Alternative synthesis of poly(hydroxymethylsiloxane) for lipase immobilization and use of the adsorbates as esterification biocatalysts

A medium molar mass poly(hydrogenomethyl- siloxane), Me₃Si(O-SiHMe)_nOSiMe₃, (PHMS), has been used for preparing poly(hydroxymethylsiloxane) supports (PHOMS) for lipase immobilization. The procedure involved the conversion of PHMS to the corresponding poly(alkoxymethylsiloxanes). Me₃Si(OSi(OR) Me/_nOSiMe₃ (PHMS), their alkaline hydrolysis to form poly(siloxanolates) which were then converted to PHOMS by neutralization. The effect of different catalysts and alcohols (methanol, ethanol, 2-propanol) on the course of poly(alkoxymethylsiloxanes) formation is reported. PHOMS supports were characterized by BET and Hg porosimetry, and the degree of their crosslinking was determined by solid-phase NMR. Fluorescence spectroscopy was used to assess surface polarity and determine lipase loading. The efficiency of lipase adsorbed on these supports was tested in the esterification of stearic acid with propanol in hexane. It was found that the activity of the adsorbates is controlled by their porosity. The addition of an inert addend (e.g. hydrotalcite) in the step of alkaline hydrolysis of poly(alkoxymethylsiloxanes) increases the adsorption efficiency of the supports as compared to PHOMS. The potential application of the biocatalysts, lipase-PHOMS adsorbates, was extended by their encapsulation into a RTV silicone rubber containing Si-substituted poly(imide) as a swelling modifier.     

The role of physicochemical interactions and FLO genes expression in the immobilization of industrially important yeasts by adhesion

One of the industrially important qualities of yeast is their ability to provide the cell-cell and cell-support interactions. This feature of yeast is responsible for technologically significant phenomena such as flocculation (brewing) and yeast biofilm formation (immobilization to supports), whereas these phenomena are time, environment, and strain dependent. Therefore, the goal of this work was to verify the possibility to predict and subsequently select yeast strains capable to colonize solid supports by using physicochemical adhesion models. Three different industrial yeast strains (Saccharomyces cerevisiae) were tested for their adhesion onto spent grain particles in the continuous gas-lift reactor. The cell adhesion energies were calculated, based on physicochemical characteristics of surfaces involved, according to three adhesion models (DLVO theory, thermodynamic approach, and extended DLVO theory). The role of physicochemical surface properties in the cell-cell and cell-support interactions was evaluated by comparing the computed predictions with experimental results. The best agreement between forecast and observation of the yeast adhesion to spent grains was achieved with the extended DLVO (XDLVO) theory, the most complex adhesion model applied in this study. Despite its relative comprehensiveness, the XDLVO theory does not take into account specific biochemical interactions. Consequently, additional understanding of the yeast adhesion mechanism was obtained by means of quantifying the expression of selected FLO genes. The presented approach provides tools to select the appropriately adhesive yeast strains and match them with solid supports of convenient surface properties in order to design immobilized biocatalysts exploitable in biotechnological processes.     

Separation and Immobilization of Lipase from Penicillium simplicissimum by Selective Adsorption on Hydrophobic Supports

Lipases are an enzyme class of a great importance as biocatalysts applied to organic chemistry. However, it is still necessary to search for new enzymes with special characteristics such as good stability towards high temperatures, organic solvents, and high stereoselectivity presence. The present work’s aim was to immobilize the lipases pool produced by Penicillium simplissicimum, a filamentous fungi strain isolated from Brazilian babassu cake residue. P. simplissicimum lipases were separated into three different fractions using selective adsorption method on different hydrophobic supports (butyl-, phenyl-, and octyl-agarose) at low ionic strength. After immobilization, it was observed that these fractions’ hyperactivation is in the range of 131% to 1133%. This phenomenon probably occurs due to enzyme open form stabilization when immobilized onto hydrophobic supports. Those fractions showed different thermal stability, specificity, and enantioselectivity towards some substrates. Enantiomeric ratio for the hydrolysis of (R,S) 2-O-butyryl-2-phenylacetic acid ranged from 1 to 7.9 for different immobilized P. simplissicimum lipase fractions. Asymmetry factor for diethyl 2-phenylmalonate hydrolysis ranged from 11.8 to 16.4 according to the immobilized P. simplissicimum lipase fractions. Those results showed that sequential adsorption methodology was an efficient strategy to obtain new biocatalysts with different enantioselectivity degrees, thermostability, and specificity prepared with a crude extract produced by a simple and low-cost technology.     

