Immobilisation of Candida rugosa lipase on polyhydroxybutyrate via a combination of adsorption and cross-linking agents to enhance acylglycerol production

In the present study, a combination of immobilisation processes was utilised to prepare robust biocatalysts. First, lipase from Candida rugosa was adsorbed on polyhydroxybutyrate (PHB) particles, followed by cross-linking with glutaraldehyde. Conditions for creating immobilised lipase involved the addition of 0.6 M glutaraldehyde and 45 U mL⁻¹ lipase while mixing at 150 rpm (4 °C) for 30 min. These conditions produced the highest yield of immobilised lipase (92 %) and the highest levels of activity (1.94 mg g⁻¹ support). At 40 °C and pH 9 the immobilised enzyme was optimally active with a K_m and V_maxat 1.2 mM and 2.5 × 10^-3 mmol min⁻¹, respectively. The use of immobilised lipase improved thermal stability, storage stability, and reusability.The immobilised lipase retained 80 % of its activity after incubation at 30–60 °C for 2 h and 4 °C for 30 d in 0.2 M sodium phosphate buffer (pH 7.0). Moreover, the immobilised enzyme retained 50 % of its activity after more than 14 cycles under optimal conditions. The immobilised lipase was used to produce monoacylglycerol MAG. The existence of a carbonyl group at 1,743 and 1,744 cm⁻¹ was identified using attenuated total reflectance (ATR)-Fourier transformed infrared spectroscopy. Results showed that 48 % MAG was produced.     

Bacterial cellulose-chitosan composite hydrogel beads for enzyme immobilization

In this work, we report the preparation of bacterial cellulose (BC)-chitosan composite hydrogel beads by co-dissolution of BC and chitosan in 1-ethyl-3-methylimidazolium acetate and subsequent reconstitution with distilled water. The BC-chitosan hydrogel beads were used as enzyme supports for immobilizing Candida rugosa lipase by physical adsorption and covalent cross-linking. BC-chitosan hydrogel beads immobilized lipase more efficiently than microcrystalline cellulose (MCC)-chitosan hydrogel beads. The amount of protein adsorbed onto BCchitosan beads was 3.9 times higher than that adsorbed onto MCC-chitosan beads, and the catalytic activity of lipase was 1.9 times higher on the BC-chitosan beads. The lipase showed the highest thermal and operational stability when covalently cross-linked on BC-chitosan hydrogel beads. The half-life time of the lipase cross-linked on BC-chitosan bead at 60°C was 22.7 times higher than that of free lipase. Owing to their inherent biocompatibility and biodegradability, the BC-chitosan composite hydrogel beads described here could be used to immobilize proteins for various biomedical, environmental, and biocatalytic applications.     

Esterification of polyglycerol with polycondensed ricinoleic acid catalysed by immobilised Rhizopus oryzae lipase

The enzymatic method for synthesising polyglycerol polyricinoleate (PGPR), a food additive named E-476, was successfully carried out in the presence of immobilised Rhizopus oryzae lipase in a solvent-free medium. The great advantage of using the commercial preparation of R. oryzae lipase is that it is ten times cheaper than the commercial preparation of R. arrhizus lipase, the biocatalyst used in previous studies. The reaction, which is really a reversal of hydrolysis, takes place in the presence of a very limited amount of aqueous phase. Immobilisation of the lipase by physical adsorption onto an anion exchange resin provided good results in terms of activity, enzyme stability and reuse of the immobilised derivative. It has been established that the adsorption of R. oryzae lipase on Lewatit MonoPlus MP 64 follows a pseudo-second order kinetics, which means that immobilisation is a process of chemisorption, while the equilibrium adsorption follows a Langmuir isotherm. The use of this immobilised derivative as catalyst for obtaining PGPR under a controlled atmosphere in a vacuum reactor, with a dry nitrogen flow intake, allowed the synthesis of a product with an acid value lower than 6 mg KOH/g, which complies with the value established by the European Commission Directive. This product also fulfils the European specifications regarding the hydroxyl value and refractive index given for this food additive, one of whose benefits, as proved in our experiments, is that it causes a drastic decrease in the viscosity (by 50 %) and yield stress (by 82 %) of chocolate, comparable to the impact of customary synthesised PGPR.     

