Production of 2-O-α-glucopyranosyl l-ascorbic acid from ascorbic acid and β-cyclodextrin using immobilized cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase) isolated and purified from Paenibacillus sp. A11 was immobilized on various carriers by covalent linkage using bifunctional agent glutaraldehyde. Among tested carriers, alumina proved to be the best carrier for immobilization. The effects of several parameters on the activation of the support and on the immobilization of enzyme were optimized. The best preparation of immobilized CGTase retained 31.2% of its original activity. After immobilization, the enzymatic properties were investigated and compared with those of the free enzyme. The optimum pH of the immobilized CGTase was shifted from 6.0 to 7.0 whereas optimum temperature remained unaltered (60°C). Free and immobilized CGTase showed similar pH stability profile but the thermal stability of the immobilized CGTase was 20% higher. Kinetic data (K _M and V _max) for the free and immobilized enzymes were determined from the rate of β-CD formation and it was found that the immobilized form had higher K _M and lower V _max. The immobilized CGTase also exhibited higher stability when stored at both 4°C and 25°C for 2 months. The enzyme immobilized on alumina was further used in a batch production of 2-O-α-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin. The yield of AA-2G was 2.92% and the immobilized CGTase retained its activity up to 74.4% of the initial catalytic activity after being used for 3 cycles. The immobilized CGTase would have a promising application in the production of various transglycosylated compounds and in the production of cyclodextrin by the hydrolysis of starch.     

Production of Xylooligosaccharides by Immobilized His-tagged Recombinant Xylanase from Penicillium occitanis on Nickel-Chelate Eupergit C

Penicillium occitanis xylanase 2 expressed with a His-tag in Pichia pastoris, termed PoXyn2, was immobilized on nickel-chelate Eupergit C by covalent coupling reaction with a high immobilization yield up to 93.49 %. Characterization of the immobilized PoXyn2 was further evaluated. The optimum pH was not affected by immobilization, but the immobilized PoXyn2 exhibited more acidic and large optimum pH range (pH 2.0–4.0) than that of the free PoXyn2 (pH 3.0). The free PoXyn2 had an optimum temperature of 50 °C, whereas that of the immobilized enzyme was shifted to 65 °C. Immobilization increased both pH stability and thermostability when compared with the free enzyme. Time courses of the xylooligosaccharides (XOS) produced from corncob xylan indicated that the immobilized enzyme tends to use shorter xylan chains and to produce more xylobiose and xylotriose initially. At the end of 24-h reaction, XOS mixture contained a total of 21.3 and 34.2 % (w/w) of xylobiose and xylotriose with immobilized xylanase and free xylanase, respectively. The resulting XOS could be used as a special nutrient for lactic bacteria.     

Immobilization of single-strand specific nuclease (S1 nuclease) fromAspergillus oryzae

S1 nuclease fromAspergillus oryzae (EC 3.1.30.1) was coupled to gelatin-alginate composite matrix using the residual free aldehyde groups on the surface of glutaraldehyde crosslinked matrix. The immobilized enzyme retained approximately 10% activity of the soluble enzyme. When partially purified enzyme was bound to the matrix, the immobilized preparation did not show any detectable enzyme activity. However, the activity could be restored when the coupling was carried out in the presence of a coprotein or substrate. The optimum pH of the immobilized S1 nuclease shifted to 3.8 from 4.3 for the soluble enzyme. Also, optimum temperature increased to 65°C after immobilization. Bound S1 nuclease showed increased pH and temperature stabilities. Immobilization brought about a twofold decrease in the Michaelis-Menton constant (K _m).     

