Asymmetric ammonolysis of (R/S)-mandelic acid by immobilized lipases via direct amidation of mandelic acid in biphasic media

Abstract

We have investigated the direct enantioselective amidation of mandelic acid with ammonia, catalyzed by a variety of commercial lipases including those from Candida rugosa, Mucor miehei, Pseudomonas sp., Rhizomucor miehei, and Thermomyces lanuginosus covalently immobilized onto Florisil^® support via glutaraldehyde and polysuccinimide spacer arms. All the immobilized lipase preparations tested preferentially amidated the R isomer of mandelic acid. The highest amide yields were obtained for immobilized Pseudomonas sp. lipase preparations under the optimized reaction conditions. After 24 h of amidation, the reaction had proceeded with an excellent yield (50%) and enantiopurity (> 99%). The immobilized Pseudomonas sp. lipase preparations catalyzed the amidation reaction with the same yield and enantioselectivity. The enzyme immobilized via a glutaraldehyde spacer arm showed better reusability than that immobilized via a polysuccinimide spacer arm.

In view of these results, it is revealed that the direct amidation of mandelic acid catalyzed by the immobilized Pseudomonas sp. lipases is a facile and effective methodology for obtaining (S)-mandelic acid and (R)-mandelamide.     

Molecular modeling studies of poly lactic acid initiation mechanisms

The two possible routes to synthesize poly (lactic acid) are polycondensation of the lactic acid and ring opening polymerization (ROP) of the lactide. This work involves molecular modeling of the polymerization initiation mechanisms using different initiators a) H₂SO₄ for polycondensation b) aluminum isopropoxide for coordination-insertion ROP c)methyl triflate for cationic ROP, and d) potassium methoxide for anionic ROP. For molecular modeling of PLA, we have benchmarked our approach using Ryner’s work on ROP of L-lactide using stannous (II) 2-ethylhexanoate (Sn(Oct)₂) and methanol as initiators. Our values of -15.2 kcal mol^-1 and -14.1 kcal mol^-1 for enthalpy changes in the two steps of activated complex formation match with Ryner’s. Geometric and frequency optimizations have been done on Gaussian’03 using B3LYP density functional theory along with the basis sets LANL2DZ for metal atoms and 6–31G* and 6–31G** for non metal atoms. The kinetic rate constant for each mechanism has been calculated using the values of energy of activation, change in enthalpy, Gibbs free energy, entropy and the partition functions from the Gaussian’03 output. Our polycondensation rate constant value of 1.07 × 10^–4 se^-1 compares well with 1.51 × 10^–4 se^-1 as reported by Wang. However, ROP rate constants could not be validated due to lack of experimental data. Figure Cationic Ring Opening Polymerization of L-Lactide

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Effects of lactic acid fermentation and gamma irradiation of barley on antinutrient contents and nutrient digestibility in mink (Mustela vison) with and without dietary enzyme supplement

Abstract

The experiment was conducted to study the effects of fermentation of barley, using two different strains of lactic acid bacteria, a Lactobacillus plantarum/pentosus strain isolated from spontaneously fermented rye sourdough (AD2) and a starch-degrading Lactobacillus plantarum (AM4), on contents of mixed-linked (1 → 3) (1 → 4)-β-glucans, α-amylase inhibitor activity, inositol phosphates, and apparent digestibility of macronutrients in mink. Effects of fermentation were compared with effects of gamma irradiation (γ-irradiation: ⁶⁰Co γ-rays at 25 kGy). The diets were fed to mink with and without a supplementary enzyme preparation. Both lactic acid fermentation and γ-irradiation followed by soaking and incubation, reduced concentrations of soluble β-glucans, phytate and α-amylase inhibitor activity. Dietary enzyme supplementation increased significantly digestibility of crude protein, fat, starch and crude carbohydrate (CHO). Fermentation of the barley increased digestibility of starch and CHO. Fermentation with lactic acid bacteria AD2 resulted in higher starch and CHO digestibility than strain AM4, and had greater effect than γ-irradiation, soaking and incubation. The highest digestibility of starch and CHO was obtained after AD2 fermentation followed by enzyme supplementation. It is concluded that both lactic acid fermentation of barley and enzyme supplementation have positive nutritional implications in the mink by limiting the effects of antinutrients and improving digestibility and energy utilization.     

