Biodiesel synthesis and conformation of lipase from Burkholderia cepacia in room temperature ionic liquids and organic solvents

Biodiesel synthesis and conformation of Burkholderia cepacia lipase (BCL) were studied in 19 different room temperature ionic liquids (RTLLs) with a range of cation and anion structures. Overall, anion selection had a greater influence on biodiesel conversion than cation choice. RTILs containing Tf2N- and PF6- anions were suitable reaction media, while RTIL of [OmPy][BF4] was the best reaction medium with a biodiesel yield of 82.2±1.2%. RTILs with strong water miscible properties showed very low biodiesel yields. Conformational analysis by FT-IR revealed that higher biodiesel conversion in RTILs was correlated with a low tendency in α-helix content of BCL. An ultrasound-assisted biocatalysis process in RTILs was used to improve mass transfer rate, leading to 83% reduction of the reaction time for biodiesel production.     

Properties of free and immobilised lipase from Burkholderia cepacia in organic media

The purified lipase from Burkholderia cepacia was immobilised on a porous polypropylene support and its biocatalytic properties were compared with those of the free enzyme in organic media. For both lipase preparations, the rate of p-nitrophenyl ester hydrolysis in n-heptane was not restricted by mass transfer limitations. The immobilisation changed neither the temperature at which the reaction rate was maximal, nor the activation energy of the reaction. The enzyme stability was slightly decreased (1.3-fold) upon immobilisation. Moreover, the immobilised enzyme displayed fewer variations of activity with fatty acid chain length. Interestingly, for all the different p-nitrophenyl esters used, the immobilised enzyme was more active (from 5.8- to 18.9-fold) than the free enzyme. Therefore, it would be very useful to use B. cepacia lipase immobilised onto porous polypropylene for applications in organic media, as it displayed high activities on a larger range of substrates. Received: 8 February 1999 / Received revision: 19 March 1999 / Accepted: 20 March 1999     

基于T7表达系统的洋葱伯克霍尔德菌G63脂肪酶同源高效表达

为实现对洋葱伯克霍尔脂肪酶的可控高效表达, 将目前被广泛使用的T7重组蛋白高效表达系统移植到洋葱伯克霍尔德菌(Burkholderia cepacia)G63中进行脂肪酶同源表达。首先采用PCR从大肠杆菌BL21(DE3)中得到T7 RNA 聚合酶基因(T7 RNAP)并将其克隆到致死质粒pJQ200SK上, 然后在T7 RNAP 前后各加入500 bp用于同源重组的片段, 再通过三亲本杂交把T7 RNAP整合到B. cepacia基因组上, 使T7 RNAP受到脂肪酶基因(lipA)启动子调控。接着把lipA和它的伴侣基因lipB单独或全部克隆到载体pUCPCM和pBBR22b上, 构建出pBBR22blipAB、pBBR22blipA、pUCPCMlipAB、pUCPCMlipA、pUCPCMΔlipAlipB、pUCPCMΔlipA、pUCPCMΔlipB七种表达质粒, 通过电转化将上述表达质粒转化到含T7 RNAP的B. cepacia宿主菌中, 最终得到一系列脂肪酶基因工程菌。通过摇瓶诱导发酵发现含表达质粒pUCPCMlipAB的工程菌脂肪酶酶活最高, 达到607 U/mg, 与野生菌相比酶活力提高2.8倍, 并且除含pUCPCMΔlipB的工程菌外, 其它工程菌的脂肪酶酶活均有不同程度提高。野生菌与工程菌pUCPCMlipAB的发酵液经硫酸铵沉淀, Sephadex G-75凝胶过滤纯化后, 比酶活分别为29 984 U/mg和30 875 U/mg。以上结果表明, 构建的基于T7表达系统的B. cepacia脂肪酶基因工程能有效提高脂肪酶的表达量, 同时说明分泌信号PelB和增强转录的核糖体接合位点对脂肪酶的表达有促进作用。     

