Impact of surfactants on solubilization and activity of the carotenoid cleavage dioxygenase,AtCCD1, in an aqueous micellar reaction system

The effect of various surfactants on both the solubilization of the carotenoid cleavage dioxygenase, AtCCD1, from cell lysates and the enzymatic activity in an aqueous micellar system was investigated. Solubilization with sodium cholate more than doubled the specific activity. Lag phases were observed when Tween surfactants were used for substrate delivery and were dependent on the surfactant and enzyme modification. In contrast to His₆- and GST-tagged AtCCD1, unmodified AtCCD1 showed a 45% increased maximum rate in the Tween 20 system compared to Triton X-100 based reference system. The results emphasize the importance of engineering the interface for the in vitro application of this enzyme family.     

Novel mannitol based non-ionic surfactants from biocatalysis: Part two: improved synthesis

The 1-O-lauroyl- -mannitol, a non-ionic surfactant, was synthesised via a chemo-enzymatic pathway starting from the 1,2:4,5-di-O-isopropylidene- -mannitol and vinyl laurate as acylation agent. The high hydrophobicity of the substrates allowed the enzymatic reaction to occur both in n-hexane and in solvent free conditions. The immobilised Candida antarctica lipase B was used as the catalyst of the enzymatic step. This enzyme acts differently depending on the position of the hydroxyls with respect to the isopropylidene groups. The acid selective hydrolysis of the isopropylidene groups gave the non-ionic surfactant without the presence of isomers.     

Adsorption of surfactants on a <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strain and the effect on cell surface lypohydrophilic property

The adsorption behavior of five surfactants, cetyltrimethylammonium bromide (CTAB), Triton X-100, Tween 80, sodium dodecyl sulfate (SDS), and rhamnolipid, on a Pseudomonas aeruginosa strain and the effect of temperature and ionic strength (IS) on the adsorption were studied. The change of cell surface lypohydrophilic property caused by surfactant adsorption was also investigated. The results showed that the adsorption kinetics of the surfactants on the cell followed the second-order law. CTAB adsorption was the fastest one under the experimental conditions, and it took longest for SDS adsorption to equilibrate because of electric repulsion. The adsorption of Triton X-100 and Tween 80 was characterized by short equilibration time, and rhamnolipid adsorption reached equilibrium in about 90 min. The adsorption isotherms of all the surfactants on the bacterium fitted Freundlich equation well, but the adsorption capacity and mode were variations for the surfactants as indicated by k and n parameters in the equations. The adsorption mode for all the surfactants except SDS is probably hydrophilic interaction because the adsorption totally turned the cell surface to be more hydrophobic. Neither the temperature nor the IS had significant effect on CTAB adsorption, but higher IS significantly enhanced SDS adsorption and modestly strengthened adsorption of Triton X-100, Tween 80, and rhamnolipid. Higher temperature strengthened adsorption of SDS but weakened the adsorption of Triton X-100, Tween 80, and rhamnolipid.     

Mixed reverse micelles facilitated downstream processing of lipase involving water‐oil‐water liquid emulsion membrane

Our earlier work for the first time demonstrated that liquid emulsion membrane (LEM) containing reverse micelles could be successfully used for the downstream processing of lipase from Aspergillus niger. In the present work, we have attempted to increase the extraction and purification fold of lipase by using mixed reverse micelles (MRM) consisting of cationic and nonionic surfactants in LEM. It was basically prepared by addition of the internal aqueous phase solution to the organic phase followed by the redispersion of the emulsion in the feed phase containing enzyme, which resulted in globules of water‐oil‐water (WOW) emulsion for the extraction of lipase. The optimum conditions for maximum lipase recovery (100%) and purification fold (17.0‐fold) were CTAB concentration 0.075 M, Tween 80 concentration 0.012 M, at stirring speed of 500 rpm, contact time 15 min, internal aqueous phase pH 7, feed pH 9, KCl concentration 1 M, NaCl concentration 0.1 M, and ratio of membrane emulsion to feed volume 1:1. Incorporation of the nonionic surfactant (e.g., Tween 80) resulted in remarkable improvement in the purification fold (3.1–17.0) of the lipase. LEM containing a mixture of nonionic and cationic surfactants can be successfully used for the enhancement in the activity recovery and purification fold during downstream processing of enzymes/proteins. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1084–1092, 2014     

