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.     

酶催化法拆分共轭亚油酸异构体

在Candida rugosa脂肪酶的催化下,采用正辛醇与共轭亚油酸(CLA)进行酯化反应,对CLA异构体进行拆分,并通过单因素实验优化酶法酯化的工艺条件.结果表明最优条件为:AY-30脂肪酶加量40.44 U/g,CLA与正辛醇摩尔比1:1,pH 6.5,温度30℃,反应时间12 h.当酯化率为41.92%时,c9,t11-CLA在两种异构体中的相对含量达到88.13%.     

米黑霉脂肪酶基因的高效表达及活性测定

为了实现米黑霉脂肪酶(Rhizomucor miehei Lipase,RML)基因在大肠杆菌中的高效表达,并得到大量的具有生物活性的脂肪酶,首先通过3种方式提高RML在大肠杆菌中可溶性蛋白的表达量:1)低温诱导(16℃)RML表达;2)新型分子伴侣(Skp)与RML N端融合表达;3)载体pET32a上的Trx·tag与RML N端融合表达.其次对各蛋白质进行纯化及活性测定,各种条件得到的RML比活在(226±10～247±10) U/mg.蛋白质表达结果显示:经低温诱导RML的效果最好,纯蛋白质量浓度为0.86 mg/mL,表明诱导温度是影响可溶性蛋白质表达量的关键因素;活性测定结果表明,Skp和Trx· tag与目的基因N端的融合没有影响RML活性中心(C末端)对底物的结合和催化能力.因此,RML不仅在大肠杆菌中得到高效表达,并且保持原有生物学活性,在工业生产中有应用价值.     

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。     

固定化米黑根毛霉脂肪酶的制备工艺优化及其酶学性质北大核心CSCD

以海藻酸钠-羧甲基纤维素钠(CMC)为复合载体,戊二醛为交联剂,探究了包埋-交联法制备固定化米黑根毛霉脂肪酶的最佳工艺条件,并对固定化米黑根毛霉脂肪酶的酶学性质进行分析。结果表明,制备固定化米黑根毛霉脂肪酶的最佳工艺条件为海藻酸钠质量分数2.5%、CMC质量分数1.5%、脂肪酶液浓度800 U/mL、CaCl_(2)质量分数5%、戊二醛质量分数0.03%、交联固定化时间30 min,在此条件下固定化米黑根毛霉脂肪酶的酶活力为245.58 U/g,与游离脂肪酶相比,固定化脂肪酶热稳定性和pH稳定性均有所提高。交联剂戊二醛的添加可以提高固定化脂肪酶的操作稳定性和储存稳定性,在重复使用7次后相对酶活力保持在57.39%,在4℃下存放7周后相对酶活力为61.89%。包埋-交联法制备的固定化米黑根毛霉脂肪酶具有更好的稳定性和适应性,为实现植物油酶法酯化脱酸工业化生产提供参考。     

双氧水对米赫根毛霉脂肪酶在米曲霉中重组表达的调控研究

米赫根毛霉脂肪酶RML是具有广泛应用价值的重要微生物脂肪酶。本研究对双氧水调控米曲霉RML转化子ONL1表达脂肪酶进行了研究。荧光定量PCR和SDS-PAGE表明,在转化子培养过程中,维持培养体系中10 mmol/L双氧水2 h,使转化子ONL1的脂肪酶活力提高5倍。在双氧水处理过程中,RML的翻译未受影响,其表达水平的提高源于双氧水对其转录水平的调控。因此,双氧水调控melO启动子控制的外源基因的表达体现在转录水平。由于双氧水易挥发,导致其效果低于延长培养时间。为了在较短培养时间内获得高酶活,应在培养液中持续添加双氧水（10 mmol/L）。基于米曲霉转化子脂肪酶活力的分析和qPCR检测,确定以下策略可用于提高米曲霉中RML的表达水平：脂肪酶RML基因的密码子优化、信号肽序列优化、连续分批培养。     

固定化脂肪酶催化合成植物甾醇油酸酯的研究

以无纺布为固定化载体,以Candida rugosa脂肪酶为催化剂,催化植物甾醇与油酸酯化反应合成植物甾醇油酸酯。研究了反应温度、反应时间、底物摩尔比(油酸与植物甾醇摩尔比)、正己烷用量和酶用量对植物甾醇酯化率的影响。在单因素实验的基础上,经响应面实验优化反应条件。结果表明,植物甾醇油酸酯最佳合成条件为:反应温度50℃,反应时间18 h,底物摩尔比2∶1,正己烷用量1 mL,酶用量62.06 U/mg。在最佳条件下,植物甾醇酯化率可达98.36%。     

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.     

