Sucrose monolaurate synthesis with Protex 6L immobilized on electrospun TiO<Subscript>2</Subscript> nanofiber

TiO₂ nanofibers with uniform diameter about 125 nm were prepared based on sol–gel process and electrospinning technology. Protex 6L, an industrial alkaline protease, was covalently immobilized on TiO₂ nanofiber through γ-aminopropyltriethoxysilane modification and glutaraldehyde crosslinking. With 2 (v/v)% glutaraldehyde as crosslinker, the enzyme loading is about 201 mg (g nanofiber membrane)⁻¹, and the specific activity of the immobilized Protex 6L is 2.45 μmol h⁻¹ ml⁻¹ mg⁻¹ protein for synthesis of sucrose monolaurate from sucrose and vinyl laurate. The optimal condition for sucrose monolaurate production is 5% (v/v) water content in DMSO/2-methyl-2-butanol solvent mixture and 50°C. Under this condition, 97% conversion was achieved within 36 h by nanofibrous Protex 6L, which is corresponding to a productivity 34 times higher than that of most widely used Novozym 435. After 10 cycles reuse, nanofibrous Protex 6L retained 52.4% of its original activity.     

Purification, immobilization and characterization of linoleic acid isomerase on modified palygorskite

Linoleic acid isomerase from Lactobacillus delbrueckii subsp. bulgaricus 1.1480 was purified by DEAE ion-exchange chromatography and gel filtration chromatography. An overall 5.1% yield and purification of 93-fold were obtained. The molecular weight of the purified protein was ~41 kDa which was analyzed by SDS-PAGE. The purified enzyme was immobilized on palygorskite modified with 3-aminopropyltriethoxysilane. The immobilized enzyme showed an activity of 82 U/g. The optimal temperature and pH for the activity of the free enzyme were 30 °C and pH 6.5, respectively; whereas those for the immobilized enzyme were 35 °C and pH 7.0, respectively. The immobilized enzyme was more stable than the free enzyme at 30–60 °C, and the operational stability result showed that more than 85% of its initial activity was retained after incubation for 3 h. The K _m and V _max values of the immobilized enzyme were found to be 0.0619 mmol l⁻¹ and 0.147 mmol h⁻¹ mg⁻¹, respectively. The immobilized enzyme had high operational stability and retained high enzymatic activity after seven cycles of reuse at 37 °C.     

Stability evaluation of 6-phosphogluconate dehydrogenase immobilized on amino-functionalized magnetic nanoparticles

Abstract

In this study, 6-phosphogluconate dehydrogenase was covalently immobilized onto the N-2-aminoethyl-3-aminopropyltriethoxysilane (APTES) modified core-shell Fe₃O₄@SiO₂ magnetic nanoparticles (ASMNPs) using glutaraldehyde (GA). Immobilization of 6PGDH on ASMNPs was confirmed using fourier transform-infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) analysis. The NADP⁺ conversion ratio, the reusability, thermal, and storage stability of the immobilized 6PGDH were determined and compared with those of the free enzyme. The maximum retention of enzyme activity reached to 96% when the enzyme was immobilized on ASMNPs activated with monomer form of GA. Although the thermal stability of free and immobilized enzymes was similar, at 30?°C, the immobilized 6PGDH showed the improved thermal stability at 40?°C and 50?°C compared with free 6PGDH. While the free 6PGDH only converted 33% of NADP⁺ in reaction medium upon 480?s, the immobilized 6PGDH performed 56% conversion of NADP⁺ at same time. The immobilized 6PGDH retained 62% of its initial activity up to the fifth cycle and 35% of its initial activity after 22?days of storage at 4?°C.     

