Candida antarctica lipase B catalyzed enantioselective acylation of pyrimidine acyclonucleoside

Abstract

The influence of solvent and acyl group donor on selectivity of the transesterification reaction of 1-[1′,3′-dihydroxy-2′-propoxymethyl]-5-methyluracil, a structural analogue of ganciclovir was examined. Lipase (EC 3.1.1.3) B from Candida antarctica (CALB) enabled desymmetrization of prochiral hydroxyl groups when 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF₆]) was used as a reaction medium. It was observed that CALB was up to 2.7–4 times more enantioselective in the ionic liquid [Bmim][PF₆] than in conventional organic solvents.     

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.     

Improved triglyceride transesterification by circular permuted Candida antarctica lipase B

Lipases represent a versatile class of biocatalysts with numerous potential applications in industry including the production of biodiesel via enzyme‐catalyzed transesterification. In this article, we have investigated the performance of cp283, a variant of Candida antarctica lipase B (CALB) engineered by circular permutation, with a series of esters, as well as pure and complex triglycerides. In comparison with wild‐type CALB, the permutated enzyme showed consistently higher catalytic activity (2.6‐ to 9‐fold) for trans and interesterification of the different substrates with 1‐butanol and ethyl acetate as acyl acceptors. Differences in the observed rates for wild‐type CALB and cp283 are believe to be related to changes in the rate‐determining step of the catalytic cycle as a result of circular permutation. Biotechnol. Bioeng. 2010;105: 44–50. © 2009 Wiley Periodicals, Inc.     

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.     

毕赤酵母表面展示南极假丝酵母脂肪酶B全细胞催化合成葡萄糖月桂酸酯

利用表面展示南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)的毕赤酵母细胞为全细胞催化剂,以葡萄糖为酰基受体,月桂酸为酰基供体,在非水相体系中催化合成糖酯。用硅胶柱层析对产物进行初提,再用制备液相色谱进一步分离纯化,并用高效液相色谱-质谱鉴定纯品性质。对该酶法合成糖脂反应体系进行了优化,其中考察了有机溶剂种类、复合溶剂体系中二甲基亚砜(DMSO)体积百分比、酶量、底物摩尔比、水活度和温度等几个影响酯化反应的因素。结果表明:在5mL反应体系中,以叔戊醇/二甲基亚砜(DMSO30%,V/V)为反应介质,添加初始水活度为0.11的全细胞催化剂0.5g,葡萄糖0.5mmol/L,月桂酸1.0mmol/L,60°C下反应72h后,葡萄糖月桂酸单酯的转化率达到48.7%。     

Catalytic properties of Candida antarctica lipase B clusters solubilized in hexane

The objective of this work was to investigate the particle size and determine the catalytic competency of a solubilized lipase in hexane. Purified Candida antarctica lipase B (CALB) was solubilized in hexane using the non-ionic surfactant Span 60. The amount of surfactant was chosen so that complete coverage of the individual enzyme molecules with surfactant was not possible. Dynamic Light Scattering (DLS) was used to directly investigate the particle size of the solubilized entities. The enzyme was found to be solubilized in the form of clusters of lipase molecules with a radius of 37±5 nm at 42°C, which we estimate to correspond to about 1200 CALB molecules. The solubilized enzyme clusters showed lower catalytic activity in a model esterification reaction in hexane compared with a commercial immobilizate of the same enzyme (Novozym 435). Further gains in catalytic activity may be possible by striving for true molecular-level dispersion of the enzyme in hexane.     

南极假丝酵母脂肪酶B基因在大肠杆菌中的表达和发酵优化

旨在利用大肠杆菌实现南极假丝酵母脂肪酶B(CALB)基因的高效可溶性表达,并降低生产成本.构建带有不同信号肽的CALB基因表达质粒,转化至不同大肠杆菌宿主中,在摇瓶中进行基础培养基、诱导条件、培养基组成成分和进程曲线的优化.结果显示,带有PelB信号肽的重组菌pET25b-CALB-1/Rosetta(DE3)在20℃...     

Immobilization of Candida antarctica lipase B (CALB) on surface-modified rice husk ashes (RHA) via physical adsorption and cross-linking methods

In the present study, the recovery of activity of Candida antarctica lipase B (CALB) immobilized onto surface-modified rice husk ash (RHA) was 90% for both cross-linking and adsorption methods. Both cross-linked and adsorbed immobilized preparations were very stable, retaining more than 48% of their activity over the range of temperatures studied. The optimum temperature and optimum pH values were 37?°C and 7.0, respectively for both immobilized preparations, while the relative activities after storage at 4.0?°C for 60 days were 55% and 65% using cross-linking and adsorption methods, respectively. Also, the activity of the immobilized lipase began to decrease after 10 cycles, more than 58% of the initial activities were still retained after 10 cycles for both immobilization methods. These results indicated that lipase immobilized by cross-linking and adsorption not only effected activity recovery, but also remarkably effected stability, reusability and application adaptability. It can be concluded that, surface-modified RHA can be used as alternative supports for immobilization of CALB for polymerization reactions.     