Development of yeast cells displaying <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B and their application to ester synthesis reaction

We isolated the lipase B from Candida antarctica CBS 6678 (CALB CBS6678) and successfully constructed CALB-displaying yeast whole-cell biocatalysts using the Flo1p short (FS) anchor system. For the display of CALB on a yeast cell surface, the newly isolated CALB CBS6678 exhibited higher hydrolytic and ester synthesis activities than the well-known CALB, which is registered in GenBank (Z30645). A protease accessibility assay using papain as a protease showed that a large part of CALB, approximately 75%, was localized on an easily accessible part of the yeast cell surface. A comparison of the lipase hydrolytic activities of yeast whole cells displaying only mature CALB (CALB) and those displaying mature CALB with a Pro region (ProCALB) revealed that mature CALB is preferable for yeast cell surface display using the Flo1p anchor system. Lyophilized yeast whole cells displaying CALB were applied to an ester synthesis reaction at 60°C using adipic acid and n-butanol as substrates. The amount of dibutyl adipate (DBA) produced increased with the reaction time until 144 h. This indicated that CALB displayed on the yeast cell surface retained activity under the reaction conditions.     

Erucic acid production using porcine pancreas lipase: Enhancement by mixed surfactants

Application of mixed surfactants coupled with statistical optimization in lipase catalyzed oil hydrolysis is presented for the first time in this study. Selective hydrolysis of brown mustard oil to erucic acid by porcine pancreas lipase was enhanced by mixed surfactants comprising of an oil-soluble nonionic surfactant (Span 80) and a watersoluble nonionic surfactant (Tween 80). The production of erucic acid was maximized using statistically designed experiments and subsequent analysis of their result by response surface methodology. The most significant variables were enzyme concentration and concentration of Tween 80. Small changes in pH and concentration of Span 80 also produced a significant change in the production of erucic acid. Temperature and speed of agitation were insignificant variables and were fixed at 35oC and 900 rpm, respectively. Under these conditions, the optimal combination of other variables were pH 9.65, 2.13 mg/g enzyme in oil, 9.8 × 10⁻³ M Span 80 (in oil), and 4 × 10⁻³ M Tween 80 (in buffer). These conditions led to formation of 99.69% of the total erucic acid in 1.25 h. Interaction of enzyme concentration with pH significantly affected erucic acid production.     

Immobilization of lipase Eversa Transform 2.0 on poly(urea–urethane) nanoparticles obtained using a biopolyol from enzymatic glycerolysis

In this work, the free lipase Eversa^® Transform 2.0 was used as a catalyst for enzymatic glycerolysis reaction in a solvent-free system. The product was evaluated by nuclear magnetic resonance (¹H NMR) and showed high conversion related to hydroxyl groups. In sequence, the product of the glycerolysis was used as stabilizer and biopolyol for the synthesis of poly(urea–urethane) nanoparticles (PUU NPs) aqueous dispersion by the miniemulsion polymerization technique, without the use of a further surfactant in the system. Reactions resulted in stable dispersions of PUU NPs with an average diameter of 190 nm. After, the formation of the PUU NPs in the presence of concentrated lipase Eversa^® Transform 2.0 was studied, aiming the lipase immobilization on the NP surface, and a stable enzymatic derivative with diameters around 231 nm was obtained. The hydrolytic enzymatic activity was determined using ρ-nitrophenyl palmitate (ρ-NPP) and the immobilization was confirmed by morphological analysis using transmission electron microscopy and fluorescence microscopy.

     

Relation between Candida maltosa Hydrophobicity and Hydrocarbon Biodegradation

Summary The growth of Candida maltosa on hydrocarbons (dodecane and hexadecane) was influenced by adding various natural and synthetic surfactants. Microbial adhesion to the hydrocarbon was used to measure the surface cell hydrophobicity of the yeast, which in the presence of a synthetic surfactant correlated with the degree of hydrocarbon biodegradation. Non-ionic surfactants caused the highest degree of hydrocarbon biodegradation corresponding the lowest hydrophobicity. A different correlation was observed with natural surfactants, of which saponin was the most effective for hydrocarbon biodegradation, though the concentration of this surfactant had no influence on surface cell hydrophobicity.     

