Surface display of active lipase in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> using Cwp2 as an anchor protein

Lipase Lip2 from Yarrowia lipolytica was displayed on the cell surface of Saccharomyces cerevisiae using Cwp2 as an anchor protein. Successful display of the lipase on the cell surface was confirmed by immunofluorescence microscopy and halo assay. The length of linker sequences was further examined to confirm that the correct conformation of Lip2 was maintained. The results showed that the displayed Lip2 exhibited the highest activity at 7.6 ± 0.4 U/g (dry cell) when using (G₄S)₃ sequence as the linker, with an optimal temperature and pH at 40°C and pH 8.0. The displayed lipase did not lose any activity after being treated with 0.1% Triton X-100 and 0.1% Tween 80 for 30 min, and it retained 92% of its original activity after incubation in 10% DMSO for 30 min. It also exhibited better thermostability than free Lip2 as reported previously.     

Native-like tertiary structure in the Mucor miehei lipase molten globule state obtained at low pH

Studies on the acid-induced denaturation of Mucor miehei lipase (E.C. 3.1.1.3) were performed by circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy and binding of hydrophobic dye, 1-anilino 8-naphthalenesulfonic acid (ANS). Acid denaturation of the lipase showed loss of secondary structure and alterations in the tertiary structure in the pH range 4 to 2 and 7 to 2 respectively, suggesting that the lipase exists as an acid-unfolded state approximately pH 2.0. A further decrease in pH (from 2.0 to 1.0) resulted in a second transition, which corresponded to the formation of both secondary and tertiary structures. The acid unfolded state at around pH 2.0 has been characterized by significant loss of secondary structure and a small increase in fluorescence intensity with a blue shift of 2 nm, indicating shift of tryptophan residues to less polar environment. Interestingly, the lipase at pH 1.0 exhibits characteristics of molten globule, such as enhanced binding of hydrophobic dye (ANS), native-like secondary structure and slightly altered tryptophanyl environments. That the molten globule of the lipase at pH 1.0 also possesses native-like tertiary structure is an interesting observation made for this lipase.     

Xylanase II from <Emphasis Type="Italic">Trichoderma reesei</Emphasis> QM 9414: conformational and catalytic stability to Chaotropes,Trifluoroethanol, and pH changes

Xylanase II, a key enzyme in the hydrolysis of xylan, was purified from cultures of Trichoderma reesei QM 9414 (anamorph of Hypocrea jecorina) grown on wheat straw as a carbon source. Xylanase treated with increasing guanidinium hydrochloride concentrations was denatured in a cooperative way regarding secondary and tertiary structures with midpoint transitions 5.6 ± 0.1 and 3.7 ± 0.1 M, respectively, whereas the enzymatic activity showed an intermediate state at 2–4 M denaturant. Treatment with urea showed that xylanase secondary structure was stabilized up to 4 M urea to be destabilized thereafter in a cooperative way with a transition midpoint Dm = 5.7 ± 0.2 M, but the ellipticity at 220 nm was greater than control in the presence of urea up to 6 M. Tertiary structure in the presence of urea showed also intermediate states with partial cooperative transitions with a midpoint: Dm = 2.7 ± 0.04 and 6.7 ± 0.3 M, respectively, whereas the enzymatic activity was enhanced about 40% at 2 M and inhibited above 4 M urea. Assays with the fluorescent probe 4,4′-bis-1-phenylamine-8-naphftalene sulfonate (bis-ANS) proved that the intermediate states had the characteristics of molten globule structures. The change of free energy for xylanase in absence of denaturants obtained from the spectral centre of mass (SCM) data at 298 K is \Updelta G_{H2 O}⁰ \Updelta G_{{{\rm H}_{2} {\rm O}}}^{0}  = ~17 kJ mol⁻¹. In the presence of increasing trifluoroethanol (TFE), the enzyme gained α-helix content and lose tertiary structure and catalytic activity. Changes in pH (2–9) had practically no effect on the secondary structure of the enzyme, whereas the SCM values indicated that tertiary structure is maintained above pH 4. Bis-ANS binds to xylanase at pH 2 and 2.5 and in the presence of 30–40% TFE (v/v) characterizing molten globule states in those environmental conditions.     