Optimization of Pseudomonas cepacia lipase preparations for catalysis in organic solvents

The activity of different lipase (from Pseudomonas cepacia) forms, such as crude powder (crude PC), purified and lyophilized with PEG (PEG + PC), covalently linked to PEG (PEG-PC), cross-linked enzyme crystals (CLEC-PC), and immobilized in Sol-Gel-AK (Sol-Gel-AK-PC) was determined, at various water activities (aw), in carbon tetrachloride, benzene and 1,4-dioxane. The reaction of vinyl butyrate with 1-octanol was employed as a model and both transesterification (formation of 1-octyl butyrate) and hydrolysis (formation of butyric acid from vinyl butyrate) rates were determined. Both rates depended on the lipase form, solvent employed, and aw value. Hydrolysis rates always increased as a function of aw, while the optimum of aw for transesterification depended on the enzyme form and nature of the solvent. At proper aw, some lipase forms such as PEG + PC, PEG-PC, and Sol-Gel-AK-PC had a total activity in organic solvents (transesterification plus hydrolysis) which was close to (39 and 48%) or even higher than (130%) that displayed by the same amount of lipase protein in the hydrolysis of tributyrin-one of the substrates most commonly used as standard for the assay of lipase activity-in aqueous buffer. Instead, CLEC-PC and crude PC were much less active in organic solvents (2 and 12%) than in buffer. The results suggest that enzyme dispersion and/or proper enzyme conformation (favored by interaction with PEG or the hydrophobic Sol-Gel-AK matrix) are essential for the expression of high lipase activity in organic media.     

海藻酸-SiO2杂化凝胶固定化洋葱伯克霍尔德茵脂肪酶的研究

利用四乙氧基硅烷（TEOS）原位水解法将SiO2掺杂于海藻酸（ALG）凝胶中,通过双交联制备出新型ALG—SiO2杂化凝胶以固定化洋葱伯克霍尔德菌脂肪酶。结果表明,固定化酶的最优条件：质量分数为2．0％的ALG、0．2mol／LCaCl2、V（ALG）／V（TEOS）为5、加酶量为1gALG加100mg酶粉、固定化60min、采用直径为0．8mm的针头滴定、真空冷冻干燥。在此条件下,酶蛋白的包埋率可达100％,酶活回收率可达91％。固定化酶的最适pH为8．0,最适作用温度为50℃,重复使用8次后,酶活性仍能保持80％以上。ALG—Si02杂化凝胶的场扫描电镜（FESEM）观察发现凝胶的整体构造仍然是海藻酸凝胶骨架;与ALG凝胶平滑的内部相比较,杂化凝胶仍具有完整的网络结构,但内部更为粗糙,结构更为致密。     

Regioselectivity-reversal in acylation of 6-azauridine catalyzed by Burkholderia cepacia lipase

3′-O-Stearoylation of 6-azauridine was achieved enzymatically for the first time. Among eight commercially available lipases, that from Burkholderia cepacia displayed a 3′-regioselectivity of 80% towards the acylation of 3-hydroxyl of 6-azauridine. Using an immobilized lipase from Burkholderia cepacia, the 3′-regioselectivities of the acylations could be reversed by lengthening the aliphatic chain of the acyl donors (C2–C18). The possible reason might be the presence of the interaction between the base moiety and the acyl group.     

Resolution of 2-nitroalcohols by Burkholderia cepacia lipase-catalyzed enantioselective acylation

Racemic 2-nitro-1-phenylethanol was resolved by via enantioselective transesterification catalyzed by Burkholderia cepacia lipase. The reaction afforded excellent E values (E > 200) and enantioselectivity (up to >99% enantiomeric excesses [ee]) of both remaining substrates and acetylated product. Moreover, the lipase displayed high enantioselectivity in the resolution of additional 2-nitroalcohols (E up to >200). This method provides an efficient alternative for obtaining enantiopure 2-nitroalcohols.     