Optimization of mono and diacylglycerols production from enzymatic glycerolysis in solvent-free systems

This work reports solvent-free enzymatic glycerolysis of olive oil with an immobilized lipase (Novozym 435) using Tween 40, Tween 65, Tween 80, Tween 85, Triton X-100, and soy lecithin as surfactants. The first step was the screening of two potential surfactants for Monoacylglycerol (MAG) and Diacylglycerol (DAG) production with a pre-established operating condition and 2 h of reaction time. Afterwards, a sequential experimental design strategy was carried out in order to optimize MAG and DAG production using Tween 65 and Triton X-100 as surfactants. The operating conditions that optimized MAG and DAG yields were 70 °C, stirring rate of 600 rpm, glycerol:olive oil molar ratio of 6:1, 16 wt% of surfactant Tween 65 and 9.0 wt% of Novozym 435, leading to a content of 26 and 17 wt% of MAG and DAG, respectively.     

Display of Bacterial Lipase on the Escherichia coli Cell Surface by Using FadL as an Anchoring Motif and Use of the Enzyme in Enantioselective Biocatalysis

We have developed a novel cell surface display system by employing FadL as an anchoring motif, which is an outer membrane protein involved in long-chain fatty acid transport in Escherichia coli. A thermostable Bacillus sp. strain TG43 lipase (44.5 kDa) could be successfully displayed on the cell surface of E. coli in an active form by C-terminal deletion-fusion of lipase at the ninth external loop of FadL. The localization of the truncated FadL-lipase fusion protein on the cell surface was confirmed by confocal microscopy and Western blot analysis. Lipase activity was mainly detected with whole cells, but not with the culture supernatant, suggesting that cell lysis was not a problem. The activity of cell surface-displayed lipase was examined at different temperatures and pHs and was found to be the highest at 50°C and pH 9 to 10. Cell surface-displayed lipase was quite stable, even at 60 and 70°C, and retained over 90% of the full activity after incubation at 50°C for a week. As a potential application, cell surface-displayed lipase was used as a whole-cell catalyst for kinetic resolution of racemic methyl mandelate. In 36 h of reaction, (S)-mandelic acid could be produced with the enantiomeric excess of 99% and the enantiomeric ratio of 292, which are remarkably higher than values obtained with crude lipase or cross-linked lipase crystal. These results suggest that FadL may be a useful anchoring motif for displaying enzymes on the cell surface of E. coli for whole-cell biocatalysis.     

Effects of monorhamnolipid and Tween 80 on the degradation of phenol by Candida tropicalis

The effects of the biosurfactant monorhamnolipid (monoRL) and the chemical surfactant Tween 80 on the degradation of phenol by Candida tropicalis CICC 1463 were studied. Both surfactants impeded the decay in cell concentrations at the beginning of the fermentation and enhanced the cell growth thereafter. They also increased the degradation efficiencies of 500 mg/L phenol from 86.9% in control to above 99.0% for all test concentrations within 30 h. The monoRL could also be degraded by the C. tropicalis. These results indicate that the surfactants could diminish the toxicity of phenol to the yeast, increase cell growth and improve phenol removal. The monoRL is better than Tween 80 because of biodegradability.     

