Production of mannosylerythritol lipids as biosurfactants by resting cells ofCandida antarctica

Summary The resting cells ofCandida antarctica strain T-34 was found to produce a large amount of mannosylerythritol lipids as biosurfactants when incubated in the medium containing only the carbon source. The resting cells prepared from different water-soluble carbon sources were able to produce the lipids abundantly from water-insoluble carbon sources. Under the optimal conditions in a shake culture, the concentration of the total lipids amounted to about 47 g/l after 6 days, and the yield of the lipids became higher than that obtained by using the growing cells of the strain.     

Effects of biosurfactants produced by Candida antarctica on the biodegradation of petroleum compounds

The effects of biosurfactants on the biodegradation of petroleum compounds were investigated. Candida antarctica T-34 could produce extracellular biosurfactant mannosylerythritol lipids (MELs) when it was cultured in vegetable oil. In addition, in our previous study, it was found that this strain could also produce a new type of biosurfactant while it grew on n-undecane (C₁₁H₂₄), and the biosurfactant was named as BS-UC. In flask culture of Candida antarctica, the addition of BS-UC could improve the biodegradation rate of some n-alkanes (e.g. 90.2% for n-decane, 90.2% for n-undecane, 89.0% for dodecane), a mixture of n-alkanes (82.3%) and kerosene (72.5%). By comparing the effects of the biosurfactants BS-UC and MEL and chemical surfactants on the biodegradation of crude oil, it was found that biosurfactants could be used to enhance the degradation of petroleum compounds instead of chemical surfactants. In a laboratory scale immobilized bioreactor, the addition of biosurfactant improved not only the emulsification of kerosene in simulated wastewater but also its biodegradation rate. The highest degradation rate of kerosene by addition of MEL and BS-UC reached 87 and 90% at 15 h, respectively. The results showed that the biosurfactant BS-UC was highly promising for work on biodegradation of hydrophobic contaminants.     

Microbial conversion of n-alkanes into glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma (Candida antarctica)

n-Alkanes ranging from C₁₂ to C₁₈ were converted into glycolipid biosurfactants, mannosylerythritol lipids (MEL), by resting cells of Pseudozyma (Candida) antarctica T-34. The highest yield (0.87 g g^–1 substrate) was obtained from 6% (v/v) of n-octadecane after 7 days reaction. The amount of MEL reached 140 g l^–1 by intermittent feeding of the substrate.     

Intracellular accumulation of mannosylerythritol lipids as storage materials by Candida antarctica

Summary Candida antarctica strain T-34, which was isolated as a biosurfactant producer, was found to produce organic acids and polyols extracellularly but not to produce biosurfactants, when grown on glucose or other carbohydrates as the sole carbon source. It was also observed microscopically that the strain contained oil globules within the cells. The intracellular lipids of the strain mainly consisted of triglycerides and mannosylerythritol lipids (MEL). The MEL content of the cells during the culture exceeded 10% of the dry cell weight, and the pattern of variation of the MEL content was very similar to that of triglycerides. All three stock strains of C. antarctica tested also accumulated a relatively large amount of MEL from glucose. These results suggested that these strains accumulated the MEL intracellularly as one of the storage materials together with triglycerides.Offprint requests to: D. Kitamoto     

Influence of medium composition and aeration on the synthesis of biosurfactants produced by Candida antarctica

Candida antarctica synthesised surface-active mannosylerythritol lipids at 46 g l^–1 by adding 80 g soybean oil l^–1 to the medium and maintaining the pO₂ at 50% with an air flow rate 1 vvm. Two-stage culturing of C. antarctica avoided medium foaming but the yield of biosurfactants synthesis was 28 g l^–1. The biosurfactants decreased the surface tension of water to 35 mN m^–1.     

Transgenic rice as bioreactor for production of the Candida antarctica lipase B

Candida antarctica lipase B (CALB) is a versatile biocatalyst used for a wide range of biotransformation. Methods for low cost production of this enzyme are highly desirable. Here, we report a mass production method of CALB using transgenic rice seeds as the bioreactor. The transgenic rice transformed with the CALB gene under the control of the promoter of the rice seed storage protein GT1 was found to have accumulated a large quantity of CALB in seeds. The transgenic line with the highest lipolytic activity reached to 85 units per gram of dry seeds. One unit is defined as the amount of lipase necessary to liberate 1 μmol p‐nitrophenol from p‐nitrophenyl butyrate in 1 min. The rice recombinant lipase (rOsCALB) from this line represents 40% of the total soluble proteins in the crude seed extracts. The enzyme purified from the rice seeds had an optimal temperature of 40 °C, and optimal pH of 8.5, similar to that of the fermentation products. Test of its conversion ability as a biocatalyst for biodiesel production suggested that rOsCALB is functionally identical to the fermentation products in its industrial application.     

