Showing papers in "Journal of Molecular Catalysis B-enzymatic in 2015"

Bovine trypsin immobilization on agarose activated with divinylsulfone: Improved activity and stability via multipoint covalent attachment

Abstract: Trypsin has been immobilized on divinyl sulfone (DVS) activated agarose at pH 5, 7 and 10. While at pH 5 and 7 immobilization was slow and presented a negative effect on enzyme activity, the immobilization at pH 10 produced a significant increment of activity (by a 24 fold factor). Using this preparation, the effect on enzyme activity/stability of different blocking reagents (used as an enzyme-support reaction end point) were evaluated, selecting ethylenediamine (EDA) because it produced an increase in enzyme activity (a 4 fold factor) and the best results in terms of stability. Next, the effect of alkaline incubation on enzyme activity/stability before the blocking step was analyzed. Activity decreased by 40% after 72 h (but it should be considered that previously it had increased by a 24 fold factor), but the stability significantly improved after this incubation. Thus, after immobilization at different pH values, the immobilized trypsin was submitted to 72 h of alkaline incubation and blocked with EDA. The most active and stable preparation was that immobilized at pH 10. This preparation was less stable than the glyoxyl preparation in thermal inactivations (by less than a twofold factor), but was more stable in organic solvent inactivation (also by less than a twofold factor). The number of groups involved in the enzyme support attachment was 6 Lys using glyoxyl and became a minimum of 13 (including Lys, Tyr and His) using the DVS-activated support (the precision of the method did not permit to analyze the implication of some of the 3 terminal amino groups). Thus, this DVS-agarose support seems to be a very promising support to permit a very intense enzyme-support multipoint covalent attachment.

Enzymatic synthesis of chitosan derivatives and their potential applications

Abstract: Chitosan (aminopolysaccharide) exhibiting excellent functional properties was found to be a good natural source in various applications. However, practical use of chitosan was generally limited due to its weak solubility in neutral pH, its poor workability and low antioxidant activity. For a breakthrough in utilization, the functionalization of chitosan will be a key point, allowing the introduction of desired properties and enlarging the field of its potential applications. Enzymatic or chemical functionalizations of chitosan were carried out based on its reactive groups (-NH2 and -OH). Due to growing safety and environmental concerns, enzymatic methods were constantly investigated as an attractive alternative to toxic and non-specific chemical approaches. This review discusses the mechanisms of enzymatic functionalization and compares the applied systems, enzymes and materials for enzymatic functionalization of chitosan. A focus is also paid concerning the current applications of enzymatically modified chitosan derivatives in the field of biomedical, pharmaceutical, food, environmental and material applications.

Lipase immobilization onto polyethylenimine coated magnetic nanoparticles assisted by divalent metal chelated ions

Abstract: In this study, polyethylenimine coated Fe3O4 magnetic nanoparticles (Fe@PEI) were prepared and used for three metal ions (Co2+, Cu2+ and Pd2+) chelation. The metal chelated magnetic nanoparticles (Fe@PEI-M) were characterized by using different spectroscopic and analytical techniques. The metal contents were analyzed by inductively coupled plasma atomic emission spectroscopy (ICP) and energy-dispersive X-ray spectroscopy (EDX). Thermomyces lanuginosa lipase (TLL) was then immobilized onto the modified magnetic supports by physical adsorption. Fe@PEI-Co and Fe@PEI-Cu showed better activity at extreme temperature and pH values than of the free enzyme. After 10 cycles of successful biocatalytic activity performed by immobilized lipase on Fe@PEI-Co, more than 60% of initial activity was reserved. Furthermore, the immobilized lipase onto Fe@PEI-Co retained about 80% of its initial activity after 14 days of storage. The immobilized lipase on the selected nanocomposite was then used for synthesis of ethyl valerate. After 24 h incubation time, the extent of esterification was found to be 70 and 60% in n-hexane and DMSO media, respectively.

Characterization of a d-psicose 3-epimerase from Dorea sp. CAG317 with an acidic pH optimum and a high specific activity

Abstract: Ketose 3-epimerase displayed an important role in not only the cyclic monosaccharides bioconversion strategy, named Izumoring, but also in the industrial biological production of d -psicose, a novel low-calorie rare sugar widely used in food and medical industries. Since the non-enzymatic side reactions could be reduced in acid conditions, slightly acidic pH optimum is one of the main issues for biological production of d -psicose. In this study, we first characterized an acidic ketose 3-epimerase, the recombinant d -psicose 3-epimerase (DPEase) from Dorea sp. CAG317. The protein exhibited high amino acid sequence identity with other reported DPEases, and was determined as a homotetramer with subunit molecular weight approximately 33 kDa, which was the same as other reported findings. The recombinant DPEase was a metal-dependent enzyme with the optimum metal cofactor as Co2+. In presence of 1 mM of Co2+, the enzyme displayed the maximal activity as 803 U/mg at pH 6.0 and 70 °C. The catalytic efficiency (kcat/Km) was measured to be 412 and 199 mM−1 min−1 toward d -psicose and d -fructose, respectively. The equilibrium ratio between d -fructose and d -psicose was approximately 30:70, and the elevated temperature did not significantly shift the equilibrium toward d -psicose. Compared to other reported DPEases, the recombinant Dorea sp. DPEase displayed significantly higher specific activity at acidic pHs and remarkably higher productivity of d -psicose at pH 6.0, indicating that it was appropriate for use as a new source of d -psicose producing enzyme.

