Immobilization of trypsin on chitosan gels: use of different activation protocols and comparison with other supports

Trypsin was immobilized on chitosan gels coagulated with 0.1 or 1 M NaOH and activated with glutaraldehyde or glycidol. The derivatives were characterized by their recovered activity, thermal (40, 55 and 70 degrees C) and alkaline (pH 11) stabilities, amount of enzyme immobilized on gels for several enzyme loads (8-14 mg(protein)/g(Gel)) and compared to agarose derivatives. Enzyme loads higher than 14 mg(protein)/g(Gel) can be immobilized on glutaraldehyde derivatives, which showed 100% immobilization yield and, for loads up to 8 mg(protein)/g(Gel), 100% recovered activity. Activation with glycidol led to lower immobilization yields than the ones obtained with glutaraldehyde, 61% for agarose-glyoxyl (AgGly) with low grade of activation and 16% for the chitosan-glyoxyl (ChGly), but allowed obtaining the most stable derivative (ChGly), that was 660-fold more stable than the soluble enzyme at 55 and 70 degrees C-approximately threefold more stable than AgGly. The ChGly derivative presented also the highest stability during incubation at pH 11. Analyses of lysine residue contents in soluble and immobilized trypsin indicated formation of multipoint bonds between enzyme and support, for glyoxyl derivatives.     

Improving the properties of chitosan as support for the covalent multipoint immobilization of chymotrypsin

Changing gel structure and immobilization conditions led to a significant improvement in the covalent multipoint attachment of chymotrypsin on chitosan. The use of sodium alginate, gelatin, or kappa-carrageenan, activation with glutaraldehyde, glycidol, or epichlorohydrin, and addition of microorganisms followed by cellular lysis allowed the modification of the gel structure. Immobilization yields, recovered activities, and stabilization factors at 55 and 65 degrees C were evaluated. Enzyme immobilization for 72 h at pH 10.05, 25 degrees C and reduction with NaBH 4 in chitosan 2.5%-carrageenan 2.5%, with addition of S. cerevisiae 5% and activation with epichlorohydrin led to the best derivative, which was 9900-fold more stable than the soluble enzyme. This support allowed an enzyme load up to 40 mg chymotrypsin x g gel (-1). The number of covalent bonds, formed by active groups in the support and lysine residues of the enzyme, can explain the obtained results. SEM images of the gel structures corroborate these conclusions.     

蒜氨酸酶的固定化及其酶学性质研究

为了提高蒜氨酸酶的稳定性并实现酶的反复利用,研究了影响蒜氨酸酶固定化的因素及固定化蒜氨酸酶的酶学性质。蒜氨酸酶的固定化以壳聚糖微球为载体,戊二醛为交联剂,固定化的最适条件为：戊二醛浓度4%,给酶量20.2U,交联时间2h。固定化蒜氨酸酶的最适pH值7.0,最适温度35℃,米氏常数Km 7.9 mmol/L,操作稳定性比较好,连续使用10次后酶活力损失低于10%。     

Immobilization of keratinolytic metalloprotease from Chryseobacterium sp. strain kr6 on glutaraldehyde-activated chitosan

Keratinases are exciting keratin-degrading enzymes; however, there have been relatively few studies on their immobilization. A keratinolytic protease from Chryseobacterium sp. kr6 was purified and its partial sequence determined using mass spectrometry. No significant homology to other microbial peptides in the NCBI database was observed. Certain parameters for immobilization of the purified keratinase on chitosan beads were investigated. The production of the chitosan beads was optimized using factorial design and surface response techniques. The optimum chitosan bead production for protease immobilization was a 20 g/l chitosan solution in acetic acid [1.5% (v/v)], glutaraldehyde ranging from 34 g to 56 g/l, and an activation time between 6 and 10 h. Under these conditions, above 80% of the enzyme was immobilized on the support. The behavior of the keratinase loading on the chitosan beads surface was well described using the Langmuir model. The maximum capacity of the support (qm) and dissociation constant (Kd) were estimated as 58.8 U/g and 0.245 U/ml, respectively. The thermal stability of the immobilized enzyme was also improved around 2-fold, when compared with that of the free enzyme, after 30 min at 65 degrees C. In addition, the activity of the immobilized enzyme remained at 63.4% after it was reused five times. Thus, the immobilized enzyme exhibited an improved thermal stability and remained active after several uses.     

