Impressive effect of immobilization conditions on the catalytic activity and enantioselectivity of Candida rugosa lipase toward S-Naproxen production

Candida rugosa lipase (CRL) was immobilized on Amberlite XAD 7 and the advantage of immobilization under the best reaction conditions in achieving high activity and enantioselectivity was shown for the hydrolysis of racemic Naproxen methyl ester. The performance of CRL was found to be better when the enzyme was immobilized at the temperature and pH values where higher conversion and enantioselectivity were obtained. The effects of immobilized lipase load, temperature, pH and substrate concentration on the conversion and enantioselectivity toward S-Naproxen production in aqueous phase/isooctane biphasic batch system were also evaluated. The increase in immobilized lipase load in 320–800 U/mL range increased the conversion of the substrate and enantioselectivity for S-Naproxen. The kinetic resolution of racemic Naproxen methyl ester conducted at the temperatures of 40, 45 and 50 °C and at the pH values of 4, 6, 7.5 and 9 resulted in the highest conversion and enantioselectivity at 45 °C and pH 6. Higher concentration of racemic Naproxen methyl ester than 10 mg/mL decreased both the conversion and enantioselectivity. CRL, which was immobilized at the temperature and pH values where the enzyme was more enantioselective, was successfully used in three successive batch runs each of 180 h. The highest enantiomeric ratio achieved in the S-Naproxen production was 174.2 with the conversion of 49%.     

Esterification of 2-methylalkanoic acids Catalysed by Lipase from Candida rugosa: Enantioselectivity as a Function of water Activity and Alcohol Chain Length

Esterifications catalysed by immobilised lipase from Candida rugosa (CRL) in cyclohexane at constant water activity (a_w = 0.76) were studied using 2-methyl substituted octa-, nona- or decanoic acids and n-alcohols of varying chain length as substrates. The importance of controlling the water activity and choosing the right alcohol for obtaining maximum enantioselectivity is demonstrated. The immobilised lipase was easily recovered without loss of activity and enantioselectivity.     

Kinetic resolution of racemic naproxen methyl ester by magnetic and non-magnetic cross-linked lipase aggregates

Abstract

In this study, the non-magnetic and the magnetic cross-linked enzyme aggregates (CLEAs) from Candida rugosa lipase were synthesized to catalyze the kinetic resolution reaction of naproxen methyl ester (NME). Magnetic iron oxide nanoparticles (MIONPs) were produced through co-precipitation method and their surfaces were modified by silanization reaction. The MIONPs were used as a platform to synthesize the magnetic CLEAs (M-CLEAs). The biocatalysts and MIONPs synthesized were characterized by FTIR spectroscopy and SEM analysis. The kinetic resolution of racemic NME was studied in aqueous buffer solution/isooctane biphasic system to compare the performance of M-CLEAs and CLEAs. The effects of reaction parameters such as temperature, pH, stirring rate on the enantiomeric excess of the substrate (ee_s%) were investigated in a batch reactor system. The activity recovery of CRL enzyme in CLEAs was higher than M-CLEAs. Compared with M-CLEAs, CLEAs biocatalysts had previously reached ee_s% values. Although both biocatalysts showed similar cavity structure from SEM analysis, the lower performance of M-CLEAs may be due to the different microenvironments of M-CLEAs from CLEAs. However, the reusability performance of M-CLEAs was higher than that of CLEAs. The optimal reaction conditions for M-CLEAs and CLEAs were found to be 37?°C, pH 7.5, and 300?rpm.     