Production and Digestibility Studies of β-Galactosyl Xylitol Derivatives Using Heterogeneous Catalysts of LacA β-Galactosidase from Lactobacillus Plantarum WCFS1

The synthesis of β-galactosyl xylitol derivatives using immobilized LacA β-galactosidase from Lactobacillus plantarum WCFS1 is presented. These compounds have the potential to replace traditional sugars by their properties as sweetener and taking the advantages of a low digestibility. The enzyme was immobilized on different supports, obtaining immobilized preparations with different activity and stability. The immobilization on agarose-IDA-Zn-CHO in the presence of galactose allowed for the conserving of 78% of the offered activity. This preparation was 3.8 times more stable than soluble. Since the enzyme has polyhistidine tags, this support allowed the immobilization, purification and stabilization in one step. The immobilized preparation was used in synthesis obtaining two main products and a total of around 68 g/L of β-galactosyl xylitol derivatives and improving the synthesis/hydrolysis ratio by around 30% compared to that of the soluble enzyme. The catalyst was recycled 10 times, preserving an activity higher than 50%. The in vitro intestinal digestibility of the main β-galactosyl xylitol derivatives was lower than that of lactose, being around 6 and 15% for the galacto-xylitol derivatives compared to 55% of lactose after 120 min of digestion. The optimal amount immobilized constitutes a very useful tool to synthetize β-galactosyl xylitol derivatives since it can be used as a catalyst with high yield and being recycled for at least 10 more cycles.     

Enzyme immobilization strategies for the design of robust and efficient biocatalysts

Different strategies for the preparation of efficient and robust immobilized biocatalysts are here reviewed. Different physico-chemical approaches are discussed.i.- The stabilization of enzyme by any kind of immobilization on pre-existing porous supports.ii.- The stabilization of enzymes by multipoint covalent attachment on support surfaces.iii.- Additional stabilization of immobilized-stabilized enzyme by physical or chemical modification with polymers.These three strategies can be easily developed when enzymes are immobilized in pre-existing porous supports. In addition to that, these immobilized-stabilized derivatives are optimal to develop enzyme reaction engineering and reactor engineering. Stabilizations ranging between 1000 and 100,000 folds regarding diluted soluble enzymes are here reported.     

Immobilization of Urease from Pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis>) on Agar Tablets and Its Application in Urea Assay

The pigeonpea urease was immobilized on agar, a common gelling substance. The tablet strips were used as moulds to cast agar tablets of uniform shape and size. The time and temperature of solidification of agar was 6 min and 44 degrees C, respectively. The 5 % agar (w/v) and 0.019 mg protein/agar tablet yielded an optimum immobilization of 51.7%. The optimum pH was shifted through 0.2 U (from 7.3 to 7.5) towards basic side upon immobilization. The optimum temperature of soluble and immobilized urease was 30 degrees C and 60 degrees C, respectively, showing the improvement in thermal stability of urease. There was an increase in K m from 3.23 to 5.07 mM after immobilization. The half-lives of soluble and immobilized urease were 21 and 53 days, respectively, at pH 7.3 and 4 degrees C. The urea was estimated in different blood samples with the help of immobilized urease and the results were consistent with those from clinical pathology laboratory through an autoanalyzer (Zydus Co., Rome, Italy).     

Immobilization of Candida antarctica Lipase B by Adsorption to Green Coconut Fiber

An agroindustrial residue, green coconut fiber, was evaluated as support for immobilization of Candida antarctica type B (CALB) lipase by physical adsorption. The influence of several parameters, such as contact time, amount of enzyme offered to immobilization, and pH of lipase solution was analyzed to select a suitable immobilization protocol. Kinetic constants of soluble and immobilized lipases were assayed. Thermal and operational stability of the immobilized enzyme, obtained after 2 h of contact between coconut fiber and enzyme solution, containing 40 U/ml in 25 mM sodium phosphate buffer pH 7, were determined. CALB immobilization by adsorption on coconut fiber promoted an increase in thermal stability at 50 and 60 °C, as half-lives (t _1/2) of the immobilized enzyme were, respectively, 2- and 92-fold higher than the ones for soluble enzyme. Furthermore, operational stabilities of methyl butyrate hydrolysis and butyl butyrate synthesis were evaluated. After the third cycle of methyl butyrate hydrolysis, it retained less than 50% of the initial activity, while Novozyme 435 retained more than 70% after the tenth cycle. However, in the synthesis of butyl butyrate, CALB immobilized on coconut fiber showed a good operational stability when compared to Novozyme 435, retaining 80% of its initial activity after the sixth cycle of reaction.     

