Immobilization on Eupergit C of cyclodextrin glucosyltransferase (CGTase) and properties of the immobilized biocatalyst

The extreme thermophilic cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. was covalently attached to Eupergit C. Different immobilization parameters (incubation time, ionic strength, pH, ratio enzyme/support, etc.) were optimized. The maximum yield of bound protein was around 80% (8.1 mg/g support), although the recovery of β-cyclodextrin cyclization activity was not higher than 11%. The catalytic efficiency was lower than 15%. Results were compared with previous studies on covalent immobilization of CGTase.

The enzymatic properties of immobilized CGTase were investigated and compared with those of the soluble enzyme. Soluble and immobilized CGTases showed similar optimum temperature (80–85 °C) and pH (5.5) values, but the pH profile of the immobilized CGTase was broader at higher pH values. The thermoinactivation of the CGTase coupled to Eupergit C was slower than the observed with the native enzyme. The half-life of the immobilized enzyme at 95 °C was five times higher than that of the soluble enzyme. The immobilized CGTase maintained 40% of its initial activity after 10 cycles of 24 h each. After immobilization, the selectivity of CGTase (determined by the ratio CDs/oligosaccharides) was notably shifted towards oligosaccharide production.     

Recycling of textile bleaching effluents for dyeing using immobilized catalase

Catalase was immobilized on alumina carrier and crosslinked with glutaraldehyde. Storing stability, temperature and pH profiles of enzyme activity were studied in a column reactor with recirculation and in a batch stirred-tank reactor. The immobilized enzyme retained 44% of its activity at pH 11, 30 °C and 90% at 80 °C, pH 7. The half-life time of the immobilized catalase was increased to 2 h at pH 12, and 60 °C. Acceptable results were achieved when the residual water from the washing process of H₂O₂-bleached fabrics was treated with the immobilized enzyme and then reused for dyeing.     

Immobilization of catalase on cotton fabric oxidized by sodium periodate

Cotton fabric was first oxidized with sodium periodate, and then employed to immobilize catalase. Optimization studies for oxidation of the fabric and immobilization of the enzyme were performed. The properties of the immobilized catalase were examined and compared with those of the free enzyme. A high activity of the immobilized enzyme was obtained when the fabric was oxidized at 40°C and pH 6.0 for 8h in a bath containing 0.20 mol L^?1 sodium periodate and the enzyme was immobilized at 4°C for 24h with a catalase dosage of 120.0 U mL^?1. The immobilized enzyme exhibited optimum activity at 40°C, while the free enzyme had optimal temperature of 30°C, suggesting that the immobilized catalase could be used in a broader temperature range. Both the immobilized and free enzyme had pH optima of 7.0. The staining test and reusability showed that the catalase was fixed covalently on the oxidized cotton fabric.     

Immobilization and kinetics of catalase onto magnesium silicate

Bovine liver catalase was immobilized covalently with glutaraldehyde, or glutaraldehyde+3-aminopropionic acid as a spacer, onto magnesium silicate. The coupling time was determined as 2 h for immobilization. The pH and temperature optima as well as the changes in the kinetics (K_m, V_max, E_a) of the immobilized catalase was observed and discussed. Immobilized catalase preparations showed higher storage stabilities than free catalase. The half-life of free catalase, catalase immobilized via glutaraldehyde and catalase immobilized via glutaraldehyde+spacer were calculated as 2, 55 and 10 days at room temperature and 4, 85 and 107 days at 5 °C, respectively. The operational stability of the catalase immobilized via glutaraldehyde was higher than the catalase immobilized via glutaraldehyde+spacer. The remaining activity of the catalase immobilized via glutaraldehyde was about 90% and that of the catalase immobilized via glutaraldeyde+spacer was about 30% after 20 cycles of batch operation.     

Immobilization of catalase into chemically crosslinked chitosan beads

Bovine liver catalase was immobilized into chitosan beads prepared in crosslinking solution. Various characteristics of immobilized catalase such as the pH–activity curve, the temperature–activity curve, thermal stability, operational stability, and storage stability were evaluated. Among them the pH optimum and temperature optimum of free and immobilized catalase were found to be pH 7.0 and 35 °C. The K_m value of immobilized catalase (77.5 mM) was higher than that of free enzyme (35 mM). Immobilization decreased in V_max value from 32,000 to 122 μmol (min mg protein)⁻¹. It was observed that operational, thermal and storage stabilities of the enzyme were increased with immobilization.     

