Improved activity and stability of Rhizopus oryzae lipase via immobilization for citronellol ester synthesis in supercritical carbon dioxide

In present work, Rhizopus oryzae lipase immobilized on a film prepared using blend of hydroxylpropyl methyl cellulose (HPMC) and polyvinyl alcohol (PVA) was investigated for synthesis of citronellol esters with supercritical carbon dioxide (Sc-CO₂) as a reaction medium. The transesterification reaction was optimized for various reaction parameters like effect of molar ratio, acyl donor, time, temperature, enzyme concentration, effect of pressure and co-solvent to achieve the maximum yield of desired product. The results obtained signify remarkable increment (about eightfold) in the yield of citronellol acetate (91%) as compared to that of free lipase (11%) in Sc-CO₂. The developed biocatalytic methodology provides a substantial advantage of low biocatalyst loading (1.5%, w/v), lower reaction temperature (45 °C) and lower pressure (8 MPa) as compared to previous reports. The immobilization method has significantly enhanced the operational stability of lipase for ester synthesis under Sc-CO₂ conditions. The developed methodology was successfully applied for synthesis of three different industrially important citronellol esters namely citronellol acetate (91%), citronellol butyrate (98%), citronellol laurate (99%) with excellent yields using vinyl esters as acyl donor under Sc-CO₂ conditions. In addition, the immobilized biocatalyst was effectively recycled for three consecutive recycles.     

Novel minor lipase from Rhizopus chinensis during solid-state fermentation: Biochemical characterization and its esterification potential for ester synthesis

Rhizopus chinensis produces two lipases that catalyze ester synthesis when cultured under solid-state fermentation. The Lip2 was purified to homogeneity by ammonium sulphate precipitation, hydrophobic interaction chromatography and gel filtration chromatography. It has an apparent molecular weight of 33 kDa estimated from SDS–PAGE and 32 kDa calculated from analytical gel permeation, with synthetic activity and purification fold of 96.8 U/mg and 138.3, respectively. Maximum hydrolytic activity was obtained at pH 8.0–8.5 and 40 °C using pNPP as substrate. Slight activation of the enzyme was observed when Mn²⁺ is present. The enzyme was most active on p-nitrophenyl laurate (C12). The purified lipase exhibited maximum synthetic activity at pH memory of 6.0 and 30 ^oC. Most of ethyl esters synthesized by lyophilized enzyme achieved good yields (>90%), and caprylic acid served as the best acyl donor. The enzyme presented a particular affinity for ethanol, n-propanol and n-hexanol, with conversion of 92%, 93% and 92%, respectively, after 20 h incubation.     

High activity and selectivity immobilized lipase on Fe3O4 nanoparticles for banana flavour synthesis

Lipase (E.C.3.1.1.3) from Thermomyces lanuginosus (TL) was directly bonded, through multiple physical interactions, on citric acid functionalized monodispersed Fe₃O₄ nanoparticles (NPs) in presence of a small amount of hydrophobic functionalities. A very promising scalable synthetic approach ensuring high control and reproducibility of the results, and an easy and green immobilization procedure was chosen for NPs synthesis and lipase anchoring. The size and structure of magnetic nanoparticles were characterized by transmission electron microscopy (TEM) and X-ray diffraction (XRD). The samples at different degree of functionalization were analysed through thermogravimetric measurements. Lipase immobilization was further confirmed by enzymatic assay and Fourier transform infrared (FT-IR) spectra. Immobilized lipase showed a very high activity recovery up to 144% at pH = 7 and 323% at pH = 7.5 (activity of the immobilized enzyme compared to that of its free form). The enzyme, anchored to the Fe₃O₄ nanoparticles, to be easy recovered and reused, resulted more stable than the native counterpart and useful to produce banana flavour. The immobilized lipase results less sensitive to the temperature and pH, with the optimum temperature higher of 5 °C and optimum pH up shifted to 7.5 (free lipase optimum pH = 7.0). After 120 days, free and immobilized lipases retained 64% and 51% of their initial activity, respectively. Ester yield at 40 °C for immobilized lipase reached 88% and 100% selectivity.     