Regioselective acylation of several polyhydroxylated natural compounds by Candida antarctica lipase B

Regioselective acylation of four polyhydroxylated natural compounds, deacetyl asperulosidic acid (1), asperulosidic acid (2), puerarin (3) and resveratrol (4) by Candida antarctica Lipase B in the presence of various acyl donors (vinyl acetate, vinyl decanoate or vinyl cinnamoate) was studied. Compounds 1, 2 and 4 were regioselectively acetylated with vinyl acetate to afford products, 3'-O-acetyl-10-O-deacetylasperulosidic acid (1a), 3',6'-O-diacetyl-10-O-deacetylasperulosidic acid (1b), 3'-O-acetylasperulosidic acid (2a), 3',6'-O-diacetylasperulosidic acid (2b), 4'-O-acetylresveratrol (4a), respectively, with yields of 22 to 50%, while reactions with vinyl decanoate and vinyl cinnamoate were slow with lower yields. Compound 3 was readily acylated with all three acyl donors and quantitatively converted to products 6'-O-acetylpuerarin (3a), 6'-O-decanoylpuerarin (3b), 6'-O-cinnamoylpuerarin (3c), respectively. The structures of these acylated products were determined by spectroscopic methods (MS and NMR).     

Regioselective acylation of several polyhydroxylated natural compounds by Candida antarctica lipase B

Regioselective acylation of four polyhydroxylated natural compounds, deacetyl asperulosidic acid (1), asperulosidic acid (2), puerarin (3) and resveratrol (4) by Candida antarctica Lipase B in the presence of various acyl donors (vinyl acetate, vinyl decanoate or vinyl cinnamoate) was studied. Compounds 1, 2 and 4 were regioselectively acetylated with vinyl acetate to afford products, 3′-O-acetyl-10-O-deacetylasperulosidic acid (1a), 3′,6′-O-diacetyl-10-O-deacetylasperulosidic acid (1b), 3′-O-acetylasperulosidic acid (2a), 3′,6′-O-diacetylasperulosidic acid (2b), 4′-O-acetylresveratrol (4a), respectively, with yields of 22 to 50%, while reactions with vinyl decanoate and vinyl cinnamoate were slow with lower yields. Compound 3 was readily acylated with all three acyl donors and quantitatively converted to products 6″-O-acetylpuerarin (3a), 6″-O-decanoylpuerarin (3b), 6″-O-cinnamoylpuerarin (3c), respectively. The structures of these acylated products were determined by spectroscopic methods (MS and NMR).     

Development of thermostable Candida antarctica lipase B through novel in silico design of disulfide bridge

Lipase B from Candida antarctica (CalB) is a versatile biocatalyst for various bioconversions. In this study, the thermostability of CalB was improved through the introduction of a new disulfide bridge. Analysis of the B‐factors of residue pairs in CalB wild type (CalB‐WT) followed by simple flexibility analysis of residues in CalB‐WT and its designated mutants using FIRST server were newly proposed to enhance the selective power of two computational tools (MODIP and DbD v1.20) to predict the possible disulfide bonds in proteins for the enhancement of thermostability. Five residue pairs (A162‐K308, N169‐F304, Q156_‐L163, S50‐A273, and S239C‐D252C) were chosen and the respective amino acid residues were mutated to cysteine. In the results, CalB A162C‐K308C showed greatly improved thermostability while maintaining its catalytic efficiency compared to that of CalB‐WT. Remarkably, the temperature at which 50% of its activity remained after 60‐min incubation (T) of CalB A162C_K308C was increased by 8.5°C compared to that of CalB‐WT (55 and 46.5°C, respectively). Additionally, the half‐life at 50°C of CalB A162C‐K308C was 4.5‐fold higher than that of CalB‐WT (220 and 49 min, respectively). The improvement of thermostability of CalB A162C‐K308C was elucidated at the molecular level by molecular dynamics (MD) simulation. Biotechnol. Bioeng. 2012; 109:867–876. © 2011 Wiley Periodicals, Inc.     