Improvement of the functional properties of a thermostable lipase from alcaligenes sp. via strong adsorption on hydrophobic supports

Lipase QL from Alcaligenes sp. is a quite thermostable enzyme. For example, it retains 75% of catalytic activity after incubation for 100 h at 55 °C and pH 7.0. Nevertheless, an improvement of the enzyme properties was intended via immobilization by covalent attachment to different activated supports and by adsorption on hydrophobic supports (octadecyl-sepabeads). This latter immobilization technique promotes the most interesting improvement of enzyme properties: (a) the enzyme is hyperactivated after immobilization: the immobilized preparation exhibits a 135% of catalytic activity for the hydrolysis of p-nitrophenyl propionate as compared to the soluble enzyme; (b) the thermal stability of the immobilized enzyme is highly improved: the immobilized preparation exhibits a half-life time of 12 h when incubated at 80 °C, pH 8.5 (a 25-fold stabilizing factor regarding to the soluble enzyme); (c) the optimal temperature was increased from 50 °C (soluble enzyme) up to 70 °C (hydrophobic support enzyme immobilized preparations); (d) the enantioselectivity of the enzyme for the hydrolysis of glycidyl butyrate and its dependence on the experimental conditions was significantly altered. Moreover, because the enzyme becomes reversibly but very strongly adsorbed on these highly hydrophobic supports, the lipase may be desorbed after its inactivation and the support may be reused. Very likely, adsorption occurs via interfacial activation of the lipase on the hydrophobic supports at very low ionic strength. On the other hand, all the covalent immobilization protocols used to immobilize the enzyme hardly improved the properties of the lipase.     

Thermo-alkali-stable catalases from newly isolated Bacillus sp. for the treatment and recycling of textile bleaching effluents

Three thermoalkaliphilic bacteria, which were grown at pH 9.3–10 and 60–65 °C were isolated out of a textile wastewater drain. The unknown micro-organisms were identified as thermoalkaliphilic Bacillus sp. Growth conditions were studied and catalase activities and stabilities compared. Catalases from Bacillus SF showed high stabilities at 60 °C and pH 9 (t_1/2=38 h) and thus this strain was chosen for further investigations, such as electron microscopy, immobilization of catalase and hydrogen peroxide degradation studies. Degradation of hydrogen peroxide with an immobilized catalase from Bacillus SF enabled the reuse of the water for the dyeing process. In contrast, application of the free enzyme for treatment of bleaching effluents, caused interaction between the denaturated protein and the dye, resulting in reduced dye uptake, and a higher color difference of 1.3 ΔE* of dyed fabrics compared to 0.9 ΔE* when using the immobilized enzyme.     

Immobilization of soybean seed coat peroxidase on polyaniline: Synthesis optimization and catalytic properties

Soybean seed coat peroxidase (SBP) was immobilized on various polyaniline-based polymers (PANI), activated with glutaraldehyde. The most reduced polymer (PANIG₂) showed the highest immobilization capacity (8.2 mg SBP g^-1 PANIG₂). The optimum pH for immobilization was 6.0 and the maximum retention was achieved after a 6-h reaction period. The efficiency of enzyme activity retention was 82%. When stored at 4°C, the immobilized enzyme retained 80% of its activity for 15 weeks as evidenced by tests performed at 2-week intervals. The immobilized SBP showed the same pH-activity profile as that of the free SBP for pyrogallol oxidation but the optimum temperature (55°C) was 10°C below that of the free enzyme. Kinetic analysis show that the K_m was conserved while the specific V_max dropped from 14.6 to 11.4 µmol min^-1 µg^-1, in agreement with the immobilization efficiency. Substrate specificity was practically the same for both enzymes. Immobilized SBP showed a greatly improved tolerance to different organic solvents; while free SBP lost around 90% of its activity at a 50% organic solvent concentration, immobilized SBP underwent only 30% inactivation at a concentration of 70% acetonitrile. Taking into account that immobilized HRP loses more than 40% of its activity at a 20% organic solvent concentration, immobilized SBP performed much better than its widely used counterpart HRP.     

