Immobilization of Bacillus licheniformis 5A1 milk-clotting enzyme and characterization of its enzyme properties

Milk-clotting enzyme from Bacillus licheniformis 5A1 was immobilized on Amberlite IR-120 by ionic binding. Almost all the enzyme activity was retained on the support. The immobilized milk-clotting enzyme was repeatedly used to produce cheese in a batch reactor. The production of cheese was repeated 5 times with no loss of activity. The specific activity calculated on a bound-protein basis was slightly higher than that of free enzyme. The free and immobilized enzyme were highly tolerant to repeated freezing and thawing. The optimum temperature for milk-clotting activity was 70 °C with the free enzyme whereas, it was ranged from 70 to 80 °C with the immobilized milk-clotting enzyme. The activation energy (E _A) of the immobilized milk-clotting enzyme was lower than the free enzyme (E _A = 1.59 and 1.99 Kcal mol⁻¹ respectively). The immobilized milk-clotting enzyme exhibited great thermal stability. The milk-clotting optimum pH was 7.0 for both free and immobilized enzyme. The Michaelis constant K _m of the immobilized milk-clotting enzyme was slightly lower than the free enzyme.     

Silanized palygorskite for lipase immobilization

Lipase from Candida lipolytica has been immobilized on 3-aminopropyltriethoxysilane-modified palygorskite support. Scanning electron micrographs proved the covalently immobilization of C. lipolytica lipase on the palygorskite support through glutaraldehyde. Using an optimized immobilization protocol, a high activity of 3300 U/g immobilized lipase was obtained. Immobilized lipase retained activity over wider ranges of temperature and pH than those of the free enzyme. The optimum pH of the immobilized lipase was at pH 7.0–8.0, while the optimum pH of free lipase was at 7.0. The retained activity of the immobilized enzyme was improved both at lower and higher pH in comparison to the free enzyme. The immobilized enzyme retained more than 70% activity at 40 °C, while the free enzyme retained only 30% activity. The immobilization stabilized the enzyme with 81% retention of activity after 10 weeks at 30 °C whereas most of the free enzyme was inactive after a week. The immobilized enzyme retains high activity after eight cycles. The kinetic constants of the immobilized and free lipase were also determined. The K_m and V_max values of immobilized lipase were 0.0117 mg/ml and 4.51 μmol/(mg min), respectively.     

Thermo-sensitive amphiphilic block copolymer poly (styrene-<Emphasis Type="Italic">b</Emphasis>-N-isopropylacrylamide) with switchable catalytic activity immobilizing pectinase

Pectinase was immobilized onto thermo-sensitive amphiphilic block copolymers poly(styrene-b-Nisopropylacrylamide) PS-b-poly(N-isopropylacrylamide) (PNIPAM) by covalent attachment. Biochemical studies have found that the stability of the PS-b-PNIPAM support is not impeded by the bound proteins despite that up to 242.5 mg of enzyme is immobilized per gram of carrier particles. The immobilized enzyme retained nearly 65% of its initial activity over 30 days, and the optimum temperature and pH also increased to the range of 60 ∼ 70°C and 4.0 ∼ 6.0, respectively. The immobilized enzyme also exhibited great operational stability, and more than 60% residual activity was observed in the immobilized enzyme after 10 batch reactions. Moreover, the lower critical solution temperature of the PS-b-PNIPAM support could be switched on or off by a small change in solution temperature. Thus, the immobilized pectinase could be recovered and showed durable activity during the recycle process.     

Improvement in stability of an immobilized fungal laccase

Summary A laccase of the basidiomyceteTrametes versicolor was immobilized on porous glass beads that were activated with 3-aminopropyltriethoxysilane and glutaraldehyde. The support immobilized 100% of the enzyme, whereupon 90% of the original activity was retained. After immobilization, the enzyme was active in a wider pH and temperature range, and its heat stability and reuse were greatly improved compared to those of the free laccase. The immobilized enzyme was found reusable in treating different substrates, either recycled alone or in a sequential order.     