Methanol-induced tertiary and secondary structure changes of granulocyte-colony stimulating factor

We have studied methanol-induced conformational changes in rmethuG-CSF at pH 2.5 by means of circular dichroism (CD), fluorescence and infrared (IR) spectroscopy, and 8-anilino-1-naphthalene sulfonic acid (ANS) binding. Methanol has little effect on the secondary and tertiary structures of rmethuG-CSF when its concentration is in the range of 0 to 20% (v/v). At 30% (v/v) methanol, rmethuG-CSF has ANS binding ability. In the methanol concentration range of 30 to 70% (v/v) the amount of alpha-helix decreases a little, and the tertiary structure decreases significantly. At methanol concentrations above 70% (v/v), a transition to a more helical state occurs, while there is little change in the tertiary structure, and no ANS binding ability. Thermal denaturation studies involving CD have demonstrated that as the methanol concentration increases the melting temperature and the cooperativity of transition decrease, and the transition covers a much wider range of temperature. It seems that the decreased cooperativity means an increase in the concentration of partially folded intermediate states during the unfolding of rmethuG-CSF.     

Structure of lysozyme dissolved in neat organic solvents as assessed by NMR and CD spectroscopies

The structure of the model protein hen egg-white lysozyme dissolved in water and in five neat organic solvents (ethylene glycol, methanol, dimethylsulfoxide (DMSO), formamide, and dimethylformamide (DMF)) has been examined by means of 1H NMR and circular dichroism (CD) spectroscopies. The NMR spectra of lysozyme reveal the lack of a defined tertiary structure in all five organic solvents, although the examination of line widths suggests the possibility of some ordered structure in ethylene glycol and in methanol. The near-UV CD spectra of the protein suggest no tertiary structure in lysozyme dissolved in DMSO, formamide, and DMF, while a distinctive (albeit less pronounced than in water) tertiary structure is seen in ethylene glycol and a drastically changed one in methanol. A highly developed secondary structure was observed by far-UV CD in ethylene glycol and methanol; interestingly, the alpha-helix content of the protein in both was greater than in water, while the beta-structure content was lower. (Solvent absorbance in the far-UV region prevents conclusions about the secondary structure in DMSO, formamide and DMF.) Copyright 1999 John Wiley & Sons, Inc.     

An improved method of lipase preparation incorporating both solvent treatment and immobilization onto matrix

A simple and effective preparation of lipases for use in organic solvents is hereby proposed. Lipases in aqueous solution were treated with isopropanol, immediately followed by immobilization onto a commercially available macroporous resin CRBO2 (crosslinked polystyrene with N-methylglucamine as a functional group). The dual modification of lipases by (1) isopropanol treatment and (2) immobilization improved the activity and stability of lipases more significantly than either of the two treatments alone. The degree of lipase activation was dependent on isopropanol–buffer (v/v) ratio and the source of lipase used. Among the lipases tested, Rhizopus oryzae lipase was more significantly activated. The maximum specific activity of R. oryzae lipase after dual modification was 94.9 mmol h⁻¹ g⁻¹, which was, respectively, 3.3-, 2.5- and 1.5-fold of untreated free, untreated immobilized and treated free lipases. The conformations of the treated and untreated free lipases were investigated by circular dichroism (CD) measurement. Changes in the far- and near-UV CD spectra of lipase indicate that lipase activation is accompanied by changes in secondary and tertiary structures of lipases. The increase in negative molar elipticity at 222 nm suggests that the α-helical content of lipase increase after pretreatment.     