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

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

Immobilization of lipase onto a photo-crosslinked polymer network: Characterization and polymerization applications

Abstract

The recovery of activity of lipases immobilized onto a photo-crosslinked polymer network was 76.0% and 41.0% for entrapment and adsorption methods, respectively. Both entrapped and adsorbed immobilized enzymes were very stable, retaining more than 60% of their activity over the range of temperatures studied. Immobilization by either method protected their relative activities nearly 96% at 70°C. The optimum pH was 8.0 for immobilized enzymes and 6.0 for the free enzyme at 40°C, while the relative activities after storage at 0–4°C for 30 days were 98% and 75% using entrapment and adsorption methods, respectively. These results indicated that lipase immobilized by entrapment and adsorption not only had good activity recovery, but also remarkable stability, better reusability and application adaptability than free lipase. Also, it can be safely stated that, photo-crosslinked polymer network can be used as alternative supports for immobilization of lipase for enzymatic polymerization reactions. In the ring-opening polymerization of ?-caprolactone, polymerization rates were clearly affected as monomer conversions were 58% and 49% and the highest molecular weights (M_n) obtained were 7890 and 5600 gmol^{? 1} for entrapment and adsorption methods, respectively.     

Immobilisation of Pseudomonas gessardii acidic lipase derived from beef tallow onto mesoporous activated carbon and its application on hydrolysis of olive oil

The study was carried out to immobilise the acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon (MAC₄₀₀) for the application of hydrolysis of olive oil. MAC₄₀₀ was prepared from rice husk by the two stages process. P. gessardii was isolated from the beef tallow acclimatised soil. The acidic lipase (ALIP) was produced from a slaughterhouse waste, namely beef tallow as a substrate and immobilised onto MAC₄₀₀. The maximum immobilisation capacity of the MAC₄₀₀ was 3570 U/g at optimum immobilisation conditions; time (180 min), pH (5.0) and temperature (30 °C). The immobilised lipase had better thermal stability and reusability than the free lipase. The immobilisation of ALIP onto MAC₄₀₀ (MAC₄₀₀–ALIP) followed pseudo-first-order rate kinetics with rate constant 0.012/min. The Michaelis–Menten constant of MAC₄₀₀–ALIP was lower than that of the ALIP, which confirmed the higher affinity between enzyme and substrate. The immobilization of acidic lipase onto MAC₄₀₀ was confirmed by Fourier transform-infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and X-ray diffraction (XRD) pattern analysis. Reusability study of MAC₄₀₀–ALIP on olive oil hydrolysis showed 82% of hydrolysis up to 13 runs and 50% of hydrolysis up to 35 cycles of reuse. This work concludes that the acidic lipase immobilised mesoporous activated carbon matrix can be considered as a potential biocatalyst for the hydrolysis of olive oil. Thus, the enzyme immobilised matrix has potential industrial applications.”     

Improving reuse cycles of Thermomyces lanuginosus lipase (NS-40116) by immobilization in flexible polyurethane

Enzyme immobilization using a low-cost support that allows increasing operational stability and reutilization arise as a great economic advantage for the industry. In this work, it was explored different methods of Thermomyces lanuginosus lipase (NS-40116) immobilization in flexible polyurethane foam (PU). PU polymer was synthesized using polyether and toluene diisocyanate as monomers. PU-NS-40116 immobilized was evaluated in terms of stability in a range of pH (7.0 and 9.0), temperature (24, 50 and 60?°C) for 24?h, and storage stability (room temperature and 4?°C) for 30?days. The results showed that after 30?days of storage immobilized enzyme kept 80% of initial enzyme activity. PU support before and after immobilization process was characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. Free and immobilized enzymes were compared in terms of hydrolysis of soybean oil. Immobilized enzyme by entrapment was evaluated in successive cycles of reuse showing catalytic activity above 50% even after 5 successive cycles of reuse, confirming the efficiency of immobilization process.     