Candida rugosa lipase encapsulated with magnetic sporopollenin: design and enantioselective hydrolysis of racemic arylpropanoic acid esters

Abstract

The aim of this study was to prepare the encapsulation of Candida rugosa lipase (CRL) with magnetic sporopollenin. The sporopollenin was covalent immobilized onto magnetic nanoparticles (Fe₃O₄), grafted amino (APTES), or epoxy groups (EPPTMS). CRL was sol-gel encapsulated in the presence of magnetic sporopollenin/Fe₃O₄ nanoparticles. The influence of activation agents ([3-(2,3-epoxypropoxy) propyl] trimethoxysilane (EPPTMS), (3-Aminopropyl)triethoxysilane (APTES) and pH and thermal stabilities of the biocatalyst were assessed. Experimental data showed the improved catalytic activity at different pH and temperature values. At 60?°C, free lipase lost its initial activity within 80?min of time, although the encapsulated lipases retained their initial activities of about 65% by APTES and 60% by EPPTMS after 120?min of heat treatment at 60?°C. The catalytic properties of the encapsulated lipases were utilized to hydrolysis of racemic aromatic carboxylic acid methyl esters (Naproxen and 2-phenoxypropionic acid). The results show that the sporopollenin-based encapsulated lipase (Fe-A-Spo-E) has greater enantioselectivity and conversion in comparison with the encapsulated lipase without supports (lipase-enc).     

Mannitol production by heterofermentative <Emphasis Type="BoldItalic">Lactobacillus reuteri</Emphasis> CRL 1101 and <Emphasis Type="BoldItalic">Lactobacillus fermentum</Emphasis> CRL 573 in free and controlled pH batch fermentations

Certain lactic acid bacteria, especially heterofermentative strains, are capable to produce mannitol under adequate culture conditions. In this study, mannitol production by Lactobacillus reuteri CRL 1101 and Lactobacillus fermentum CRL 573 in modified MRS medium containing a mixture of fructose and glucose in a 6.5:1.0 ratio was investigated during batch fermentations with free pH and constant pH 6.0 and 5.0. Mannitol production and yields were higher under constant pH conditions compared with fermentations with free pH, the increase being more pronounced in the case of the L. fermentum strain. Maximum mannitol production and yields from fructose for L. reuteri CRL 1101 (122 mM and 75.7 mol%, respectively) and L. fermentum CRL 573 (312 mM and 93.5 mol%, respectively) were found at pH 5.0. Interestingly, depending on the pH conditions, fructose was used only as an alternative external electron acceptor or as both electron acceptor and energy source in the case of the L. reuteri strain. In contrast, L. fermentum CRL 573 used fructose both as electron acceptor and carbon source simultaneously, independently of the pH value, which strongly affected mannitol production by this strain. Studies on the metabolism of these relevant mannitol-producing lactobacilli provide important knowledge to either produce mannitol to be used as food additive or to produce it in situ during fermented food production.     

Large Scale Production and Crystallization of Acid Carboxypeptidase from Submerged Culture of Penicillium janthinellum,and Stability of the Crystalline Enzyme

Acid carboxypeptidase of Penicillium janthinellum IFO–8070 was produced effectively in submerged culture on a medium of 4 ~ 5% rice bran. The enzyme production was enlarged to volume cultivation of 150-liters in a 200-liters jar fermentor, and the yield of acid carboxypeptidase per milliliter filtrate reached to the maximum 3 days after inoculation.

Acid carboxypeptidase of low molecular weight (M.W. = 51,000) produced in the liquid culture broth was purified and crystallized in a large scale. Purification steps include Amberlite CG–50 treatment, ammonium sulfate precipitation, dialysis using “Diaflow,” activated charcoal treatment, and condensation using collodion-bag, or condensation and dialysis using “Diaflow.”

The crystals of the acid carboxypeptidase suspended in 50 mm acetate buffer (pH 3.7) were completely stable for six months at 5°C. On the other hand, at low enzyme concentration (0.01 U/ml) in 50 mm acetate buffer (pH 3.7), crystallized enzyme was somewhat labile, whereas, this inactivation was completely depressed by covering enzyme solution with toluene.     

Production of Extracellular Alkaline α‐Amylase by Solid State Fermentation with a Newly Isolated Bacillus sp.