Substitution of Asp189 residue alters the activity and thermostability of <Emphasis Type="Italic">Geobacillus</Emphasis> sp. NTU 03 lipase

Error-prone PCR was used to create more thermoactive and/or thermostable variants of thermoalkalophilic lipases. A variant of the α6 helix (lid domain), with an 189E to V substitution at residue 189, lost its thermostability but exhibited higher activity than that of its wild-type predecessor (r03Lip). Site-saturation mutagenesis was used to explore the sequence-function relationship. Five other mutants also lost thermostability (20–40%) but exhibited enhanced thermoactivity (6.3–79-fold). The mutant E189I showed the highest activity retaining 50% activity after maintaining it at 65°C for 24 h. In comparison to r03Lip, the mutant E189I had a higher affinity for p-nitrophenyl palmitate and p-nitrophenyl stearate (61 and 56% decreased Km) and catalytic efficiency (42-fold and 18-fold increased kcat/Km). The mutant lipase retained its tolerance to n-hexane, but had an improved transesterification activity. The results suggest that residue Glu189 plays a significant role in the thermostability and activity of this thermoalkalophilic lipase.     

圆弧青霉PG37脂肪酶的生物信息学分析

利用生物信息学软件对GenBank上登录的圆弧青霉PG37碱性脂肪酶(LipⅠ)进行预测和分析。结果表明,PG37LipⅠ全肽含多个疏水区域,无明显跨膜结构域,定位于胞外,N-末端20个氨基酸为信号肽;PG37LipⅠ成熟肽是等电点为6.16的疏水性稳定蛋白质,包含一个Lipase_3的结构域,属于α/β水解酶超家族,含磷酸化位点等多种功能性位点,具有脂肪酸代谢的功能;α-螺旋和不规则卷曲是其蛋白质二级结构的主要结构元件,在三级结构中,Ser132-Asp188-His2413个氨基酸残基组成酶活性中心。     

Novel Strategy of Using Methyl Esters as Slow Release Methanol Source during Lipase Expression by mut+ Pichia pastoris X33

One of the major issues with heterologous production of proteins in Pichia pastoris X33 under AOX1 promoter is repeated methanol induction. To obviate repeated methanol induction, methyl esters were used as a slow release source of methanol in lipase expressing mut⁺ recombinant. Experimental design was based on the strategy that in presence of lipase, methyl esters can be hydrolysed to release their products as methanol and fatty acid. Hence, upon break down of methyl esters by lipase, first methanol will be used as a carbon source and inducer. Then P. pastoris can switch over to fatty acid as a carbon source for multiplication and biomass maintenance till further induction by methyl esters. We validated this strategy using recombinant P. pastoris expressing Lip A, Lip C from Trichosporon asahii and Lip11 from Yarrowia lipolytica. We found that the optimum lipase yield under repeated methanol induction after 120 h was 32866 U/L, 28271 U/L and 21978 U/L for Lip C, Lip A and Lip 11 respectively. In addition, we found that a single dose of methyl ester supported higher production than repeated methanol induction. Among various methyl esters tested, methyl oleate (0.5%) caused 1.2 fold higher yield for LipA and LipC and 1.4 fold for Lip11 after 120 h of induction. Sequential utilization of methanol and oleic acid by P. pastoris was observed and was supported by differential peroxisome proliferation studies by transmission electron microscopy. Our study identifies a novel strategy of using methyl esters as slow release methanol source during lipase expression.     

Characterization of a thermostable lipase showing loss of secondary structure at ambient temperature