Cooperativity of covalent attachment and ion exchange on alcalase immobilization using glutaraldehyde chemistry: Enzyme stabilization and improved proteolytic activity

Alcalase was scarcely immobilized on monoaminoethyl-N-aminoethyl (MANAE)-agarose beads at different pH values (<20% at pH 7). The enzyme did not immobilize on MANAE-agarose activated with glutaraldehyde at high ionic strength, suggesting a low reactivity of the enzyme with the support functionalized in this manner. However, the immobilization is relatively rapid when using low ionic strength and glutaraldehyde activated support. Using these conditions, the enzyme was immobilized at pH 5, 7, and 9, and in all cases, the activity vs. Boc-Ala-ONp decreased to around 50%. However, the activity vs. casein greatly depends on the immobilization pH, while at pH 5 it is also 50%, at pH 7 it is around 200%, and at pH 9 it is around 140%. All immobilized enzymes were significantly stabilized compared to the free enzyme when inactivated at pH 5, 7, or 9. The highest stability was always observed when the enzyme was immobilized at pH 9, and the worst stability occurred when the enzyme was immobilized at pH 5, in agreement with the reactivity of the amino groups of the enzyme. Stabilization was lower for the three preparations when the inactivation was performed at pH 5. Thus, this is a practical example on how the cooperative effect of ion exchange and covalent immobilization may be used to immobilize an enzyme when only one independent cause of immobilization is unable to immobilize the enzyme, while adjusting the immobilization pH leads to very different properties of the final immobilized enzyme preparation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2768, 2019.     

Immobilization and characterization of a thermostable β-galactosidase from a thermophilic anaerobe on a porous ceramic support

A thermostable -galactosidase (EC 3.2.1.23) from a thermophilic anaerobe, strain NA10, was purified from the crude extract of the Escherichia coli transformant harboring the lacN gene. The purified enzyme was physically and covalently immobilized to a porous ceramic support, SM-10. Among the supports tested, the highest residual activity after 3 h incubation at 70° C was obtained when the enzyme was covalently immobilized to silanized SM-10 with 3-[2-(2-amino-ethylaminoethylamino)propyl]trimethoxysilane. The amount of the enzyme immobilized was about 60 mg/g of this support. The enzymatic properties were almost the same as those of the free enzyme. The half-life of this immobilized enzyme was estimated to be approximately 450 h at the pasteurization temperature (65° C).On leave from Aichi Institute of Technology, Yakusa-cho, Toyota 470-03, Japan Correspondence to: T. Saito     

Immobilization and stabilization of cephalosporin C acylase on aminated support by crosslinking with glutaraldehyde and further modifying with aminated macromolecules

In this work, cephalosporin C acylase (CA), a heterodimeric enzyme of industrial potential in direct hydrolysis of cephalosporin C (CPC) to 7‐aminocephalosporanic acid (7‐ACA), was covalently immobilized on the aminated support LX1000‐HA (HA) with two different protocols. The stability of CA adsorbed onto the HA support followed by crosslinking with glutaraldehyde (HA–CA–glut) was better than that of the CA covalently immobilized on the glutaraldehyde preactivated HA support (HA–glut–CA). The thermostabilization factors (compared with the free enzyme) of these two immobilized enzymes were 11.2‐fold and 2.2‐fold, respectively. In order to improve the stability of HA–CA–glut, a novel strategy based on postimmobilization modifying with aminated molecules was developed to take advantage of the glutaraldehyde moieties left on the enzyme and support. The macromolecules, such as polyethyleneimine (PEI) and chitosan, had larger effects than small molecules on the thermal stability of the immobilized enzyme perhaps due to crosslinking of the enzymes and support with each other. The quaternary structure of the CA could be much stabilized by this novel approach including physical adsorption on aminated support, glutaraldehyde treatment, and macromolecule modification. The HA–CA–glut–PEI20000 (the HA–CA–glut postmodified with PEI M_w = 20,000) had a thermostabilization factor of 20‐fold, and its substrate affinity (K_m = 14.3 mM) was better than that of HA–CA–glut (K_m = 33.4 mM). The half‐life of the immobilized enzymes HA–CA–glut–PEI20000 under the CPC‐catalyzing conditions could reach 28 cycles, a higher value than that of HA–CA–glut (21 cycles). © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:387–395, 2015     

A novel support for the immobilization of lipase and the effects of the details of its preparation on the hydrolysis of triacylglycerides

Summary The lipase from Candida cylindracea was immobilized by its adsorption on the internal surface of hydrophobic microporous poly(styrene-divinylbenzene) supports prepared by the concentrated emulsion polymerization method. The prepared supports have a surface area of the order of 200 m²/g. The immobilized enzyme catalyst is used for the hydrolysis of triacylglycerides. The effects of the amounts of surfactant and divinylbenzene used in the preparation of the hydrophobic support on the adsorption capacity for lipase and on the activity of the immobilized lipase have been investigated. The activity of the immobilized enzyme per enzyme molecule can be higher than that of the free lipase.     