南极假丝酵母脂肪酶B在离子液体/超临界流体体系中的稳定性模拟

脂肪酶在离子液体/超临界流体体系中的结构稳定性是影响其活性的重要因素。本文采用分子动力学方法分别研究了南极假丝酵母脂肪酶B(CALB)在离子液体CYPHOS IL-201/极性超临界流体CHF_3两相体系和离子液体CYPHOS IL-201/非极性超临界流体CO_2两相体系中的结构稳定性,揭示影响CALB结构稳定性的因素。研究结果表明,在超临界CHF_3中,CHF_3破坏蛋白维持α螺旋结构的氢键是蛋白结构不稳定的主要原因;在超临界CO_2中,CALB蛋白的结构紧密性降低,有序二级结构发生了变化,导致稳定性下降。离子液体和两种超临界流体均形成了两相体系,蛋白处于离子液体相中,离子液体不溶于超临界流体,但超临界流体部分进入离子液体相,降低了离子液体相的黏度。其中,相比于CYPHOS IL-201/CO_2体系,CYPHOS IL-201/CHF_3体系的黏度降低多。在离子液体CYPHOS IL-201与超临界流体(CHF_3、CO_2)形成的两相体系中,离子液体CYPHOS IL-201具有保护蛋白结构的作用,使CALB蛋白结构更加稳定。     

Substrate specificity of lipase B from Candida antarctica in the synthesis of arylaliphatic glycolipids

Arylaliphatic glycolipids are known for their pharmaceutical and medicinal properties. We found that a great variety of arylaliphatic esters can be synthesized from non-activated substrates like glucose or the natural occurring drug salicin using lipase B from Candida antarctica (CAL-B). However, esters based on aromatic carboxylic acids or unsaturated arylaliphatic acids, like cinnamic acid and its derivatives, which are known to display anticancer activity, could not be obtained. In this work, we performed computer-aided molecular modeling based on data of our work published recently and syntheses of new glycolipids to understand why some substances are accepted by CAL-B while some are not. For this purpose, we investigated the accessibility of the lipase binding site for the arylaliphatic acyl donors as well as the steric interactions between the aglycons of glucosides and the residues of the alcohol binding pocket in order to elucidate potentials and limitations of CAL-B for the synthesis of aromatic glycolipids.     

Enantioselectivity in Candida antarctica lipase B: a molecular dynamics study

A major problem in predicting the enantioselectivity of an enzyme toward substrate molecules is that even high selectivity toward one substrate enantiomer over the other corresponds to a very small difference in free energy. However, total free energies in enzyme-substrate systems are very large and fluctuate significantly because of general protein motion. Candida antarctica lipase B (CALB), a serine hydrolase, displays enantioselectivity toward secondary alcohols. Here, we present a modeling study where the aim has been to develop a molecular dynamics-based methodology for the prediction of enantioselectivity in CALB. The substrates modeled (seven in total) were 3-methyl-2-butanol with various aliphatic carboxylic acids and also 2-butanol, as well as 3,3-dimethyl-2-butanol with octanoic acid. The tetrahedral reaction intermediate was used as a model of the transition state. Investigative analyses were performed on ensembles of nonminimized structures and focused on the potential energies of a number of subsets within the modeled systems to determine which specific regions are important for the prediction of enantioselectivity. One category of subset was based on atoms that make up the core structural elements of the transition state. We considered that a more favorable energetic conformation of such a subset should relate to a greater likelihood for catalysis to occur, thus reflecting higher selectivity. The results of this study conveyed that the use of this type of subset was viable for the analysis of structural ensembles and yielded good predictions of enantioselectivity.     

Enzymatic synthesis of new aromatic and aliphatic esters of flavonoids using Candida antarctica lipase as biocatalyst

Enzymatic acylation of rutin and esculin with aromatic, aliphatic and aryl-aliphatic acids using Candida antarctica lipase in tert amyl alcohol as solvent was investigated under low water content. Whatever the acyl donor used, the conversion yields and initial rates for esculin were higher than for rutin. For a given flavonoid, the performance of these reactions depended on the acyl donor structures. For aliphatic acids, conversion yields and initial rates of both flavonoids were respectively in the ranges of 68-90% and of 9.5×10^-2-72×10^-2 mmol l^-1 h^-1. For aromatic acids, the reaction occurred only with the aryl subgroup (cinnamic, hydrocinnamic, 3,4-dihydroxyhydrocinnamic and 4-hydroxyphenyl acetic acids) and was drastically influenced by the presence of side chain and substitution patterns of the aromatic ring. Except for hydrocinnamic acid (75%, 23.4×10^-2 mmol l^-1 h^-1), with these acids the conversion yields and initial rates were lower and in the range of 10-45% and of 0.7×10^-2 to 12.1×10^-2 mmol l^-1 h^-1. Unsaturation of the side chain of the hydrocinnamic acid decreased the esculin conversion rate from 75 to 13% and initial rate from 23.4 to 1.76×10^-2 mmol l^-1 h^-1. The presence of hydroxyl or nitro-groups on the aromatic ring of the aryl aliphatic acid also reduced conversion yields and initial rates. Even without a spacer, the non-phenolic ring acid (quinic acid) was reactive and lead to conversion yields of about 20 and 23% respectively for rutin and esculin.     