A gene encoding extracellular lipase was cloned and characterized from metagenomic DNA extracted from hot spring soil. The recombinant gene was expressed in E. coli and expressed protein was purified to homogeneity using hydrophobic interactions chromatography. The mature polypeptide consists of 388 amino acids with apparent molecular weight of 43 kDa. The enzyme displayed maximum activity at 50°C and pH 9.0. It showed thermal stability up to 40°C without any loss of enzyme activity. Nearly 80% enzyme activity was retained at 50°C even after incubation for 75 min. However above 50°C the enzyme displayed thermal instability. The half life of the enzyme was determined to be 5 min at 60°C. Interestingly the CD spectroscopic study carried out in the temperature range of 25–95°C revealed distortion in solution structure above 35°C. However the intrinsic tryptophan fluorescence spectroscopic study revealed that even with the loss of secondary structure at 35°C and above the tertiary structure was retained. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K _m , V _max and K _cat of 0.73 ± 0.18 μM, 239 ± 16 μmol/ml/min and 569 s⁻¹ respectively. Enzyme activity was strongly inhibited by CuCl₂, HgCl₂ and DEPC but not by PMSF, eserine and SDS. The protein retained significant activity (~70%) with Triton X-100. The enzyme displayed 100% activity in presence of 30% n-Hexane and acetone.     

Vertebrate endothelial lipase: comparative studies of an ancient gene and protein in vertebrate evolution

Endothelial lipase (gene: LIPG; enzyme: EL) is one of three members of the triglyceride lipase family that contributes to lipoprotein degradation within the circulation system and plays a major role in HDL metabolism in the body. In this study, in silico methods were used to predict the amino acid sequences, secondary and tertiary structures, and gene locations for LIPG genes and encoded proteins using data from several vertebrate genome projects. LIPG is located on human chromosome 18 and is distinct from other human ‘neutral lipase’ genes, hepatic lipase (gene: LIPC; enzyme: HL) and lipoprotein lipase (gene: LPL; enzyme: LPL) examined. Vertebrate LIPG genes usually contained 10 coding exons located on the positive strand for most primates, as well as for horse, bovine, opossum, platypus and frog genomes. The rat LIPG gene however contained only 9 coding exons apparently due to the presence of a ‘stop’ codon’ within exon 9. Vertebrate EL protein subunits shared 58–97% sequence identity as compared with 38–45% sequence identities with human HL and LPL. Four previously reported human EL N-glycosylation sites were predominantly conserved among the 10 potential N-glycosylation sites observed for the vertebrate EL sequences examined. Sequence alignments and identities for key EL amino acid residues were observed as well as conservation of predicted secondary and tertiary structures with those previously reported for horse pancreatic lipase (PL) (Bourne et al. 1994). Several potential sites for regulating LIPG gene expression were observed including CpG islands near the LIPG gene promoter and a predicted microRNA binding site near the 3’-untranslated region. Promoter regions containing functional polymorphisms that regulate HDL cholesterol in baboons were conserved among primates but not retained between primates and rodents. Phylogenetic analyses examined the relationships and potential evolutionary origins of the vertebrate LIPG gene subfamily with other neutral triglyceride lipase gene families, LIPC and LPL. It is apparent that the triglyceride lipase ancestral gene for the vertebrate LIPG gene predated the appearance of fish during vertebrate evolution >500 million years ago.     

High catalytic performances of Pseudomonas fluorescens lipase adsorbed on a new type of cyclodextrin-based nanosponges

Lipases are water-soluble enzymes that catalyze the hydrolysis of triacylglycerols (in aqueous media) or trans-esterification reactions (in microaqueous media) and are involved in a number of industrial applications. As a limit to lipase application is represented by the need for interfacial activation, the search for suitable solid supports able to fulfill this requirement is always ongoing. In the present work, we report the preliminary characterization of a system obtained by adsorbing Pseudomonas fluorescens lipase on a newly synthesized cyclodextrin-based carbonate nanosponge (CD–NS–1:4). The activity and structural stability of lipase adsorbed on this new support were evaluated by checking the effect of temperature, pH changes and organic solvents (methanol) on the enzyme structure and function, which were compared with those of the free enzyme in solution. Our data show that the non-covalent interaction of Ps. fluorescens lipase with CD–NS–1:4 results in enzyme structural and functional stabilization, as it was still active after 66 days of incubation at T ～ 18 °C. Stabilization with respect to T, pH and the presence of organic solvent was observed as well as, unlike the solubilized enzyme, the adsorbed lipase was active at T > 40 °C, at pH 5 and after 24-h incubation with 70% (v/v) methanol (13% residual activity).     

