Candida rugosa lipases: Molecular biology and versatility in biotechnology

This review describes how the versatile Candida rugosa lipases (CRL) have extended the frontiers of biotechnology. As evidenced by the current literature, CRL claims more applications than any other biocatalyst. This review comprises a detailed discussion on the molecular biology of CRL, its versatile catalytic reactions, broad specificities and diverse immobilization strategies. It also discusses its role in the food and flavour industry, the production of ice cream and single cell protein, biocatalytic resolution of life-saving pharmaceuticals, carbohydrate esters and amino acid derivatives unobtainable by conventional chemical synthesis, potent biocide making, biosensor modulations, eco-friendly approach and bioremediation, biosurfactants in detergent making, and recently, cosmetics and perfumery. © 1998 John Wiley & Sons, Ltd.     

Response surface methodological study of esterification of lactic acid with palmitic acid catalyzed by immobilized lipases from Mucor miehei and porcine pancreas

X _s), enzyme/substrate (E/S) ratios (X _e) and incubation time (X _t) on the preparation of palmitoyl lactic acid using Mucor miehei lipase (MML) and porcine pancreas lipase (PPL). Experimental ester yields were found to be in good agreement with predictions. In the case of MML, the ester yield was found to increase with X _e and X _t and a maximum yield of 27 mM was obtained at X _e=128 activity units(AU)/mM of substrate, X _s=64–74 mM and X _t=72 h. In the case of PPL, the ester yield was found to increase with increases in X _e and X _s. Optimum conditions for PPL were X _e=45 AU/mM, X _s=85–90 mM and X _t=72 h, at which the maximum ester yield of 15 mM was obtained. Received: 14 April 1999 / Revised version: 2 September 1999     

米黑根毛霉脂肪酶基因在毕赤酵母中的高效表达

脂肪酶是主要工业用酶制剂之一,在食品、洗涤剂和制药等领域广泛应用。将编码米黑根毛霉(Rhizo-mucor miehei)脂肪酶RML的基因克隆到pPIC9K载体中,构建了分泌型表达载体pPIC9K-RML,载体经线性化后转化Pichia pastorisGS115,G418梯度筛选获得了分泌表达RML的重组毕赤酵母工程菌,SDS-PAGE分析显示表达的脂肪酶分子量大小与预期一致。初步研究表明,重组脂肪酶最适温度为40℃,最适pH值为8.0,以橄榄油为底物时,发酵上清液酶活最大可达102 U/mL,表明构建的重组毕赤酵母工程菌具有较好的工业化生产潜力。     

Use of lipase from Rhizomucor miehei in dry fermented sausages elaboration: Microbial, chemical and sensory analysis

Three different amounts of lipase (0.075, 0.100 and 0.150 LU/g) from Rhizomucor miehei (Palatase M 200L Novo Nordisk) were used to determine the correct amount to use in dry fermented sausages. Determination of acidity values through fourteen days of ripening showed that 0.100 LU/g was the most appropriate. Two types of fermented sausages were manufactured, addition of the enzyme being the only difference between them. Addition of Palatase did not affect product stability (pH and A(w)), and the growth of micro-organisms. In spite of the increase in acidity value, no rancidity developed as determined by both chemical and sensory analysis. Increases in the liberation of palmitic, palmitoleic, stearic, oleic and linoleic acids were found when lipase was used. Juiciness and taste were slightly better in the sausages with Palatase than in those without, but these differences were not reflected in the overall acceptability.     

皱褶假丝酵母产脂蛋白酯酶的发酵条件优化及性质研究

皱褶假丝酵母经发酵培养可合成分泌脂蛋白酯酶,对该菌种进行了发酵条件的优化,其最适培养基条件为:pH值6.0,250mL三角瓶中装量25mL,所接菌种种龄为24h,接种量为10%,摇床转速为200r/min,28℃培养27h。酶学性质研究结果表明,该酶的最适温度为45℃,最适pH值为7.5。     

Influence of buffer and l-phenylalanine concentration in the Rhizomucor miehei lipase catalysed synthesis of l-phenylalanyl-d-glucose ester investigated through response surface methodology