Enzymatic synthesis of new aromatic and aliphatic esters of flavonoids using Candida antarctica lipase as biocatalyst

Enzymatic acylation of rutin and esculin with aromatic, aliphatic and aryl-aliphatic acids using Candida antarctica lipase in tert amyl alcohol as solvent was investigated under low water content. Whatever the acyl donor used, the conversion yields and initial rates for esculin were higher than for rutin. For a given flavonoid, the performance of these reactions depended on the acyl donor structures. For aliphatic acids, conversion yields and initial rates of both flavonoids were respectively in the ranges of 68–90% and of 9.5×10^?2–72×10^?2 mmol l^?1 h^?1. For aromatic acids, the reaction occurred only with the aryl subgroup (cinnamic, hydrocinnamic, 3,4-dihydroxyhydrocinnamic and 4-hydroxyphenyl acetic acids) and was drastically influenced by the presence of side chain and substitution patterns of the aromatic ring. Except for hydrocinnamic acid (75%, 23.4×10^?2 mmol l^?1 h^?1), with these acids the conversion yields and initial rates were lower and in the range of 10–45% and of 0.7×10^?2 to 12.1×10^?2 mmol l^?1 h^?1. Unsaturation of the side chain of the hydrocinnamic acid decreased the esculin conversion rate from 75 to 13% and initial rate from 23.4 to 1.76×10^?2 mmol l^?1 h^?1. The presence of hydroxyl or nitro-groups on the aromatic ring of the aryl aliphatic acid also reduced conversion yields and initial rates. Even without a spacer, the non-phenolic ring acid (quinic acid) was reactive and lead to conversion yields of about 20 and 23% respectively for rutin and esculin.     

Synthesis of novel d-glucuronic acid fatty esters using Candida antarctica lipase in tert-butanol

Glucuronic acid n-alkyl esters, a novel class of promising biosurfactants and their corresponding glucose esters with the same side-chain length, were synthesized by direct esterification in a non-aqueous phase (tert-butanol) using an immobilized lipase.     

Kinetic Resolution of Profens by Enantioselective Esterification Catalyzed by Candida antarctica and Candida rugosa Lipases

Profens (2‐arylpropionic acids) are known as one of the major nonsteroidal antiinflammatory drugs (NSAIDs) used in the treatment of inflammation associated with tissue injury. The inflammatory activity of profens is mainly due to their (S)‐enantiomer, whereas they are commercially available not only as pure enantiomers, but as racemates as well. There are several methods widely used in order to obtain enantiomerically pure compounds, however, the kinetic resolution with the application of lipases as biocatalysts may have an added advantage in the production of optically pure active pharmaceutical ingredients, such as milder reaction conditions, reduced energy requirements, and production costs. The aim of this study was to compare the results described in the literature in the case of the influence of reaction medium, alcohol moiety, and reaction temperature on the catalytic activity of lipases from Candida antarctica and Candida rugosa. Chirality 26:663–669, 2014. © 2014 Wiley Periodicals, Inc.     

Kinetic resolution of drug intermediates catalyzed by lipase B from Candida antarctica immobilized on immobead‐350

Novozyme 435, which is a commercial immobilized lipase B from Candida antarctica (CALB), has been proven to be inadequate for the kinetic resolution of rac‐indanyl acetate. As it has been previously described that different immobilization protocols may greatly alter lipase features, in this work, CALB was covalently immobilized on epoxy Immobead‐350 (IB‐350) and on glyoxyl‐agarose to ascertain if better kinetic resolution would result. Afterwards, all CALB biocatalysts were utilized in the hydrolytic resolution of rac‐indanyl acetate and rac‐(chloromethyl)‐2‐(o‐methoxyphenoxy) ethyl acetate. After optimization of the immobilization protocol on IB‐350, its loading capacity was 150 mg protein/g dried support. Furthermore, the CALB‐IB‐350 thermal and solvent stabilities were higher than that of the soluble enzyme (e.g., by a 14‐fold factor at pH 5–70°C and by a 11‐fold factor in dioxane 30%–65°C) and that of the glyoxyl‐agarose‐CALB (e.g., by a 12‐fold factor at pH 10–50°C and by a 21‐fold factor in dioxane 30%–65°C). The CALB‐IB‐350 preparation (with 98% immobilization yield and activity versus p‐nitrophenyl butyrate of 6.26 ± 0.2 U/g) was used in the hydrolysis of rac‐indanyl acetate using a biocatalyst/substrate ratio of 2:1 and a pH value of 7.0 at 30°C for 24 h. The conversion obtained was 48% and the enantiomeric excess of the product (e.e._p) was 97%. These values were much higher than the ones obtained with Novozyme 435, 13% and 26% of conversion and e.e.p, respectively. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:878–889, 2018