Immobilization of α-galactosidase on galactose-containing polymeric beads

Industrial application of α-galactosidase requires efficient methods to immobilize the enzyme, yielding a biocatalyst with high activity and stability compared to free enzyme. An α-galactosidase from tomato fruit was immobilized on galactose-containing polymeric beads. The immobilized enzyme exhibited an activity of 0.62 U/g of support and activity yield of 46%. The optimum pH and temperature for the activity of both free and immobilized enzymes were found as pH 4.0 and 37 °C, respectively. Immobilized α-galactosidase was more stable than free enzyme in the range of pH 4.0–6.0 and more than 85% of the initial activity was recovered. The decrease in reaction rate of the immobilized enzyme at temperatures above 37 °C was much slower than that of the free counterpart. The immobilized enzyme shows 53% activity at 60 °C while free enzyme decreases 33% at the same temperature. The immobilized enzyme retained 50% of its initial activity after 17 cycles of reuse at 37 °C. Under same storage conditions, the free enzyme lost about 71% of its initial activity over a period of 7 months, whereas the immobilized enzyme lost about only 47% of its initial activity over the same period. Operational stability of the immobilized enzyme was also studied and the operational half-life (t_1/2 was determined as 6.72 h for p-nitrophenyl α-d-galactopyranoside (PNPG) as substrate. The kinetic parameters were determined by using PNPG as substrate. The K_m and V_max values were measured as 1.07 mM and 0.01 U/mg for free enzyme and 0.89 mM and 0.1 U/mg for immobilized enzyme, respectively. The synthesis of the galactose-containing polymeric beads and the enzyme immobilization procedure are very simple and also easy to carry out.     

Synthesis of Protein-Inorganic Nanohybrids with Improved Catalytic Properties Using Co<Subscript>3</Subscript>(PO<Subscript>4</Subscript>)<Subscript>2</Subscript>

In the present study, a method for easy and rapid synthesis of lipase nanohybrids was evaluated using cobalt chloride as an encapsulating agent. The synthesized nanohybrids exhibited higher activity (181%) compared to free lipase and improved catalytic properties at higher temperature and in harsh conditions. The nanohybrids retained 84% of their residual activity at 25 °C after 10 days. In addition, these nanohybrids also exhibited high storage stability and reusability. Collectively, the synthesis of carrier-free immobilized biocatalysts was performed rapidly within 24 h at 4 °C. Their high reusability and catalytic activities highlight the broad applicability of this method for catalysis in organic and aqueous media.     

A novel nonionic surfactant- and solvent-stable alkaline serine protease from <Emphasis Type="Italic">Serratia</Emphasis> sp. SYBC H with duckweed as nitrogen source: production,purification, characteristics and application

A novel nonionic surfactant- and hydrophilic solvent-stable alkaline serine protease was purified from the culture supernatant of Serratia sp. SYBC H with duckweed as nitrogen source. The molecular mass of the purified protease is about 59 kDa as assayed via SDS-PAGE. The protease is highly active over the pH range between 5.0 and 11.0, with the maximum activity at pH 8.0. It is also fairly active over the temperature range between 30 and 80°C, with the maximum activity at 40°C. The protease activity was substantially stimulated by Mn²⁺ and Na⁺ (5 mM), up to 837.9 and 134.5% at 40°C, respectively. In addition, Mn²⁺ enhanced the thermostability of the protease significantly at 60°C. Over 90% of its initial activity remained even after incubating for 60 min at 40°C in 50% (v/v) hydrophilic organic solvents such as DMF, DMSO, acetone and MeOH. The protease retained 81.7, 83.6 and 76.2% of its initial activity in the presence of nonionic surfactants 20% (v/v) Tween 80, 25% (v/v) glycerol and Triton X-100, respectively. The protease is strongly inhibited by PMSF, suggesting that it is a serine protease. Washing experiments revealed that the protease has an excellent ability to remove blood stains.     

Stabilization of ficin extract by immobilization on glyoxyl agarose. Preliminary characterization of the biocatalyst performance in hydrolysis of proteins

A protein extract containing ficin was immobilized on glyoxyl agarose at pH 10 and 25 °C. The free enzyme remained fully active after 24 h at pH 10. However the enzyme immobilized on the support retained only 30% of the activity after this time using a small substrate. After checking the stability of ficin preparations obtained after different enzyme-support multi-interaction times, it was found that it reached a maximum at 3 h (40-folds more stable than the free enzyme at pH 5). The immobilized enzyme was active in a wide range of pH (e.g., retained double activity at pH 10 than the free enzyme) and temperatures (e.g., at 80 °C retained three-folds more activity than the free enzyme). The activity versus casein almost matched the results using the small substrate (60%) at 55 °C. However, in the presence of 2 M of urea, it became three times more active than the free enzyme. The immobilized enzyme could be reused five cycles at 55 °C without losing activity.     