Eco-friendly methodology for efficient synthesis and scale-up of 2-ethylhexyl-p-methoxycinnamate using Rhizopus oryzae lipase and its biological evaluation

Lipase-mediated synthesis of phenolic acid esters is a green and economical alternative to current chemical methods. Octyl methoxycinnamate, an important UVB-absorbing compound, was synthesized by the esterification of p-methoxycinnamic acid with 2-ethyl hexanol using Rhizopus oryzae lipase. A molar ratio of 1:2 of p-methoxycinnamic acid and 2-ethyl hexanol was found to give an optimum yield using cyclo-octane (50 ml) as reaction solvent, at a temperature of 45 °C, and 750 U of lipase, resulting in a yield of 91.3 % in 96 h. This reaction was successfully scaled up to 400-ml reaction size where 88.6 %bioconversion was achieved. The synthesized compound was found to have superior antioxidant activity as compared to ascorbic acid. The synthesized compound also exhibited good antimicrobial activity against Escherichia coli, Klebsiella pneumonia, Salmonella typhi, Staphylococcus aures, Candida albicans (yeast), Aspergillus niger, Alternaria solani, and Fussarium oxysporum by well diffusion method in terms of zone of inhibitions (in mm).     

Immobilization and stability of a Rhizopus oryzae lipase expressed in Pichia pastoris: comparison between native and recombinant variants

The stability of a soluble extract containing a recombinant lipase from Rhizopus oryzae (Cursive) lipase (rROL) produced by Pichia pastoris (Cursive), as well as that for the commercial extract containing the lipase produced by the native organism (nROL), was investigated. The results showed higher residual activity values of the commercial protein compared with the recombinant one. Moreover, two different kinds of support, the polypropylene powder EP100 and Eupergit®C, were tested to immobilize the enzymes. The residual activity of the immobilizated derivatives was also tested to determine whether their stability was enhanced. The results showed a slight improvement in rROL using both supports but a decrease in nROL using Eupergit®C. The study of the residual activity of soluble and immobilized enzymes was performed by means of a central composite rotatable experiment design. In addition, EP100 adsorption isotherms were determined. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Immobilization of lipase B from Candida antarctica on porous styrene-divinylbenzene beads improves butyl acetate synthesis

A new biocatalyst of lipase B from Candida antarctica (MCI-CALB) immobilized on styrene-divinylbenzene beads (MCI GEL CHP20P) was compared with the commercial Novozym 435 (immobilized lipase) in terms of their performances as biocatalysts for the esterification of acetic acid and n-butanol. The effects of experimental conditions on reaction rates differed for each biocatalyst, showing different optimal values for water content, temperature, and substrate molar ratio. MCI-CALB could be used at higher acid concentrations, up to 0.5 M, while Novozym 435 became inactivated at these acid concentrations. Although Novozym 435 exhibited 30% higher initial activity than MCI-CALB for the butyl acetate synthesis, the reaction course was much more linear using the new preparation, meaning that the MCI-CALB allows for higher productivities per cycle. Both preparations produced around 90% of yield conversions after only 2 h of reaction, using 10% (mass fraction) of enzyme. However, the main advantage of the new biocatalyst was the superior performance during reuse. While Novozym 435 was fully inactivated after only two batches, MCI-CALB could be reused for six consecutive cycles without any washings and keeping around 70% of its initial activity. It is proposed that this effect is due to the higher hydrophobicity of the new support, which does not retain water or acid in the enzyme environment. MCI-CALB has shown to be a very promising biocatalyst for the esterification of small-molecule acids and alcohols.     