Application of cellulose/lignin hydrogel beads as novel supports for immobilizing lipase

Abstract: Lignocellulose-based hydrogels may have many potential applications in biomedical and biotechnological fields owing to their attractive properties including low cost, biodegradability, and biocompatibility. In this study, cellulose/lignin composite hydrogel beads were prepared by co-dissolution of cellulose and lignin in 1-ethyl-3-methylimidazolium acetate and then reconstitution with distilled water. Lipase from Candida rugosa was immobilized on various cellulose/lignin hydrogel beads. The results showed that lipase immobilized on cellulose/lignin beads showed higher activity and stability than those of lipase immobilized on pure cellulose beads. The activity and stability of immobilized lipase increased with the increase in the lignin content in the cellulose/lignin beads. The activity, protein loading, and specific activity of lipase immobilized on the optimal cellulose/lignin beads were 2.6, 2.2, and 1.2 times higher than those of lipase immobilized on cellulose beads, respectively. The effect of lignin on the activity of lipase immobilized on cellulose/lignin beads was statistically well predicted. The residual activity of lipase immobilized on the optimal cellulose/lignin beads after incubation for 12 h at 40 °C was 3.2 and 1.9 times higher than that of free lipase and lipase immobilized on cellulose beads, respectively. Interestingly, the half-life time of lipase immobilized on cellulose/lignin beads at pH 3.0 was 24 and 3 times higher than that of free lipase and that of lipase immobilized on cellulose beads, respectively. These results show that cellulose/lignin hydrogels may offer many potential applications in the biocatalytic, biomedical, and bioelectronic fields owing to their high biocompatibility, biodegradability, and controllable properties.

Characterization of a novel amine transaminase from Halomonas elongata

Abstract: Chiral amines are indispensable building blocks in the production of biologically active compounds. They are fundamental for the pharmaceutical industry, both as active molecules themselves and as chiral auxiliaries in asymmetric synthesis; however, the available synthetic strategies often present disadvantages. ω -Transaminases ( ω -TAs) appear as an attractive alternative by driving the stereoselective amination of prochiral ketones. HEWT is a novel amine transaminase from the moderate halophilic bacterium, Halomonas elongata DSM 2581, which is highly ( S )-selective, being able to fully convert ( S )-1-phenylethylamine to acetophenone and showing no activity with the corresponding ( R )-1-phenylethylamine. HEWT has a broad substrate scope, active with a range of amino donors and acceptors, and naturally accepts isopropylamine (IPA) as amino donor in asymmetric synthesis providing a 41% conversion of pyruvate in 24 h at 37 °C starting with 1:1 molar ratio between the reagents. HEWT also accepts ortho -xylylenediamine as amino donor in for amine synthesis, in particular, with benzaldehyde yielding high conversions between 90 and 95%. The enzyme is also tolerant to the presence of cosolvents up to 20% making it a promising candidate for industrial applications.

Levan versus fructooligosaccharide synthesis using the levansucrase from Zymomonas mobilis: effect of reaction conditions

Abstract: This work addresses the effect of sucrose concentration and temperature on the three activities displayed by the levansucrase from Zymomonas mobilis : formation of levan (polymerization), production of short-chain fructooligosaccharides (FOS), and sucrose hydrolysis. Of the conditions tested, levan formation reached the highest value at 4 °C and 100 g/L sucrose. The increase of temperature (40 °C) and sucrose concentration (600 g/L) caused a significant decrease of the levan concentration and a higher production of FOS. However, an increase of the temperature also caused an enhancement of the undesired hydrolytic activity. Several inulin-type FOS (1-kestose, nystose, 1 F-fructosylnystose), neoFOS (blastose, neokestose, neonystose) and levan-type FOS (6-kestose, 6,6-nystose) were synthesized by levansucrase. The latter compound was purified and characterized by mass spectrometry and 2D NMR. Using 600 g/L sucrose at 40 °C, the maximum yield of FOS was reached at 85% sucrose conversion; at this point, the reaction mixture contained (in weight basis) 31% glucose, 14% fructose, 15% sucrose and 40% FOS (including a small contribution of levan).