Abstract

Production of alkaline α‐amylase employing our laboratory isolate, Bacillus sp., under solid state fermentation, was optimized. The effect of wheat bran and lentil husk was examined. Lentil husk exhibited the highest enzyme production. The appropriate incubation time, inoculum size, moisture level, and buffer solution level were determined. Maximum yields of 216,000 and 172,800 U/g were achieved by employing lentil husk and wheat bran as substrates in 0.1 M carbonate/bicarbonate buffer at pH 10.0 with 30% initial moisture level at 24 h. Inoculum size and buffer solution level were found to be 20% and 1:0.5 for two solid substrates.     

<Emphasis Type="Italic">Burkholderia cenocepacia</Emphasis>: a new biocatalyst for efficient bioreduction of ezetimibe intermediate

Ezetimibe is a selective acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor used in hypercholesterolemia. Synthesis of ezetimibe requires enantiopure 3-[5-(4-fluorophenyl)-5(S)-hydroxypentanoyl]-4(S)-4-phenyl-1,3-oxazolidin-2-one (FOP alcohol) as a crucial intermediate which is produced by reduction of the corresponding prochiral ketone (FOP dione). A new biocatalyst from acclimatized soil was screened for bioreduction of the above prochiral ketone. The microorganism was identified by 16S mRNA sequencing, as Burkholderia cenocepacia. Various physicochemical conditions were optimized to increase cellmass and enzyme activity. The overall increase in cellmass concentration and enzyme activity was 2.06 and 1.85-fold, respectively. Various reaction conditions, for example pH, temperature, agitation, and cellmass concentration, were optimized for maximum product yield (chiral alcohol) with excellent enantioselectivity. Best reduction was achieved in phosphate buffer (50 mM, pH 8.0) at 40°C (200 rpm) and the yield of enantiopure alcohol from the corresponding prochiral ketone was 54%. This biocatalyst was also used for the reduction of various other prochiral ketones.     

Improved production of (R)-2-octanol via asymmetric reduction of 2-octanone with Oenococcus oeni CECT4730 in a biphasic system

Abstract

Oenococcus oeni CECT4730, which catalyses the asymmetric reduction of 2-octanone to (R)-2-octanol with high enantioselectivity, was further studied to exploit its potential for production of (R)-2-octanol in an aqueous/organic solvent biphasic system. Variables such as the volume ratio of aqueous to organic phase (V_a/V_o), buffer pH, reaction temperature, shaking speed, co-substrates and the ratio of biocatalyst to substrate were examined with respect to the molar conversion, the initial reaction rate and the product enantiomeric excess (e.e.). Under the optimized conditions (V_a/V_o=1:1 (v/v), buffer pH=8.0, reaction temperature=30°C, shaking speed=150 rev/min, ratio of glucose to biomass=5.4:l (w/w), ratio of biocatalyst to substrate=0.51:l (g/mol)), the highest space time yield of (R)-2-octanol, 24 mmol L^?1 per h, and >98% product e.e. were obtained at a substrate concentration close to 1.0 mol L^?1 after 24 h reduction.     

Effect of several factors on soluble lipase-mediated biodiesel preparation in the biphasic aqueous-oil systems

Biodiesel is increasingly perceived as an important component of solutions to the important current issues of fossil fuel shortages and environmental pollution. Utilization of soluble lipase offers an alternative approach to lipase-catalyzed biodiesel production using immobilized enzyme or whole-cell catalysis. Soluble lipase NS81020, produced by submerged fermentation of genetically modified Aspergillus oryzae microorganism, was first proposed here as the catalyst of biodiesel preparation with oleic acid in the biphasic aqueous-oil systems. The effect factors such as enzyme concentration, water content, temperature, molar ratio of methanol to oil, stirring rate and pH of buffer solution on the esterification rate were investigated systematically. The reaction time could be shortened with the increasing of enzyme concentration as long as the maximum enzyme absorptive capacity on the interface in the biphasic aqueous-oil systems was not achieved. The optimal water content in the biphasic aqueous-oil systems was 10 wt% by oleic acid weight. The reaction rate was enhanced with the increasing molar ratio of methanol to oil, the increasing stirring rate or the decreasing temperature. Although soluble lipase NS81020 had lower activity at pH 10.55, hydroxyl ion conduced to restrain hydrolysis of methyl ester and facilitated the reaction toward the methyl ester formation.     