Production of Ricinoleic Acid from Castor Oil by Immobilised Lipases

Abstract

Porcine pancreatic lipase (PPL), Candida rugosa lipase (CRL), and Castor bean lipase (CBL) were immobilized on celite by deposition from aqueous solution by the addition of hexane. Lipolytic performance of free and immobilized lipases were compared and optimizations of lipolytic enzymatic reactions conditions were performed by free and immobilized derivatives using olive oil as substrate. Afterwards, the influence on lipolysis of castor oil of free lipases and immobilized lipase derivatives have been studied in the case of production of ricinoleic acid. All of the lipases performances were compared and enzyme derivative was selected to be very effective on the production of ricinoleic acid by lipolysis reaction. Various reaction parameters affecting the production of ricinoleic acid were investigated with selected the enzyme derivative.

The maximum ricinoleic acid yield was observed at pH 7–8, 50°C, for 3 hours of reaction period with immobilized 1,3-specific PPL on celite. The kinetic constants K_m and V_max were calculated as 1.6 × 10^?4 mM and 22.2 mM from a Lineweaver–Burk plot with the same enzyme derivative. To investigate the operational stability of the lipase, the three step lipolysis process was repeated by transferring the immobilized lipase to a substrate mixture. As a result, the percentange of conversion after usage decreased markedly.     

Immobilization and stabilization of papain on poly(hydroxyethyl methacrylate-ethylenglycol dimethacrylate) beads grafted with epoxy functional polymer chains via surface-initiated-atom transfer radical polymerization (SI-ATRP)

Poly(hydroxyethyl methacrylate–ethylen glycol dimethacrylate), p(HEMA–EGDMA), beads were prepared by suspension polymerization, and were decorated with fibrous poly(glycidyl methacrylate), p(GMA), via surface initiated-atom transfer radical polymerization (SI-ATRP). The functional epoxy groups of the beads were used for covalent immobilization of papain. The average amount of immobilized enzyme was 18.7 mg/g beads. The immobilized enzyme was characterized by temperature, pH, operational and storage stability experiments. The maximum velocity of the free and immobilized enzymes (V_max) and Michaelis–Menten constant (K_m) values were determined as 10.7 and 8.3 U/mg proteins and 274 and 465 μM, respectively. The immobilized papain was operated in a batch reactor, and it was very effective for hydrolysis of different proteins (i.e., casein and cytochrom c).     

Improvement of lipase activity by synergistic immobilization on polyurethane and its application for large-scale synthesizing vitamin A palmitate

Abstract

We have developed an improved and effective method to immobilize lipase on hydrophobic polyurethane foam (PUF) with different modifications. PUF was treated with hydrochloric acid to increase the active sites and then the active carboxyl groups and amino groups were exposed. Enzyme activity of lipase immobilized on PUF-HCL (8000?U/g) was 50% higher than that of lipase immobilized on PUF (5300?U/g). There is an increase in the activity of the immobilized lipase on AA/PEI-modified support (115,000?U/g), a 2.17-fold increase compared to lipase immobilized on the native support was observed. The activity of immobilized lipases was dependent on the PEI molecular weight, with best results from enzyme immobilized on PUF-HCL-AA/PEI (MW 70,000?Da, 12,800?U/g)), which was 2.41 times higher compared to that of the same enzyme immobilized on PUF. These results suggest that the activity of immobilized lipase is influenced by the support surface properties, and a moderate support surface micro-environment is crucial for improving enzyme activity. Finally, the immobilized lipase was used for the production of vitamin A palmitate. The immobilized lipase can be reused for up to 18 times with a conversion rate above 90% for 12?h in a 3?L bioreactor.

