = 90.79 cm^?1, F(000) = 519.89, T = 295 K, final R_F = 0.036 and R_G = 0.044 for 566 observed reflections. The discrete [UO₂F₄(H₂0)]^2? anion has site symmetry m2m, its virtually linear uranyl moiety being surrounded by fluoro and aquo ligands occupying the vertices of a pentagon in the equatorial plane. Watet molecules serve to link the complex anions by hydrogen bonds into layers, between which the organic cations are accommodated.     

Application of advanced materials as support for immobilisation of lipase from Candida rugosa

Ni/Al-layered double hydroxides (Ni-LDHs) and Ni/Al-sodium dodecyl sulfonate layered double hydroxide nanocomposites (Ni-SDS-LDHs) with a molar ratio of Ni:Al (4:1) have been prepared by a co-precipitation (or salt-base) method. Their structures were determined using Powder X-Ray Diffractometer (PXRD) and the spectra showed that basal spacings for Ni-LDHs and Ni-SDS-LDHs synthesised were around 8.1?Å and 34.8?Å, respectively. Lipase from Candida rugosa was immobilised onto these advanced materials, by physical adsorption. The activity of immobilised lipase was investigated through esterification of palmitic acid and isopropyl alcohol in hexane. The effects of reaction temperature, thermostability, stability in organic solvent, operational stability, leaching and storage studies of the immobilised lipase were investigated. These biocatalysts exhibited higher activities than the native lipase with an optimum temperature of 40°C. Immobilised lipases showed higher storage stability than native lipase (up to 60 days) and during operational studies at 30°C for 5?h, more than 50% of its activity was retained. Leaching studies showed that physical adsorption is suitable for the attachment of enzymes onto LDHs.     

Substrate selectivity of bacterial monoacylglycerol lipase based on crystal structure

Lipases, which are conserved from bacteria to mammals, catalyze the hydrolysis of acylglycerol to free fatty acids and glycerol. Monoacylglycerol lipase (MGL) specifically catalyzes the hydrolysis of monoacylglycerol. Although there have been numerous studies of the structure of lipases, there have been few studies of MGL. Here, we report the crystal structure of authentic MGL isolated from Bacillus sp. H257 (bMGL). The crystal diffracts to 1.96 Å resolution. It belongs to space group P2₁2₁2, and the unit cell parameters are a = 99.7 Å, b = 106.1 Å and c = 43.0 Å. As in other lipases, three structural features for lipase activity are conserved in bMGL: the glycine-X-serine-X-glycine motif, catalytic triad and cap region. The structure of bMGL appears to be closed, as the cap region covers the active site entrance. The isolated bMGL hydrolyzed 2-AG, a known human MGL-specific substrate. Based on a 2-AG bound model, we discuss the substrate selectivity. The functional and structural features of bMGL provide insight how its substrate selectivity is determined and how specific inhibitors of bacterial MGL could be designed, which may be useful for development of novel antibiotics.     

Crystal structure of cytochrome c' from Rhodocyclus gelatinosus and comparison with other cytochromes c'

Cytochromes c' are heme proteins found in photosynthetic and denitrifying bacteria, where they are presumably involved in electron transport. The cytochrome c' isolated from the bacterium Rhodocyclus gelatinosus (RGCP) forms a homodimer with each polypeptide containing 129 residues. It has been crystallised in ammonium sulfate at pH?6. Crystals belong to space group P3₁21 with cell parameters a?=?70.2?Å and c?=?126.8?Å, which corresponds to a dimer in the asymmetric unit (VM?=?3.5?ų?/?Da). The crystal structure of RGCP was solved by the molecular replacement method and refined using data to 2.5-Å resolution. The final crystallographic R factor was 17.9% for all reflections (above 2?σ) in the resolution range 27.4 to 2.5?Å. The refined model includes 1876 non-hydrogen protein atoms and 56 water molecules. As typical of c–type cytochromes, the heme group is covalently bound to Cys-X-Y-Cys-His through thio-ether bonds, and His123 occupies the fifth axial coordination position. On the vacant "distal" site, Phe16 blocks the direct access to the sixth coordination site, which is in a predominantly hydrophobic environment. In spite of the low sequence homology among cytochromes c' the overall fold is similar. The monomer structure consists of 4 anti-parallel α-helices and has random coils in the loops between the helices, and at the N- and C-termini. The subunits cross each other to form an X shape.     

Bioactive peptides: Conformational studies of [TYR4] cyclolinopeptide A

The solid state conformational analysis of [Tyr⁴] cyclolinopeptide A has been carried out by x-ray diffraction studies. The crystal structure of the monoclinic form, grown from a dioxane-water mixture [a = 9.849 (5) Å, b = 20.752 (4) Å, c = 16.728 (5) Å, β = 98.83 (3)°, space group P2₁, Z = 2], shows the presence of five intramolecular N-H? O?C hydrogen bonds, with formation of one C₁₇ ring structure, one α-turn (C₁₃), one inverse γ-turn (C₇), and two β-turns (C₁₀, one of type III and one of type 1). The Pro¹-Pro² peptide unit is cis (ω = 5°) all others are trans. The structure is almost superimposable with that of cyclolinopeptide A. The rms deviation for the atoms of the backbones is on the average 0.33 Å. © 1995 John Wiley & Sons, Inc.     

The effect of substitution of Phe181 and Phe182 with Ala on activity,substrate specificity and stabilization of substrate at the active site of Bacillus thermocatenulatus lipase

Steric hindrance leads to limitation in the access of substrate into the enzyme active site. In order to decrease steric hindrance, two conserved residues, Phe¹⁸¹ and Phe¹⁸², in the lid domain of Bacillus thermocatenulatus lipase were substituted with alanine by using site-directed mutagenesis. As a result, three mutant lipases were produced. Circular dichroism (CD) spectroscopy showed that the secondary structure of all lipases is similar to one another. F181A mutation increased the distance between phe¹⁸¹ and catalytic ser¹¹⁴, which is buried in the active site by 3.24 Å. It can be suggested that such an increase in distance may lead to a decrease in steric hindrance. F181A mutation increased overall lipase activity by up to 2.6-fold (4670 U mg⁻¹) toward C8 substrate. It also resulted in optimal lipase activity at 65 °C rather than 55 °C. F182A mutation increased the distance between phe¹⁸² and catalytic ser¹¹⁴ by 1.54 Å but failed to induce any significant effect on lipase activity. However, F181A–F182A mutation significantly decreased the activity due to decreased van der Waals interactions between the phenyl group of phenylalanines and the acyl chain of triacylglycerol. These results indicate that presence of one of the two residues, Phe¹⁸¹ or Phe¹⁸², is important for stabilizing triacylglycerols in active site.