Modified procedures for extraction and analysis of carrageenan applied to the red alga Hypnea musciformis

Dried powder of Hypnea musciformis was extracted with water at pH 7 after an initial short pre-treatment with cold, diluted HCl. Carrageenans were isolated by alcohol precipitation after an amylase treatment and a filtration of the extracts. The yields at 25 and 90 °C were 25 and 75% (w/w) of the dry alga, with molecular weights (M_w) corresponding to 194 000 and 245 000, respectively. The chemical structure was dominated by G4S-DA-(kappa-carrageenan or carrageenose 4-sulphate). A simple fractionation procedure for kappa-carrageenase hydrolysates, based on stirring in different enthanol/water mixtures, is introduced. NMR analysis showed that oligosaccharides with a repeating DA-G4S structure were the main constituents in the enzymic hydrolysates of the carrageenans from Hypnea musciformis. These oligosaccharides were solubilized in an ethanol concentration from 96 to 48% (v/v). In some enzyme resistant fractions D6S-G4S and DA2S-G4S sequences and D2S,6S unites were detected by ¹³C-NMR.Author for correspondence     

Enantioselective resolution of racemic flurbiprofen methyl ester by lipase encapsulated mercapto calix[4]arenes capped Fe3O4 nanoparticles

This work presents the synthesis of new mercapto calix[4]arenes derivatives (4 and 5). These derivatives were capped on Fe₃O₄ magnetic nanoparticles and subsequently encapsulated with Candida rugosa through sol–gel method to furnish enc-4 and enc-5, respectively, to enhance catalytic activity and enantioselectivity of lipase for hydrolysis reaction of racemic flurbiprofen methyl ester. Catalytic activity and enantioselectivity of enc-4 and enc-5 were assayed at different pH and temperature conditions and it was found that the resultant encapsulated enzyme exhibited higher thermal and operational stabilities compared to the free lipase in which enc-5 showed the excellent rate of enantioselectivity (E = 176) for S-flurbiprofen better than free lipase (E = 137) at pH 7 and 35 °C for 48 h. The time study shows that enantioselectivity reached the maximum value of E = 244 after 72 h. Catalytic activity  of these materials was hardly affected by 20 and 23% after five usages of enc-4 and enc-5, respectively.

     

Enantioselectivity of Candida Rugosa Lipase Toward Carboxylic Acids: A Predictive Rule from Substrate Mapping and X-Ray Crystallography

We used substrate mapping to develop a rule that predicts which enantiomer of chiral carboxylic acid esters reacts faster in hydrolyses catalyzed by lipase from Candida rugosa (CRL, triacylglycerol hydrolase, E. C. 3.1.1.3). This rule, based on the size of the substituents at the stereocenter, is not reliable for crude CRL. It predicts the favoured enantiomer for only 23 out of 34 examples, 68% reliability. However, this rule is completely reliable for purified CRL; it predicts the favoured enantiomer for all 16 examples correctly. The examples include arylpropanoicacids, aryloxypropanoic acids, α-halophenylacetic acids, mandelic acid and O-methylmandelic acid. Further, purified CRL did not catalyse the hydrolysis of N-CBZ-phenylalanine methyl ester and N-CBZ-norleucine methyl ester. These two substrates were exceptions to the rule with crude CRL as the catalyst. Besides eliminating several exceptions, purification also raised the enantioselectivity of CRL toward carboxylic acid esters. To provide a structural basis for this proposed rule we examined the x-ray crystal structure of CRL containing transition state analogs of ester hydrolysis. We suggest that the large substituent of chiral carboxylic acids binds in a tunnel that normally binds the alkyl chain of a fatty acid. The phenyl rings of Phe 345 and Phe 415 lie close to the stereocenter, thereby fixing the orientation of the medium substituent. The three-dimensional orientation of these proposed binding sites is consistent with the rule derived from substrate mapping.     