Mannose production from fructose by free and immobilized <Emphasis Type="SmallCaps">d</Emphasis>-lyxose isomerases from <Emphasis Type="Italic">Providencia stuartii</Emphasis>

A recombinant d-lyxose isomerase from Providencia stuartii was immobilized on Duolite A568 beads which gave the highest conversion of d-fructose to d-mannose among the various immobilization beads evaluated. Maximum activities of both the free and immobilized enzymes for fructose isomerization were at pH 7.5 and 45°C in the presence of 1 mM Mn²⁺. Enzyme half-lives were 14 and 30 h at 35°C and 3.4 and 5.1 h at 45°C, respectively. The immobilized enzyme in 300 g fructose/l (replaced hourly), produced 75 g mannose/l at 35°C = 25% (w/w) yield with a productivity of 75 g mannose l⁻¹ h⁻¹ after 23 cycles.     

Production of high fructose syrup from <Emphasis Type="Italic">Asparagus</Emphasis> inulin using immobilized exoinulinase from <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> YS-1

Extracellular exoinulinase from Kluyveromyces marxianus YS-1, which hydrolyzes inulin into fructose, was immobilized on Duolite A568 after partial purification by ethanol precipitation and gel exclusion chromatography on Sephadex G-100. Optimum temperature of immobilized enzyme was 55 °C, which was 5 °C higher than the free enzyme and optimal pH was 5.5. Immobilized biocatalyst retained more than 90% of its original activity after incubation at 60 °C for 3 h, whereas in free form its activity was reduced to 10% under same conditions, showing a significant improvement in the thermal stability of the biocatalyst after immobilization. Apparent K _m values for inulin, raffinose and sucrose were found to be 3.75, 28.5 and 30.7 mM, respectively. Activation energy (E _a) of the immobilized biocatalyst was found to be 46.8 kJ/mol. Metal ions like Co²⁺ and Mn²⁺ enhanced the activity, whereas Hg²⁺ and Ag²⁺ were found to be potent inhibitors even at lower concentrations of 1 mM. Immobilized biocatalyst was effectively used in batch preparation of high fructose syrup from Asparagus racemosus raw inulin and pure inulin, which yielded 39.2 and 40.2 g/L of fructose in 4 h; it was 85.5 and 92.6% of total reducing sugars produced, respectively.     

Preparative-scale kinetic resolution of racemic styrene oxide by immobilized epoxide hydrolase

Epoxide hydrolase from Aspergillus niger was immobilized onto the modified Eupergit C 250 L through a Schiff base formation. Eupergit C 250 L was treated with ethylenediamine to introduce primary amine groups which were subsequently activated with glutaraldehyde. The amount of introduced primary amine groups was 220 μmol/g of the support after ethylenediamine treatment, and 90% of these groups were activated with glutaraldehyde. Maximum immobilization of 80% was obtained with modified Eupergit C 250 L under the optimized conditions. The optimum pH was 7.0 for the free epoxide hydrolase and 6.5 for the immobilized epoxide hydrolase. The optimum temperature for both free and immobilized epoxide hydrolase was 40 °C. The free epoxide hydrolase retained 52 and 33% of its maximum activity at 40 and 60 °C, respectively after 24 h preincubation time whereas the retained activities of immobilized epoxide hydrolase at the same conditions were 90 and 75%, respectively. Immobilized epoxide hydrolase showed about 2.5-fold higher enantioselectivity than that of free epoxide hydrolase. A preparative-scale (120 g/L) kinetic resolution of racemic styrene oxide using immobilized preparation was performed in a batch reactor and (S)-styrene oxide and (R)-1-phenyl-1,2-ethanediol were both obtained with about 50% yield and 99% enantiomeric excess. The immobilized epoxide hydrolase was retained 90% of its initial activity after 5 reuses.     

Comparative biochemical characterization of soluble and chitosan immobilized β-galactosidase from Kluyveromyces lactis NRRL Y1564

An investigation was conducted on the production of β-galactosidase (β-gal) by different strains of Kluyveromyces, using lactose as a carbon source. The maximum enzymatic activity of 3.8 ± 0.2 U/mL was achieved by using Kluyveromyces lactis strain NRRL Y1564 after 28 h of fermentation at 180 rpm and 30 °C. β-gal was then immobilized onto chitosan and characterized based on its optimal operation pH and temperature, its thermal stability and its kinetic parameters (K_m and V_max) using o-nitrophenyl β-d-galactopyranoside as substrate. The optimal pH for soluble β-gal activity was found to be 6.5 while the optimal pH for immobilized β-gal activity was found to be 7.0, while the optimal operating temperatures were 50 °C and 37 °C, respectively. At 50 °C, the immobilized enzyme showed an increased thermal stability, being 8 times more stable than the soluble enzyme. The immobilized enzyme was reused for 10 cycles, showing stability since it retained more than 70% of its initial activity. The immobilized enzyme retained 100% of its initial activity when it was stored at 4 °C and pH 7.0 for 93 days. The soluble β-gal lost 9.4% of its initial activity when it was stored at the same conditions.     