Physical and Covalent Immobilization of <Emphasis Type="Italic">Lipase</Emphasis> onto Amine Groups Bearing Thiol-Ene Photocured Coatings

In this study, amine groups containing thiol-ene photocurable coating material for lipase immobilization were prepared. Lipase (EC 3.1.1.3) from Candida rugosa was immobilized onto the photocured coatings by physical adsorption and glutaraldehyde-activated covalent bonding methods, respectively. The catalytic efficiency of the immobilized and free enzymes was determined for the hydrolysis of p-nitrophenyl palmitate and also for the synthesis of p-nitrophenyl linoleate. The storage stability and the reusability of the immobilized enzyme and the effect of temperature and pH on the catalytic activities were also investigated. The optimum pH for free lipase and physically immobilized lipase was determined as 7.0, while it was found as 7.5 for the covalent immobilization. After immobilization, the optimum temperature increased from 37 °C (free lipase) to 50–55 °C. In the end of 15 repeated cycles, covalently bounded enzyme retained 60 and 70 % of its initial activities for hydrolytic and synthetic assays, respectively. While the physically bounded enzyme retained only 56 % of its hydrolytic activity and 67 % of its synthetic activity in the same cycle period. In the case of hydrolysis V _max values slightly decreased after immobilization. For synthetic assay, the V _max value for the covalently immobilized lipase was found as same as free lipase while it decreased dramatically for the physically immobilized lipase. Physically immobilized enzyme was found to be superior over covalent bonding in terms of enzyme loading capacity and optimum temperature and exhibited comparable re-use values and storage stability. Thus, a fast, easy, and less laborious method for lipase immobilization was developed.     

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.     

Immobilization and Kinetics of Catalase on Calcium Carbonate Nanoparticles Attached Epoxy Support

A novel hybrid epoxy/nano CaCO₃ composite matrix for catalase immobilization was prepared by polymerizing epoxy resin in the presence of CaCO₃ nanoparticles. The hybrid support was characterized using scanning electron microscopy and Fourier transform infrared spectroscopy. Catalase was successfully immobilized onto epoxy/nano CaCO₃ support with a conjugation yield of 0.67?±?0.01 mg/cm² and 92.63?±?0.80 % retention of activity. Optimum pH and optimum temperature of free and immobilized catalases were found to be 7.0 and 35 °C. The value of K _m for H₂O₂ was higher for immobilized enzyme (31.42 mM) than native enzyme (27.73 mM). A decrease in V _max value from 1,500 to 421.10 μmol (min mg protein)^?1 was observed after immobilization. Thermal and storage stabilities of catalase improved immensely after immobilization. Immobilized enzyme retained three times than the activity of free enzyme when kept at 75 °C for 1 h and the half-life of enzyme increased five times when stored in phosphate buffer (0.01 M, pH 7.0) at 5 °C. The enzyme could be reused 30 times without any significant loss of its initial activity. Desorption of catalase from the hybrid support was minimum at pH 7.0.     

Inulin Hydrolysis by Immobilized Inulinase on Functionalized Magnetic Nanoparticles Using Soy Protein Isolate and Bovine Serum Albumin

Inulin hydrolysis was performed by inulinase from Aspergillus niger covalently immobilized on magnetite nanoparticles (Fe₃O₄) covered with soy protein isolate (Fe₃O₄/SPI) functionalized by bovine serum albumin (Fe₃O₄/SPI/BSA) nanoparticles as a new bio‐functional carrier. The specific activity and protein content of the immobilized enzyme were 25.99 U/mg and 3.52 mg/mL, respectively, with 80% enzyme loading. The immobilized inulinase showed maximum activity at 45 °C, which is 5 °C higher than the optimum temperature of the free enzyme. Also, the optimum pH of the immobilized enzyme shifted from 6 to 5.5, which is more acidic compared to that of the free enzyme. The K_m value of immobilized inulinase decreased to 2.03 mg/mL. Thermal stability increased considerably at 65 and 75 °C, and a 5.13‐fold rise was detected in the enzyme half‐life at 75 °C after immobilization. Moreover, 80% of initial activity of immobilized inulinase remained after 10 cycles of hydrolysis.     

Immobilization of a Recombinant Esterase from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> on Polypropylene Accurel MP1000

A recombinant esterase from Lactobacillus plantarum was immobilized on hydrophobic support polypropylene Accurel MP1000 by adsorption. Adsorption efficiency was 83%, and the immobilized protein was 12.4 mg/g of support. Esterase activity was determined using p-nitrophenyl butyrate as substrate, and highest activities were observed at 50 °C for immobilized enzyme and 30 °C for free enzyme extract. Concerning thermal stability, after enzyme incubation at 80 °C for 30 min, immobilized and free enzyme retained 91% and 56% of initial activity, respectively. Immobilized enzyme presented lower V _max and higher K _m than free enzyme. Protein was not released from the support, and esterase activity increased after 3 cycles of reuse.     