Synthesis of cinnamyl acetate catalysed by highly reusable cotton-immobilized Pseudomonas fluorescens lipase

Cinnamyl acetate as an important fragrance ingredient could be synthesized by lipase-catalysed transesterification in organic systems, but enzyme proteins tended to denature and inactivate for no water lubrication. To improve the non-aqueous stability of lipases, absorbent cotton was taken as an alternative “water” phase to stabilize enzyme proteins. In a mass ratio of 1:1, Pseudomonas fluorescens lipase was immobilized on cotton fibres by physical absorption in a column glass bottle, forming a facile cotton-lipase bioreactor in which the transesterification between cinnamyl alcohol and vinyl acetate processed efficiently. From the molar conversions after reaction for 2?h at 37?°C and 160?rpm, the ability of cotton-lipase to transform substrate was more than 5-folds of native lipase. And even in static state and at 4?°C, the conversion of reaction catalysed by cotton-PFL had 11-fold increase relative to native lipase after 8?h. Recycles showed that the cotton-lipase had an extra-long half-life of activity (t_1/2?=?693?h) and a negligible decay rate in the ability to transform substrate (D_r?=?0.08% h^?1). All these showed that this lipase had been effectively activated and stabilized by cotton fibres for the numerous hydroxyl groups and fluffy structure.     

X-ray structure and characterization of a thermostable lipase from Geobacillus thermoleovorans

Thermo-alkalophilic bacterium, Geobacillus thermoleovorans secrets many enzymes including a 43?kDa extracellular lipase. Significant thermostability, organic solvent stability and wide substrate preferences for hydrolysis drew our attention to solve its structure by crystallography. The structure was solved by molecular replacement method and refined up to 2.14?Å resolution. Structure of the lipase showed an alpha-beta fold with 19 α-helices and 10 β-sheets. The active site remains covered by a lid. One calcium and one zinc atom was found in the crystal. The structure showed a major difference (rmsd 5.6?Å) from its closest homolog in the amino acid region 191 to 203. Thermal unfolding of the lipase showed that the lipase is highly stable with T_m of 76?°C. ¹³C NMR spectra of products upon triglyceride hydrolysate revealed that the lipase hydrolyses at both sn-1 and sn-2 positions with equal efficiency.     

Geranyl acetate synthesis catalyzed by Thermomyces lanuginosus lipase immobilized on electrospun polyacrylonitrile nanofiber membrane

Isolated Thermomyces lanuginosus lipase (TLL) was immobilized by different protocols on the polyacrylonitrile nanofibers membrane. The conditions for immobilization of TLL were optimized by investigating effect of protein concentration, time and temperature on the extent of immobilization. The effect of immobilization on the catalytic activity and stability of lipase was studied thoroughly. The immobilized TLL was used as biocatalyst for geranyl acetate synthesis with geraniol and vinyl acetate as substrates and their performance was compared with free enzyme. The TLL immobilized by physical adsorption shows higher transesterification and hydrolytic activities than that of covalently linked or native TLL. There was 32 and 9 fold increase in transesterification activity of TLL immobilized by adsorption and covalent bonding, while hydrolytic activity increases only by 3.6 and 1.8 fold respectively. The optimum conditions for immobilization in both the cases were immobilization time 90–150 min, temperature 45 °C and protein concentration of 2 mg/ml. The percentage conversion of ester was more than 90% and 66% in case of physically adsorbed and covalently bonded enzyme respectively as compared to native one. However, covalently immobilized TLL shows higher operational stability than native and physically adsorbed TLL.     