Purification and properties of Amycolatopsis mediterranei DSM 43304 lipase and its potential in flavour ester synthesis

An extracellular thermostable lipase from Amycolatopsis mediterranei DSM 43304 has been purified to homogeneity using ammonium sulphate precipitation followed by anion exchange chromatography and hydrophobic interaction chromatography. This protocol resulted in a 398-fold purification with 36% final recovery. The purified A. mediterranei DSM 43304 lipase (AML) has an apparent molecular mass of 33 kDa. The N-terminal sequence, AANPYERGPDPTTASIEATR, showed highest similarity to a lipase from Streptomyces exfoliatus. The values of K(m)(app) and V(max)(app) for p-nitrophenyl palmitate (p-NPP) at the optimal temperature (60°C) and pH (8.0) were 0.099±0.010 mM and 2.53±0.06 mmol/min mg, respectively. The purified AML displayed significant activity towards a range of short and long chain triglyceride substrates and p-nitrophenyl esters. Hydrolysis of glycerol ester bonds occurred non-specifically. The purified AML displayed significant stability in the presence of organic solvents (40%, v/v) and catalyzed the synthesis of the flavour ester isoamyl acetate in free and immobilized states.     

Immobilization of Candida rugosa lipase on glass beads for enantioselective hydrolysis of racemic Naproxen methyl ester

Candida rugosa lipase (CRL) was immobilized on glutaraldehyde-activated aminopropyl glass beads by using covalent binding method or sol-gel encapsulation procedure and improved considerably by fluoride-catalyzed hydrolysis of mixtures of RSi(OCH₃)₃ and Si(OCH₃)₄. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e. the hydrolysis of p-nitrophenylpalmitate (p-NPP). It has been observed that the percent activity yield of the encapsulated lipase was 166.9, which is 5.5 times higher than that of the covalently immobilized lipase. The enantioselective hydrolysis of racemic Naproxen methyl ester by immobilized lipase was studied in aqueous buffer solution/isooctane reaction system and it was noticed that particularly, the glass beads based encapsulated lipases had higher conversion and enantioselectivity compared to covalently immobilized lipase. In short, the study confirms an excellent enantioselectivity (E > 400) for the encapsulated lipase with an ee value of 98% for S-Naproxen.     

Impact of NH4+ nitrogen source on the production of Rhizopus oryzae lipase in Pichia pastoris

BackgroundPichia pastoris is a highly successful system for heterologous expression. During the induction stage, the ammonium ion released into the fermentation broth has a deep impact on cell growth and protein expression. The impact of NH₄⁺ concentration on the expression of the Rhizopus oryzae lipase proAROL in P. pastoris was investigated.ResultsThe lipase activity under the optimum NH₄⁺ concentration of 440 mmol/L reached 12,019 U/mL. Increased concentrations of NH₄⁺ in the broth prevented the protease production, resulting in higher specific lipase activity in the supernatant. Furthermore, analysis of carbon metabolism and energy regeneration pattern revealed that under the definite NH₄⁺ concentrations more carbon source (methanol) was consumed with surged AOX activity and then the higher energy and amino acid precursors demand for recombinant protein synthesis is compensated for by the TCA cycle.ConclusionsIn this study, the R. oryzae lipase activity reaches the highest level ever reported under optimized NH₄⁺ concentration and the analysis of the carbon metabolism provides useful information for future optimization of protein production by P. pastoris in a molecular level.     

Evaluation of Fe(III)EDTA and Fe(II)EDTA-NO reduction in a NO x scrubber solution by magnetic Fe3O4-chitosan microspheres immobilized microorganisms

A two-stage bioreduction system containing magnetic-microsphere-immobilized denitrifying bacteria and iron-reducing bacteria was developed for the regeneration of scrubbing solutions for NO _x removal. In this process, a higher bioreduction rate and a better tolerance of inhibition of bacteria were achieved with immobilized bacteria than with free bacteria. This work focused on evaluation of the effects of the main components in the scrubbing solution on Fe(III)EDTA (EDTA: ethylenediaminetetraacetate) and Fe(II)EDTA-NO reduction, with an emphasis on mass transfer and the kinetic model of Fe(III)EDTA and Fe(II)EDTA-NO reduction by immobilized bacteria. It was found that Fe(II)EDTA-NO had a strong inhibiting effect, but Fe(II)EDTA had no effect, on Fe(III)EDTA reduction. Fe(II)EDTA accelerated Fe(II)EDTA-NO reduction, whereas Fe(III)EDTA had no effect. This showed that the use of the two stages of regeneration was necessary. Moreover, the effect of internal diffusion on Fe(III)EDTA and Fe(II)EDTANO reduction could be neglected, and the rate-limiting step was the bioreduction process. The reduction of Fe(III)EDTA and Fe(II)EDTA-NO using immobilized bacteria was described by a first-order kinetic model. Bioreduction can therefore be enhanced by increasing the cell density in the magnetic chitosan microspheres.     