Polyelectrolyte complex formation mediated immobilization of chitosan-invertase neoglycoconjugate on pectin-coated chitin

Saccharomyces cerevisiae invertase, chemically modified with chitosan, was immobilized on pectin-coated chitin support via polyelectrolyte complex formation. The yield of immobilized enzyme protein was determined as 85% and the immobilized biocatalyst retained 97% of the initial chitosan-invertase activity. The optimum temperature for invertase was increased by 10 °C and its thermostability was enhanced by about 10 °C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was 4-fold more resistant to thermal treatment at 65 °C than the native counterpart. The biocatalyst prepared retained 96 and 95% of the original catalytic activity after ten cycles of reuse and 74 h of continuous operational regime in a packed bed reactor, respectively.     

Immobilization of urease on gelatin — poly (HEMA) copolymer preparation and characterization

Urease was immobilized onto gelatin-poly (HEMA) copolymer by covalent linkage. Maximum amount of urease was immobilized onto the support at a pH of 8.5. The optimal pH of the immobilized urease was similar to that of free urease; the optimal temperature showed an increase of 10 °C over the free enzyme. The stability of the immobilized urease for a range of pH, temperature and shelf life was greater than the corresponding values for the free enzyme. The same result was obtained for k _m also.Grateful acknowledgement is made to CSIR, Govt. of India for the research associateship conferred on Dr. M. Chellapandian which helped the progress of this piece of research investigation.     

Characterization of a novel immobilized biocatalyst obtained by matrix-assisted refolding of recombinant polyhydroxyoctanoate depolymerase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2442 isolated from inclusion bodies

Purification and matrix-assisted refolding of recombinant His-tagged polyhydroxyalkanoate (PhaZ) depolymerase from Pseudomonas putida KT2442 was carried out. His-tagged enzyme was overproduced as inclusion bodies in recombinant E. coli M15 (pREP4, pPAZ3), which were denatured by 8 M urea, immobilized on Ni²⁺-nitrilotriacetate-agarose matrix, and refolded by gradual removal of the chaotropic agent. The refolded enzyme could not be eluted with 1 M imidazole buffer, leading to an immobilized biocatalyst where PhaZ depolymerase was homogeneously distributed in the agarose support as shown by confocal scanning microscopy. Polyhydroxyoctanoate could not be hydrolyzed by this novel immobilized biocatalyst, whereas the attached enzyme was active in the hydrolysis of p-nitrophenyl alkanoate esters, which differed in their alkyl chain length. Taking advantage of the observed esterase activity on p-nitrophenylacetate, functional characterization of immobilized PhaZ depolymerase was carried out. The immobilized enzyme was more stable than its soluble counterpart and showed optimal hydrolytic activity at 37°C and 50 mM phosphate buffer pH 8.0. Kinetic parameters were obtained with both p-nitrophenylacetate and p-nitrophenyloctanoate, which had not been described so far for the soluble enzyme, representing an attractive and alternative chromogenic assay for the study of this paradigmatic enzyme.     

Adsorption of peroxidase on Celite 545 directly from ammonium sulfate fractionated white radish (Raphanus sativus) proteins

This paper demonstrates the direct immobilization of peroxidase from ammonium sulfate fractionated white radish proteins on an inorganic support, Celite 545. The adsorbed peroxidase was crosslinked by using glutaraldehyde. The activity yield for white radish peroxidase was adsorbed on Celite 545 was 70% and this activity was decreased and remained 60% of the initial activity after crosslinking by glutaraldehyde. The pH and temperature-optima for both soluble and immobilized peroxidase was at pH 5.5 and 40°C. Immobilized peroxidase retained higher stability against heat and water-miscible organic solvents. In the presence of 5.0 mM mercuric chloride, immobilized white radish peroxidase retained 41% of its initial activity while the free enzyme lost 93% activity. Soluble enzyme lost 61% of its initial activity while immobilized peroxidase retained 86% of the original activity when exposed to 0.02 mM sodium azide for 1 h. The K_m values were 0.056 and 0.07 mM for free and immobilized enzyme, respectively. Immobilized white radish peroxidase exhibited lower V_max as compared to the soluble enzyme. Immobilized peroxidase preparation showed better storage stability as compared to its soluble counterpart.     