Aggregation properties of cold-active lipase produced by a psychrotolerant strain of Pseudomonas palleroniana (GBPI_508)

Abstract

The present study aims to exploit microbial potential from colder region to produce lipase enzyme stable at low temperatures. A newly isolated bacterium GBPI_508 from Himalayan environment, was investigated for the production of cold-active lipase emphasizing on its aggregation properties. Plate based assays followed by quantitative production of enzyme was estimated under different culture conditions. Further characterization of partially purified enzyme was done for molecular weight determination and activity and stability under varying conditions of pH, temperature, and in presence of organic solvents, inhibitors, and metal ions. The psychrotolerant bacterium was identified as Pseudomonas palleroniana following 16S rRNA gene sequencing. Maximum lipase production by GBPI_508 was recorded in 7?days at 25?°C utilizing yeast extract as nitrogen source and olive oil as substrate in the lipase production medium. Triton X-100 (1%) in the medium as emulsifier significantly enhanced the lipase production. Lipase produced by bacterium showed aggregation which was confirmed by dynamic light scattering and native PAGE. SDS-PAGE followed by zymogram analysis of partially purified enzyme showed two active bands of ～50?kDa and ～54?kDa. Optimum activity of partially purified enzymatic preparation was recorded at 40?°C while the activity remained nearly consistent from pH 7.0 to 12.0, whereas, maximum stability was recorded at pH values 7.0 and 11.0 at 25?°C. Interestingly, lipase in the partially purified fraction retained 60% enzyme activity at 10?°C. Medium chain pNP ester (C10) was the most preferred substrate for the lipase of GBPI_508. The lipase possessed >50% residual activity when incubated with different organic solvents (25% v/v) except toluene and dichloromethane which inhibited the activity below 50%. Partially purified enzyme was also stable in the presence of metal ions and inhibitors. The study suggests applicability of GBPI_508 lipase in low temperature conditions such as cold-active detergent formulations and cold bioremediation.     

Cellulosic exopolysaccharide produced by Zoogloea sp. as a film support for trypsin immobilisation

Trypsin (E.C. 3.4.21.4) was covalently immobilised onto a membrane of a cellulosic exopolysaccharide produced by Zoogloea sp. in sugarcane molasses. Carbonyl groups were introduced into the matrix by sodium metaperiodate oxidation and the enzyme was immobilised either directly or through bovine serum albumin (BSA) as a spacer. The trypsin-membrane and trypsin–BSA-membrane retained, respectively, 37.2% and 9.16% of the specific activity of the native enzyme acting on N-benzoil-dl-arginine-p-nitroanilide (BAPNA). No activity decrease was observed in both preparations after seven reutilisations as well as they showed to be more thermal stable than the native enzyme. The trypsin–BSA-membrane presented the same initial activity (99%) after 54 days stored in 0.1 M Tris–HCl buffer, pH 8.0, at 4 °C but the trypsin-membrane lost 15% of activity. Furthermore, the trypsin–BSA-membrane lost 31% of activity after reuse at 9 days interval during 54 days of storage at 4 °C whereas the trypsin-membrane lost 69% of activity under the same conditions. These results showed an additional application for this biofilm, namely, to act as a reusable matrix for trypsin immobilisation and the presence of BSA improved the derivative performance.     

Biodiesel production from waste cooking oil by immobilized lipase on superparamagnetic Fe3O4 hollow sub-microspheres

Superparamagnetic Fe₃O₄ hollow sub-microspheres (FHSM) with strong response to an external magnet were prepared via a solvothermal method, followed by acid etching. Lipase from Candida sp. 99–125 was directly immobilized onto the amino-functional FHSM by simple adsorption, without glutaraldehyde linkage. The immobilized lipase was used to catalyze the esterification/transesterification of waste cooking oil with methanol to produce fatty acid methyl ester (FAME), a major source of biodiesel. FAME yield exceeded 93.4% over a wide range of temperatures from 10 to 40?°C. Notably, stability was clearly improved at the lower temperatures, in particular, giving a FAME yield of 89.6% after eight cycles of use at 10?°C.     