Use of a Novel Sub‐2 µm Silica Hydride Vancomycin Stationary Phase in Nano‐Liquid Chromatography. II. Separation of Derivatized Amino Acid Enantiomers

A novel vancomycin silica hydride stationary phase was synthesized and the particles of 1.8 µm were packed into fused silica capillaries of 75 µm internal diameter (I.D.). The chiral stationary phase (CSP) was tested for the separation of some derivatized amino acid enantiomers by using nano‐liquid chromatography (nano‐LC). Some experimental parameters such as the type and the content of organic modifier, the pH, and the concentration of the buffer added to the mobile phase were modified and the effect on enantioselectivity, retention time, and enantioresolution factor was studied. The separation of selected dansyl amino acids (Dns‐AAs), e.g., Asp, Glu, Leu, and Phe in their enantiomers was initially achieved utilizing a mobile phase containing 85% (v/v) methanol (MeOH) and formate buffer measuring the enantioresolution factor and enantioselectivity in the range 1.74–4.17 and 1.39–1.59, respectively. Better results were obtained employing a more polar organic solvent as acetonitrile (ACN) in the mobile phase. Optimum results (Rs 1.41–6.09 and α 1.28–2.36) were obtained using a mobile phase containing formate buffer pH 2.5/water/MeOH/ACN 6:19:12.5:62.5 (v/v/v/v) in isocratic elution mode at flow rate of 130 nL/min. Chirality 27:767–772, 2015. © 2015 Wiley Periodicals, Inc.     

A Simple Purification Method for Chloroperoxidase and Its Use in Organic Media

A simple two-step purification method for chloroperoxidase from Caldariomyces fumago has been developed. After filtration of the mycelium the enzyme was bound to a DEAE Sepharose fast flow column. The enzyme was eluted with a 20–200 mM phosphate buffer, pH=5.8. After gel filtration on a Superose 12 HPLC column pure enzyme was obtained. Instead of gel filtration it was also possible to purify the enzyme by concentration over a membrane filter, 10 K cutoff. Concentration to 8% of the original volume yielded an enzyme preparation with R_z=1.31^b in 77% yield. The enzyme was active in t-butyl alcohol/water mixtures up to 70% t-butyl alcohol. The sulfoxidation of thioanisole proceeded readily (conversion > 99%) and with high enantioselectivity (>99%) in t-butyl alochol/water mixtures.     

Immobilization conditions of ketoreductase on enantioselective reduction in a gas-solid bioreactor

The immobilization conditions of commercial ketoreductase for continuous enantioselective reduction in the gas-phase reaction were investigated with respect to the immobilization efficiency (residual activity and protein loading) and the gas-phase reaction efficiency (initial reaction rate, half-life, and enantioselectivity). For the analyses, ketoreductase was first immobilized by physical deposition on glass supports and the reduction of 2-butanone to (S)-2-butanol with the concomitant regeneration of NADH by 2-propanol was used as a model reaction. The optimal conditions of enzyme immobilization were obtained using an absolute pressure of 100 hPa for drying, a pH between 6.5 and 7.0, and a buffer concentration of 50 mM. The buffer concentration in particular had a strong effect on both the enzyme activity and enantioselectivity. Under optimal immobilization conditions, the thermostability of ketoreductase in the gas-phase system was enhanced compared to the aqueous-phase system, while the enantioselectivity was successfully maintained at a level identical to that of the native enzyme. These results indicate that the gas-phase reaction has a great potential for industrial production of chiral compounds, but requires careful optimization of immobilization conditions for the reaction to progress effectively.     