Immobilization–stabilization of the lipase from Thermomyces lanuginosus: Critical role of chemical amination

This paper describes the immobilization and stabilization of the lipase from Thermomyces lanuginosus (TLL) on glyoxyl agarose. Enzymes attach to this support only by the reaction between several aldehyde groups of the support and several Lys residues on the external surface of the enzyme molecules at pH 10. However, this standard immobilization procedure is unsuitable for TLL lipase due to the low stability of TLL at pH 10 and its low content on Lys groups that makes that the immobilization process was quite slow. The chemical amination of TLL, after reversible immobilization on hydrophobic supports, has been shown to be a simple and efficient way to improve the multipoint covalent attachment of this enzyme. The modification enriches the enzyme surface in primary amino groups with low pKb, thus allowing the immobilization of the enzyme at lower pH values. The aminated enzyme was rapidly immobilized at pH 9 and 10, with activities recovery of approximately 70%. The immobilization of the chemically modified enzyme improved its stability by 5-fold when compared to the non-modified enzyme during thermal inactivation and by hundreds of times when the enzyme was inactivated in the presence of organic solvents, being both glyoxyl preparations more stable than the enzyme immobilized on bromocyanogen.     

An efficient system for catalyzing ester synthesis using a lipase from a newly isolated Burkholderia cepacia strain

The synthesis of ethyl-oleate by the lipase from the newly isolated strain Burkholderia cepacia LTEB11 in three different systems has been studied – immobilization on a hydrophobic support (Accurel EP 100®), encapsulation in reverse micelles, and direct addition of powdered free enzyme to the reaction medium. The immobilized enzyme performed best, giving a 70% ester yield in 10 h, this yield being five-fold greater than that obtained for reversed micelles, and two and a half times greater than that obtained for direct addition. An increase in the amount of immobilized enzyme preparation added gave a 100% ester yield in 3 h. The immobilized preparation was quite stable, giving a 100% yield of ethyl-oleate during 11 repeated reactions, and 50% yield after 24 reactions. These results suggest that the lipase of our strain of B. cepacia LTEB11 immobilized on Accurel has good potential for application in biocatalysis in organic media.     

Stabilization of immobilized l-arabinose isomerase for the production of d-tagatose from d-galactose

The aim of this work was to develop a stable immobilized enzyme biocatalyst for the isomerization of d -galactose to d -tagatose at high temperature. l -Arabinose isomerase from the hyperthermophilic bacterium Thermotoga maritima (TMAI) was produced as a (His)₆-tagged protein, immobilized on a copper–chelate epoxy support and subjected to several postimmobilization treatments aimed at increasing its operational and structural stability. Treatment with glutaraldehyde and ethylenediamine resulted in a more than twofold increase in the operational stability and in all enzyme subunits linked, directly or indirectly, to the support via covalent bonds. A postimmobilization treatment of the immobilized derivatives with mercaptoethanol for the removal of any remaining copper ions, determined a further increase of the operational biocatalytic activity. Immobilized derivatives subjected to both treatments were used for the bioconversion of 18 g/L d -galactose to d -tagatose at 80°C in a packed bed reactor in three repeated cycles and showed a better operational stability compared with the literature data. This study shows that a postimmobilization stabilization treatment with glutaraldehyde and ethylenediamine can stabilize the multi-subunit structure of an enzyme immobilized on a metal-chelate epoxy support with an increase of its operational stability, results that are not easily achievable with the sole immobilization on epoxy or metal chelate-epoxy supports in the case of complex multimeric enzymes with geometric incongruence with the support.     

Performance of Aspergillus niger B 03 β-xylosidase immobilized on polyamide membrane support

The dynamics of β-xylosidase biosynthesis from Aspergillus niger B 03 was investigated in laboratory bioreactor. Maximum xylosidase activity 5.5 U/ml was achieved after 80 h fermentation at medium pH 4.0. The isolated β-xylosidase was immobilized on polyamide membrane support and the basic characteristics of the immobilized enzyme were determined. Maximum immobilization and activity yield obtained was 30.0 and 6.8%, respectively. A shift in temperature optimum and pH optimum was observed for immobilized β-xylosidase compared to the free enzyme. Immobilized enzyme exhibited maximum activity at 45 °C and pH 4.5 while its free counterpart at 70 °C and pH 3.5, respectively. Thermal stability at 40 and 50 °C and storage stability of immobilized β-xylosidase were investigated at pH 5.0. Kinetic parameters K_m, V_max and K_i were determined for both enzyme forms. Free and immobilized β-xylosidase were tested for xylose production from birchwood xylan. The substrate was preliminarily depolymerized with xylanase to xylooligosaccharides and the amount of xylose obtained after their hydrolysis with free and immobilized β-xylosidase was determined by HPLC analysis. Continuous enzyme hydrolysis of birchwood xylan was performed with xylanase and free or immobilized β-xylosidase. The maximum extent of hydrolysis was 25 and 30% with free and immobilized enzyme, respectively. Immobilized preparation was also examined for reusability in 20 consecutive cycles at 40 °C.     