Enhanced activity and stability of <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase by immobilization on aminopropyl glass

Immobilization of Bacillus licheniformis l-arabinose isomerase (BLAI) on aminopropyl glass modified with glutaraldehyde (4 mg protein g support⁻¹) was found to enhance the enzyme activity. The immobilization yield of BLAI was proportional to the quantity of amino groups on the surface of support. Reducing particle size increased the adsorption capacity (q _m) and affinity (k _a). The pH and temperature for immobilization were optimized to be pH 7.1 and 33°C using response surface methodology (RSM). The immobilized enzyme was characterized and compared to the free enzyme. There is no change in optimal pH and temperature before and after immobilization. However, the immobilized BLAI enzyme achieved 145% of the activity of the free enzyme. Correspondingly, the catalytic efficiency (k _cat/K _m) was improved 1.47-fold after immobilization compared to the free enzyme. The thermal stability was improved 138-fold (t _1/2 increased from 2 to 275 h) at 50°C following immobilization.     

Immobilization of the recombinant invertase INVB from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> on Nylon-6

The recombinant invertase INVB (re-INVB) from Zymomonas mobilis was immobilized on microbeads of Nylon-6, by means of covalent bonding. The enzyme was strongly and successfully bound to the support. The activity of the free and immobilized enzyme was determined, using 10% (w/v) sucrose, at a temperature ranging between 15 and 60 °C and a pH ranging between 3.5 and 7. The optimal pH and temperature for the immobilized enzyme were 5.5 and 25 °C, respectively. Immobilization of re-INVB on Nylon-6 showed no significant change in the optimal pH, but a difference in the optimal temperature was evident, as that for the free enzyme was shown to be 40 °C. The values for kinetic parameters were determined as: 984 and 98 mM for of immobilized and free re-INVB, respectively. values for immobilized and free enzymes were 6.1 × 10² and 1.2 × 10⁴ s⁻¹, respectively, and immobilized re-INVB showed of 158.73 μmol h min⁻¹ mg⁻¹. Immobilization of re-INVB on Nylon-6 enhanced the thermostability of the enzyme by 50% at 30 °C and 70% at 40 °C, when compared to the free enzyme. The immobilization system reported here may have future biotechnological applications, owing to the simplicity of the immobilization technique, the strong binding of re-INVB to the support and the effective thermostability of the enzyme.     

High-level expression of the <Emphasis Type="Italic">Penicillium notatum</Emphasis> glucose oxidase gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using codon optimization

The glucose oxidase (GOD) gene from Penicillium notatum was expressed in Pichia pastoris. The 1,815 bp gene, god-w, encodes 604 amino acids. Recombinant GOD-w had optimal activity at 35–40°C and pH 6.2 and was stable, from pH 3 to 7 maintaining >75% maximum activity after incubation at 50°C for 1 h. GOD-w worked as well as commercial GODs to improve bread making. To achieve high-level expression of recombinant GOD in P. pastoris, 272 nucleotides involving 228 residues were mutated, consistent with the codon bias of P. pastoris. The optimized recombinant GOD-m yielded 615 U ml⁻¹ (2.5 g protein l⁻¹) in a 3 l fermentor—410% higher than GOD-w (148 U ml⁻¹), and thus is a low-cost alternative for the bread baking industry.     

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.     

Preparation of regenerable magnetic nanoparticles for cellulase immobilization: Improvement of enzymatic activity and stability

To obtain regenerable magnetic nanoparticles, triethoxy(3-isocyanatopropyl)silane and iminodiacetic acid (IZ) were used as the starting material and immobilized on Fe₃O₄ nanoparticles. Copper ions (Cu²⁺ ions) were loaded on the Fe-IZ nanoparticles and used for cellulase immobilization. The support was characterized by spectroscopic methods (FTIR, NMR) and thermogravimetric analysis, transmission electron microscopy, scanning electron microscope, X-ray diffraction, energy dispersive X-ray analysis, and vibrating sample magnetometer techniques. As a result of experiments, the amount of protein bound to immobilized cellulase (Fe-IZ-Cu-E) and cellulase activity was found to be 33.1 mg/g and 154 U/g at pH 5, 50°C, for 3 h. The results indicated that the free cellulase had kept only 50% of its activity after 2 h, while the Fe-IZ-Cu-E was observed to be around 77%, at 60°C. It was found that the immobilized cellulase maintained 93% of its initial catalytic activity after its sixth use. Furthermore, the Fe-IZ-Cu-E retained about 75% of its initial activity after 28 days of storage. To reuse the support material (Fe-IZ-Cu), it was regenerated by thorough washing with ammonia or imidazole.     