Oxidation with galactose oxidase: Multifunctional enzymatic catalysis

Abstract: Galactose oxidase (EC 1.1.3.9) is a single copper metalloenzyme, having a molecular weight of 65–68 kDa. Galactose oxidase catalyzes the oxidation of primary alcohols to corresponding aldehydes with strict regioselectivity, and the selectivity is high for the galactose C-6 primary hydroxyl group. The oxidation of alcohols to carbonyl compounds is one of the most important reactions in synthetic chemistry, thus enzymatic biocatalysis requiring only molecular oxygen as an oxidant is a valuable alternative to chemical reagents. We review here the various mono-, oligo- and polysaccharide derivatives that have been synthesized by galactose oxidase-catalyzed reactions, and the several either qualitative or quantitative analytical techniques utilized to follow of the reaction, determine the conversion, and identify the products. The optimal reaction conditions, the formation of side products, and the oxidation of substrates other than carbohydrates are discussed. Finally, we summarize engineering efforts of galactose oxidase that have improved recombinant enzyme expression and yield, or altered substrate specificity.

Cholinium-based deep eutectic solvents and ionic liquids for lipase-catalyzed synthesis of butyl acetate

Abstract: The aim of this study was to analyze the advantages and limitations of cholinium-based ionic liquids (ILs) and deep eutectic solvents (DESs) used as green solvents for immobilized Candida antarctica lipase B-catalyzed synthesis. The reaction of acetic anhydride with 1-butanol to give short chain ester butyl acetate was chosen as a model reaction. Results showed that selected ILs (choline glycinate, choline alaninate, choline asparaginate, choline malate) and DESs (choline chloride mixtures with glycerol Gly, ethylene glycol EG, and urea U as hydrogen bond donors in molar ratio 1:2) are poor media for tested reaction if applied as pure solvents (yield 5 mM) for both cell lines. Obtained results suggest that DESs are promising candidates for green biocatalysis.

(Chemo)enzymatic cascades - Nature's synthetic strategy transferred to the laboratory

Abstract: The astonishing efficiency with which living organisms build complex molecules from simple starting materials has inspired chemists for centuries. Among the synthetic strategies that nature uses to achieve this efficiency, the combination of several enzymatic transformations in cascading sequences is of outstanding importance. With the rise of biocatalysis, researchers now have the tools at hand to mimic this strategy and develop artificial enzyme cascades of impressive complexity. This editorial review aims to introduce the reader to some key aspects of (chemo)enzymatic cascades, as well as to put the submissions to the present Special Issue into a broader context.

Multi-step synthesis strategies towards 1,2-amino alcohols with special emphasis on phenylpropanolamines

Abstract: Chiral vicinal amino alcohols are molecules with broad applications in the pharmaceutical as well as in the chemical industry. Due to their high potential, various multi-step chemical, chemo-enzymatic and enzymatic reaction synthesis strategies have been developed within the last decades. This review summarises the asymmetric synthetic routes towards vicinal amino alcohols in general and provides exemplary in-depth looks into multi-step phenylpropanolamine synthesis with special focus on recently published cascade approaches.

Enhanced pectinase production by optimizing fermentation conditions of Bacillus subtilis growing on hazelnut shell hydrolyzate

Abstract: This study describes optimization of polygalacturonase (PG) production using Bacillus subtilis in submerged fermentation by Plackett–Burman (PB) design and response surface methodology (RSM). Five variables (pH, time, temperature, yeast extract concentration and K 2 HPO 4 ), which were determined to be significant by the PB analysis, were further optimized using Box–Behnken response surface method. The optimization results indicated that a maximal PG activity of 5.60 U mL −1 was achieved at pH 7.0, 72 h, and 30 °C using 0.5% (w/v) yeast extract and 0.02% (w/v) K 2 HPO 4 in the fermentation medium. The results implied a 2.7-fold increase in PG activity of B. subtilis under the optimized conditions. Thus, it was concluded that hazelnut shell hydrolyzate have remarkable potential for low cost commercial PG production.

Synthesis of long-chain isomaltooligosaccharides from tapioca starch and an in vitro investigation of their prebiotic properties

Abstract: The recombinant wild-type (WT) or Y101S-mutated amylomaltase prepared from the gene screened from soil DNA was used in combination with Aspergillus niger transglucosidase to produce isomaltooligosaccharides (IMOs) from tapioca starch. The highest IMO yield was obtained when 30% (w/v) soluble tapioca starch was incubated with 120 Units of both amylomaltases and 6 Units of transglucosidase at 40 °C for 30 min with WT or for 1 h with Y101S-mutated enzymes. Mass spectrometry and 1H NMR analysis of the IMOs synthesized from WT and Y101S-mutated enzymes showed the presence of α-1,4 and α-1,6 glycosidic bonds with a polymerization degree of ≤9. HPAEC-PAD analysis showed that the sizes of the IMOs synthesized from both enzymes were significantly larger than those of commercial IMOs. A smaller proportion of long-chain IMOs was observed when Y101S-amylomaltase was used. As for physical and biological properties, the degree of sweetness of both synthesized IMOs was lower than those of sucrose and commercial IMOs, while the brown color from the Maillard reaction, the viscosity and the hygroscopicity were higher than those of commercial IMOs. The prebiotic properties of both synthesized IMOs showed that they were able to tolerate acidic conditions, heat, and human digestive enzymes. In addition, these IMOs could stimulate the growth of Lactobacillus casei and resulted in a decrease of the culture pH, similar to the effect of commercial IMOs.