Use of different adsorbents for sorption and Bacillus polymyxa protease immobilization

Proteases constitute one of the most important groups of industrial enzymes, accounting for at least 25% of the total enzyme sales, with two-thirds of the proteases produced commercially being of microbial origin (1). Immobilized enzymes are currently the subject of considerable interest because of their advantages over soluble enzymes or alternative, technologies, and the steadily increasing number of applications for immobilized enzymes. The general application of immobilized proteins and enzymes has played a central role in the expansion of biotechnology and synthesis-related industries. Proteases have been immobilized on natural and synthetic supports (2,3). In the present work, a protease from Bacillus polymyxa was partially purified with 80% ammonium sulfate precipitation followed by dialysis and chromatography using a diethylaminoethyl (DEAE)-cellulose ion exchange column. Immobilization was evaluated by using different adsorbents (chitin, chitosan, alginate, synthetic zeolite, and raw zeolite) and the storage stability and recycle of the immobilized protease determined. Immobilization yields were estimated to be 96% and 7.5%, by using alginate and chitosan, respectively, after, 24 h. The yield of the immobilization was 17% for alginate at 16h and the enzyme did not adsorb on the chitin, chitosan, synthetic zeolite, and raw zeolite.     

A novel lipase-based stationary phase in liquid chromatography

In this paper, a new chiral stationary phase (CSP) based on Candida antarctica lipase B (CALB) bounding to the surface of macroporous silica gel was developed and its stereoselectivity in enantioseparation and asymmetrical hydrolysis was evaluated. Three CALB-based HPLC columns with different amounts of enzyme immobilized were prepared by employing the immobilization method, namely “in batch”. In this technique two chromatographic supports epoxy silica and aminopropyl silica were considered. This novel CSP was proven capable of hydrolyzing chiral esters asymmetrically as bioreactor and separating several aromatic alcohols and diniconazole enantiomers.     

General characterization of noncommercial microbial lipases in hydrolytic and synthetic reactions

Fourteen noncommercial preparations of microbial lipases were investigated with respect to their catalytic activity for hydrolysis and synthesis of ester bonds. Six of the lipases were derived from microorganisms that have not previously been described as lipase producers, and another four were characterized for the first time. The synthetic reactions were carried out in two solvents of different polarities (n-heptane and acetone) using a series of fatty acids and primary and secondary alcohols with different chain lengths. Under the culture conditions employed, Pseudomonas cepacia produced more active enzyme than the other microorganisms. The lipase preparations produced using Ovadendron sulphureo-ochraceum, Monascus mucoroides, Monascus sp., Fusarium oxysporum, Penicillium chrysogenum, Rhodotorula araucariae, Pseudomonas cepacia, Streptomyces halstedii, and Streptomyces sp. were the most efficient catalysts for hydrolysis at lipid-water interfaces. Enzyme preparations from P. cepacia, Streptomyces sp., S. halstedii, and R. araucariae were good biocatalysts for esterification in the polar medium (acetone). When the lipase preparations with the greatest activity for hydrolytic, reactions were excluded, regression analysis of the data for the hydrolytic and synthetic activities of the remaining lipase preparations yielded high multiple correllation coefficients for these reactions in both n-heptane and acetone (R=0.82 and 0.91, respectively).     

New methodology for tosylation of hydroxylic supports as exemplified by the immobilization of micrococcal endonuclease on agarose

Improvement have been made in a simplified procedure we previously reported (J.Mol.Catal. (1986),38,227 for the activation of tosyl chloride of supports possessing primary hydroxyl groups. The method is simple, can be completed in less than 90 min, yields a broad range of activation degrees, and, since it involves no toxic reagents, may be used for preparing immobilized enzymes to be utilized in food manufacturing and processing. The immobilization ofStophylococcal Nuclease has been carried out by this method. The insolubilized derivatives are more active than the native enzyme in the hydrolysis of DNA. The thermal stability of nuclease derivatives is greater than that of the native enzyme. These derivatives remain active at 50°C, and the native enzyme, 39°C. The insolubilized nuclease is more stable against organic solvents such as, dimethylsulfoxide (DMSO) or tetrahydroduran (THF) than the native enzyme.     