Highly Efficient Method Towards In Situ Immobilization of Invertase Using Cryogelation

A novel method was developed for the immobilization of Saccharomyces cerevisiae invertase within supermacroporous polyacrylamide cryogel and was used to produce invert sugar. First, the cross-linking of invertase with soluble polyglutaraldehyde (PGA) was carried out prior to immobilization in order to increase the bulkiness of invertase and thus preventing the leakage of the cross-linked enzyme after immobilization by entrapment. And then, in situ immobilization of PGA cross-linked invertase within cryogel synthesis was achieved by free radical polymerization in semi-frozen state. The method resulted in 100 % immobilization and 74 % activity yields. The immobilized invertase retained all the initial activity for 30 days and 30 batch reactions. Immobilization had no effect on optimum temperature and it was 60 °C for both free and immobilized enzyme. However, optimum pH was affected upon immobilization. Optimum pH values for free and immobilized enzyme were 4.5 and 5.0, respectively. The immobilized enzyme was more stable than the free enzyme at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined. The newly developed method is simple yet effective and could be used for the immobilization of some other enzymes and microorganisms.     

Highly Thermostable Xylanase of the Thermophilic Fungus Talaromyces thermophilus: Purification and Characterization

A thermostable xylanase from a newly isolated thermophilic fungus Talaromyces thermophilus was purified and characterized. The enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl cellulose anion exchange chromatography, P-100 gel filtration, and Mono Q chromatography with a 23-fold increase in specific activity and 17.5% recovery. The molecular weight of the xylanase was estimated to be 25kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and gel filtration. The enzyme was highly active over a wide range of pH from 4.0 to 10.0. The relative activities at pH5.0, 9.0, and 10.0 were about 80%, 85.0%, and 60% of that at pH7.5, respectively. The optimum temperature of the purified enzyme was 75°C. The enzyme showed high thermal stability at 50°C (7days) and the half-life of the xylanase at 100°C was 60min. The enzyme was free from cellulase activity. K _m and V _max values at 50°C of the purified enzyme for birchwood xylan were 22.51mg/ml and 1.235μmol min⁻¹ mg⁻¹, respectively. The enzyme was activated by Ag⁺, Co²⁺, and Cu²⁺; on the other hand, Hg²⁺, Ba²⁺, and Mn²⁺ inhibited the enzyme. The present study is among the first works to examine and describe a secreted, cellulase-free, and highly thermostable xylanase from the T. thermophilus fungus whose application as a pre-bleaching aid is of apparent importance for pulp and paper industries.     

Efficient Immobilization of Porcine Pancreatic α-Amylase on Amino-Functionalized Magnetite Nanoparticles: Characterization and Stability Evaluation of the Immobilized Enzyme

The potential of the modified magnetic nanoparticles for covalent immobilization of porcine pancreatic α-amylase has been investigated. The synthesis and immobilization processes were simple and fast. The co-precipitation method was used for synthesis of magnetic iron oxide (Fe₃O₄) nanoparticles (NPs) which were subsequently coated with silica through sol–gel reaction. The amino-functionalized NPs were prepared by treating silica-coated NPs with 3-aminopropyltriethoxysilane followed by covalent immobilization of α-amylase by glutaraldehyde. The optimum enzyme concentration and incubation time for immobilization reaction were 150 mg and 4 h, respectively. Upon this immobilization, the α-amylase retained more than 50 % of its initial specific activity. The optimum pH for maximal catalytic activity of the immobilized enzyme was 6.5 at 45 °C. The kinetic studies on the immobilized enzyme and its free counterpart revealed an acceptable change of K_m and V_max. The Km values were found as 4 and 2.5 mM for free and immobilized enzymes, respectively. The V_max values for the free and immobilized enzymes were calculated as 1.75 and 1.03 μmol mg^?1 min^?1, in order, when starch was used as the substrate. A quick separation of immobilized amylase from reaction mixture was achieved when a magnetically active support was applied. In comparison to the free enzyme, the immobilized enzyme was thermally stable and was reusable for 9 cycles while retaining 68 % of its initial activity.     