Reaction conditions for Rhizomucor miehei lipase (RML) catalysed synthesis of l-phenylalanyl-d-glucose using unprotected l-phenylalanine and d-glucose were optimized using response surface methodology (RSM). A central composite rotatable design (CCRD) was employed involving 32 experiments of five variables (l-phenylalanine concentration in mmol, amount of RML in mg, pH, incubation period in h and buffer concentration in mM) at five levels. A second-order polynomial equation was developed in terms of linear, quadratic and cross product terms to study the effects of variables on esterification yields. A R ² value of 0.7 was obtained for this complex reaction. From the surface and contour plots it was found that higher yields were observed for a very narrow pH range of 4.5–6.5 at l-phenylalanine concentrations above 3 mmol. The extent of esterification decreased with increase in RML concentration for incubation periods below 60 h. However, longer incubation periods above 60 h, enhanced esterification at all RML concentrations. Lower conversions below 0.5 mmol required less than 3.5 mmol l-phenylalanine concentration and a broad range of buffer concentration from 0.1 mM (0.1 ml, 0.1 M buffer of pH 6.0) to 0.5 mM (0.5 ml, 0.1 M buffer of pH 6.0). However, higher conversions from 0.6 to 0.8 mmol required a buffer concentration above 0.25 mM (0.25 ml, 0.1 M buffer of pH 6.0) at l-phenylalanine concentrations above 3.5 mmol. An optimum predicted yield of 1.01 mmol for l-phenylalanyl-d-glucose at 3 mmol l-phenylalanine, 100 mg of RML, 24 h incubation period, 0.5 mM (0.5 ml of 0.1 M buffer), pH 4.8 acetate buffer was found to agree with 0.97 mmol obtained under these experimental conditions. Validation experiments carried out under random conditions also exhibited good correspondence between predicted and experimental yields.     

Screening of lipases for the enantiomer resolution of R,S-2-octanol by esterification in organic medium

Summary Eleven commercially available lipase preparations of microbial and animal origin were screened for enantiomer resolution of R,S-2-octanol by esterification with dodecanoic acid in heptane. The influence of temperature, substrate/enzyme ratio and reaction time was studied. Among the lipases used, three microbial (2212 D, Röhm,Aspergillus niger; MY, Meito Sangyo,Candida cylindracea; S 80000, Gist-Brocades,Rhizopus arrhizus) and three porcine pancreas lipases (MKC, Sigma, Röhm) showed the highest rates of ester formation (13%–67%), in which (R)-(–)-2-octanol was predominantly selected by the enzymes. Quantitative capillary gas chromatography (HRGC) revealed ee values for the ester, ranging from 10% to 83%. Enantioselectivity was influenced, in part, by the temperature used. For microbial lipases, increasing the temperature led to a decrease of the enantioselectivity, whereas at 40 °C and 70 °C similar ee values were observed for porcine pancreas lipases.

---

Einsatz von Lipasen zur Enantiomerentrennung von R,S-2-Octanol durch Veresterung in organischem Medium

Zusammenfassung Elf handelsiibliche Lipase-Präparate mikrobiellen und tierischen Ursprungs wurden zur Enantiomerentrennung von R,S-2-Octanol durch Veresterung mit Dodecansäure in Heptan untersucht. Der Einfluß von Temperatur, Substrat-/Enzymverhältnis und Reaktionszeit auf Esterbildung und Enantioselektivität wurde überprüft. Unter diesen Lipasen zeigten drei mikrobielle Enzyme (2212 D, Röhm,Aspergillus niger; MY, Meito Sangyo,Candida cylindracea; S 80000, Gist-Brocades,Rhizopus arrhizus) und drei Schweinepankreas-Lipasen (MKC; Sigma; Röhm) die höchsten Esterbildungsraten (13%–67%) bei vorwiegender Selektivität für (R)-(–)-2-Octanol. Die capillargaschromatographisch ermittelten ee-Werte variierten zwischen 10 bis 83%. Die Enantioselektivität wurde teilweise durch die Temperatur beeinflußt; bei den mikrobiellen Lipasen führte eine Temperaturerhöhung zur Abnahme der Enantioselektivität, während bei den Pankreas-Lipasen bei 40 °C und 70 °C ähnliche ee-Werte festgestellt wurden.

     

Inhibition of L-alanine uptake into bovine jejunal brush border membrane vesicles by L-amino acids

Jejunal epithelial cells from slaughtered Holstein cows were fractionated to obtain purified brush border membranes from which membrane vesicles were prepared for use in amino acid uptake studies. Uptake of alanine was determined by incubation of vesicles with a solution containing radiolabelled alanine, isolation of vesicles and accumulated alanine by filtration, and detection of accumulated alanine by liquid scintillation counting. Uptake studies were conducted under conditions shown to provide linear rates of accumulation. Sodium-dependent active transport was determined as the difference between uptake measured in the presence and absence of sodium in the extravesicular buffer. Inhibition of alanine uptake increased with increasing extra-vesicular inhibitor concentration until a plateau value was reached. Inhibition of sodium-dependent alanine uptake by 100 mM glycine was 72%; 25 mM isoleucine, valine, or methionine completely inhibited initial alanine uptake. These results indicate the existence of at least two sodium-dependent transport systems, one capable and one incapable of accepting glycine for transport. At concentrations designed to represent expected concentrations of free amino acids in intestinal digesta, several equimolar mixtures (.2 to 5 mM) of 20 amino acids inhibited alanine uptake, suggesting that significant interaction among amino acids for uptake may be occurring under in vivo conditions.     