• Research highlights

• An efficient immobilization protocol on polyurethane foam was developed

• Polyethyleneimine and acetic acid were used to regulate the micro-environment concurrently

• The activity of lipase immobilized on PUF-HCL-AA/PEI was improved by 2.41 times

• Immobilized lipase exhibited excellent operational stability for vitamin A palmitate synthesis

     

Stabilization of Immobilized Enzymes Against Water-Soluble Organic Cosolvents and Generation of Hyper-Hydrophilic Micro-Environments Surrounding Enzyme Molecules

Enzymes usually undergo rapid inactivation in the presence of organic media. In some cases, the mechanism is quite simple. For example, when an enzyme, fully dispersed and immobilized inside porous supports, is inactivated, at neutral p^H and moderate temperature, in the presence of medium-high concentrations of water-miscible organic cosolvents, the unique cause of inactivation is the interaction of the enzyme with cosolvent molecules and the only inactivating effect is the promotion of conformational changes on enzyme structure.

On this basis, two distinct strategies for stabilization of enzymes against organic solvents are proposed:

a. reduction of the causes of inactivation: generation of hyper-hydrophilic micro-environments having a very open structure and fully surrounding every enzyme molecule;

b. reduction of the effects of inactivation: “rigidification of enzymes” via multipoint covalent immobilization.

By using penicillin G acylase (PGA) as a model enzyme, both strategies have been evaluated and compared. Both stabilizing strategies had significant effects. In this case, hydrophilization of the enzyme nano-environment was found to be more effective than rigidification of the enzyme via multipoint covalent attachment. The combined effect of both stabilizing strategies was also tested: multipoint covalently immobilized enzyme molecules were completely surrounded by hyper-hydrophilic microenvironments. In this way, native PGA that was unstable against organic cosolvents (completely inactivated in less than 3 min in 95% dioxane) was transformed into a very stable immobilized derivative (preserving more than 80% of activity after 40 days under the same conditions).     

Production of l (+) lactic acid by Lactobacillus delbrueckii immobilized in functionalized alginate matrices

The role of functionalized alginate gels as immobilized matrices in production of l (+) lactic acid by Lactobacillus delbrueckii was studied. L. delbrueckii cells immobilized in functionalized alginate beads showed enhanced bead stability and selectivity towards production of optically pure l (+) lactic acid in higher yields (1.74Yp/s) compared to natural alginate. Palmitoylated alginate beads revealed 99% enantiomeric selectivity (ee) in production of l (+) lactic acid. Metabolite analysis during fermentation indicated low by-product (acetic acid, propionic acid and ethanol) formation on repeated batch fermentation with functionalized immobilized microbial cells. The scanning electron microscopic studies showed dense entrapped microbial cell biomass in modified immobilized beads compared to native alginate. Thus the methodology has great importance in large-scale production of optically pure lactic acid.     

A convenient preparation of 6-oligo(lactic acid)cyclomaltoheptaose as kinetically degradable derivative for controlled release of amoxicillin

6-Oligo(lactic acid)cyclomaltoheptaose (6-OLA-βCD) with an average substitution of about 7.0 lactic acid units was prepared as a new water-soluble cyclomaltoheptaose (βCD) derivative (solubility of about 70.7-fold that of βCD), based on the ring-opening polymerization of 3,6-dimethyl-1,4-dioxane-2,5-dione (lactide). The product was characterized by ¹H NMR, ¹³C NMR, IR, and MS spectroscopy. The complexation of amoxicillin with 6-OLA-βCD was found to be much stronger than that with βCD at first, and then 6-OLA-βCD was shown to decompose moderately into βCD and lactic acid. 6-OLA-βCD might be greatly valuable in a controlled release system for Amoxicillin (AMX).     