Stabilization of a raw starch digesting amylase from Aspergillus carbonarius via immobilization on activated and non-activated agarose gel

Applications of raw starch digesting amylases (RSDAs) are limited due to instability, product inhibition of enzyme and contamination. RSDA from Aspergillus carbonarius was stabilized through immobilization on agarose gel by adsorption, spontaneous crosslinking and conjugation using glycidol, glutaraldehyde or polyglutaraldehyde. Effects of immobilization on kinetics, catalytic, storage and operational stability of immobilized enzyme were evaluated. Polyglutaraldehyde activated agarose RSDA (PGAg-RSDA) gave the highest immobilization yield (100%) with expressed activity of 86.7% while that of glycidol activated RSDA (GlyAg-RSDA) was 80.4%. A shift in pH from optimum of 5 for the soluble enzyme to 6 for RSDA adsorbed on agarose followed by crosslinking with glutaraldehyde (AgRSDA-CROSS) and simultaneous adsorption and crosslinking (AgRSDA-RET), and pH 7 for PGAg-RSDA was seen. PGAg-RSDA and AgRSDA-CROSS were most pH stable and retained over 82% of their activities between pH 3.5 and 9 compared to 59% for the soluble enzyme. Thermoinactivation studies showed that immobilized RSDAs with the exception of GAg-RSDA retained over 90% of their activities at 60°C for 120 min while soluble enzyme retained only 76% activity under the same condition. AgRSDA-CROSS, PGAg-RSDA, Gly-RSDA and GAg-RSDA retained approximately 100% of their activities after 30 days storage at 4°C. GlyAg-RSDA retained 99.6%, PGAg-RSDA 94%, AgRSDA-CROSS 90%, GAg-RSDA 86.5% and Ag-RSDA-RET 80% activity after 10 batch reactions. Immobilization stabilized RSDA and permits processing at higher temperatures to reduce contamination.     

桦褶孔菌漆酶固定化及其对染料的降解

采用壳聚糖交联法和海藻酸钠-壳聚糖包埋交联法固定化桦褶孔菌产生的漆酶,探讨最佳固定化条件,固定化漆酶的温度,pH稳定性及操作稳定性,并以两种固定化酶分别对4种染料进行了降解.结果表明:(1)壳聚糖交联法固定化漆酶的最佳条件为:壳聚糖2.5%,戊二醛7%,交联时间2h,固定化时间5h,给酶量1g壳聚糖小球:1mL酶液(1U/mL),固定化效率56%;(2)海藻酸钠-壳聚糖包埋交联法固定化漆酶的最佳条件为:海藻酸钠浓度4%,壳聚糖浓度0.7%,氯化钙浓度5%,戊二醛浓度0.6%,给酶量4mL 4%海藻酸钠:1mL酶液(1U/mL),固定化效率高达86%;(3)固定化的漆酶相比游离漆酶有更好的温度和pH稳定性;(4)比较两种固定化漆酶,海藻酸钠-壳聚糖包埋交联法固定化酶的温度及酸度稳定性要优于壳聚糖固定化酶,但可重复操作性要弱于后者,两者重复使用8次后的剩余酶活比率分别为71%及64%;(5)两种固定化酶对所选的4种不同结构的合成染料均有较好的降解效果,其中壳聚糖固定化酶对茜素红的降解效果及重复使用性极佳,重复降解40mg/L的茜素红10次,降解率仍保持在100%.     

Immobilization of phosphorylase B and study of its enzymatic and immunological properties]

The properties of phosphorylase B (PhB) immobilized on an agar derivative were studied. It was shown that the enzyme activity makes up to 15-20% as compared to that of the soluble enzyme, the Km value for glucose-1-phosphate is increased 1.5-fold and the pH optimum remains unchanged, whereas the thermostability of enzyme shows a considerable increase. PhB immobilized on a highly activated sorbent completely losses its enzymatic activity but retains its antigenic properties and binds 1.6-2 mol antibodies (per monomer). Using immunosorbents, purified antibodies homogeneous during electrophoresis in polyacrylamide gel were isolated. The immunosorbent capacity is 500-800 mg of antibodies per 1 g of dry weight. The purified antibodies are characterized by a lower inhibitory power upon interaction with soluble PhB. The type of inhibition of both immobilized and soluble enzyme is similar. It is assumed that immobilization produces conformational changes only at the active site of enzyme, which is spatially separated from the antibody binding site.     