Immobilized Cyclodextrin Glycosyl Transferase for the Continuous Production of Cyclodextrins

Cyclodextrin glycosyl transferase (E.C: 2.4.1.19) from Bacillus, macerans and from a Bacillus sp. isolate was immobilized by two methods, viz. to epoxy-activated Sepharose and to alkylamine silica treated with glutaraldehyde. Because of the ready availability, low cost ($0.01/g), good surface area (30 M²/g) and ease of operation of a continuous cylindrical reactor, the high silica fabric was chosen. The immobilized enzyme had a pH optimum shifted to the alkaline side (from 6.5 to 7.5) and had a reduced temperature optimum (from 60°C to 50-55°C). Reuse efficiency showed 65% reduction in the overall activity of the immobilized enzyme after 10 cycles of 48 h each. Continuous operation at 55°C of a cylindrical reactor of 141 ml capacity, using the immobilized enzyme (80 g of high - silica fabric containing 114 mg of purified enzyme) gave a maximum productivity of 10.2 g of cyclodextrins L^-1 h,^-1, at a dilution rate of 0.32 h^-1 and a substrate concentration of 20 g L^-1. The half life of the biocatalyst was found to be 22 days, which could be further improved by using a lower operating temperature. Over the useful life time of the immobilized biocatalyst (22 days), the total Cyclodextrin produced was of the order of 88 Kg.     

Purification and partial characterization of manganese peroxidase from immobilized Phanerochaete chrysosporium

Solid-state culture of the white-rot fungus Phanerochaete chrysosporium BKMF-1767 (ATCC 24725) has been carried out, using an inert support, polystyrene foam. Suitable medium and culture conditions have been chosen to favor the secretion of manganese peroxidase (MnP). The enzyme was isolated and purified from immobilized P. chrysosporium and partially characterized. Partial protein precipitation in crude enzyme was affected using ammonium sulphate, polyethylene glycol, methanol, and ethanol methods. Fractionation of MnP was performed by DEAE-Sepharose ion exchange chromatography followed by Ultragel AcA 54 gel filtration chromatography. This purification attained 23.08% activity yield with a purification factor of 5.8. According to data on gel filtration chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of the enzyme was 45 000±1000 Da. The optimum pH and temperature of purified MnP were 4.5 and 30 °C, respectively. This enzyme was stable in the pH range 4.5–6.0, at 25 °C and also up to 35 °C at pH 4.5 for 1 h incubation period. MnP activity was inhibited by 2 mM NaN₃, ascorbic acid, β-mercaptoethanol and dithreitol. The K_m values of MnP for hydrogen peroxide and 2.6-dimetoxyphenol were 71.4 and 28.57 μM at pH 4.5, respectively. The effects of possible inhibitors and activators of enzyme activity were investigated.     

Hydrogen peroxide degradation by immobilized cells of alkaliphilic Bacillus halodurans

Whole cells of Bacillus halodurans LBK 261 were used as a source of catalase for degradation of hydrogen peroxide. The organism, B. halodurans grown at 55°C and pH 10, yielded a maximum catalase activity of 275 U g^-1 (wet wt.) cells. The catalase in the whole cells was active over a broad range of pH with a maximum at pH 8-9. The enzyme was optimally active at 55°C, but had low stability above 40°C. The whole cell biocatalyst exhibited a K_m of 6.6 mM for H₂O₂ and V_max of 707 mM H₂O₂ min^-1 g^-1 wet wt. cells, and showed saturation kinetics at 50 mM H₂O₂. The cells were entrapped in calcium alginate and used for H₂O₂ degradation at pH 9 in batch and continuous mode. In the batch process, the immobilized preparation containing 1.5 g (wet wt.) cells could be recycled at least four times for complete degradation of the peroxide in 50 mL solution at 25°C. An excess of immobilized biocatalyst could be used in a continuous stirred tank reactor for an average of 9 days at temperatures upto 55°C, and in a packed bed reactor (PBR) for 5 days before the beads started to deform.     