Capsaicin hydrolysis by Candida antarctica lipase

Capsaicin was hydrolysed by lipase B from Candida antarctica into vanillylamine and 8-methyl-6-trans-nonenoic acid. Conversions of 70% were obtained after 72 h at 70 °C in water but decreased to only 15% when capsaicin was solubilized in 15% (v/v) ethanol/water after 72 h at 45 °C. No activity occurred in chloroform/water mixtures. According to our knowledge, this is the first report concerning amide hydrolysis by a lipase.     

Effect of cerulenin and sodium butyrate on chitin synthesis in Candida albicans

Experiments were conducted to gain insight concerning the mechanism(s) whereby cerulenin and sodium butyrate affect chitin synthesis in Candida albicans. In vitro studies with isolated membrane-bound chitin synthase from C. albicans, strain 4918, showed that neither agent affected the level of either unactivated or trypsin-activated enzyme activity. Subsequent studies utilizing protoplasts revealed that early in the cell wall regeneration process, cells treated with cerulenin or butyrate synthesized chitin at a rate equal to untreated controls, as measured by the incorporation of [3H]-N-acetylglucosamine (GlcNAc) into acid-alkali insoluble material. However, after 40 min of incubation, the incorporation of [3H]GlcNAc into chitin is reduced in cells treated with either agent. On the other hand, samples taken during the same time intervals and analyzed by flow cytometry suggested that the amount of chitin synthesis in treated and untreated cells was identical. A marked decrease in fluorescence was observed in similar experiments using polyoxin D, a direct inhibitor of chitin synthase activity. Experiments that measured uptake of [3H]GlcNAc into both whole cells and protoplasts demonstrated that cerulenin and butyrate had no effect on the transport of the chitin precursor.     

Effect of additives on the esterification activity of immobilized Candida antarctica lipase

In this work, the stabilizing effect of bovine serum albumin (BSA), peptone (PEP), and polyethylene glycol (PEG) during immobilization of Candida antarctica lipase on activated carbon was investigated. The influence of enzyme concentration and type of additive, added during the immobilization procedure, was studied using a 2² factorial central composite design. The goal was to maximize the synthetic activity of butyl butyrate, using butyric acid and butanol as substrate in n-heptane. An increase of 31–58% in the esterification activity was obtained when enzyme concentration on the supernatant was enhanced from 86.50 U m L⁻¹ to 226.80 U mL⁻¹. An enhancement in esterification activity of 38–68.95% was observed, depending on the initial enzyme concentration, when PEP was used instead of BSA. No significant increase in the esterification activity was observed when PEP was replaced by PEG. However, thermal stability tests at 50 °C showed that PEG had a higher stabilizing effect.     

添加生物素和浅蓝菌素对裂殖壶菌发酵产DHA的影响

为提高发酵产量,根据裂殖壶菌生物合成二十二碳六烯酸(DHA)的途径,考察添加代谢途径关键酶的辅酶及酶的抑制剂对发酵裂殖壶菌的影响.结果表明:添加生物素可促进油脂积累,添加浅蓝菌素有利于DHA及不饱和脂肪酸含量的提高.添加生物素0.3 mg/L时,DHA占细胞干质量分数达11.26％,相对于对照提高了13％;当添加浅蓝菌素0.1mg/L时,DHA占细胞干质量分数可达12.2％.     

Glycerol triacetate as solvent and acyl donor in the production of isoamyl acetate with Candida antarctica lipase B

Glycerol triacetate was successfully used as a green solvent and as the acyl donor in the transesterification of isoamyl alcohol to produce isoamyl acetate using free and immobilized Candida antarctica lipase B. Immobilized lipase was more catalytically active than free lipase and could be easily separated from the reaction mixture by filtration. In addition, it was found that increasing either the reaction temperature or the enzyme to substrate ratio increased the conversion of isoamyl alcohol. Using triacetin as the solvent also enabled the separation of product by simple extraction with petroleum ether and catalyst recycling.     