Enzymatic production and characterization of potential biolubricants from castor bean biodiesel

Abstract: Biodegradable lubricants (esters) are a logical solution to avoid the environmental threat of mineral lubricants disposal. This research reports the enzymatic synthesis and characterization of new biodegradable lubricants obtained via the transesterification of different methyl fatty acids esters (biodiesels produced from castor, soybean and jatropha oils) and 2-(hydroxymethyl)-2-ethylpropane-1,3-diol or trimethylolpropane (TMP). Different lipases have been compared in their performance to catalyze these reactions. The best lipase depended on the substrate, but a lipase from Candida rugosa and castor oil were selected due to the promising properties of the final product. After optimizing the experimental parameters of the processes, the optimal values were: enzyme content: 4.0%, water content: 1.0%, molar ratio castor oil biodiesel/TMP: 3.915:1, temperature: 40 °C and residence time: 24 h. Under these conditions, a conversion yield higher than 95% was obtained. This product is a promising biolubricant (very rich in ricinoleic acid) which was characterized and exhibited better lubricant properties than the products achieved using the other materials: viscosities of 290.2 mm2/s (at 40 °C) and 28.46 mm2/s (at 100 °C), and a viscosity index of 132. This work has indicated the feasibility of the proposed route for a new biolubricant with better properties than the previously described.

Biocompatible cellulose nanocrystals as supports to immobilize lipase

Abstract: Cellulose nanocrystals (CNCs) are rod-like cellulose nanomaterials that can be economically prepared from various cellulosic materials by the elimination of amorphous regions of cellulose. CNCs can be used as supports to immobilize enzymes because of their inherent biocompatibility, high specific-surface area, and exceptional mechanical properties. In this study, CNCs obtained from cotton linter cellulose (CLC) and bacterial cellulose (BC) were used as supports to immobilize Candida rugosa lipase. The protein loading and immobilization yield of the lipase immobilized onto the CNC obtained from CLC (CNC⿿CLC) were 1.8 and 2.2 times higher, respectively, than those of the lipase immobilized onto CLC. The lipase immobilized onto CNC⿿CLC showed significantly enhanced thermal stability. The half-life time of the immobilized lipase during incubation at 60 °C was 27 times higher than that of free lipase. The lipase immobilized onto CNC⿿CLC also showed increased pH stability at an alkaline pH. The residual activity of the immobilized lipase after 5 h incubation at pH 10 was 8.8 times higher than that of free lipase. In addition, the lipase immobilized onto CNC⿿CLC was able to perform homogeneous-like biocatalysis without shaking or agitation because the immobilized lipase remained well-dispersed during the aqueous reaction. The CNCs as enzyme supports have many potential applications in the biomedical, bioelectronic, and biocatalytic fields.

Surfactant-activated magnetic cross-linked enzyme aggregates (magnetic CLEAs) of Thermomyces lanuginosus lipase for biodiesel production

Abstract: Novel surfactant-activated magnetic cross-linked enzyme aggregates of Thermomyces lanuginosus lipase (TLL-magnetic-CLEAs) were developed and provided an efficient approach to improve the activity and stability of lipase for biodiesel production. In the methanolysis of Jatropha oil for biodiesel synthesis, the maximum yield in isopropyl ether was 88% after 48 h at 40 °C, representing 3.5-fold and 2.5-fold higher activity than that exhibited by free TLL and TLL CLEAs, respectively. Moreover, Tween 80-activated TLL-magnetic-CLEAs retained their activity during storage at 4 °C for 11 weeks and 10 cycles of repeated 48 h biodiesel reactions at 40 °C (over 30 days). Additionally, the surface morphology, particle size and loading of lipase aggregates were confirmed by Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). The combination of interfacial activation, high specific enzyme activity, improved stability and easy recovery of magnetic CLEAs presents an attractive process for lipase immobilization and provides a promising catalyst for biodiesel production.