Structural analysis of phospholipase A2 from functional perspective. 2. Characterization of a molten globule-like state induced by site-specific mutagenesis

Previous NMR studies have shown that many phospholipase A2 (PLA2, from bovine pancreas, overexpressed in Escherichia coli) mutants display some properties reminiscent of a molten globule state. Further NMR analyses for some of the mutants indicated that formation of the "molten globule-like state" is a pH-dependent phenomenon. The mutants I9Y and I9F showed perturbed NMR properties throughout the pH range studied, while the mutants H48A and C44A/C105A displayed native-like spectra at neutral pH but molten globule-like ones under acidic conditions, with a "transition pH" around 4. On the other hand, wild-type PLA2 exhibits exceptional pH stability and turns into a similar molten globule-like state only under highly acidic conditions such as 1 M HCl. The H48A mutant was used to rigorously establish the property of the molten globule-like state of PLA2 mutants. The results of far-UV CD, near-UV CD, and ANS-binding fluorescence suggest that H48A retains native-like secondary structures but loses tertiary structure during the conformational transition. However, the tertiary structure is not completely lost, as evidenced by the retention of some long-range NOEs in two-dimensional NOESY spectra. The conclusion was further substantiated by three-dimensional NOESY-HSQC experiments on a 15N-labeled H48A sample. It was revealed that the molten globule-like state at mildly acidic pH retained some rigid tertiary structure, which consisted of partial alpha-helix II (Y52-L58), alpha-helix III (D59-V63), beta-wing (S74-S85) and partial alpha-helix IV (A90-N97). These residual tertiary structures grouped in half of the protein could be attributed to stabilization by some of the disulfide bonds. The extreme sensitivity of the PLA2 structure to site-directed mutagenesis is unprecedented. It is interesting to note that most of the functional residues (the active site, the hydrophobic channel, the interfacial binding site, and the calcium-binding loop) are located in the remainder of the protein, which is well disrupted in tertiary interactions.     

A novel cold-adapted lipase from <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. XMZ-26: gene cloning and characterisation

Acinetobacter sp. XMZ-26 (ACCC 05422) was isolated from soil samples obtained from glaciers in Xinjiang Province, China. The partial nucleotide sequence of a lipase gene was obtained by touchdown PCR using degenerate primers designed based on the conserved domains of cold-adapted lipases. Subsequently, a complete gene sequence encoding a 317 amino acid polypeptide was identified. Our novel lipase gene, lipA, was overexpressed in Escherichia coli. The recombinant protein (LipA) was purified by Ni-affinity chromatography, and then deeply characterised. The LipA resulted to hydrolyse pNP esters of fatty acids with acyl chain length from C2 to C16, and the preferred substrate was pNP octanoate showing a k _cat = 560.52 ± 28.32 s⁻¹, K _m = 0.075 ± 0.008 mM, and a k _cat/K _m = 7,377.29 ± 118.88 s⁻¹ mM⁻¹. Maximal LipA activity was observed at a temperature of 15°C and pH 10.0 using pNP decanoate as substrate. That LipA peaked at such a low temperature and remained most activity between 5°C and 35°C indicated that it was a cold-adapted enzyme. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents, including Ninol, Triton X-100, methanol, PEG-600, and DMSO. This cold-adapted lipase may find applications in the detergent industry and organic synthesis.     