Immobilization of Urease on (HEMA/IA) Hydrogel Prepared by Gamma Radiation

In the present study, the copolymeric hydrogels based on 2-hydroxyethyl methacrylate (HEMA) and itaconic acid (IA) were synthesized by gamma radiation induced radical polymerization, in order to examine the potential use of these hydrogels in immobilization of Citrullus vulgaris urease. Gelation and Swelling properties of PHEMA and copolymeric P (HEMA/IA) hydrogels with different IA contents (96.5/3.5, 94.4/5.6 and 92.5/7.5 mol) were studied in a wide pH range. Initial studies of so-prepared hydrogels show interesting pH sensitivity in swelling and immobilization. C. vulgaris urease was immobilized on HEMA/IA (92.5/7.5) at 6 kGy with 41.3% retention of activity. The properties of free and immobilized urease were compared. Immobilized urease maintained a higher relative activity than free urease at both lower and higher pH levels, indicating that the immobilized urease was less sensitive to pH changes than the free urease. The K_m value of the immobilized urease was approximately 2 times higher than that of the free urease. Temperature stability was improved for immobilized enzyme. The free form exhibited a loss about 80% of activity upon incubation for 15 min at 80°C. The influence of various heavy metal ions at the concentration of l mM was improved after enzyme immobilization. The immobilization of C. vulgaris urease on HEMA/IA (92.5/7.5) at 6 kGy showed a residual activity of 47 % after 4 reuses.     

Immobilization of <Emphasis Type="Italic">Aspergillus niger</Emphasis> Xylanase on Chitosan Using Dialdehyde Starch as a Coupling Agent

Dialdehyde starch (DAS) was used as a novel coupling agent to prepare chitosan carrier to immobilize the xylanase from Aspergillus niger A-25. Compared with glutaraldehyde-cross-linked chitosan (CS-GA) and pure chitosan beads, the DAS-cross-linked chitosan (CS-DAS) beads exhibited the highest xylanase activity recovery. The DAS adding amount and cross-linking time in CS-DAS preparation process were optimized with respect to activity recovery to the values of 1.0 g (6.7% w/v concentration) and 16 h, respectively. The optimum temperature of both the CS-DAS- and CS-GA-immobilized xylanase was observed to be 5 °C higher than that of free enzyme (50 °C). The CS-DAS-immobilized xylanase had the highest thermal and storage stability as compared to the CS-GA-immobilized and free xylanase. The apparent K _m and V _max values of the CS-DAS-immobilized xylanase were estimated to be 1.29 mg/ml and 300.7 μmol/min/mg protein, respectively. The CS-DAS-immobilized xylanase could produce from birchwood xylan high-quality xylo-oligosaccharides, mainly composed of xylotriose, as free xylanase did. The proposed CS-DAS carrier was more advantageous over the CS-GA or pure chitosan carrier for xylanase immobilization application.     

Screening and Immobilization <Emphasis Type="Italic">Burkholderia</Emphasis> sp. GXU56 Lipase for Enantioselective Resolution of (<Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">S</Emphasis>)-Methyl Mandelate

Microorganisms producing lipase were isolated from soil and sewage samples and screened for enantioselective resolution of (R,S)-methyl mandelate to (R)-mandelic acid. A strain designated as GXU56 was obtained and identified as Burkholderia sp. Preparing immobilized GXU56 lipase by simple adsorption on octyl sepharose CL-4B, the optimum temperature was shifted from 40 °C (free lipase) to 50 °C (immobilized lipase), and the optimum pH was shifted from 8.0 (free lipase) to 7.2 (immobilized lipase). The immobilized enzyme displayed excellent stability in the pH range of 5.0–8.0, at the temperatures below 50 °C and in organic solvents compared with free enzyme. Enantioselectivity ratio for (R)-mandelic acid (E) was dramatically improved from 29.2 to more than 300 by applying immobilized lipase in the resolution of (R,S)-methyl mandelate. After five cycles of use of immobilized lipase, conversion and enantiomeric excess of (R)-mandelic acid were 34.5% and 98.5%, respectively, with enantioselectivity ratio for (R)-mandelic acid (E) of 230. Thus, octyl-sepharose-immobilized GXU56 lipase can be used as a bio-resolution reagent for producing (R)-mandelic acid.     