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

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

     

共表达伴侣蛋白对重组毕赤酵母产米根霉(Rhizopus oryzae)脂肪酶发酵过程的影响

[目的]通过共表达伴侣蛋白Erolp和PDI获得米根霉(Rhizopus oryzae)脂肪酶在毕赤酵母中的高效表达.[方法]利用毕赤酵母基因重组菌高密度发酵的方法,在7L发酵罐水平上分析共表达伴侣蛋白菌株(BH128)与非共表达伴侣蛋白菌株(H238)对脂肪酶表达的差异.[结果]在相同条件下,BH128发酵产酶能力高于H238,最高酶活可达到2 338.7U/mL,最大比生长速率达到0.02 h-1,最大产物比形成速率达到944.5 U/(gDCW·h),最大底物比消耗速率也能达到0.15 gmethanol/(gDCW·h),分别是H238的1.7、0.5、4.1和1.3倍,且发酵周期缩短了20h.[结论]毕赤酵母基因重组菌BH128通过共表达伴侣蛋白Ero1p和PDI,提高了米根霉脂肪酶的产量,而且缩短了发酵周期,为工业化生产奠定了基础.     

Immobilization of Pseudomonas delafieldii with magnetic polyvinyl alcohol beads and its application in biodesulfurization

Pseudomonas delafieldii was immobilized in magnetic polyvinyl alcohol (PVA) beads using a hydrophilic magnetic fluid, which was prepared by a co-precipitation method. The beads had distinct super-paramagnetic properties and were compared with immobilized cells in non-magnetic PVA beads. Their desulfurizing activity was increased slightly from 8.7 to 9 mmol sulfur kg(-1) (dry cell) h(-1). The main advantages was that the magnetic immobilized cells maintain a high desulfurization activity and remain in good shape after 7 times of repeated use, while the non-magnetic immobilized cells could only be used for 5 times. Furthermore, the magnetic immobilized cells could be easily collected or separated magnetically from the biodesulfurization reactor.     

Immobilization of proteases to porous chitosan beads and their catalysis for ester and peptide synthesis in organic solvents.

alpha-Chymotrypsin (CT), subtilisin BPN' (STB), and subtilisin Carlsberg (STC) were immobilized by adsorption to porous chitosan beads (Chitopearl, CP). The immobilized enzymes showed higher catalytic activities than free enzymes for amino acid esterification in many hydrophilic organic solvents except for methanol and DMF. In ethanol, the initial rate of the esterification increased with water content, whereas in ethyl acetate, the maximum rate was obtained at 2%-3% water. CP-immobilized CT also catalysed transesterification of Ac-Tyr-OMe in ethanol and peptide synthesis in acetonitrile from Ac-Tyr-OH or its ethyl ester and amino acid amides. The immobilized enzymes are highly stable in organic solutions, and can easily be separated from the reaction solutions. Repeated esterifications of Ac-Tyr-OH in acetonitrile by a CP-immobilized CT gave almost constant yields of the ester for more than 3 weeks.     

Immobilization of lipase from Pseudomonas fluorescens on glyoxyl-octyl-agarose beads: Improved stability and reusability

The lipase from Pseudomonas fluorescens (PFL) has been immobilized on glyoxyl-octyl agarose and compared to the enzyme immobilized on octyl-agarose. Thus, PFL was immobilized at pH 7 on glyoxyl-octyl support via lipase interfacial activation and later incubated at pH 10.5 for 20 h before reduction to get some enzyme-support covalent bonds. This permitted for 70% of the enzyme molecules to become covalently attached to the support. This biocatalyst was slightly more stable than the octyl-PFL at pH 5, 7 and 9, or in the presence of some organic solvents (stabilization factor no higher than 2). The presence of phosphate anions produced enzyme destabilization, partially prevented by the immobilization on glyoxyl-octyl (stabilization factor became 4). In contrast, the presence of calcium cations promoted a great PFLstabilization, higher in the case of the glyoxyl-octyl preparation (that remained 100% active when the octyl-PFL preparations had lost 20% of the activity). However, it is in the operational stability where the new biocatalyst showed the advantages: in the hydrolysis of 1 M triacetin in 60% 1.4 dioxane, the octyl biocatalyst released >60% of the enzyme in the first cycle, while the covalently attached enzyme retained its full activity after 5 reaction cycles.     