Gene analysis,optimized production and property of marine lipase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> B106 associated with South China Sea sponge <Emphasis Type="Italic">Halichondria rugosa</Emphasis>

Gene cloning, optimized production and property of marine lipase from Bacillus pumilus B106 associated with South China Sea sponge Halichondria rugosa were investigated in this paper. A lipase gene with whole ORF encoding 215 amino acids was obtained by PCR, protein domain prediction suggested that the deduced lipase belongs to α/β hydrolases family. Based on single factor Seriatim-Factorial test and Plackett–Burman experimental design, the optimal medium consisted of (per l) 12.5 ml maize oil, 5.0 g beef extract, 2.0 g PO₄ ³⁻ (0.6 g KH₂PO₄, 1.4 g K₂HPO₄), 17.15 g Mg²⁺, 5.0 g yeast extract, 2.282 g CaCl₂ and 5.0 ml Tween80 with artificial sea water. Using this optimum medium, lipase activity and cell concentration were increased by 3.54- and 1.31-fold over that of the basal medium, respectively. This lipase showed tolerance to high salinity, pH and temperature. About 10–20% methanol exhibited a stimulatory effect on the lipase activity, while activity was inhibited by 30–40% methanol, 2-propanol, DMSO, and ethanol. This study provides a valuable resource for marine lipase production and extends our understanding of the possible role of sponge-associated bacteria in the biotransformation of chemical compounds for the sponge host.     

Functional stability and structural transitions of Kallikrein: spectroscopic and molecular dynamics studies

Kallikrein, a physiologically vital serine protease, was investigated for its functional and conformational transitions during chemical (organic solvents, Gdn-HCl), thermal, and pH induced denaturation using biochemical and biophysical techniques and molecular dynamics (MD) simulations approach. The enzyme was exceptionally stable in isopropanol and ethanol showing 110% and 75% activity, respectively, after 96 h, showed moderate tolerance in acetonitrile (45% activity after 72 h) and much lower stability in methanol (40% activity after 24 h) (all the solvents [90% v/v]). Far UV CD and fluorescence spectra indicated apparent reduction in compactness of KLKp structure in isopropanol system. MD simulation studies of the enzyme in isopropanol revealed (1) minimal deviation of the structure from native state (2) marginal increase in radius of gyration and solvent accessible surface area (SASA) of the protein and the active site, and (3) loss of density barrier at the active site possibly leading to increased accessibility of substrate to catalytic triad as compared to methanol and acetonitrile. Although kallikrein was structurally stable up to 90 °C as indicated by secondary structure monitoring, it was functionally stable only up to 45 °C, implicating thermolabile active site geometry. In GdnHCl [1.0 M], 75% of the activity of KLKp was retained after incubation for 4 h, indicating its denaturant tolerance. A molten globule-like structure of KLKp formed at pH 1.0 was more thermostable and exhibited interesting structural transitions in organic solvents. The above results provide deeper understanding of functional and structural stability of the serine proteases at molecular level.     

Molten globule-like state of bovine carbonic anhydrase in the presence of acetonitrile

We have evaluated the effects of acetonitrile on the structure and function of bovine carbonic anhydrase II. The potential structural and functional changes in carbonic anhydrase in the presence of different acetonitrile/buffer ratios (0%, 17.5% and 47.5% v/v) were determined using a variety of methods. These included simple spectrophotometric methods to record enzyme velocity, fluorescence measurements and calculation of accessible surface area (ASA) to identify possible alterations in tertiary structure of the protein, CD measurements to search for secondary structure conversions, and thermal scanning to determine structural stability of the protein in different media. The Far-UV CD studies indicated that carbonic anhydrase, for the most part, retains its secondary structure in the presence of acetonitrile. Fluorescence measurements using iodide ion and ANS along with ASA calculations revealed that in the presence of acetonitrile some degree of conformational change occurs in the carbonic anhydrase structure. In addition to the hydrophobic pockets, two additional tryptophanyl residues become exposed to the solvent, thereby increasing the surface hydrophobicity of the protein. These alterations dramatically reduce the catalytic activity, thermal stability, and aggregation velocity of the enzyme. Thus, our results support a molten globule-like structure of carbonic anhydrase in the presence of acetonitrile.     

Characterization of acid-induced unfolding intermediates of glucose/xylose isomerase.