Cross-linked esterase aggregates (CLEAs) using nanoparticles as immobilization matrix

Abstract

The present study focusses on the enhancement of the catalytic activity and stability of an acetylesterase enzyme isolated from Staphylococcus spp. as Cross-Linked Enzyme Aggregates (CLEAs). The various parameters governing the activity of CLEAs were optimized. The magnetite and graphene oxide nanoparticles were successfully prepared via the chemical co-precipitation and Hummer's method, respectively. These nanoparticles supported the preparation as magnetite nanoparticle-supported cross-Linked Enzyme Aggregates (MGNP-CLEAs) and graphene oxide-supported Cross-Linked Enzyme Aggregates (GO-CLEAs). The activity and stability of these immobilized CLEAs were compared with the free enzyme at various temperature, pH, and organic solvents along with its storage stability and reusability. The immobilized preparations were analyzed by Scanning Electron Microscopy (SEM) and Fourier Transform Infrared spectroscopy (FT-IR) techniques. Acetylesterase precipitated with 60% saturated ammonium sulfate salt (SAS) solution and cross-linked with 100?mM glutaraldehyde for 4?h at 30?°C was found to be optimal to produce CLEAs with highest activity recovery of 99.8%. The optimal pH at 8.0 and temperature at 30?°C remained the same for both the free and immobilized enzyme, respectively. Storage stability significantly improved for the immobilized enzyme as compared to free enzyme. SEM showed type-I aggregate and FT-IR revealed the successful immobilization of the enzyme. MGNP-CLEAs were found to have better activity and stability in comparison to other immobilized preparations.     

L-Lactic acid production from raw cassava starch in a circulating loop bioreactor with cells immobilized in loofa (Luffa cylindrica)

l-Lactic acid was produced from raw cassava starch, by simultaneous enzyme production, starch saccharification and fermentation in a circulating loop bioreactor with Aspergillus awamori and Lactococcus lactis spp. lactis immobilized in loofa sponge. A. awamori was immobilized directly in cylindrical loofa sponge while the L. lactis was immobilized in a loofa sponge alginate gel cube. In the loofa sponge alginate gel cube, the sponge serves as skeletal support for the gel with the cells. The alginate gel formed a hard outer layer covering the soft porous gel inside. By controlling the rate and frequency of broth circulation between the riser and downcomer columns, the riser could be maintained under aerobic condition while the downcomer was under anaerobic condition. Repeated fed-batch l-lactic acid production was performed for more than 400 h and the average lactic acid yield and productivity from raw cassava starch were 0.76 g lactic acid g^–1 starch and 1.6 g lactic acid l^–1 h^–1, respectively.     

Studies on the Proteolytic Action of Dairy Lactic Acid Bacteria

In sterilized skim milk or sterilized 10% solution of dry skim milk at 120°C for 15 min, Lactobacillus bulgaricus, Lactobacillus helveticus and Streptococcus lactis were cultivated for 7 days at given temperature.

Both NCN (non casein type nitrogen) content and pH in each culture of lactic acid bacteria were rapidly decreased until 2 days after cultivation, But NCN content increased and the pH change got small after 3 days cultivation.

Caseins prepared from the cultures of these three kinds of lactic acid bacteria were examined electrophoretically. From the results of electrophoresis of these caseins, we have concluded that α-casein could be hydrolyzed by these lactic acid bacteria. And, it seemed that β-casein could not be hydrolyzed by these lactic acid bacteria.

Rennet easily hydrolyzed casein treated with L. bulgaricus and L. helveticus but hardly hydrolyzed that treated with S. lactis compared with control-casein. Caseins treated with L. bulgaricus and L. helveticus were hydrolyzed easier than control-casein.

Particle weights of caseins prepared from fermented milk by lactic acid bacteria, Streptococcus cremoris, Streptococcus lactis, Lactobacillus bulgaricus and Lactobacillus helveticus, and of hydrolyzed casein by rennet, trypsin or pepsin were measured according to the light scattering experiment.

Particle weights of various treated caseins were larger than that of raw native casein at both pH 7.0 and 12.0. And the heating caused the polymerization of casein to large particle.     