High catalytic activity of immobilized laccase on core–shell magnetic nanoparticles by dopamine self-polymerization

Abstract: A facile and efficient method is developed for enzyme immobilization on silica-coated magnetic nanoparticles (Fe3O4@SiO2 NPs) via dopamine (DA) self-polymerization process. The scanning electron microscope images indicate that Fe3O4@SiO2 NPs have a spherical and uniform size distribution, and the high saturation magnetization (14.68 emu g(-1)) makes it easily to be separated from the reaction system under an extra magnetic field. Fourier-transform infrared spectroscopy and thermogravimetric analysis reveal that polydopamine (PDA) has been successfully coated on Fe3O4@SiO2 NPs surface. During in situ polymerization of DA, laccase is also firmly immobilized on Fe3O4@SiO2 NPs, and the total activity recovery can reach to 43.28%. However, the laccase immobilized by glutaraldehyde (GA) crosslinking method only keeps 3.33% of the total activity recovery under the optimized condition. Compared with free laccase and laccase immobilized by GA, the laccase immobilized by DA exhibits superior resistance to a broader pH value and obviously enhanced stability. After 10 times reusing cycles, the activity of laccase immobilized by DA still retains 65% of its initial activity, whereas the laccase immobilized by GA has 35% of its original activity. After 70 days of storage at 4 degrees C, the laccase immobilized by DA keeps about 80% of its initial activity, but the free laccase and the laccase immobilized by GA only remained 7.8% and 37%, respectively. Thus, this work provides a method for laccase immobilization with advantages of environmentally friendly, low cost and high catalytic activity. (C) 2014 Elsevier B.V. All rights reserved.

Monitoring the Rhizopus oryzae lipase catalyzed hydrolysis of castor oil by ATR-FTIR spectroscopy

Abstract: A rapid and environmental friendly Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopic method was developed for monitoring the Rhizopus oryzae lipase (ROL) catalyzed hydrolysis of castor oil in oil-in-water emulsion system. A calibration curve was constructed using partial least square (PLS) model by gravimetric addition of oleic acid (10–50%) in castor oil to detect the carbonyl absorption of free fatty acids (FFA) in the region (1690–1730 cm−1). The correlation co-efficient (R2) and root mean square error of calibration (RMSEC) by PLS model were found to be 0.999 and 0.316, respectively. ROL was found to be an efficient biocatalyst to produce free fatty acids (FFA) from castor oil. Factors affecting the rate of hydrolysis such as enzyme concentration (0.01%, w/v), pH (7), temperature (37 °C), oil–water ratio (1:4) and reaction time (12 h) were optimized. Under all set of conditions the ROL effectively hydrolyzed castor oil up to 90% yield of fatty acids. The methodology is fairly environmental friendly in both cases, i.e. using lipase for hydrolysis of castor oil and analyzing the product through FTIR spectroscopy.

Purification strategies and properties of a low-molecular weight xylanase and its application in agricultural waste biomass hydrolysis

Abstract: Xylanases are hydrolytic enzymes responsible for the depolymerization of xylan, and they are of great interest because of their wide industrial applications. Extracellular low molecular weight xylanase was purified from a submerged culture of a thermophilic fungus, Scytalidium thermophilum ATCC No. 16454 grown on corn cobs, and the enzyme was characterized. Among the investigated low-cost purification techniques, the highest recovery (79%) with 2.7-fold purification, was obtained using an aqueous two phase system. The maximal purity (4.3-fold) was achieved by ultrafiltration and generated a yield of 25%. A two-step chromatography procedure consisting of gel filtration and anion exchange without any additional pre-purification steps yielded ca . 21.8-fold xylanase purification to apparent homogeneity with 9.6% recovery as demonstrated by SDS-PAGE. This study is the first to report a complement purification procedure and the corresponding results for S. thermophilum low molecular weight xylanase isolation from culture medium. The enzyme has a molecular weight of 21 kDa and an isoelectric point of pH 8.6, indicating that it belongs to GH family 11. The low molecular weight of the enzyme provides the important advantage of making it easy to access in the lignocellulosic network, and it efficiently degrades hemicellulose. The optimum temperature and pH values of purified xylanase were 65 °C and 6.5, respectively. Xylanolytic activity was most stable at pH 7.0 and 40 °C. K m and V max values of xylanase on beechwood xylan were predicted as 2.4 mg xylan/ml and 168.6 IU/ml, respectively. The potential of xylanase in biomass hydrolysis was also investigated, as lignocellulosic biomass has attracted increasing attention as an important raw material for second generation biofuel production. Among the investigated commercial and lignocellulosic substrates, purified xylanase showed the maximum specificity for beechwood xylan and wheat bran. The hydrolysis of an inedible and waste lignocellulosic substrate, corn cob, by xylanase was studied. A release of reducing sugars and apparent morphological changes in the corn cob structures were observed by SEM analysis after enzymatic treatment.