Immobilized Lipase from Candida sp. 99–125 on Hydrophobic Silicate: Characterization and Applications

Lipase Candida sp. 99–125 has been proved to be quite effective in catalyzing organic synthesis reactions and is much cheaper than commercial lipases. Mesoporous silicates are attractive materials for the immobilization of enzymes due to their unique structures. The present research designed a hydrophobic silicate with uniform pore size suitable for the comfort of lipase Candida sp. 99–125 for improving its activity and stability. The resulting immobilized lipase (LP@PMO) by adsorption was employed to catalyze hydrolysis, esterification, and transesterification reactions, and the performances were compared with the lipase immobilized on hydrophilic silicate (LP@PMS) and native lipase. The LP@PMO showed as high activity as that of native lipase in hydrolysis and much increased catalytic activity and reusability in the reactions for biodiesel production. Besides, LP@PMO also possessed better organic stability. Such results demonstrate that immobilization of lipase onto hydrophobic supports is a promising strategy to fabricate highly active and stable biocatalysts for applications.     

Ethanol production from corn starch in a fluidized-bed bioreactor

The production of ethanol from industrial dry-milled corn starch was studied in a laboratory-scale fluidized-bed bioreactor using immobilized biocatalysts. Saccharification and fermentation were carried out either simultaneously or separately. Simultaneous saccharification and fermentation (SSF) experiments were performed using small, uniform κ-carrageenan beads (1.5–2.5 mm in diameter) of co-immobilized glucoamylase and Zymomonas mobilis. Dextrin feeds obtained by the hydrolysis of 15% drymilled corn starch were pumped through the bioreactor at residence times of 1.5–4h. Single-pass conversion of dextrins ranged from 54–89%, and ethanol concentrations of 23–36 g/L were obtained at volumetric productivities of 9–15 g/L-h. Very low levels of glucose were observed in the reactor, indicating that saccharification was the rate-limiting step. In separate hydrolysis and fermentation (SHF) experiments, dextrin feed solutions of 150–160 g/L were first pumped through an immobilized-glucoamylase packed column. At 55°C and a residence time of 1 h, greater than 95% conversion was obtained, giving product streams of 162–172 g glucose/L. These streams were then pumped through the fluidized-bed bioreactor containing immobilized Z. mobilis. At a residence time of 2 h, 94% conversion and ethanol concentration of 70 g/L were achieved, resulting in an overall process productivity of 23 g/L-h. Atresidence times of 1.5 and 1 h, conversions of 75 and 76%, ethanol concentrations of 49 and 47 g/L, and overall process productivities of 19 and 25 g/L-h, respectively, were achieved.     

Lipase biosensor for tributyrin and pesticide detection

Potentiometric biosensors based on Candida rugosa lipase was described for the detection of organophosphorus pesticide; methyl-parathion and tributyrin. Lipase was immobilized on the glass electrode by means of a gelatin membrane, which is then cross-linked with glutaraldehyde. The principle of the biosensor is based on the measurement of pH variation which was recorded in millivolts due to the enzymatic hydrolysis of tributyrin to butyric acid. For the inhibitor detection, biosensor responses were measured after pesticide treatment, which caused a drop in enzyme activity because of the irreversible inhibition. Reactivation conditions of the reused enzyme electrodes were also investigated by pyridine-2-aldoxime methiodide (2-PAM). The limit of detection for tributyrin was estimated as 93?µM for lipase sensor within the linear range of 65–455?µM.     

绿豆环氧水解酶催化对硝基苯乙烯氧化物的对映归一性水解

利用绿豆环氧水解酶催化(R,S)-对硝基苯乙烯氧化物水解,产物(R)-对硝基苯乙二醇的产率超过常规酶促拆分的极限产率(50%), 表明发生了对映体会聚. 采用绿豆粉剂和粗酶制剂考察了不同条件下的生物催化反应,结果表明,添加吐温-80 作为分散剂对反应有促进作用. 对该酶的固定化进行了初步研究,发现酶经硅藻土吸附固定化后稳定性显著增加. 在克级规模制备实验中,产物的总产率达73.3%;产物经一步重结晶,得到了光学纯度大于99%的(R)-对硝基苯乙二醇.     