Acid-induced unfolding of the tetrameric glucose/xylose isomerase (GXI) from Streptomyces sp. NCIM 2730 has been investigated using intrinsic fluorescence, fluorescence quenching, second derivative spectroscopy, hydrophobic dye (1-anilino-8-naphthalene-sulfonate) binding and CD techniques. The pH dependence of tryptophanyl fluorescence of GXI at different temperatures indicated the presence of two stable intermediates at pH 5.0 and pH 3.0. The pH 3.2 intermediate was a dimer and exhibited molten globule-like characteristics, such as the presence of native-like secondary structure, loss of tertiary structure, increased exposure of hydrophobic pockets, altered microenvironment of tyrosine residues and increased accessibility to quenching by acrylamide. Fluorescence and CD studies on GXI at pH 5.0 suggested the involvement of a partially folded intermediate state in the native to molten globule state transition. The partially folded intermediate state retained considerable secondary and tertiary structure compared to the molten globule state. This state was characterized by its hydrophobic dye binding capacity, which is smaller than the molten globule state, but was greater than that of the native state. This state shared the dimeric status of the molten globule state but was prone to aggregate formation as evident by the Rayleigh light scattering studies. Based on these results, the unfolding pathway of GXI can be illustrated as: N-->PFI-->MG-->U; where N is the native state at pH 7.5; PFI is the partially folded intermediate state at pH 5.0; MG is the molten globule state at pH 3.2 and U is the monomeric unfolded state of GXI obtained in the presence of 6 M GdnHCl. Our results demonstrate the existence of a partially folded state and molten globule state on the unfolding pathway of a multimeric alpha/beta barrel protein.     

Exploring the conformational states and rearrangements of Yarrowia lipolytica Lipase

We report the 1.7 Å resolution crystal structure of the Lip2 lipase from Yarrowia lipolytica in its closed conformation. The Lip2 structure is highly homologous to known structures of the fungal lipase family (Thermomyces lanuginosa, Rhizopus niveus, and Rhizomucor miehei lipases). However, it also presents some unique features that are described and discussed here in detail. Structural differences, in particular in the conformation adopted by the so-called lid subdomain, suggest that the opening mechanism of Lip2 may differ from that of other fungal lipases. Because the catalytic activity of lipases is strongly dependent on structural rearrangement of this mobile subdomain, we focused on elucidating the molecular mechanism of lid motion. Using the x-ray structure of Lip2, we carried out extensive molecular-dynamics simulations in explicit solvent environments (water and water/octane interface) to characterize the major structural rearrangements that the lid undergoes under the influence of solvent or upon substrate binding. Overall, our results suggest a two-step opening mechanism that gives rise first to a semi-open conformation upon adsorption of the protein at the water/organic solvent interface, followed by a further opening of the lid upon substrate binding.     

Higher order structure of Mucor miehei lipase and micelle size in cetyltrimethylammonium bromide reverse micellar system

The higher order structure of Mucor miehei lipase and micelle size in a cationic cetyltrimethylammonium bromide (CTAB) reverse micellar system was investigated. Circular dichroic (CD) measurement revealed that the lipase far-UV CD spectra changed markedly, going from buffer solution to the reverse micellar solution, and were very similar for any organic solvent used. The ellipticity of the solubilized lipase in the far-UV region markedly decreased with increasing water content (W(0): molar ratio of water to CTAB), indicating that the secondary structure of lipase changed with the water content. The linear correlation between the W(0) and the micelle size was obtained by measuring dynamic light scattering. From the linear correlation between the micelle size and W(0), the higher order structure of the solubilized lipase appears to be affected directly by the micellar interface. The species and concentration of alcohol as a cosurfactant had an inferior effect on lipase structure. Especially, at ratios of 1-pentanol to CTAB of less than 8, the secondary and tertiary structures of lipase were preserved in the reverse micelles. The CTAB concentration had little effect on the lipase structure in the micelles. The catalytic activity of the lipase solubilized in the CTAB reverse micelles increased with increasing the W(0).