Influence of substrate supply on the production of sophorose lipids by Candida bombicola ATCC 22214

Summary Candida (Torulopsis) bombicola ATCC 22214 produces 180 g/l sophorose lipids using glucose and oleic acid (technical grade) as combined substrates in an extended fed-batch cultivation. Excess of oleic acid generated a paste-like product. However, only when oleic acid was not detectable during the whole run of cultivation, a microcrystalline product precipitated. The unsaturated C-18 fatty acids of the technical grade oleic acid were transferred unchanged into the sophorose lipid.     

Kinetic model of biodiesel production using immobilized lipase Candida antarctica lipase B

We have designed a kinetic model of biodiesel production using Novozym 435 (Nz435) with immobilized Candida antarctica lipase B (CALB) as a catalyst. The scheme assumed reversibility of all reaction steps and imitated phase effects by introducing various molecular species of water and methanol. The global model was assembled from separate reaction blocks analyzed independently. Computer simulations helped to explore behavior of the reaction system under different conditions. It was found that methanolysis of refined oil by CALB is slow, because triglycerides (T) are the least reactive substrates. Conversion to 95% requires 1.5–6 days of incubation depending on the temperature, enzyme concentration, glycerol inhibition, etc. Other substrates, free fatty acids (F), diglycerides (D) and monoglycerides (M), are utilized much faster (1–2 h). This means that waste oil is a better feedstock for CALB. Residual enzymatic activity in biodiesel of standard quality causes increase of D above its specification level because of the reaction 2M ↔ D + G. Filtration or alkaline treatment of the product prior to storage resolves this problem. The optimal field of Nz435 application appears to be decrease of F, M, D in waste oil before the conventional alkaline conversion. Up to 30-fold reduction of F-content can be achieved in 1–2 h, and the residual enzyme (if any) does not survive the following alkaline treatment.     

Green enzymatic production of glyceryl monoundecylenate using immobilized Candida antarctica lipase B

Enzymatic synthesis of glyceryl monoundecylenate (GMU) was performed using indigenously immobilized Candida anatarctica lipase B preparation (named as PyCal) using glycerol and undecylenic acid as substrates. The effect of molar ratio, enzyme load, reaction time, and organic solvent on the reaction conversion was determined. Both batch and continuous processes for GMU synthesis with shortened reaction time were developed. Under optimized batch reaction conditions such as 1:5 molar ratio of undecylenic acid and glycerol, 2?h of reaction time at 30% substrate concentration in tert-butyl alcohol, conversion of 82% in the absence of molecular sieve, and conversion of 93% in the presence of molecular sieve were achieved. Packed bed reactor studies resulted in high conversion of 86% in 10-min residence time. Characterization of formed GMU was performed by FTIR, MS/MS. Enzymatic process resulted in GMU as a predominant product in high yield and shorter reaction time periods with GMU content of 92% and DAG content of 8%. Optimized GMU synthesis in the present study can be used as a useful reference for industrial synthesis of fatty acid esters of glycerol by the enzymatic route.     

Effect of temperature on siderophore production by Candida albicans

The purpose of this study was to examine the effect of elevated temperature on growth and siderophore production by Candida albicans. The results showed that an increase in incubation temperature from 37 degrees C to 41 degrees C produced a marked decrease in both the rate and quantity of siderophore production. Elevated temperature was unable to suppress growth of C. albicans in either a control culture medium or a deferrated culture medium. A significant suppression of growth compared to the controls was observed in the deferrated media at both 37 degrees C and 41 degrees C. However with time, the growth of cells in the deferrated media showed partial recovery which was followed by an increase in siderophore production. Thus, elevation of temperature to suppress growth and siderophore production by C. albicans appears to be an ineffective host defense mechanism.     

Solvent as a competitive inhibitor for Candida antarctica lipase B

In enzyme-catalyzed reactions, the choice of solvent often has a marked effect on the reaction outcome. In this paper, it is shown that solvent effects could be explained by the ability of the solvent to act as a competitive inhibitor to the substrate. Experimentally, the effect of six solvents, 2-pentanone, 3-pentanone, 2-methyl-2-pentanol, 3-methyl-3-pentanol, 2-methylpentane and 3-methylpentane, was studied in a solid/gas reactor. As a model reaction, the CALB-catalyzed transacylation between methyl propanoate and 1-propanol, was studied. It was shown that both ketones inhibited the enzyme activity whereas the tertiary alcohols and the hydrocarbons did not. Alcohol inhibition constants, K(i)(I) were changed to "K(i)", determined in presence of 2-pentanone, 3-pentanone, and 3-methyl-3-pentanol, confirmed the marked inhibitory character of the ketones and an absence of inhibition of 3-methyl-3-pentanol. The molecular modeling study was performed on three solvents, 2-pentanone, 2-methyl-2-pentanol and 2-methyl pentane. It showed a clear inhibitory effect for the ketone and the tertiary alcohol, but no effect for the hydrocarbon. No change in enzyme conformation was seen during the simulations. The study led to the conclusion that the effect of added organic component on lipase catalyzed transacylation could be explained by the competitive inhibitory character of solvents towards the first binding substrate methyl propanoate.     