Surface display of active lipase in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> using Cwp2 as an anchor protein

Lipase Lip2 from Yarrowia lipolytica was displayed on the cell surface of Saccharomyces cerevisiae using Cwp2 as an anchor protein. Successful display of the lipase on the cell surface was confirmed by immunofluorescence microscopy and halo assay. The length of linker sequences was further examined to confirm that the correct conformation of Lip2 was maintained. The results showed that the displayed Lip2 exhibited the highest activity at 7.6 ± 0.4 U/g (dry cell) when using (G₄S)₃ sequence as the linker, with an optimal temperature and pH at 40°C and pH 8.0. The displayed lipase did not lose any activity after being treated with 0.1% Triton X-100 and 0.1% Tween 80 for 30 min, and it retained 92% of its original activity after incubation in 10% DMSO for 30 min. It also exhibited better thermostability than free Lip2 as reported previously.     

Immobilization of alliinase with a water soluble–insoluble reversible N-succinyl-chitosan for allicin production

N-Succinyl-chitosan (NSC), a pH-sensitive polymer of reversibly soluble–insoluble characteristics with pH change, was prepared by modification of the chitosan backbone with succinic anhydride and employed as carrier for alliinase immobilization. The obtained NSC is soluble at pH above 4.8 and insoluble at pH below 4.4. The characteristics of NSC were evaluated using Fourier transform IR spectrophotometer, the X-ray diffraction spectrometry and thermogravimetric analyzer. Under an optimized condition (glutaraldehyde 0.8% (v/v), 31.2 U alliinase), the enzyme immobilization yield was 75.6%. The maximum activity of NSCA was achieved at 40 °C, pH 7, while the free enzyme exhibited maximum activity at 30 °C, pH 6. The Michaelis–Menten constant of NSCA was lower than that of free alliinase, indicating higher affinity of immobilized enzyme toward its substrate. The NSCA retained 85% of its initial activity even after being recycled 5 times. The immobilized alliinase in reversibly soluble NSC is suitable to catalyze the conversion of alliin to allicin, as active ingredient of pharmaceutical compositions and food additive.     

Covalent immobilization of ω-transaminase from Vibrio fluvialis JS17 on chitosan beads

Chitosan beads were prepared by emulsion method and used for the immobilization of ω-transaminase of Vibrio fluvialis. The yield of enzyme immobilization (54.3%) and its residual activity (17.8%) were higher than those obtained with other commercial beads. ω-Transaminase was effectively immobilized on the chitosan beads at pH 6.0. The optimal pH of the immobilized enzyme was pH 9.0, which is the same as that of the free enzyme. The immobilized enzyme on chitosan beads retained ca. 77% of its conversion after five consecutive reactions with the 25 mM substrate, while the immobilized enzyme on Eupergit^® C retained 12%. Also, the immobilized ω-transaminase on chitosan bead retained 70% of initial activity when it's stored at 4 °C for 3.5 weeks. Addition of the co-factor, pyridoxal 5-phosphate (PLP), was needed to maintain the stability of the immobilized ω-transaminase.     

Heterologous expression and characterization of a novel halotolerant,thermostable, and alkali-stable GH6 endoglucanase from <Emphasis Type="Italic">Thermobifida halotolerans</Emphasis>

A novel endoglucanase gene was cloned from Thermobifida halotolerans YIM 90462^T, designated as thcel6A for being a member of glycoside hydrolase family 6. The gene was 1332 bp long and encoded a 443-amino-acid protein with a molecular mass of 45.9 kDa. The purified recombinant endoglucanase had optimal activity at 55 °C and pH 8.5. Thcel6A showed high hydrolytic activities at 25–55 °C and retained 58 % of initial activity after incubation at 90 °C for 1 h. It retained more than 80 % of activity after incubation for 12 h at pH values from 4 to 12. Thcel6A displayed higher hydrolytic activities in 5–15 % NaCl (w/v) than at 0 % NaCl. Activity increased 2.5-fold after incubation with 20 % (w/v) NaCl at 37 °C for 10 min. These properties suggest that this novel endoglucanase has potential for specific industrial application.