Purification, biochemical characterization and structural modeling of a potential htrA-like serine protease from Bacillus subtilis DR8806

Abstract: The production of an extracellular htrA-like serine protease by Bacillus subtilis DR8806 was studied in this study. The enzyme was purified using ammonium sulphate precipitation and Sephacryl S-200 size exclusion chromatography. The analysis by SDS-PAGE and zymogram of enzyme showed a molecular weight of 37 kDa. Isoelectric focusing revealed a p I value of 6.6 for the enzyme. By the use of casein as substrate, the enzyme was active and stable at the wide range of temperatures with maximum activity at 45 °C and pH 8. The enzyme activity was increased by Ca 2+ , K + , Mg 2+ , Fe 2+ , dimethylsulfoxide (DMSO), whereas its activity was decreased by Hg 2+ , Ba 2+ , Cu 2+ , Zn 2+ , H 2 O 2 , CTAB (cetyltrimethylammonium bromide) and SDS (sodium dodecyl sulphate). In addition, Mn 2+ , Na + , Triton X-100, β-mercaptoethanol, EDTA (ethylenediaminetetraacetic acid) had no significant effect on the enzyme activity. Among organic solvents, ethanol and methanol enhanced the activity. The gene of the protease showed a 1200 bp open reading frame with 97% similarity to other htrA-like proteases. The computational modeling of the protease showed two distinct domains: a PDZ domain and protease core domain. The catalytic triad also demonstrated a degree of discrepancy in comparison to other serine proteases. It is composed of a serine residue as a nucleophile and a proline as a base center, while the acidic center was not fully identified. The obtained results suggested a new type of htrA-like protease with no previous records in bacillaceae family.

Immobilisation of ω-transaminase for industrial application: Screening and characterisation of commercial ready to use enzyme carriers

Abstract: Despite of the advantages that enzyme immobilisation can bring to industrial biocatalysis, its utilisation is still limited to a small number of enzymes and processes. Transaminase catalysed processes are a good example where immobilisation can be of major importance and even decisive for economic feasibility. This work presents results obtained for screening of enzyme carriers for immobilisation of ω-transaminase for industrial application. A total of 6 commercial enzyme carriers (polymeric resins) were screened and two suitable enzyme carriers were selected for immobilisation of both (S)- and (R)-selective ω-transaminases. These carriers allowed the re-use of the immobilised enzyme for 8 cycles of 24 h each, under relevant process conditions, corresponding to approximately 250 h of operation, with more than 50% of the initial activity retained. Likewise the stability towards higher temperatures and possibility to store the biocatalyst for more than 70 days (at room temperature) were obtained as result of the immobilisation on the selected supports.

Continuous production of fructooligosaccharides and invert sugar by chitosan immobilized enzymes: Comparison between in fluidized and packed bed reactors

Abstract: In this work, β-fructofuranosidase and β-fructosyltransferase were covalently immobilized on chitosan spheres, using glutaraldehyde as a coupling agent, in order to produce invert sugar and fructooligosaccharides (FOS). Maxinvert L was used to make β-fructofuranosidase biocatalyst yielding 7000 HU/g. A partial purified β-fructosyltransferase from Viscozyme L was used to prepare the other biocatalyst yielding 2100 TU/g. The production of invert sugar and FOS was evaluated using different continuous enzymatic reactors: two packed bed reactors (PBR) and two fluidized bed reactors (FBR). The invert sugar production achieved a yield of 98% (grams of product per grams of initial sucrose) in the PBR and 94% in the FBR, whereas FOS production achieved a yield of 59% in the PBR and 54% in the FBR. It was also observed in both cases that varying the flow rate it is possible to modulate the FOS composition in terms of nystose and kestose concentrations. The operational stability of FOS produced in the PBR was evaluated for 40 days showing no reductions in yields.

Hydrolysis of soybean oil to produce diacylglycerol by a lipase from Rhizopus oryzae

Abstract: A lipase from Rhizopus oryzae (rProROL) was produced by recombinant Pichia pastoris in high productivity with a maximum lipase activity of 15,900 U/mL, and was employed to hydrolyze soybean oil for the production of diacylglycerol (DAG). Changes in triacylglycerol (TAG) conversion and DAG content were observed under various factors, like pH, water content, temperature and enzyme loading. A polynomial equation ( R 2 = 0.9797) between DAG content and TAG conversion was modeled and showed good agreement with the experimental results. The highest content of DAG at 32.09% was observed when TAG conversion was between 68% and 73%. Besides, 13 C nuclear magnetic resonance (NMR) was used to identify the 1,2(2,3)-DAG and 1,3-DAG, and during hydrolysis, rProROL was discovered to show 1,3-regioselectivity in the hydrolysis. Moreover, rProROL exhibited high hydrolytic activity and capacity in the accumulation of DAG, indicating that rProROL is a prospective enzyme which could be used in the oils and fats industries.