Immobilization of hemoglobin on chitosan films as mimetic peroxidase

Hemoglobin (Hb) was immobilized on the chitosan films using glutaraldehyde as a bifunctional agent. Atomic force microscopy (AFM) was used to examine the film surface in order to image the presence of Hb and Fourier transform infrared spectroscopy (FT-IR) was detected to elucidate the structural change of the immobilized Hb. The influences of several immobilization parameters were investigated, the optimum concentration of glutaraldehyde, pH and binding time were determined as 0.7%, 4.5 and 6 h, respectively. The enzymatic assay indicates that the immobilized Hb showed a higher thermal stability than that of free Hb, and the catalytic activity in organic solvents was also enhanced.     

Thermal inactivation and reactivity of beta-glucosidase immobilized on chitosan-clay composite

The dried and wet chitosan-clay composite beads were prepared by mixing equal weights of cuttlebone chitosan and activated clay and then spraying drop-wise through a syringe, with and without freeze-drying, respectively. These beads were then immersed in 5 g/L of glutaraldehyde solution at a dosage of 0.5 g/L and were cross-linked, which were finally used as supports for beta-glucosidase immobilization. The properties of the enzyme immobilized on wet- and dried-composite beads were compared. Kinetic modeling of thermal inactivation of free and immobilized enzymes was also investigated. For a given enzymatic reaction, the rate constant related to the decomposition of the enzyme-substrate complex to final product and the uncomplexed enzyme using dried-composite immobilized enzyme was larger than those using both free and wet-composite immobilized enzymes.     

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.     

Immobilization of Aspergillus oryzae β-galactosidase in ion exchange resins by combined ionic-binding method and cross-linking

The main objective of the present work is to study the immobilization process of Aspergillus oryzae β-galactosidase using the ionic exchange resin Duolite A568 as carrier. Initially, the immobilization process by ionic binding was studied through a central composite design (CCD), by analyzing the simultaneous influences of the enzyme concentration and pH on the immobilization medium. The results indicate that the retention of enzymatic activity during the immobilization process was strongly dependant of those variables, being maximized at pH 4.5 and enzyme concentration of 16 g/L. The immobilized enzyme obtained under the previous conditions was subjected to a cross-linking process with glutaraldehyde and the conditions that maximized the activity were a glutaraldehyde concentration of 3.83 g/L and cross-linking time of 1.87 h. The residual activity of the immobilized enzyme without glutaraldehyde cross-linking was 51% of the initial activity after 30 uses, while the enzyme with cross-linking immobilization was retained 90% of its initial activity. The simultaneous influence of pH and temperature on the immobilized β-galactosidase activity was also studied through a central composite design (CCD). The results indicate a greater stability on pH variations when using the cross-linking process.     

漆酶在磁性壳聚糖微球上的固定及其酶学性质研究

以磁性壳聚糖微球为载体,戊二醛为交联剂,共价结合制备固定化漆酶。探讨了漆酶固定化的影响因素,并对固定化漆酶的性质进行了研究。确定漆酶固定化适宜条件为：50 mg磁性壳聚糖微球,加入10mL 0.8mg/mL 漆酶磷酸盐缓冲液（0.1mol/L,pH 7.0）,在4℃固定2h。固定化酶最适pH为3.0, 最适温度分别为10℃和55℃,均比游离酶降低5℃。在pH 3.0,温度37℃时,固定化酶对ABTS的表观米氏常数为171.1μmol/L。与游离酶相比,该固定化漆酶热稳定性明显提高,并具有良好的操作和存储稳定性。     

Tuning of Lecitase features via solid-phase chemical modification: Effect of the immobilization protocol

Lecitase Ultra (a quimeric fosfolipase commercialized by Novozymes) has been immobilized via two different strategies: mild covalent attachment on cyanogen bromide agarose beads and interfacial activation on octyl-agarose beads. Both immobilized preparations have been submitted to different individual or cascade chemical modifications (amination, glutaraldehyde or 2,4,6-trinitrobenzensulfonic acid (TNBS) modification) in order to check the effect of these modifications on the catalytic features of the immobilized enzymes (including stability and substrate specificity under different conditions). The first point to be remarked is that the immobilization strongly affects the enzyme catalytic features: octyl-Lecitase was more active versus p-nitrophenylbutyrate but less active versus methyl phenylacetate than the covalent preparations. Moreover, the effects of the chemical modifications strongly depend on the immobilization strategy used. For example, using one immobilization protocol a modification improves activity, while for the other immobiled enzyme is even negative. Most of the modifications presented a positive effect on some enzyme properties under certain conditions, although in certain cases that modification presented a negative effect under other conditions. For example, glutaraldehyde modification of immobilized or modified and aminated enzyme permitted to improve enzyme stability of both immobilized enzymes at pH 7 and 9 (around a 10-fold), but only the aminated enzyme improved the enzyme stability at pH 5 by glutaraldehyde treatment. This occurred even though some intermolecular crosslinking could be detected via SDS-PAGE. Amination improved the stability of octyl-Lecitase, while it reduced the stability of the covalent preparation. Modification with TNBS only improved enzyme stability of the covalent preparation at pH 9 (by a 10-fold factor).     