Effect of the immobilization protocol on the properties of lipase B from Candida antarctica in organic media: Enantiospecifc production of atenolol acetate

In this work, we intend to check the effect of the immobilization protocol on the performance of lipase B from Candida antarctica (CalB) in organic medium. To this purpose, CalB has been immobilized on Eupergit C (EC) under different conditions and on EC partially modified with ethylenediamine (EDA), iminodiacetic acid (IDA) or metal chelate (IDA-Cu²⁺) and used for kinetic resolution of (R/S) 4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]benzeneacetamide (rac-atenolol). Enantiomeric resolution of atenolol was carried out by a transesterification reaction using vinyl acetate as acylant agent and an organic solvent as reaction medium. After a preliminary optimization of the reaction to obtain satisfactory yields, toluene was selected as the optimal solvent and the performances of the different CalB biocatalysts were compared. The R enantiomer was preferred in all cases but their performances were substantially different, with high differences in reaction rates, reaction yields in this kinetically controlled synthesis (EC-CalB gave a conversion of 76% while EC-IDA-Cu²⁺-CalB gave just a 27%), and enantiospecificities (EC-CalB gave an E value of 65 while EC-IDA-Cu²⁺-CalB gave a value of 13). Replacing toluene with hexane caused a decrease in enzyme activity, reaction yields and enantiospecificity of the reaction. It was remarkable that some preparations were much more sensitive to this solvent change than others. Considering that the activity decreased by less than 10% per reaction cycle, these differences are likely associated with the differences in the enzyme catalytic properties caused by the different immobilization protocols and not by inactivation of immobilized enzyme preparations during the reaction. These results confirmed that use of different immobilization protocols may be a powerful tool for altering enzyme properties when used in organic media.     

Effect of molecular chaperones on the expression of Candida antarctica lipase B in Pichia pastoris

One of the reasons for limited heterologous protein secretion in Pichia pastoris is the suboptimal folding conditions inside the cell. The Hsp70 and Hsp40 chaperone families in the cytoplasm or the ER regulate the folding and secretion of heterologous proteins. Here, we have studied the effect of chaperones Ydj1p, Ssa1p, Sec63p and Kar2p on the secretory expression of Candida antarctica lipase B (CalB) protein. Expression of CalB in P. pastoris resulted in the induction of Kar2p secretion into the medium surpassing the retrieval capacity of the cell. Individual overexpression of Ydj1p, Ssa1p and Sec63p in recombinant P. pastoris increased CalB expression level by 1.6-, 1.4- and 1.4-fold respectively compared to the control strain harboring only the CalB gene. However, overexpression of Kar2p had a negative effect on the expression of CalB. Moreover, Western blot analysis indicated accumulation and secretion of Kar2p in the ER, Golgi and extracellular medium in the chaperone coexpression strains. When expressed in combinations such as Ydj1p–Ssa1p, Ydj1p–Sec63p, Kar2p–Ssa1p, Kar2p–Sec63p, the expression level of CalB was increased by 2.5-, 1.5-, 1.5- and 1.5-fold respectively. Contrastingly, the Kar2p–Ydj1p combination resulted in decreased CalB secretion in the supernatant. From these results, we conclude that overexpression of Kar2p is not required for the secretion of CalB. Also, our work confirmed the synergistic effect of Ssa1p and Ydj1p chaperones in the expression of CalB.     

Aminolysis of 2-hydroxy esters catalyzed by Candida antarctica lipase

Candida antarctica lipase catalyzed the aminolysis of 2-hydroxy esters with amines in organic solvents to yield the corresponding 2-hydroxy amides. The reactions proceeded at 28–30 °C in dioxane for 6 h with 3 mM substrates with yields ranging between 45% (w/w) (for branched substrates) to 88% (w/w) (for linear substrates). Although the reaction was not enantioselective, because of its simplicity it represents an alternative method for the synthesis of functionalised amides.