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.     

Understanding the effect of tert-butanol on Candida antarctica lipase B using molecular dynamics simulations

The use of organic solvents as reaction media for enzymatic reactions has many advantages. Several organic solvents have been proposed as reaction media, especially for transesterifications using Candida antarctica lipase B (CalB). Among organic solvents, tert-butanol is associated with an enhanced conversion rate in bio-diesel production. Thus, it is necessary to understand the effect of tert-butanol on CalB to explain the high-catalytic efficiency compared with the reaction in other hydrophilic organic solvents. In this study, the effects of tert-butanol on the structure of CalB were investigated by MD simulations. The overall flexibility was increased in the presence of tert-butanol. The substrate entrance and the binding pocket size of CalB in tert-butanol were maintained as in TIP3P water. The distance between the catalytic residues of CalB in tert-butanol indicated a higher likelihood of forming hydrogen bonds. These structural analyses could be useful for understanding the effect of tert-butanol on lipase transesterification.     

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.     

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.     

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.     

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.     

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%。     

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     

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℃...     

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).     

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.     

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.     

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.     

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.     

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.     

Continuous synthesis of alkyl ferulate by immobilized Candida antarctica lipase at high temperature

1-Pentyl, 1-hexyl and 1-heptyl ferulates were continuously synthesized at 60–90°C using a reactor system in which a column packed with ferulic acid powders and another column packed with immobilized Candida antarctica lipase particles were connected in series. Conversions greater than 0.9 were achieved for the synthesis of the 1-hexyl and 1-heptyl ferulates at 90°C. The system could be stably operated for the 1-heptyl ferulate synthesis at 90°C for at least two weeks.     

Regioselective acylation of flavonoids catalyzed by immobilized Candida antarctica lipase under reduced pressure

A single-step acylation of rutin and naringin, catalyzed by immobilized Candida antarctica lipase B in 2-methyl-2-butanol, occurred preferentially on the primary hydroxyl group. Using palmitic methyl ester as acyl donor, the acylation rate of naringin was 10-fold higher than that of rutin. Under optimal conditions, i.e. a molar ratio acyl donor/naringin of 7:1 and 200 mbar, 92% naringin was acylated.     

Biocatalytic synthesis of vitamin A palmitate using immobilized lipase produced by recombinant Pichia pastoris

In this work, the Candida antarctica lipase B (CALB), produced by recombinant Pichia pastoris , was immobilized and used to synthesize vitamin A palmitate by transesterification of vitamin A acetate and palmitic acid in organic solvent. The reaction conditions including the type of solvent, temperature, rotation speed, particle size, and molar ratio between the two substrates were investigated. It turned out that the macroporous resin HPD826 serving as a carrier showed the highest activity (ca. 9200 U g^?1) among all the screened carriers. It was found that the transesterification kinetic of the immobilized CALB followed the ping pong Bi‐Bi mechanism and the reaction product acetic acid inhibited the enzymatic reaction with an inhibition factor of 2.823 mmol L^?1. The conversion ability of the immobilized CALB was 54.3% after 15 cycles. In conclusion, the present work provides a green route for vitamin A palmitate production using immobilized CALB to catalyze the transesterification of vitamin A acetate and palmitic acid.