From lactic acid to poly(lactic acid) (PLA): characterization and analysis of PLA and its precursors

The quality of the monomers lactic acid and lactide as well as the chemical changes induced during polymerization and processing are crucial parameters for controlling the properties of the resulting poly(lactic acid) (PLA) products. This review presents the most important analysis and characterization methods for quality assessment of PLA and its precursors. The impurities typically present in lactic acid or lactide monomers and their possible origins and effects on resulting PLA products are discussed. The significance of the analyses for the different polymer production stages is considered, and special applications of the methods for studying features specific for PLA-based materials are highlighted.     

Applicability of pectate-entrapped <Emphasis Type="Italic">Lactobacillus casei</Emphasis> cells for <Emphasis Type="SmallCaps">l</Emphasis>(+) lactic acid production from whey

Lactic acid is a versatile organic acid, which finds major application in the food, pharmaceuticals, and chemical industries. Microbial fermentation has the advantage that by choosing a strain of lactic acid bacteria producing only one of the isomers, an optically pure product can be obtained. The production of l(+) lactic acid is of significant importance from nutritional viewpoint and finds greater use in food industry. In view of economic significance of immobilization technology over the free-cell system, immobilized preparation of Lactobacillus casei was employed in the present investigation to produce l(+) lactic acid from whey medium. The process conditions for the immobilization of this bacterium using calcium pectate gel were optimized, and the developed cell system was found stable during whey fermentation to lactic acid. A high lactose conversion (94.37%) to lactic acid (32.95 g/l) was achieved with the developed immobilized system. The long-term viability of the pectate-entrapped bacterial cells was tested by reusing the immobilized bacterial biomass, and the entrapped bacterial cells showed no decrease in lactose conversion to lactic acid up to 16 batches, which proved its high stability and potential for commercial application.     

Selective synthesis of panthenyl esters by a kinetically controlled enzymatic process

Abstract

Panthenyl esters (panthenyl monoacetate and panthenyl diacetate) were synthesized in high yields (≈100%) by a kinetic reaction control using a commercial immobilized Candida Antarctica lipase B (Novozyme 435) in acetonitrile. The enzyme showed excellent synthetic activity, regioselectivity, and operational stability under the conditions used.     

Improved oxygen transfer and increased l-lactic acid production by morphology control of Rhizopus oryzae in a static bed bioreactor

Rhizopus oryzae was immobilized on a cotton matrix in a static bed bioreactor. Compared with free cells in a stirred tank bioreactor, immobilized R. oryzae in this bioreactor gave higher lactic acid production but lower ethanol production. The highest lactic acid production rate (2.09 g/L h) with the final concentration of 37.83 g/L from 70 g/L glucose was achieved when operating the bioreactor at 700 rpm and 0.5 vvm air. To better understand the relationship between shear effects (agitation and aeration) and R. oryzae morphology and metabolism, oxygen transfer rate, fermentation kinetics, and lactate dehydrogenase activity were determined. In immobilized cell culture, higher oxygen transfer rate and lactic acid production were achieved but lower lactate dehydrogenase activity was found as compared with those in free cell culture operated at the same conditions. These results clearly imply that mass transport was the rate controlling step in lactic acid fermentation by R. oryzae.     

Entrapment of purified novel dextransucrase obtained from newly isolated Acetobacter tropicalis and its comparative study of kinetic parameters with free enzyme

Abstract

Purified Acetobacter tropicalis dextransucrase was immobilized in different matrices viz. calcium-alginate, κ-carrageenan, agar, agarose and polyacrylamide. Calcium-alginate was proved to be superior to the other matrices for immobilization of dextransucrase enzyme. Standardization of immobilization conditions in calcium-alginate resulted in 99.5% relative activity of dextransucrase. This is the first report with such a large amount of relative activity as compared to the previous reports. The immobilized enzyme retained activity for 11 batch reactions without a decrease in activity which suggested that enzyme can be used repetitively for 11 cycles. The dextransucrase was also characterized, which revealed that enzyme worked best at pH 5.5 and 37?°C for 30?min in both the free as well as immobilized state. Calcium-alginate immobilized dextransucrase of A. tropicalis showed the K_m and V_max values of 29?mM and 5000?U/mg, respectively. Free and immobilized enzyme produced 5.7?mg/mL and 2.6?mg/mL of dextran in 2?L bench scale fermenter under optimum reaction conditions. This immobilization method is very unconventional for purified large molecular weight dextran-free dextransucrase of A. tropicalis as this method is used usually for cells. Such reports on entrapment of purified enzyme are rarely documented.     