Water activity dependence of performance of surface-displayed lipase in yeast cells: a unique water requirement for enzymatic synthetic reaction in organic media

Water activity (a(w)) is a crucial parameter affecting enzymatic synthetic reactions in organic media. In this paper, we report on the a(w) dependence of surface-displayed lipases, genetically immobilized on yeast cells via fusion with cell wall proteins. When Saccharomyces cerevisiae displaying Rhizopus oryzae lipase was used for esterification in n-hexane, equilibrating the dried cells with water prior to the reaction markedly increased the reaction rate. An equilibration of the cells with various saturated salt solutions showed that the reaction rate increased with increasing a(w) of the salt solution, to give the best performance at a(w) of 1.0. Interestingly, this trend was extremely different from those of lipases in powder or resin-immobilized form. To determine whether the cell surface is responsible for the unique a(w) profiles, an investigation was carried out similarly using other lipase sources and yeast strains, which indicated that, in all the cells examined, a higher a(w) resulted in a higher reaction rate. Moreover, increasing a(w) was found to increase the cell surface hydrophobicity determined by an aqueous-hydrocarbon biphasic partitioning assay. These results indicate that lipases displayed on yeast cells show a unique a(w) dependence probably because of the variation in cell surface characteristics.     

A novel thermostable lipase from Basidiomycete <Emphasis Type="Italic">Bjerkandera adusta </Emphasis>R59: characterisation and esterification studies

Microbial lipases are widely diversified in their enzymatic properties and substrate specificities, which make them very attractive for industrial application. Partially purified lipase from Bjerkandera adusta R59 was immobilized on controlled porous glass (CPG) and its properties were compared with those of the free enzyme. The free and immobilized lipases showed optimal activities at 45 and 50°C, respectively. Both enzyme forms were highly thermostable up to 60°C. The enzymes were stable at pH from 6.0 to 9.0 and their optimal pH for activity was 7.0. The free lipase was more thermostable in n-hexane than in aqueous environment. Both lipase preparations had good stabilities in non-polar solvents and were capable of hydrolysing a variety of synthetic and natural fats. Non-immobilized lipase activity was inhibited by disulphide bond reagents, serine and thiol inhibitors, while EDTA and eserine had no effect on enzyme activity. All anionic detergents tested in experiments inhibited lipase activity. The free lipase showed good stability in the presence of commercial detergents at laundry pH and temperatures. Applications of free and immobilized lipases for esterification were also presented.     

Effect of a synthetic surfactant on phenanthrene and n-eicosane utilization by two pure marine strains grown separately in batch cultures with or without sand particles

The effect of Tween 80, a nonionic surfactant, on the extent of biodegradation of phenanthrene by Sphingomonas sp. 2MPII and n-eicosane by Corynebacterium sp. 8 was investigated. This surfactant was beneficial only for phenanthrene biodegradation, where it increased the extent of degradation from 54 to 74%. It appears to be used as carbon source by Corynebacterium sp. 8 but the extent of biodegradation decreases from 43 to 20%. In sand-containing cultures, the phenanthrene sorption observed only in the presence of Tween reduced the biodegradation extent to 35%. On the other hand, for n-eicosane, which remains in the aqueous phase, the biodegradation extent was markedly enhanced to 74%.     

Transesterification of phosphatidylcholine in <Emphasis Type="Italic">sn</Emphasis>-1 position through direct use of lipase-producing <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> cells as whole-cell biocatalyst

The enzymatic process presents an advantage of producing specified phospholipids that rarely exist in nature. In this study, we investigated the regiospecific modification of phosphatidylcholine (PC) in the sn-1 position using immobilized Rhizopus oryzae. In a reaction mixture containing egg yolk PC and exogenous lauric acid (LA) in n-hexane, lipase-producing R. oryzae cells immobilized within biomass support particles (BSPs) showed a much higher transesterification activity than lipase powders. To improve the product yield, several parameters including substrate ratio and reaction time were investigated, resulting in the incorporation of 44.2% LA into the product PC after a 48-h reaction. The analysis of the molecular structure showed that a large proportion of exogenous LA (>90%) was incorporated in the sn-1 position of the enzymatically modified PC. Moreover, the BSP-immobilized R. oryzae maintained its activity for more than 12 batch cycles. The presented results, therefore, suggest the applicability of BSP-immobilized R. oryzae as a whole-cell biocatalyst for the regiospecific modification of phospholipids.     