Bioaffinity Based Oriented Immobilization of Stem Bromelain

Bromelain is a basic, 23.8 kDa thiol proteinase obtained from stem of the pineapple plant (Ananas comosus) and is unique in containing a single oligosaccharide chain attached to the polypeptide. This property allowed its affinity binding and favorable orientation on a Sepharose support pre-coupled with the lectin, concanavalin A (Con A). For comparison, bromelain was also immobilized by covalently coupling to the CNBr-activated Sepharose. The preparation obtained was more resistant to thermal inactivation as evident from the retention of over 50% activity after incubation at 60 for 100 min (as compared to 20% retained by the native enzyme and 30% retained by the covalently immobilized enzyme), exhibited a broader pH-activity profile with the enzyme retaining over 60% activity at pH 11 (as compared to over 25% retained by native and the enzyme immobilized covalently). The native, covalently-coupled and affinity-bound bromelains had apparent K _m values of 1.1, 2 and 0.54 mg/ml, respectively using casein as the substrate. The V _max values remained unaffected on immobilization.     

Poly(styrene-divinylbenzene) beads surface functionalized with di-block polymer grafting and multi-modal ligand attachment: performance of reversibly immobilized lipase in ester synthesis

Fibrous poly(styrene-b-glycidylmethacrylate) brushes were grafted on poly(styrene–divinylbenzene) (P(S–DVB)) beads using surface-initiated atom transfer radical polymerization. Tetraethyldiethylenetriamine (TEDETA) ligand was incorporated on P(GMA) block. The ligand attached beads were used for reversible immobilization of lipase. The influences of pH, ionic strength, and initial lipase concentration on the immobilization capacities of the beads have been investigated. Lipase adsorption capacity of the beads was about 78.1 mg/g beads at pH 6.0. The K _m value for immobilized lipase was about 2.1-fold higher than that of free enzyme. The thermal, and storage stability of the immobilized lipase also was increased compared to the native lipase. It was observed that the same support enzyme could be repeatedly used for immobilization of lipase after regeneration without significant loss in adsorption capacity or enzyme activity. A lipase from Mucor miehei immobilized on styrene–divinylbenzene copolymer was used to catalyze the direct esterification of butyl alcohol and butyric acid.     

Specific immobilization of laccase onp-benzoquinone-activated supports

Summary A specific immobilization of laccase (EC 1.10.3.2) onto a ready-to-usep-benzoquinone-activated agarose support is described. The single-step procedure leads to a laccase protein coupling of I8% and an enzyme activity immobilization yield of 27%, while the retained specific activity of the immobilized enzyme was 150% of the specific activity of the free laccase. This peculiar result is thought to be related to the fact that during the process of support activation byp-benzoquinone, a significant amount of the hydroquinone by-product of the activation process is coupled to the support. These coupled derivatives constitute substrate (hydroquinone) analogues for which laccase exhibits a high affinity. Therefore, simultaneous affinity retention on the hydroquinone groups and covalent coupling on the p-benzoquinone groups allow the binding of the enzyme in an advantageous conformation which can generate this increase specific activity by immobilization. The entire process can be considered as an affinity immobilization. The immobilized enzyme is much more stable to the inhibitory action of chloride and azide ions, with a recovery of 100% of the activity, than the free laccase, with a recovery of 67% and 32%, respectively, after removal of the inhibitors by dialysis. The stability was 95% after storage for 14 months at 4° C.Abbreviations HQ hydroquinone - p-BQ p-benzoquinone - U enzyme units Part of the work was presented at the Satellite FEBS 1989 Symposium onBiochemical and biophysical approaches to the study of copper proteins, Camerino, Italy.     

Study of lipase immobilization on zeolitic support and transesterification reaction in a solvent free-system

In order to understand the role of the acid–base, electrostatic and covalent interactions between enzyme and support, the catalytic behavior of the Rhizomucor miehei lipase (RML) immobilized on zeolite materials has been studied. The highest lipase activities were obtained when this enzyme, immobilized by adsorption, interacts through acid–base binding forces with the support surface, resulting in activation of the enzyme catalytic center. Due to the interest in biodiesel production by mild enzymatic transesterification, this heterogeneous biocatalyst has been used in transesterification of fatty acids contained in olive oil. The results show a high oleic acid conversion for several reaction cycles with a higher total biodiesel productivity compared to that using the free enzyme.