Modified chitosan as a spacer for protein immobilization

Abstract

Three new, water-soluble, N-modified chitosan derivatives containing poly(ethylene glycol), dextran or inulin side chains were used as spacers for enzyme immobilization on a natural silk carrier. Amylolytic enzymes Maltogenase L and Promozyme D2, lipolytic enzyme Resinase HT and a complex of proteolytic enzymes from Streptomyces flavus 197 were immobilized. The activity of the immobilized enzymes and their stability during storage were similar to that obtained with synthetic polyamine—poly(ethylene imine) as a spacer. High operational stability of co-immobilized amylolytic enzymes Maltogenase L and Promozyme D2 in a continuous flow mini-reactor was demonstrated.     

7 The Molecular Biology of Tryptophan Synthase: A Model for Protein–Protein Interaction

Abstract

The use of plastic produced from non-renewable resources constitutes a major environmental problem of the modern society. Polylactide polymers (PLA) have recently gained enormous attention as one possible substitution of petroleum derived polymers. A prerequisite for high quality PLA production is the provision of optically pure lactic acid, which cannot be obtained by chemical synthesis in an economical way. Microbial fermentation is therefore the commercial option to obtain lactic acid as monomer for PLA production. However, one major economic hurdle for commercial lactic acid production as basis for PLA is the costly separation procedure, which is needed to recover and purify the product from the fermentation broth. Yeasts, such as Saccharomyces cerevisiae (bakers yeast) offer themselves as production organisms because they can tolerate low pH and grow on mineral media what eases the purification of the acid. However, naturally yeasts do not produce lactic acid. By metabolic engineering, ethanol was exchanged with lactic acid as end product of fermentation. A vast amount of effort has been invested into the development of yeasts for lactic acid production since the first paper on this topic by Dequin and process insight. If pH stress is used as basis for DNA microarray analyses, in order to improve the host, what exactly is addressed? Growth? Or productivity? They might be connected, but can be negatively correlated. A better growing strain might not be a better producer. So if the question was growth, the answer might not be what was initially intended (productivity).

A major task for the future is to learn to ask the right questions – a lot of studies intended to lead to better productivity, did lead to interesting results, but NOT to better production strains.

Taking together what we learned from lactic acid production with yeasts, we see a bright future for bulk and fine chemical production with these versatile hosts.     

Alteration of cofactor specificity of the acrylyl-CoA reductase from Escherichia coli

Objectives

Alteration of the cofactor specificity of acrylyl-CoA reductase (AcuI) catalyzing the NAD(P)H-dependent reduction of acrylyl-CoA to propionyl-CoA is often desirable for designing of artificial metabolic pathways of various appointments.

Results

Several variants of AcuIs from Escherichia coli K-12 with multiple amino acid substitutions to alter the cofactor preference were obtained by site directed mutagenesis and the modified enzymes as His₆-tagged proteins were characterized. The simultaneous substitutions of arginine-180, arginine-198 and serine-178 residues by alanine in the enzyme pocket sequence as well as other amino acid changes decreased both NADPH- and NADH-dependent activities in comparison to the wild-type enzyme. The replacement of serine-156 by glutamic acid decreased NADPH-dependent activity at least 7000-fold but NADH-dependent activity only by threefold. The replacement of serine-156 by aspartic acid decreased NADPH-dependent activity 70-fold with fair preservation of activity and specificity to NADH.

Conclusions

These results demonstrated a relevance of Asp156 in the interaction of AcuI from E. coli K-12 with NADH as a coenzyme. These findings may provide reference information for shifting coenzyme specificity of acrylyl-CoA reductases.