Characterization of bioconversion of fumarate to succinate by alginate immobilized Enterococcus faecalis RKY1

In this study, the immobilization characteristics of Enterococcus faecalis RKY1 for succinate production were examined. At first, three natural polymers—agar, κ-carrageenan, and sodium alginate—were tried as immobilizing matrices. Among these, sodium alginate was selected as the best gel for immobilization of E. faecalis RKY1. Efficient conditions for immobilization were established to be with a 2% (w/v) sodium alginate solution and 2-mm-diameter bead. The bioconversion characteristics of the immobilized cellsat various pH values and temperatures were examined and compared with those of free cells. The optimum pH and temperature of the immobilized cells were the same as for free cells, 7.0 and 38°C respectively, but the conversion ratio was higher by immobilization for all the other pH and temperature conditions tested. When the seed volume of the immobilized cells was adjusted to 10% (v/v), 30 g/L of fumarate was completely converted tosuccinate (0.973 g/g conversion ratio) after 12 h. In addition, the immobilized cells maintained a conversion ratio of >0.95 g/g during 4wk of storageat 4°C in a 2% (w/v) CaCl₂ solution. In repetitive bioconversion experiments, the activity of the immobilized cells decreased linearly according to the number of times of reuse.     

Design and Properties of an Immobilization Enzyme System for Inulin Conversion

A commercial inulinase could convert inulin into fructose, which was optimized to be entrapped in the calcium alginate-gelatin beads with the immobilization yield of 86% for free inulinase activities. The optimum pH values and temperatures were 4.5 and 40 °C for the free enzyme and 5.0–5.5 and 45–50 °C for the immobilized enzyme. The kinetic parameters of V _max and K _m were 5.24 μmol/min and 57.6 mg/mL for the free inulinase and 4.32 μmol/min and 65.8 mg/mL for the immobilized inulinase, respectively. The immobilized enzyme retained 80% of its initial activities at 45 °C for 4 days, which could exhibit better thermal stability. The reuse of immobilized inulinase throughout the continuous batch operations was explored, which had better reusability of the immobilized biocatalyst. At the same time, the stability of immobilized enzyme in the continuous packed-bed bioreactor was estimated, which showed the better results and had its potential scale-up fructose production for inulin conversion.     

Pigeonpea (Cajanus cajan L.) urease immobilized on glutaraldehyde-activated chitosan beads and its analytical applications

Urease from pigeonpea (Cajanus cajan L.) was covalently linked to crab shell chitosan beads using glutaraldehyde. The optimum immobilization (64% activity) was observed at 4°C, with a protein concentration of 0.24 mg/bead and 3% glutaraldehyde. The immobilized enzyme stored in 0.05 M Trisacetate buffer, pH 7.3, at 4°C had a t _1/2 of 110 d. There was practically no leaching of enzyme (<3%) from the immobilized beads in 30 d. The immobilized urease was used 10 times at an interval of 24 h between each use with 80% residual activity at the end of the period. The chitosan-immobilized urease showed a significantly higher Michaelis constant (8.3 mM) compared to that of the soluble urease (3.0 mM). Its apparent optimum pH also shifted from 7.3 to 8.5. Immobilized urease showed an optimal temperature of 77°C, compared with 47°C for the soluble urease. Time-dependent kinetics of the thermal denaturation of immobilized urease was studied and found to be monophasic in nature compared to biphasic in nature for soluble enzyme. This immobilized urease was used to analyze blood urea of some of the clinical samples from the clinical pathology laboratories. The results compared favorably with those obtained by the various chemical/biochemical methods employed in the clinical pathology laboratories. A column packed with immobilized urease beads was also prepared in a syringe for the regular and continuous monitoring of serum urea concentrations.     