Hydrolysis of Jatropha curcas oil for biodiesel synthesis using immobilized Candida cylindracea lipase

Abstract: Environmental degradation and the diminishing reserves of fossil fuel are the major drives for the search for biodiesel as alternative fuel. Jatropha curcas oil has been well researched as non-edible vegetable oil for the production of biodiesel using conventional processes which generate huge obnoxious wastes and consequently incur additional cost for treatment and disposal of the generated waste. This paper presents the hydrolytic biosynthesis of J. curcas hydrolysate (FFA) for the production of biodiesel using locally produced Candida cylindracea lipase immobilized on functionalized activated carbon support. Up to 78% of free fatty acid (FFA) was achieved through hydrolytic reaction relative to the source and activity of the enzyme employed. Optimum dose of the catalyst loading was 8% (w/w) of the immobilized matrix at 40 °C reaction temperature and 200 rpm agitation. All reactions were allowed to proceed for 24 h, and catalyst could be recycled four times for optimal FFA yield.

Application of diethyl ethoxymethylenemalonate (DEEMM) derivatization for monitoring of lysine decarboxylase activity

Abstract: Aliphatic diamines have been used as chemical reagents for the monomers of polyamides ( i.e. , Nylon 46, Nylon 510, Nylon 66, Nylon 610 and so on), and can be made from lysine, arginine or ornithine by the metabolism of microbes through decarboxylases. However, the conventional derivatization methods for the HPLC-based quantitative analysis of aliphatic diamines exhibit poor sensitivity and efficiency for the monitoring of enzyme activity. In this study, a chemical derivatization method using diethyl ethoxymethylenemalonate (DEEMM) was applied to monitor lysine decarboxylase activity for the production of cadaverine by measuring the levels of intracellular and secreted lysine and cadaverine. The calibration graphs for the determination of 2,4-diaminobutyrate, 2,6-diaminopimelic acid, ornithine, arginine, lysine, 1,3-diaminopropane, putrescine dihydrochloride, cadaverine, hexamethylenediamine, and 1,7-diaminoheptane were measured by the highly sensitive method using DEEMM, and a linear relationship was observed for each dimaine compound, with limits of detection up to 0.001 mM. Application of this method will be useful for the sensitive monitoring of lysine decarboxylase reactions through the detection of substrates containing amine groups and products with diamines using HPLC and a UV detector.

Laccase isozymes from Ganoderma lucidum MDU-7: Isolation, characterization, catalytic properties and differential role during oxidative stress

Abstract: Strain of Ganoderma lucidum MDU-7 produce multiple extracellular isoforms of laccase in submerged culture condition using malt extract as a carbon source and copper sulfate as an inducer. SDS–PAGE followed by MALDI–TOF peptide fingerprinting confirmed laccase isozyme with molecular mass of 24–66 kDa. Two laccase isozymes (Glac H1 and Glac L1) were purified from native-PAGE protein purification method and a comparative catalytic and antioxidant study has been performed. Both of the laccase isozymes have optimum temperature and pH at 50 °C and 4.0, respectively. Glac L1 has higher stability in comparison to Glac H1, over wide range of temperature, pH, divalent metal ions and surfactants. The Km values of Glac L1 and Glac H1 determined for guaiacol, ABTS and O-tolidine were 98 μM, 26 μM, 320 μM and 281 μM, 29 μM, 338 μM, respectively. Glac H1, irrespective to its laccase activity and stability, acts as a better antioxidant than Glac L1.

Effect of water, organic solvent and adsorbent contents on production of biodiesel fuel from canola oil catalyzed by various lipases immobilized on epoxy-functionalized silica as low cost biocatalyst

Abstract: Enzymatic production of biodiesel from canola oil using self-made covalently immobilized lipase from Candida antarctica (CALB), Thermomyces lanuginosus (TLL) and Rhizomucor miehei (RML) on epoxy-functionalized silica as low cost catalyst was investigated. In this optimization study, the effect of water, t-butanol and water adsorbent content on the yield of fatty acid methyl ester (FAME) was considered. Complete conversion to FAMEs was achieved under optimum conditions for CALB immobilized on epoxy-functionalized silica (Silica-CALB); 30% (w/w) t-butanol by substrate weight, reaction time of 96 h, 50 °C and molar ratio of methanol to oil 3:1, which was added to the reaction mixture in three steps. In general by adding t-butanol to the reaction medium, the conversion of oil to FAME increased for RML immobilized on epoxy-functionalized silica (Silica-RML) and 50 wt.% t-butanol by substrate weight gave the best yield. TLL immobilized on epoxy-functionalized silica (Silica-TLL) reach to 100% yield at 10% t-butanol. Water suppresses the methanolysis reaction catalyzed by Silica-CALB but significantly increased FAME yield for Silica-TLL and Silica-RML. It was observed that at high water adsorbent amounts (more than 55%, w/w), conversion to FAME decreased possibly due to mass transfer limitation. The immobilized TLL was quite stable and can be reused for 16 cycles without significant loss in activity (95%). The immobilized preparations of RML and CALB also presented a good reusability, keeping 85% of their initial activities after 16 cycles of the reaction.