壳聚糖固定化琼脂酶的研究

采用壳聚糖微球对琼脂酶进行固定化,在单因素实验的基础上用正交试验法确定最佳固定化工艺。结果表明:在戊二醛体积分数为2.5%,交联时间为6 h,加酶量为15 mL,固定时间为3 h时固定酶的活力最高;固定化酶的最适反应温度及最适pH分别为50℃和8.5,高于游离酶;同时其热稳定性及操作稳定性均高于游离酶。     

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.     

Multipoint covalent immobilization of lipase on chitosan hybrid hydrogels: influence of the polyelectrolyte complex type and chemical modification on the catalytic properties of the biocatalysts

This work aimed at the production of stabilized derivatives of Thermomyces lanuginosus lipase (TLL) by multipoint covalent immobilization of the enzyme on chitosan-based matrices. The resulting biocatalysts were tested for synthesis of biodiesel by ethanolysis of palm oil. Different hydrogels were prepared: chitosan alone and in polyelectrolyte complexes (PEC) with κ-carrageenan, gelatin, alginate, and polyvinyl alcohol (PVA). The obtained supports were chemically modified with 2,4,6-trinitrobenzene sulfonic acid (TNBS) to increase support hydrophobicity, followed by activation with different agents such as glycidol (GLY), epichlorohydrin (EPI), and glutaraldehyde (GLU). The chitosan-alginate hydrogel, chemically modified with TNBS, provided derivatives with higher apparent hydrolytic activity (HA_app) and thermal stability, being up to 45-fold more stable than soluble lipase. The maximum load of immobilized enzyme was 17.5 mg g⁻¹ of gel for GLU, 7.76 mg g⁻¹ of gel for GLY, and 7.65 mg g⁻¹ of gel for EPI derivatives, the latter presenting the maximum apparent hydrolytic activity (364.8 IU g⁻¹ of gel). The three derivatives catalyzed conversion of palm oil to biodiesel, but chitosan-alginate-TNBS activated via GLY and EPI led to higher recovered activities of the enzyme. Thus, this is a more attractive option for both hydrolysis and transesterification of vegetable oils using immobilized TLL, although industrial application of this biocatalyst still demands further improvements in its half-life to make the enzymatic process economically attractive.     

吸附-交联法固定D-泛解酸内酯水解酶的研究

选择6种吸附树脂和离子交换树脂对D-泛解酸内酯水解酶进行固定化,筛选出了固定化效果较好的大孔弱碱性丙烯酸系阴离子交换树脂D-380为载体,用先吸附后交联的方法固定化。通过实验对固定化条件进行了优化,得出最佳的固定化条件为：加酶量6U／g树脂、吸附pH7．5、吸附时间4h、吸附温度30℃、交联剂戊二醛终浓度0．1％、交联时间2h。实验表明在此条件下制得的固定化酶有很好的稳定性：固定化酶在连续20次的底物水解反应后,剩余酶活达到71％。当温度达到80℃时游离酶几乎失去酶活,而固定化酶剩余酶活为60％以上。游离酶的pH稳定性范围为pH7～8,而固定化酶为pH6．5～8．5。     

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.     

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.     

Urea potentiometric biosensor based on modified electrodes with urease immobilized on polyethylenimine films

The development of a new electrochemical sensor consisting in a glass-sealed metal microelectrode coated by a polyethylenimine film is described. The use of polymers as the entrapping matrix for enzymes fulfils all the requirements expected for these materials without damaging the biological material. Since enzyme immobilization plays a fundamental role in the performance characteristics of enzymatic biosensors, we have tested four different protocols for enzyme immobilization to determine the most reliable one. Thus the characteristics of the potentiometric biosensors assembled were studied and compared and it appeared that the immobilization method leading to the most efficient biosensors was the one consisting in a physical adsorption followed by reticulation with dilute aqueous glutaraldehyde solutions. Indeed, the glutaraldehyde immobilized urease sensor provides many advantages, compared to the other types of sensors, since this type of urea biosensor exhibits short response times (15–30 s), sigmoidal responses for the urea concentration working range from 1×10^−2.5 to 1×10^−1.5 M and a lifetime of 4 weeks.