Structural effects of amphiphiles on Candida rugosa lipase activation by freeze-drying of aqueous solution of enzyme and amphiphile

Lipases co-lyophilized with water-soluble gemini-type amphiphiles were found to have high enzyme activity in nonaqueous media without washing out of the amphiphile with anhydrous organic solvent. In this study, we obtained freeze-dried complexes of Candida rugosa lipase (CRL) with six water-soluble twin glusitol-headed amphiphiles bearing different types of hydrophobic tails, including newly synthesized ones, and their transesterification activity in organic solvent was evaluated. The results indicate that the increased enzyme activity upon CRL modification at 200 molar ratio of amphiphile/CRL, which are restricted to the ester-containing amphiphiles, is probably due to the surface activation by the interaction between ester-carbonyl of the amphiphile and phenyl group of the tyrosine residue situated on the surface of the lid in the CRL.     

Lipase-catalyzed synthesis of hydrophobically modified dextrans: activity and regioselectivity of lipase from Candida rugosa

Vinyl decanoate-modified dextran macromolecules (DexT40-VD) were synthesized in dimethyl sulfoxide at 50°C using lipase AY from Candida rugosa for catalyzing transesterification between polysaccharide and vinyl fatty esters. The extent of dextran modification (quantified by the molar ratio of attached alkyl tails to sugar repeat units) with native-, pH-adjusted-, 18-crown-6 ether pretreated pH-adjusted-, and stepwise addition of pretreated lipase AY yielded <3%, 49%, 64% and 96% modified dextran respectively. Lipase AY accelerated the transesterification of DexT40 from 2- to 63-fold higher than the non-catalyzed system. This procedure was extended to other acyl donors showing that modification pattern exhibited regioselectivity depending on acyl donor structure. Regioselectivity equaled between 2- and 3-OH with saturated fatty acyl donors. The 2-OH was favored for unsaturated fatty acyl donors, while sterically hindered acyl donors oriented modification toward 3-OH position. DexT40-VD at 96% modification was a water-insoluble polymer forming 150nm diameter nanoparticles in water which can be used as drug carrier systems.     

玫瑰花提取物与几种抗氧化物质之间的相互作用

目的:为玫瑰花的综合开发应用提供理论基础。方法:以玫瑰花提取物、没食子酸、儿茶素、槲皮素、VC、芦丁和BHT为材料,研究了它们之间混合后对DPPH·清除活性的增效或减效作用。结果:玫瑰花提取物和没食子酸、槲皮素、VC在低浓度下起到了增效作用,而其余材料或高浓度下,玫瑰花和不同抗氧化剂表现为减效作用。结论:低浓度的某些抗氧化物质与玫瑰花粗提物的混合其混合物溶液的抗氧化活性增强。     

Short communication: Epidemiology and genotyping of Candida rugosa strains responsible for persistent intramammary infections in dairy cows

The present study was undertaken during an outbreak of clinical and subclinical mastitis in 14 dairy cows caused by Candida rugosa, in which high somatic cell counts were seen and cases did not respond to antibiotic treatment. Intramammary infection cured spontaneously in 10 cows, whereas 4 cows were culled as a result of persistent infections. Repeated sampling of these cows and biomolecular analysis of the isolates showed that the infections were caused by the same genotype, even over a period of 2 lactations. Random amplification of the genome of C. rugosa milk isolates gave 3 different DNA banding patterns (genotypes G1, G2, and G3). Viable cells of C. rugosa were also isolated from various environmental sources and were present in high concentrations in total mixed ration samples, which could be considered the primary source of diffusion of viable yeast cells in the environment, as demonstrated by genotyping. The proven capacity of these microorganisms to survive in the environment of the cow, such as the total mixed ration, bedding, water, and cow skin, and to cause persistent intramammary infections highlights the importance of mycotic spread in dairy herds.     

Production of Lipase from Candida rugosa Using Cheese Whey through Experimental Design and Surface Response Methodology

The present work investigates the production of lipases from Candida rugosa in a culture medium containing cheese whey and also determines the importance of the components of the used culture medium using two experimental design and surface response methodology. When pure cheese whey was used as culture medium, the lipolytic activity measured in the broth, after 120 h of fermentation, was 5.18 U/mL. From the first experimental design, it was possible to conclude that the combination of brewery co-product, yeast and malt extract, Tween 80, and olive oil with cheese whey provided an average increase of 15 U/mL in the enzymatic activity, which represented 287.5% in relation to the activity obtained using only with cheese whey as substrate. Nevertheless, this essay did not provide a predictive model for the lipase production using the studied components. From the second experimental design, with suppression of yeast and malt extract, the best values of enzymatic activity were, on average, 28% lesser than the observed in the first optimization, but yet 180% higher than the obtained values when only cheese whey was present as substrate. A second-order model correlating the used components could be achieved, indicating that high concentrations of brewery co-product and Tween 80 enhanced the production of C. rugosa lipases in a medium incorporating cheese whey, which, at high concentrations, can substitute olive oil that may have its concentration diminished in this condition.     