Efficient regioselective acylation of andrographolide catalyzed by immobilized Burkholderia cepacia lipase

For the first time, PSL-C, an immobilized lipase from Burkholderia cepacia, was successfully applied to the regioselective acylation of andrographolide by vinyl acetate in acetone. FT-IR spectra demonstrated the occurrence of acylation reaction. The ¹³C NMR, ESI-MS and elemental analysis confirmed that the 14-acetylandrographolide was formed exclusively. Water activity and reaction temperature had a significant effect on the initial rate and the substrate conversion, but little effect on the regioselectivity of the reaction. The optimal water activity and reaction temperature were 0.11 and 50 °C, respectively. Under these conditions, the initial rate and substrate conversion were 50.2 mM h⁻¹ and 99.0%, respectively, after a reaction time of around 4 h. Besides, immobilized lipase also displayed higher operational stability and 83.5% of its original activity was maintained after being reused for eight batches.     

Effect of pretreatment by different organic solvents on esterification activity and conformation of immobilized Pseudomonas cepacia lipase

Experiments were carried out to investigate the effect of pretreatment by organic solvents with different hydrophobicities, functional groups and molecular constitutions as activation agents on initial esterification activity and secondary structure of immobilized Pseudomonas cepacia lipase. The results showed that esterification activity of immobilized P. cepacia lipase treated with organic solvents containing –CO– and –CN– functional groups was highest, followed by the one treated with –C–C– functional groups but the lowest with –OH and aromatics functional groups. An organic solvent with a branched structure was more favorable compared with a straight chain in terms of enhancing enzyme activity. Conformational studies via Fourier transform-infrared spectroscopy indicated that the catalytic activity variance was attributed to the secondary structure changes for immobilized P. cepacia lipase treated with organic solvents. Moreover, the effects of moisture, pH and temperature on the esterification activity of immobilized P. cepacia lipase were also addressed.     

Burkholderia cepacia lipase: A versatile catalyst in synthesis reactions

The lipase from Burkholderia cepacia, formerly known as Pseudomonas cepacia lipase, is a commercial enzyme in both soluble and immobilized forms widely recognized for its thermal resistance and tolerance to a large number of solvents and short‐chain alcohols. The main applications of this lipase are in transesterification reactions and in the synthesis of drugs (because of the properties mentioned above). This review intends to show the features of this enzyme and some of the most relevant aspects of its use in different synthesis reactions. Also, different immobilization techniques together with the effect of various compounds on lipase activity are presented. This lipase shows important advantages over other lipases, especially in reaction media including solvents or reactions involving short‐chain alcohols.     

Insights into lid movements of Burkholderia cepacia lipase inferred from molecular dynamics simulations

The interfacial activation of many lipases at water/lipid interface is mediated by large conformational changes of a so‐called lid subdomain that covers up the enzyme active site. Here we investigated using molecular dynamic simulations in different explicit solvent environments (water, octane and water/octane interface) the molecular mechanism by which the lid motion of Burkholderia cepacia lipase might operate. Although B. cepacia lipase has so far only been crystallized in open conformation, this study reveals for the first time the major conformational rearrangements that the enzyme undergoes under the influence of the solvent, which either exposes or shields the active site from the substrate. In aqueous media, the lid switches from an open to a closed conformation while the reverse motion occurs in organic environment. In particular, the role of a subdomain facing the lid on B. cepacia lipase conformational rearrangements was investigated using position‐restrained MD simulations. Our conclusions indicate that the sole mobility of α9 helix side‐chains of B. cepacia lipase is required for the full completion of the lid conformational change which is essentially driven by α5 helix movement. The role of selected α5 hydrophobic residues on the lid movement was further examined. In silico mutations of two residues, V138 and F142, were shown to drastically modify the conformational behavior of B. cepacia lipase. Overall, our results provide valuable insight into the role played by the surrounding environment on the lid conformational rearrangement and the activation of B. cepacia lipase. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Production of biodiesel by Burkholderia cepacia lipase as a function of process parameters