Lactose Hydrolysis by β-Galactosidase Covalently Immobilized to Thermally Stable Biopolymers

Lactose has been hydrolyzed using covalently immobilized β-galactosidase on thermally stable carrageenan coated with chitosan (hydrogel). The hydrogel’s mode of interaction was proven by Fourier transform infrared spectroscopy, differential scanning calorimetry (DSC), and Schiff’s base formation. The DSC thermogram proved the formation of a strong polyelectrolyte complex between carrageenan and chitosan followed by glutaraldehyde as they formed one single peak. The modification of carrageenan improved the gel’s thermal stability in solutions from 35 °C to 95 °C. The hydrogel has been proven to be efficient for β-galactosidase immobilization where 11 U/g wet gel was immobilized with 50% enzyme loading capacity. Activity and stability of free and immobilized β-galactosidase towards pH and temperature showed marked shifts in their optimum pH from 4.5–5 to 5–5.5 and temperature from 50 °C to 45–55 °C after immobilization, which reveals higher catalytic activity and reasonable stability at wider pHs and temperatures. The apparent K _m of the immobilized enzyme increased from 13.2 to 125 mM, whereas the V _max increased from 3.2 to 6.6 μmol/min compared to the free enzyme, respectively. The free and immobilized enzymes showed lactose conversion of 87% and 70% at 7 h, respectively. The operational stability showed 97% retention of the enzyme activity after 15 uses, which demonstrates that the covalently immobilized enzyme is unlikely to leach. The new carrier could be suitable for immobilization of other industrial enzymes.     

Immobilization of Naringinase in PVA–Alginate Matrix Using an Innovative Technique

A synthetic polymer, polyvinyl alcohol (PVA), a cheap and nontoxic synthetic polymer to organism, has been ascribed for biocatalyst immobilization. In this work PVA–alginate beads were developed with thermal, mechanical, and chemical stability to high temperatures (<80 °C). The combination of alginate and bead treatment with sodium sulfate not only prevented agglomeration but produced beads of high gel strength and conferred enzyme protection from inactivation by boric acid. Naringinase from Penicillium decumbens was immobilized in PVA (10%)–alginate beads with three different sizes (1–3 mm), at three different alginate concentrations (0.2–1.0%), and these features were investigated in terms of swelling ratio within the beads, enzyme activity, and immobilization yield during hydrolysis of naringin. The pH and temperature optimum were 4.0 and 70 °C for the PVA–alginate-immobilized naringinase. The highest naringinase activity yield in PVA (10%)–alginate (1%) beads of 2 mm was 80%, at pH 4.0 and 70 °C. The Michaelis constant (K _Mapp) and the maximum reaction velocity (V _maxapp) were evaluated for both free (K _Mapp = 0.233 mM; V _maxapp = 0.13 mM min⁻¹) and immobilized naringinase (K _Mapp = 0.349 mM; V _maxapp = 0.08 mM min⁻¹). The residual activity of the immobilized enzyme was followed in eight consecutive batch runs with a retention activity of 70%. After 6 weeks, upon storage in acetate buffer pH 4 at 4 °C, the immobilized biocatalyst retained 90% of the initial activity. These promising results are illustrative of the potential of this immobilization strategy for the system evaluated and suggest that its application may be effectively performed for the entrapment of other biocatalysts.     

Improved Thermal and Reusability Properties of Xylanase by Genipin Cross-Linking to Magnetic Chitosan Particles

Enzymes are gradually increasingly preferred over chemical processes, but commercial enzyme applications remain limited due to their low stability and low product recovery, so the application of an immobilization technique is required for repeated use. The aims of this work were to produce stable enzyme complexes of cross-linked xylanase on magnetic chitosan, to describe some characteristics of these complexes, and to evaluate the thermal stability of the immobilized enzyme and its reusability. A xylanase was cross-linked to magnetite particles prepared by in situ co-precipitation of iron salts in a chitosan template. The effect of temperature, pH, kinetic parameters, and reusability on free and immobilized xylanase was evaluated. Magnetization, morphology, size, structural change, and thermal behavior of immobilized enzyme were described. 1.0?±?0.1 μg of xylanase was immobilized per milligram of superparamagnetic chitosan nanoparticles via covalent bonds formed with genipin. Immobilized xylanase showed thermal, pH, and catalytic velocity improvement compared to the free enzyme and can be reused three times. Heterogeneous aggregates of 254 nm were obtained after enzyme immobilization. The immobilization protocol used in this work was successful in retaining enzyme thermal stability and could be important in using natural compounds such as Fe₃O₄@Chitosan@Xylanase in the harsh temperature condition of relevant industries.