Human probiotic bacteria attenuate Pseudomonas aeruginosa biofilm and virulence by quorum-sensing inhibition

Abstract

This work investigated chloroform extracts from culture supernatants of two human probiotic bacteria, Lactobacillus casei CRL 431 and Lactobacillus acidophilus CRL 730 for the production of virulence factors and quorum sensing (QS) interference against three Pseudomonas aeruginosa strains. Both extracts inhibited biofilm biomass (up to 50%), biofilm metabolic activity (up to 39%), the production of the enzyme elastase (up to 63%) and pyocyanin (up to 77%), and decreased QS, without presenting any antibacterial acgivity. In addition, the chloroform extracts of both strains disrupted preformed biofilms of the three strains of P. aeruginosa analyzed (up to 40%). GC-MS analysis revealed that the major compounds detected in the bioactive extracts were four diketopiperazines. This study suggests that the metabolites of L. casei and L. acidophilus could be a promising alternative to combat the pathogenicity of P. aeruginosa.     

Determination of residues of the β-agonist clenbuterol in liver of medicated farm animals by gas chromatography–mass spectrometry using diphasic dialysis as an extraction procedure

A method has been developed for the rapid confirmation of clenbuterol in cow liver using gas chromatography coupled with detection by mass spectrometry of the trimethylsilyl derivatives of clenbuterol. The technique used for the extraction was diphasic dialysis. It was observed that the best suitable solution to homogenize the liver the barium hydroxide–barium chloride buffer, the optimal extraction solvent was tert.-butylmethyl ether at an extraction temperature of 37°C, and stirring should be applied at 150 rpm for 4 h. This extraction method improves clenbuterol recovery up to values of 99.3%. With the use of the barium buffer, derivatization is performed more efficiently and the detection and quantification limits can be decreased to values close to 250 ppt and 500 ppt, respectively.     

Immobilization of Candida rugosa lipase on poly(3-hydroxybutyrate-co-hydroxyvalerate): a new eco-friendly support

The overall objective of this study is to evaluate the morphological [scanning electron microscopy (SEM)], physicochemical [differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), chemical composition analysis, Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR)], and biochemical properties of Candida rugosa lipase (CRL) immobilized on a natural biopolymer poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) in aqueous solution. CRL was immobilized by physical adsorption with efficiency of 30%. Compared with free CRL enzyme, there were slight changes in immobilized CRL activity as a function of temperature (from 37°C to 45°C), but a similar optimal pH value of 7.0. Inactivation rate constants for immobilized CRL enzyme were 0.009 and 0.334 h⁻¹, and half-lives were 77 and 2 h at 40°C and 60°C, respectively. Kinetic parameters obtained for immobilized CRL include the Michaelis–Menten constant of K _m = 213.18 mM and maximum reaction velocity of V _max = 318.62 U/g. The operational stability of immobilized CRL was tested repeatedly, and after 12 cycles of reuse, the enzyme retained 50% activity. Based on our results, we propose that PHBV-immobilized CRL could serve as a promising biocatalyst in several industrial applications.     

Green and scalable synthesis of chiral aromatic alcohols through an efficient biocatalytic system

Chiral aromatic alcohols have received much attention due to their widespread use in pharmaceutical industries. In the asymmetric synthesis processes, the excellent performance of alcohol dehydrogenase makes it a good choice for biocatalysts. In this study, a novel and robust medium-chain alcohol dehydrogenase RhADH from Rhodococcus R6 was discovered and used to catalyse the asymmetric reduction of aromatic ketones to chiral aromatic alcohols. The reduction of 2-hydroxyacetophenone (2-HAP) to (R)-(-)-1-phenyl-1,2-ethanediol ((R)-PED) was chosen as a template to evaluate its catalytic activity. A specific activity of 110 U mg⁻¹ and a 99% purity of e.e. was achieved in the presence of NADH. An efficient bienzyme-coupled catalytic system (RhADH and formate dehydrogenase, CpFDH) was established using a two-phase strategy (dibutyl phthalate and buffer), which highly raised the tolerated substrate concentration (60 g l⁻¹). Besides, a broad range of aromatic ketones were enantioselectively reduced to the corresponding chiral alcohols by this enzyme system with highly enantioselectivity. This system is of the potential to be applied at a commercial scale.