Despite the already established route of chemically catalyzed transesterification reaction in biodiesel production, due to some of its shortcomings, biocatalysts such as lipases present a vital alternative. Namely, it was noticed that one of the key shortcomings for the optimization of the enzyme catalyzed biodiesel synthesis process is the information on the lipase activity in the reaction mixture. In addition to making optimization difficult, it also makes it impossible to compare the results of the independent research. This article shows how lipase intended for use in biodiesel synthesis can be easily and accurately characterized and what is the enzyme concentration that enables achievement of the desired level of fatty acid methyl esters (FAME) in the final product mixture. Therefore, this study investigated the effect of two different activity loads of Burkholderia cepacia lipase on the biodiesel synthesis varying the pH and temperature optimal for lipase activity. The optimal lipase pH and temperature were determined by two different enzyme assays: spectrophotometric and titrimetric. The B. cepacia lipase pH optimum differentiated between assays, while the lipase optimally hydrolyzed substrates at 50°C. The analysis of FAME during 24 hr of biodiesel synthesis, at two different enzyme concentrations, pH 7, 8, and 10, and using two different buffers, revealed that the transesterification reaction at optimal pH, 1 hr reaction time and lipase activity load of 250 U per gram of reaction mixture was sufficient to produce more than 99% FAME.     

Inactivation of Toluene 2-Monooxygenase in Burkholderia cepacia G4 by Alkynes

High concentrations of acetylene (10 to 50% [vol/vol] gas phase) were required to inhibit the growth of Burkholderia cepacia G4 on toluene, while 1% (vol/vol) (gas phase) propyne or 1-butyne completely inhibited growth. Low concentrations of longer-chain alkynes (C₅ to C₁₀) were also effective inhibitors of toluene-dependent growth, and 2- and 3-alkynes were more potent inhibitors than their 1-alkyne counterparts. Exposure of toluene-grown B. cepacia G4 to alkynes resulted in the irreversible loss of toluene- and o-cresol-dependent O₂ uptake activities, while acetate- and 3-methylcatechol-dependent O₂ uptake activities were unaffected. Toluene-dependent O₂ uptake decreased upon the addition of 1-butyne in a concentration- and time-dependent manner. The loss of activity followed first-order kinetics, with apparent rate constants ranging from 0.25 min⁻¹ to 2.45 min⁻¹. Increasing concentrations of toluene afforded protection from the inhibitory effects of 1-butyne. Furthermore, oxygen, supplied as H₂O₂, was required for inhibition by 1-butyne. These results suggest that alkynes are specific, mechanism-based inactivators of toluene 2-monooxygenase in B. cepacia G4, although the simplest alkyne, acetylene, was relatively ineffective compared to longer alkynes. Alkene analogs of acetylene and propyne—ethylene and propylene—were not inactivators of toluene 2-monooxygenase activity in B. cepacia G4 but were oxidized to their respective epoxides, with apparent K_s and V_max values of 39.7 μM and 112.3 nmol min⁻¹ mg of protein⁻¹ for ethylene and 32.3 μM and 89.2 nmol min⁻¹ mg of protein⁻¹ for propylene.     

Magnetic lipase active in organic solvents

Magnetic lipase (magnetite particles coated with polyethylene glycol-modified lipase) was prepared in two steps: Lipase was coupled with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine, activated PEG2, to obtain polyethylene glycol-modified lipase, PEG-lipase. The PEG-lipase was added to the solution of ferrous (Fe2+)- and ferric(Fe3+)-ions with the pH value adjusted to 8.0-8.5 to obtain magnetic lipase. The magnetic lipase was dispersed in organic solvents such as benzene and 1,1,1-trichloroethane with the particle size of 120 +/- 60 nm. The colloidal solution was very stable and no aggregation occurred even after 5 days. A high enzymic activity (11.6 mumol/min/mg protein) for lauryl laurate synthesis was observed in 1,1,1-trichloroethane. The magnetic lipase was readily recovered from the organic solvents in a magnetic field of 6000 Oe without loss of the enzymic activity.     