帆布固定化脂肪酶及其性能的研究

研究了用高碘酸钠氧化帆布纤维,使其纤维衍生化成为醛基,与脂肪酶交联进行固定化的过程。通过醛基被交联程度来评价交联过程的优劣。首先对纤维的氧化过程进行了简单优化,进而通过反复交联法与酶蛋白交联。以大豆油和橄榄油水解作为固定化酶的性能评价指标。实验结果表明,通过采用反复交联的方法,可提高载体表面酶蛋白质量分数30％左右。酶活力平均达到4U／cm^2,其对温度、pH的耐受性相比游离酶均有不同程度提高。同时利用油脂在固定化酶过程对酶进行保护,使其对温度、pH等的耐受性进一步增强。在维持较高水解率条件下,可在温和条件下连续反应7批,反应半衰期达140h以上。     

Enhanced reactivity of Rhizopus oryzae lipase displayed on yeast cell surfaces in organic solvents: potential as a whole-cell biocatalyst in organic solvents

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with alpha-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 x 10(4)-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 x 10(4)-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H2O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.     

Immobilization of lipase on chitin and its use in nonconventional biocatalysis

Chitin was functionalized with hexamethylenediamine followed by glutaraldehyde activation, and its capacity to bind Candida rugosa lipase was investigated. The loading of 250 units g(-1) support showed to be effective, resulting in a uniform enzyme fixation with high catalytic activity. Both free and immobilized lipases were characterized by determining the activity profile as a function of pH, temperature, and thermal stability. For the immobilized lipase, the influence of the reaction temperature and substrate polarity in nonconventional biocatalysis was also analyzed. Production of butyl esters was found to be dependent on the substrate partition coefficient, which accounts the greatest value for the system butanol and butyric acid. The highest enzyme activity was found for the system butanol and caprylic acid at a reaction temperature of 40 degrees C. Under such conditions, the operational stability tests indicated that a small enzyme deactivation occurs after 12 batches, revealing a biocatalyst half-life of 426.7 h.     

3D in silico study of magnetic fluid hyperthermia of breast tumor using Fe3O4 magnetic nanoparticles

Modeling and simulation of the temperature distribution, the mass concentration, and the heat transfer in the breast tissue are hot issues in magnetic fluid hyperthermia treatment of cancer. The breast tissue can be visualized as a porous matrix with saturated blood. In this paper, 3D in silico study of breast cancer hyperthermia using magnetic nanoparticles (MNPs) is conducted. The 3D FEM models are incorporated to investigate the infusion and backflow of nanofluid in the breast tumor, the diffusion of nanofluid, temperature distribution during the treatment, and prediction of the fraction of tumor necrosis while dealing with the thermal therapy. All the hyperthermia procedures are simulated and analyzed on COMSOL Multiphysics. The sensitivity of frequency and amplitude of the applied magnetic field (AMF) is investigated on the heating effect of the tumor. The mesh dependent solution of Penne's bioheat model is also analyzed. The simulated results demonstrate successful breast cancer treatment using MNPs with minimum side effects. Validation of current simulations results with experimental studies existing in literature advocates the success of our therapy. The increase in the amplitude and frequency of the AMF increases of the temperature in the tumor. The variation of mesh from coarser to finer increased the temperature through small fractions. We have also simulated the magnetic induction problem where the magnetic field is generated by current-carrying coil conductors induce heat in nearby breast tumors due to excitation of MNPs by magnetic flux. This research will aid treatment protocols and real-time clinical breast cancer treatments.