Exploring the synthetic applicability of a new carboxylic acid reductase from Segniliparus rotundus DSM 44985

Abstract: A new carboxylic acid reductase (CAR) gene from Segniliparus rotundus DSM 44985 was overexpressed in Escherichia coli . The recombinant enzyme exhibited high activity toward a variety of aromatic and aliphatic carboxylic acids. Especially, it effectively reduced 4-hydroxybenzoic acid ( 8a ) and 4-nitrobenzoic acid ( 19a ), toward which the known Nocardia CAR exhibited no or little activity. The recombinant E. coli cells co-expressing the Segniliparus CAR and Nocardia PPTase genes catalyzed the reductions of vanillic acid ( 20a ) and 3,4-dihydroxyphenylacetic acid ( 25a ) to give vanillyl alcohol ( 20c ) and 3-hydroxytyrosol ( 25c ) with high yield, respectively. The endogenous aldehyde reductases of E. coli should be responsible for the further reduction of the produced aldehydes. These results demonstrated that Segniliparus CAR was a useful addition to the biocatalyst tool-box for the reduction of carboxylic acids and might find applications in the synthesis of valuable bio-based chemicals from renewable resources.

Strategic optimization of xylanase–mannanase combi-CLEAs for synergistic and efficient hydrolysis of complex lignocellulosic substrates

Abstract: Cost-effective application of lignocellulolytic enzymes holds the key towards commercialization of enzymatic hydrolysis of lignocellulosic biomass. Carrier free immobilization of enzyme(s) offers a lucrative prospect. Combined-cross linked enzyme aggregates (combi-CLEAs) are a novel prospective and this present study addresses the preparation, characterization and application of xylanase–mannanase combi-CLEAS on lime-preteated sugarcane bagasse and milled corn stover. X6-CLEAs, X7-CLEAs, L1-CLEAs and L7-CLEAs were prepared after elaborative optimization of the precipitating agent and glutaraldehyde concentration. The highest activity after precipitation was observed with acetone but following cross-linking with glutaraldehyde less than 60% activity was retained, while more than 60% activity was retained after precipitation with ammonium sulphate and cross-linking with glutaraldehyde. Accessory enzyme activities including α-arabinofuranosidase, β-xylosidase, esterases, β-mannosidase, α-galactosidase and β-glucosidase were also determined. More than an 1.5 fold increase in thermostability compared to the free enzyme was observed over a broad temperature range (50–70 °C). Tri-synergy studies and quad synergy studies were used to generate combi-CLEAs with different protein ratios. Hydrolysis of lime pre-treated bagasse with combi-CLEAs at protein ratios corresponding to X6 (33.0%):X7 (17.0%):L1 (17.0%):L7 (33.0%) resulted in a 1.68 fold higher sugar release compared to the quad synergy model using free enzymes. Similarly, hydrolysis of corn stover with combi-CLEAs at protein ratios corresponding to X6 (40.0%):X7 (10.0%):L1 (10.0%):L7 (40.0%) resulted in an 1.58 fold higher sugar release compared to the sugar release observed with the quad synergy model using free enzymes. Monomeric sugars constituted 70–75% of reducing sugars released during hydrolysis. The role of accessory enzymes in improving enzyme synergy was clearly shown. The efficiency of combi-CLEAs compared to free enzymes makes them ideal candidates for the prudent and cost-effective commercialization of lignocellulolytic enzymes.

Medium and reaction engineering for the establishment of a chemo-enzymatic dynamic kinetic resolution of rac-benzoin in batch and continuous mode

Abstract: The effect of the reaction parameters water activity and reaction solvent was investigated for the dynamic kinetic resolution (DKR) of rac-benzoin with immobilized Lipase TL as biocatalyst for transesterification and the heterogeneous chemo-catalyst Zr-TUD-1 (Si/Zr = 25) for in situ racemization. Overall dry reaction conditions led to the best results for both catalysts. The immobilized lipase in a more environmentally benign solvent like cyclopentyl methyl ether (CPME) exhibited a 1.6-fold higher activity and an up to 1.5-fold higher half-life time than in the standard solvents such as toluene and 2-methyltetrahydrofuran (2-MeTHF). Among a variety of deep eutectic solvents (DESs) choline chloride:isosorbide (ChCl:Iso) was found to be suitable for the reaction system. The activity was lower than in the aforementioned solvents, but the very low solubility of the product (S)-benzoin butyrate in ChCl:Iso compared to the investigated organic solvents possesses great potential with respect to downstream processing. Optimized reaction parameters (dry CPME) were applied for DKR in batch and continuous mode yielding comparable or slightly better results than in toluene or 2-MeTHF.