Statistical medium optimization and production of a hyperthermostable lipase from Burkholderia cepacia in a bioreactor

AIM: Statistical medium optimization for maximum production of a hyperthermostable lipase from Burkholderia cepacia and its validation in a bioreactor. METHODS AND RESULTS: Burkholderia cepacia was grown in shake flasks containing 1% glucose, 0.1% KH2PO4, 0.5% NH4Cl, 0.24% (NH4)2HPO4, 0.01% MgSO4.7H2O and 1% emulsified palm oil, at 45 degrees C and pH 7.0, agitated at 250 rev min(-1) with 6-h-old inoculum (2% v/v) for 20 h. A fourfold enhancement in lipase production (50 U ml(-1)) and an approximately three fold increase in specific activity (160 U mg(-1)) by B. cepacia was obtained in a 14 litre bioreactor within 15 h after statistical optimization following shake flask culture. The statistical model was obtained using face centred central composite design (FCCCD) with five variables: glucose, palm oil, incubation time, inoculum density and agitation. The model suggested no interactive effect of the five factors, although incubation period, inoculum and carbon concentration were the important variables. CONCLUSIONS: The maximum lipase production was 50 U ml(-1), with specific activity 160 U mg(-1) protein, in a 14 litre bioreactor after 15 h in a medium obtained after statistical optimization in shake flasks. Further, the model predicted reduction in time for lipase production with reduction in total carbon supply. SIGNIFICANCE AND IMPACT OF THE STUDY: Statistical optimization allows quick optimization of a large number of variables. It also provides a deep insight into the regulatory role of various parameters involved in enzyme production.     

Improving esterification activity of Burkholderia cepacia lipase encapsulated in silica by bioimprinting with substrate analogues

Bioimprinting is a promising, though relatively unexplored, approach to improving the performance of enzymes. In this study, bioimprinting with substrate analogues of fatty acids was systematically conducted to improve the esterification activity of Burkholderia cepacia lipase that had undergone a sol–gel immobilization procedure with methyltrimethoxysilane (MTMS) and tetramethoxysilane (TMOS) as the precursors. The specific activity of the bioimprinted lipases was 3682.0 μmol h^?1 mg protein, which was a 47.9- and 2.5-fold increase over the free and non-imprinted immobilized lipases, respectively. Compared to the free and non-imprinted immobilized lipases, bioimprinted lipases exhibited better thermal stability, and their activity did not change after being incubated at 60 °C for 12 h. Bioimprinted lipases were more easily affected by alcohol than the non-imprinted ones, whose specific activity could be markedly enhanced by ethanol, isopropanol and n-butanol by factors of 1.23-, 1.28- and 1.12-fold, respectively. The reasons for the improvement of imprinted enzyme activity are also discussed based on the surface structure, specific surface area and average pore diameter of the silane particles.     

Covalent immobilization of lipase in organic solvents

Lipase from Rhizopus sp. has been immobilized covalently on tresyl activated silica. Three different coupling media were evaluated: aqueous buffer, n-hexane, and a microemulsion based on n-hexane, aqueous buffer, and the nonionic surfactant triethylene glycol monododecyl ether. In addition, coupling via a very long, hydrophilic spacer arm, polyethylene glycol 1500 (PEG 1500), was compared with attachment to the silica via a short silane bridge only. The enzyme preparations were tested in hydrolysis and transesterification reactions. In the hydrolysis no marked differences in activity were found between the coupling media used. In the transesterification, on the other hand, the choice of immobilization medium had a very large effect on lipase activity, the preparation from microemulsion being the most active one. The use of the hydrophilic spacer had a large effect on activity in the hydrolysis reaction. Whereas direct coupling gave an activity of immobilized lipase of 26-34% of that of free enzyme, depending on the reaction medium, lipase bound via the spacer exhibited 56-67% activity. The latter values are considerably higher than previously reported in the literature for covalently immobilized lipase. The hydrophilic spacer had no effect on enzyme activity in the transesterification, however, a fact which is attributed to the hydrophobic medium of this reaction. The spacer is incompatible with the reaction medium and will, therefore, adsorb on the particles rather than stretch out into the bulk phase. The stability of the bound lipase was extremely good, no loss in activity being observed after a period of three weeks in aqueous solution of 37 degrees C.     