Long-term stability of oxidative stress biomarkers in human serum

The storage time and storage temperature might affect stability of oxidative stress biomarkers, therefore, they have to be analyzed after long-term storage of serum samples. The stability of three biomarkers reflecting oxidative stress: reactive oxygen metabolites (ROM) for hydroperoxides, total thiol levels (TTL) for the redox status and biological antioxidant potency (BAP) for the antioxidant status, was investigated at several time points during 60 months of storage at ?20 and ?80?°C. Biomarkers ROM and BAP showed a very good stability during storage for 60 months at both temperatures. In addition, the correlation of the data after 60 months of storage compared with the starting data was very good with correlation coefficients >0.9. The TTL assay showed good results in serum samples stored at ?80?°C, but not in samples stored at ?20?°C. Serum samples for analysis of the set of oxidative stress biomarkers ROM, BAP and TTL can be stored up to 60 months at ?80?°C. ROM and BAP can also be stored at ?20?°C during this period. The present results are very important for the biomarker-related epidemiological studies that make use of biobanks with samples stored for many years and for new project planning, including sample storage conditions.     

Screening of porous and cellular materials for covalent immobilisation of Agaricus bisporus tyrosinase

We conducted a systematic study of covalent immobilisation of Agaricus bisporus tyrosinase onto typical enzyme carriers. Acrylic beads, two commercial silica gels with different pore structures and mesoporous silica foam (MCFs) beads functionalised using different organosilanes showed that only aminated MCFs offer active preparations with immobilisation efficiencies greater than 100% and a similar ratio of diphenolase (L-DOPA) to monophenolase (L-tyrosine) activities as the free enzyme. The native enzyme was entirely inactivated during incubation at 55°C for 30 min, whereas the enzyme immobilised on acrylic carrier or MCF retained 46 and 35%, respectively, of the initial activity after similar treatment. Susceptibility of native and immobilised tyrosinase to suicide inactivation in the presence of L-tyrosine and L-DOPA was tested in repeated batch tests. However, none of the preparations obtained in the L-DOPA solution was operationally stable enough to be used for practical applications.     

Immobilisation of the Thermostable l -aminoacylase from Thermococcus litoralis to Generate a Reusable Industrial Biocatalyst

A thermostable archaeal l -aminoacylase from Thermococcus litoralis has been used in immobilisation trials to optimise its application in industrial biotransformation reactions. Immobilisation techniques used included direct adsorption and crosslinking of the enzyme onto solid supports, bioencapsulation, and covalent bonding onto a variety of activated matrices. The most successful immobilisation methods were covalent binding of the enzyme onto glyoxyl-Sepharose and Amberlite XAD7. These methods yielded an average of 15 and 80 mg of protein bound per gram of support (wet weight for glyoxyl-Sepharose), respectively, with nearly 80% activity recovery in both cases. Enzyme immobilised onto glyoxyl-agarose was stabilised 106-fold under aqueous conditions and 142-fold in 100% acetonitrile when activity was measured after 24 h at 90°C. A column bioreactor containing the recombinant l -aminoacylase immobilised onto Sepharose beads was constructed with the substrate, N -acetyl- dl -Trp, continuously flowing at 60°C for 10 days. No loss of activity was detected over five days, with 32% activity remaining after 40 days at 60°C. These results show the potential of the use of immobilised l -aminoacylase in biotransformation reactions for the production of fine chemicals.     