皱褶假丝酵母脂肪酶催化水解大豆油工艺优化及机制研究北大核心CSCD

为了降低脂肪酶催化水解植物油脂的应用成本,以皱褶假丝酵母脂肪酶(CRL)为催化用酶,对比了CRL催化水解不同植物油脂的效果,同时以大豆油为研究对象,采用单因素实验考察pH、反应温度、反应时间、水油体积比和酶用量对水解率的影响,并运用响应面法优化工艺条件,进而对CRL催化甘油三酯水解的机制进行了总结。结果表明:CRL催化水解C脂肪酸甘油酯的选择性较高;CRL催化大豆油水解的最优工艺条件为反应温度39.5℃、pH 6.7、水油体积比1∶2.2、酶用量0.6%(以大豆油质量计),在此条件下反应2 h水解率可达77.21%,反应12 h水解率可达96.33%;CRL催化水解甘油三酯的反应机制主要为亲核催化三联体中的丝氨酸对甘油三酯的羰基进行亲核反应以夺取脂肪酸前体,然后水分子对该前体进行亲核反应释放脂肪酸。CRL能够在温和条件下高效催化植物油脂生产脂肪酸,是一种具有广阔应用前景的生物催化剂。     

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.

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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.     

Production of n-3 polyunsaturated fatty acid concentrate from sardine oil by immobilized Candida rugosa lipase

ABSTRACT:  This study was conducted to develop an immobilized-enzyme system to entrap lipase in chitosan-alginate-CaCl₂ beads for the purpose of concentrating n-3 polyunsaturated fatty acids (n-3 PUFAs) from sardine oil. Lipase was immobilized by an ionotropic gelatin method analyzed for characteristics. Optimum pH of immobilized lipase shifted from pH 7.0 to 6.0 and immobilized lipase showed higher stability against pH and temperature changes. Original sardine oil contained 38.1% n-3 PUFAs (25.2% 20:5n3 and 7.20% 22:6n3), and the concentration was significantly increased to 65.3% (40.2% 20:5n3 and 15.5% 22:6n3) with free lipase and to 64.8% (39.6% 20:5n3 and 15.3% 22:6n3) with immobilized lipase after 90 min of repeated hydrolysis. Fatty acid content of the free fatty acid (FFA) fraction of hydrolyzed oil showed that lipase preferably hydrolyzed 16:0, 16:1n7 and 18:0 accounting for 76.6% and 69.5% of total FFAs (after 1st and 2nd hydrolysis, respectively). This study shows that use of immobilized lipase systems for increasing n-3 PUFA concentration in sardine oil provides new processing opportunities for the industry.     

大孔树脂负载南极假丝酵母脂肪酶B催化甘油解和酯化反应制备甘油二酯

目的 采用3种大孔树脂(DA-201,ADS-17,DM-301)对CALB进行固定化,研究固定化脂肪酶催化甘油解和酯化反应制备甘油二酯(diacylglycerol,DAG)。方法 以大孔树脂为载体,采用吸附法制备固定化脂肪酶;通过单因素实验对固定化酶催化甘油解和酯化反应制备DAG的反应条件进行优化。结果 CALB@DA-201催化甘油解反应制备DAG的效率最高,当反应温度为70℃,甘油解反应24 h后产物中的DAG含量最高为64.52%;CALB@DM-301催化酯化反应制备DAG的效率最高,当反应温度为80℃,酯化反应24 h后产物中DAG含量高达72.06%。此外,CALB@ADS-17催化酯化反应具有选择性合成甘油三酯(TAG)的特性,TAG含量可达89.74%。结论 大孔树脂负载的CALB能够有效催化甘油解和酯化反应,并获得可观含量的DAG,具有较好的应用前景。     

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。