Cytotoxicity associated with trichloroethylene oxidation in Burkholderia cepacia G4

The effects of trichloroethylene (TCE) oxidation on toluene 2-monooxygenase activity, general respiratory activity, and cell culturability were examined in the toluene-oxidizing bacterium Burkholderia cepacia G4. Nonspecific damage outpaced inactivation of toluene 2-monooxygenase in B. cepacia G4 cells. Cells that had degraded approximately 0.5 micromol of TCE (mg of cells(-1)) lost 95% of their acetate-dependent O(2) uptake activity (a measure of general respiratory activity), yet toluene-dependent O(2) uptake activity decreased only 35%. Cell culturability also decreased upon TCE oxidation; however, the extent of loss varied greatly (up to 3 orders of magnitude) with the method of assessment. Addition of catalase or sodium pyruvate to the surfaces of agar plates increased enumeration of TCE-injured cells by as much as 100-fold, indicating that the TCE-injured cells were ultrasensitive to oxidative stress. Cell suspensions that had oxidized TCE recovered the ability to grow in liquid minimal medium containing lactate or phenol, but recovery was delayed substantially when TCE degradation approached 0.5 micromol (mg of cells(-1)) or 66% of the cells' transformation capacity for TCE at the cell density utilized. Furthermore, among B. cepacia G4 cells isolated on Luria-Bertani agar plates from cultures that had degraded approximately 0.5 micromol of TCE (mg of cells(-1)), up to 90% were Tol(-) variants, no longer capable of TCE degradation. These results indicate that a toxicity threshold for TCE oxidation exists in B. cepacia G4 and that once a cell suspension has exceeded this toxicity threshold, the likelihood of reestablishing an active, TCE-degrading biomass from the cells will decrease significantly.     

Cytotoxicity Associated with Trichloroethylene Oxidation in Burkholderia cepacia G4

The effects of trichloroethylene (TCE) oxidation on toluene 2-monooxygenase activity, general respiratory activity, and cell culturability were examined in the toluene-oxidizing bacterium Burkholderia cepacia G4. Nonspecific damage outpaced inactivation of toluene 2-monooxygenase in B. cepacia G4 cells. Cells that had degraded approximately 0.5 μmol of TCE (mg of cells⁻¹) lost 95% of their acetate-dependent O₂ uptake activity (a measure of general respiratory activity), yet toluene-dependent O₂ uptake activity decreased only 35%. Cell culturability also decreased upon TCE oxidation; however, the extent of loss varied greatly (up to 3 orders of magnitude) with the method of assessment. Addition of catalase or sodium pyruvate to the surfaces of agar plates increased enumeration of TCE-injured cells by as much as 100-fold, indicating that the TCE-injured cells were ultrasensitive to oxidative stress. Cell suspensions that had oxidized TCE recovered the ability to grow in liquid minimal medium containing lactate or phenol, but recovery was delayed substantially when TCE degradation approached 0.5 μmol (mg of cells⁻¹) or 66% of the cells' transformation capacity for TCE at the cell density utilized. Furthermore, among B. cepacia G4 cells isolated on Luria-Bertani agar plates from cultures that had degraded approximately 0.5 μmol of TCE (mg of cells⁻¹), up to 90% were Tol⁻ variants, no longer capable of TCE degradation. These results indicate that a toxicity threshold for TCE oxidation exists in B. cepacia G4 and that once a cell suspension has exceeded this toxicity threshold, the likelihood of reestablishing an active, TCE-degrading biomass from the cells will decrease significantly.