Ethyl esterification of docosahexaenoic acid in an organic solvent-free system with immobilized Candida antarctica lipase

Ethyl docosahexaenoate (EtDHA) is regarded as a potentially useful pharmaceutical substance on account of its beneficial physiological activities. We attempted the ethyl esterification of docosahexaenoic acid (DHA) in an organic solvent-free system using Candida antarctica lipase, which acts strongly on DHA and ethanol. Esterification of 88% was attained by shaking a mixture of DHA/ethanol (1:1, mol/mol) and 2 wt% immobilized C. antarctica lipase at 30 degrees C for 24 h. However, even in the presence of an excess amount of ethanol, the extent of esterification could not be raised above 90%. To attain a higher level of esterification, a two-step reaction was found to be effective. The first step was performed in a mixture of DHA/ethanol (1:1, mol/mol), and the reaction mixture was then dehydrated. In the second step, the resulting mixture was shaken at 30 degrees C for 24 h with 5 molar equivalents of ethanol against the remaining DHA using 2 wt% immobilized lipase. By means of this two-step procedure, 96% esterification was attained. Repetition of the first and second reactions showed that the immobilized lipase was reusable for at least 50 cycles. In addition, DHA remaining in the second-step reaction mixture was removed by a conventional alkali refining process, giving purified EtDHA with a high yield.     

Synthesis of hexyl butyrate (apple and citrus aroma) by Candida rugosa lipase immobilized on Diaion HP-20 using the Box-Behnken design

Food Science and Biotechnology - This study aims at the synthesis of hexyl butyrate by Candida rugosa lipase (CRL) immobilized on Diaion HP 20. The lipase load used was...     

Interesterification of Butter Fat by Lipase from Mucor miehei in Organic Solvent Media

Summary

Butter fat was interesterified in three organic solvent media, hexane, hexane‐chloroform (70:30, v/v), and hexane‐ethyl acetate (70:30, v/v), at different water contents, using commercial immobilized lipase from Mucor miehei. The results indicate that the addition of 30% of either chloroform or ethyl acetate to the hexane resulted in a 25% increase in the interesterification yield; moreover, the addition of 0.8 to 1% water shifted the hydrolytic affinity of lipase towards low molecular weight fatty acids. However, the addition of a small portion of water in hexane, 0.4%, increased the amount of free fatty acids from 0.30 to 7.98 mmole per gram of butter.     

Production of D-lyxose from D-glucose by microbial and enzymatic reactions

D-arabitol was first prepared from D-glucose using Candida famata R28. The reaction gave 5.0% D-arabitol from 10.0% D-glucose. D-arabitol was then almost completely converted to D-xylulose using Acetobacter aceti IFO 3281. Finally, D-lyxose was prepared from D-xylulose enzymatically using L-ribose isomerase from toluene-treated cells of Acinetobacter sp. strain DL-28. The isomerization reaction progressed steadily and the concentration of D-xylulose increased from 1.0 to 10.0%. About 70% of D-xylulose was converted to D-lyxose in all cases. Separation of residual D-xylulose from the reaction mixture is very difficult to achieve by column chromatography, but D-xylulose could be selectively degraded easily using Saccharomyces cerevisiae IFO 0841. The product was crystallized and was confirmed to be D-lyxose by HPLC, 13C-NMR spectra, IR spectra analysis, and optical rotation measurement.     

脉冲电场下乙酸乙醇酯化反应机理研究

主要研究不同场强下脉冲电场(PEF)对乙酸和乙醇酯化反应的影响,并通过红外光谱法探讨电场作用下酯化反应的机理.结果表明:常温无催化剂条件下PEF对乙酸和乙醇的酯化反应有一定的促进作用,且随着场强的增强而增加.比较PEF处理前后样品的红外光谱图,发现O-H、C=O伸缩振动峰均向高频移动,体系内的氢键缔合被破坏,电场下碳氧极性键被进一步极化.     

三聚磷酸钠对魔芋葡甘聚糖酯化交联改性研究

三聚磷酸钠对魔芋葡甘聚糖进行酯化交联改性。实验结果表明,反应最佳条件为三聚磷酸钠∶魔芋葡甘聚糖=1∶9(W/W),反应pH为2,反应温度60℃,反应时间2h。改性KGM透明度、粘度、冻融稳定性均比未改性KGM明显改善,并具有一定的耐酸、耐高温能力,且有相当的抑菌效果。