ImmobilizedE. coli Alkaline Phosphatase

We have immobilized E.coli alkaline phosphatase (EC 3.1.3.1) by linking it covalently to sepharose 4B. This preparation has several advantages over the soluble enzyme. The immobilized enzyme is easily separable from other constituents in incubation mixtures. The immobilized enzyme can be reused repeatedly and is more stable than the soluble enzyme to heat treatment in the presence of 10 mM Mg²⁺. The insoluble and soluble phosphatases removed 75 and77%, respectively, of the inorganic phosphorus from casein. The immobilized enzyme inactivated two enzymes believed to be active in the phosphorylated state, acyl-CoA : cholesterol acyltransferase (ACAT) by 39% and NADPH-cytochrome P-450 reductase by 89%. The utility of immobilized alkaline phosphatase for studying the phosphorylation and dephosphorylation of soluble or membrane-bound enzymes and proteins is discussed.     

Determination de l'Acide l-Ascorbique a l'Aide d'Electrodes Bacteriennes,Tissulaires et Enzymatiques

Abstract

L-ascorbic acid membrane electrodes based upon the use of four classes of biocatalysts immobilized at an oxygen electrode are evaluated and compared in terms of electrode properties and operating requirements. Isolated ascorbate oxydase enzyme in soluble form and in covalent binding matrices, peel of cucumber and living bacterial cells of Enterobacter agglomerans strain, respectively, are employed as biocatalytic layers. The physico-chemical factors, life times and interferences are discussed in details. The low stability of the soluble enzyme sensor does not allow its analytical utilization, but the immobilized enzyme, bacterial and tissue electrodes can be used, even in multivitamin pharmaceutical formulations. The common linear range of those three biosensors are of 4.10^?6 M to 7.10^?4 M with a precision and a reproducibility of ± 3%.     

Affinity chromatographic purification of lentil lectin using immobilized yeast cells

A model system for evaluating macroporous supports containing immobilized whole cells in affinity Chromatographic applications is described. Whole cells were immobilized in a polyacrylamide network in a two-step polymerization process. The affinity system discussed consists of immobilized cells ofSaccharomyces cervisiae in the purification of lentil lectin fromLens culinaris.     

Kinetic Study of the Alcoholic Fermentation Process, in the Presence of Free and Immobilized Saccharomyces Cerevisiae Cells, at Different Initial Glucose Concentrations by Reversed Flow GC

Reversed flow gas chromatography (RFGC) was applied for the kinetic study of the alcoholic fermentation processes conducted with cells of the alcohol-resistant and psychrophilic Saccharomyces cerevisiae AXAZ-1 yeast strain, either free or immobilized on wheat, barley and corn grains as well as on potato pieces. Repeated alcoholic fermentations with must of varying initial glucose concentrations were performed in order to estimate the catalytic efficiency of the biocatalysts used in the present study. With the RFGC method, the distinction of the duration of alcoholic fermentation phases was achieved, which may be correlated to the phases of AXAZ-1 cells growth cycle. The rate constants of ethanol production for each phase of the alcoholic fermentations, conducted with free and immobilized cells, were also determined with the aid of RFGC, confirming the predominance of the immobilized against free cells in the fermentation process. Comparing the supports used for immobilization, wheat and barley grains seemed to be more efficient than corn grains and potato pieces, as they provided a higher number of immobilized cells and rate constant values.     

Immobilization of Xylanase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> Strain MK001 and its Application in Production of Xylo-oligosaccharides

Xylanase from Bacillus pumilus strain MK001 was immobilized on different matrices following varied immobilization methods. Entrapment using gelatin (GE) (40.0%), physical adsorption on chitin (CH) (35.0%), ionic binding with Q-sepharose (Q-S) (45.0%), and covalent binding with HP-20 beads (42.0%) showed the maximum xylanase immobilization efficiency. The optimum pH of immobilized xylanase shifted up to 1.0 unit (pH 7.0) as compared to free enzyme (pH 6.0). The immobilized xylanase exhibited higher pH stability (up to 28.0%) in the alkaline pH range (7.0–10.0) as compared to free enzyme. Optimum temperature of immobilized xylanase was observed to be 8 °C higher (68.0 °C) than free enzyme (60.0 °C). The free xylanase retained 50.0% activity, whereas xylanase immobilized on HP-20, Q-S, CH, and GE retained 68.0, 64.0, 58.0, and 57.0% residual activity, respectively, after 3 h of incubation at 80.0 °C. The immobilized xylanase registered marginal increase and decrease in K _m and V _max values, respectively, as compared to free enzyme. The immobilized xylanase retained up to 70.0% of its initial hydrolysis activity after seven enzyme reaction cycles. The immobilized xylanase was found to produce higher levels of high-quality xylo-oligosaccharides from birchwood xylan, indicating its potential in the nutraceutical industry.