Lipase-catalyzed synthesis of monoacylglycerol in a homogeneous system

The 1,3-regiospecifique lipase, Lipozyme IM, catalyzed the esterification of lauric acid and glycerol in a homogeneous system. To overcome the drawback of the insolubility of glycerol in hexane, which is extensively used in enzymatic synthesis, a mixture of n-hexane/tert-butanol (1:1, v/v) was used leading to a monophasic system. The conversion of lauric acid into monolaurin was 65% in 8 h, when a molar ratio of glycerol to fatty acid (5:1) was used with the fatty acid at 0.1 M, and the phenomenon of acyl migration was minimized.     

Improvement of direct ethanol fermentation from woody biomasses by the Antarctic basidiomycetous yeast, Mrakia blollopis, under a low temperature condition

The Antarctic basidiomycetous yeast Mrakia blollopis SK-4 can quite uniquely ferment various sugars under low temperature conditions. When strain SK-4 fermented lignocellulosic biomass using the direct ethanol fermentation (DEF) technique, approximately 30% to 65% of the theoretical ethanol yield was obtained without and with the addition of the non-ionic surfactant Tween 80, respectively. Therefore, DEF from lignocellulosic biomass with M. blollopis SK-4 requires the addition of a non-ionic surfactant to improve fermentation efficiency. DEF with lipase converted Eucalyptus and Japanese cedar to 12.6 g/l, and 14.6 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80 and 5 U/g-dry substrate lipase, ethanol concentration increased about 1.4- to 2.4-fold compared to that without Tween 80 and lipase. We therefore consider that the combination of M. blollopis SK-4 and DEF with Tween 80 and lipase has good potential for ethanol fermentation in cold environments.     

A New Cold-Adapted,Organic Solvent Stable Lipase from Mesophilic Staphylococcus epidermidis AT2

The gene encoding a cold-adapted, organic solvent stable lipase from a local soil-isolate, mesophilic Staphylococcus epidermidis AT2 was expressed in a prokaryotic system. A two-step purification of AT2 lipase was achieved using butyl sepharose and DEAE sepharose column chromatography. The final recovery and purification fold were 47.09 % and 3.45, respectively. The molecular mass of the purified lipase was estimated to be 43 kDa. AT2 lipase was found to be optimally active at pH 8 and stable at pH 6–9. Interestingly, this enzyme demonstrated remarkable stability at cold temperature (<30 °C) and exhibited optimal activity at a temperature of 25 °C. A significant enhancement of the lipolytic activity was observed in the presence of Ca²⁺, Tween 60 and Tween 80. Phenylmethylsulfonylfluoride, a well known serine inhibitor did not cause complete inhibition of the enzymatic activity. AT2 lipase exhibited excellent preferences towards long chain triglycerides and natural oils. The lipolytic activity was stimulated by dimethylsulfoxide and diethyl ether, while more than 50 % of its activity was retained in methanol, ethanol, acetone, toluene, and n-hexane. Taken together, AT2 lipase revealed highly attractive biochemical properties especially because of its stability at low temperature and in organic solvents.     

Extractive fermentation in cloud point system for lipase production by Serratia marcescens ECU1010

Extractive microbial fermentation for production of lipase by Serratia marcescens ECU1010 has been carried out in cloud point system. The cloud point system is composed of mixture nonionic surfactants with a ratio of Triton X-114 to Triton X-45 4:1 in aqueous solution. The lipase prefers to partition into the surfactant rich phase (coacervate phase) whereas the cells and other hydrophilic proteins retain in the dilute phase of cloud point system. Thus, a concentration factor 4.2-fold and a purification factor 1.3-fold of the lipase have been achieved in the extractive fermentation process. This is the first report about extractive fermentation of proteins in cloud point system.     

Application of solvent engineering to optimize lipase-catalyzed 1,3-diglyacylcerols by mixture response surface methodology

1,3-Diacylglycerol has been introduced in Japan as a cooking oil under the trade name of Econa to reduce body fat accumulation. Solvent engineering was applied to determine the optimum solvent mixtures for the lipase-catalyzed synthesis of 1,3-DAG by mixture response surface methodology. n-Hexane was required to maintain the lipase activity and the product selectivity could be adjusted by changing the hydrophobicity of reaction medium. The optimum yield (40%) of 1,3-DAG synthesis was obtained with n-hexane/octane (1:1, v/v).