Biocatalysis of silica gel-immobilized chlorophyllase in a ternary micellar system

A partially purified enzymic extract from Phaeodactylum tricornutum was immobilized on silica gel and the specific activity of chlorophyllase in its free and immobilized states were compared in a ternary micellar system. The storage stability of the free and immobilized chlorophyllase extracts, maintained at temperatures ranging from 4 to 35°C for a period of 0–20 h, was temperature-dependent. The results also showed that the specific activity of the free and immobilized chlorophyllase extracts was highest at 30°C for long-term incubation, using chlorophyll and pheophytin as substrates and that a three-fold increase in the specific activity of the immobilized chlorophyllase was observed in comparison to that obtained with the free counterpart. The findings indicated that when free and immobilized chlorophyllase extracts were recovered and reused with both substrates, the immobilized chlorophyllase extract could be recycled for longer periods of time, while the free enzyme extract showed no activity after the first cycle.     

Biosynthesis and properties of an extracellular metalloprotease from the Antarctic marine bacterium Sphingomonas paucimobilis

An extracellular protease from the marine bacterium Sphingomonas paucimobilis, strain 116, isolated from the stomach of Antarctic krill, Euphausia superba Dana, was purified and characterized. The excretion of protease was maximal at temperatures from 5 to 10°C, i.e. below the temperature optimum for the strain growth (15°C). The highly purified enzyme was a metalloprotease [sensivity to ethylenediaminetetraacetic acid (EDTA)] and showed maximal activity against proteins at 20–30°C and pH 6.5–7.0, and towards N-benzoyl-tyrosine ethyl ester (BzTyrOEt) at pH 8.0. At 0°C the enzyme retained as much as 47% of maximal activity in hydrolysis of urea denatured haemoglobin (Hb) (at pH 7.0), and at −5 and −10°C, 37 and 30%, respectively. The metalloprotease was stable up to 30°C for 15 min and up to 20°C for 60 min. These results indicate that the proteinase from S. paucimobilis 116 is a cold-adapted enzyme.     

Kerase Immobilization by Covalent Attachment to Porous Glass

Kerase, a serine protease from Streptomyces fradiae, was immobilized on porous glass (SIKUG®) by covalent attachment, through amino groups on the enzyme. Modifications of four lysine residues (44·4% of the accessible or superficial amino groups) results in a loss of 6·5% of the enzymic activity. After immobilization, the optimal reaction pH changed from a range of 7·5-8·5 to 9-10. The immobilized protease was stable in a broad pH range, 6-12, while the soluble protease was irreversibly denaturated at alkaline pHs (pH>8). The optimal reaction temperature was displaced from 55 to 65°C, showing a higher thermal stability of the immobilized enzyme. Kerase immobilized onto porous glass was stable for at least 28 days, working in a repeated-batch process of three cycles per day, with an activity loss of 22·1 ± 3·1%.     

Immobilization of RNase Rs via its carbohydrate moiety to aminoethyl-Bio-Gel P-2 and its application for the hydrolysis of RNA to 2′,3′ cyclic nucleotides

Purified RNase Rs, from Rhizopus stolonifer, when covalently coupled to aminoethyl (AE) Bio-Gel P-2, via its carbohydrate moiety, retained 35–40% activity of the soluble enzyme. Optimization of coupling conditions showed that the most active immobilized preparations are obtained when 400 units of 100 μM periodate oxidized enzyme are allowed to react with 1 ml (packed volume) of AE-Bio-Gel P-2 at 6±1°C for 15 h. Immobilization did not change the pH and temperature optima of the enzyme but it increased the temperature stability. Immobilization did not bring about a change in the K_m but resulted in a 2·5-fold decrease in the V_max. Substrate concentrations as high as 25 mg of RNA could be converted to more than 80% 2′,3′ cyclic nucleotides in 14 h, at pH 5·5 and 37°C. On repeated use, the bound enzyme retained 70% of its initial activity after six cycles of use. The bound enzyme could be stored in wet state for 60 days without any significant loss in its initial activity.     

以Fe(III) 改性胶原纤维为载体固定过氧化氢酶

将胶原纤维用三价铁改性后作为载体,通过戊二醛的交联作用将过氧化氢酶固定在该载体上。制备的固定化过氧化氢酶蛋白固载量为16.7 mg/g,酶活收率为35％。研究了固定化酶与自由酶的最适pH、最适温度、热稳定性、贮存稳定性及操作稳定性。结果表明：过氧化氢酶经此法固定化后,最适pH及最适温度与自由酶相同,分别为pH 7.0和25 ℃;但固定化酶的热稳定性显著提高,在75 ℃保存5 h后,仍能保留30％的活力,而自由酶则完全失活;固定化酶在室温下保存12 d后,酶活力仍保持在88％以上,而自由酶在此条件下则完全失     