Stability improvement of immobilized lactoperoxidase using polyaniline polymer

Enzyme engineering via immobilization techniques is perfectly compatible against the other chemical or biological approximate to improve enzyme functions and stability. In this study lactoperoxidase was immobilized onto polyaniline polymer activated with glutaraldehyde as a bifunctional agent, to improve enzyme properties. Polyaniline polymer was used due its unique physical and chemical properties to immobilize lactoperoxidase (LPO). The optimum activity of immobilized LPO was observed at pH 6 and 55?°C, which has been increased about 10?°C for the immobilized enzyme. The immobilized enzyme maintained absolutely active for 60?days whereas the native enzyme lost 80?% of its initial activity within this period of time. Moreover, the immobilized enzyme can be reused for several times without loss of activity. The kinetic parameter studies showed slight differences between free and immobilized enzymes. The K_m and K_m.app were calculated to be 0.6 and 0.4; also V_max and V_max.app were 1.3 and 0.9 respectively.     

Adsorption and immobilisation of human insulin on graphene monoxide,silicon carbide and boron nitride nanosheets investigated by molecular dynamics simulation

The adsorption and immobilisation of human insulin onto the bio-compatible nanosheets including graphene monoxide, silicon carbide and boron nitride nanosheets were studied by molecular dynamics simulation at the temperature of 310 K. After equilibration, heating and 100 ns production molecular dynamic runs, it was found that the insulin was adsorbed and immobilised onto the considered surfaces in a native folded state. The structural parameters, including root-mean-square deviation and fluctuation, surface accessible solvent area, radius of gyration (R_g) and the distance between the centre of the mass of immobilised protein and the surface of the considered nanosheets, were measured, analysed and discussed. The energetics of the studied systems such as the interaction energy between protein and nanosheet was also measured and addressed. The discussions were centred on the structural and energetic parameters of the protein and nanosheets, including charge density, hydrophobicity, hydrophilicity and residue polarity. The results also showed that the active site of C-termini of chain B played an important role in the adsorption process and this could be helpful in the protection of insulin in its smart delivery and release applications.     

Comparison of differently modified Pseudomonascepacia lipases in enantioselective preparation of a chiral alcohol for agrochemical use

Three methods for enzyme modification/immobilization were compared to enhance the catalytic performance of a commercially available lipase, Lipase PS from Pseudomonascepacia, in highly enantioselective transesterification of an agrochemically useful sec-alcohol, (R,?S)-HMPC [=(R,?S)-4-hydroxy-3-methyl-2-(2′-propenyl)-2-cyclopenten-1-one], with vinyl acetate as both acyl donor and reaction medium. The stearic acid-coated lipase showed the highest catalytic activity, with a specific activity improved by 54 times over the native lipase. The microcrystal salt-supported lipase and celite-adsorbed lipase also displayed much better performance as compared with the native lipase. All the three modified lipase preparations showed a similar thermal stability to that of the native enzyme. The enantioselectivity (E-value) was also quite satisfactory in all the cases (E>100 at 30°C), though a trend of slight decline was also observed with the temperature increase in the range of 25–60°C. The optimum aqueous pH, from which the modified lipases were prepared, was 6.0–7.0. A low water activity (a_w) of ca. 0.1 was favorable for all the three modified lipases. The stearic acid-coated lipase displayed prominent advantages in catalyzing the transesterification reaction at a very high (R,?S)-HMPC concentration up to 1.0?M.     

Stability of niosomes with encapsulated vitamin D3 and ferrous sulfate generated using a novel supercritical carbon dioxide method

Niosomes were prepared using a novel supercritical carbon dioxide based method to simultaneously encapsulate ferrous sulfate and vitamin D₃ as hydrophilic and hydrophobic cargo, respectively. Vesicle particle size was determined to be bimodal with peak diameters of 1.44?±?0.16?μm and 7.21?±?0.64?μm, with the smaller peak comprising 98.8% of the total niosomal volume. Encapsulation efficiency of ferrous sulfate was 25.1?±?0.2% and encapsulation efficiency of vitamin D₃ was 95.9?±?1.47%. Physical stability of the produced niosomes was assessed throughout a storage period of 21 days. Niosomes showed good physical stability at 20?°C, but storage at 4?°C showed an initial burst release, indicating possible rupture of the niosomal membrane. The Korsmeyer–Peppas equation was used to model the release of ferrous sulfate over time at both storage temperatures.