Isolation of a thermostable uricase-producing bacterium and study on its enzyme production conditions

A uricase-producing bacterium was isolated from soil with a medium containing uric acid as the only carbon source. Based on its morphological and physiological characteristics, as well as 16S rDNA sequence and phylogenetic tree analysis, this new isolate belong to the genus Microbacterium. After heat treatment at 70 °C for 30 min, the uricase retained about 100% of the initial activity. The enzyme activity remained largely unchanged when it was stored in borate buffer at pH 8.5 at 37 °C for 40 days. The effects of different factors on the enzyme production were studied. Maize milk was the best C and N resources, and the uric acid showed to be an inducer for uricase production. When the strain was cultured at 30 °C at pH 7.5 for 30–36 h, the uricase activity peaked at 1.0 U/ml.     

Immobilization of thermostable β-glucosidase from Sulfolobus shibatae by cross-linking with transglutaminase

Thermostable β-glucosidase from Sulfolobus shibatae was immobilized on silica gel modified or not modified with 3-aminopropyl-triethoxysilane using transglutaminase as a cross-linking factor. Obtained preparations had specific activity of 3883 U/g of the support, when measured at 70 °C using o-nitrophenyl β-d-galactopyranoside (GalβoNp) as substrate. The highest immobilization yield of the enzyme was achieved at pH 5.0 in reaction media. The most active preparations of immobilized β-glucosidase were obtained at a transglutaminase concentration of 40 mg/ml at 50 °C. The immobilization was almost completely terminated after 100 min of the reaction and prolonged time of this process did not cause considerable changes of the activity of the preparations. The immobilization did not influence considerably on optimum pH and temperature of GalβoNp hydrolysis catalyzed by the investigated enzyme (98 °C, pH 5.5). The broad substrate specifity and properties of the thermostable β-glucosidase from S. shibatae immobilized on silica-gel indicate its suitability for hydrolysis of lactose during whey processing.     

Improving of Catalase Stability Properties by Encapsulation in Alginate/Fe3O4 Magnetic Composite Beads for Enzymatic Removal of H2O2

The aim of this study was enhancing of stability properties of catalase enzyme by encapsulation in alginate/nanomagnetic beads. Amounts of carrier (10–100 mg) and enzyme concentrations (0.25–1.5 mg/mL) were analyzed to optimize immobilization conditions. Also, the optimum temperature (25–50°C), optimum pH (3.0–8.0), kinetic parameters, thermal stability (20–70°C), pH stability (4.0–9.0) operational stability (0–390 min), and reusability were investigated for characterization of the immobilized catalase system. The optimum pH levels of both free and immobilized catalase were 7.0. At the thermal stability studies, the magnetic catalase beads protected 90% activity, while free catalase maintained only 10% activity at 70°C. The thermal profile of magnetic catalase beads was spread over a large area. Similarly, this system indicated the improving of the pH stability. The reusability, which is especially important for industrial applications, was also determined. Thus, the activity analysis was done 50 times in succession. Catalase encapsulated magnetic alginate beads protected 83% activity after 50 cycles.     

Characteristics of galactomannanase for degrading konjac gel

Galactomannanase (Glmnase) is an enzyme product derived from Aspergillus niger. The activity of Glmnase degrading (hydrolyzing) the konjac gel were investigated. Significant loss in the enzyme activity was found when the temperature above 60 °C. Similar observations were obtained when the reaction pH above 5. Further increase in the pH value resulted in entirely loss of enzyme activity at the alkaline pH region (pH 8.0 and above). The optimal hydrolyzing temperature and pH were at 60 °C and 5.0, respectively. For the stability test, the purified Glmnase increased its thermostability up to 70 °C at pH 5.0, but it retained only about 60% activity after 60 min incubation at this temperature and its activity became zero after 20 min incubation at 80 °C. The Glmnase was stable at the pH range from 3.0 to 7.0 at room temperature and retained at least 80% activity for 60 min. For the storage temperature test, the lyophilized Glmnase still conserved about 90% activity during 7 days at 30 °C, and was higher than about 80% at 4 °C. The K_m and V_max, were 0.018 mg/ml konjac powder and 0.20 mg/ml reducing sugar per min, respectively.