Covalent crowding strategy for trypsin confined in accessible mesopores with enhanced catalytic property and stability

Chemically modified macromolecules were assembled with adsorptive trypsin in mesoporous silica foams (MCFs) to establish covalent linkage. Effects of catalytic properties and stability of immobilized trypsin were examined. The addition of chemically modified protein (BSA) and polysaccharide (ficoll) to the immobilized trypsin exhibited high coupled yield (above 90%) and relative activities (174.5% and 175.9%, respectively), showing no protein leaching after incubating for 10 h in buffers. They showed broader pH and temperature profiles, while the half life of thermal stability of BSA-modified preparation at 50 °C increased to 1.3 and 2.3 times of unmodified and free trypsin, respectively. The modified trypsin in aqueous-organic solvents exhibited 100% activity after 6 h at 50 °C. The kinetic parameters of trypsin preparations and suitable pore diameter of MCFs warranted compatibility of covalent modification for substrate transmission. The covalent crowding modification for immobilized trypsin in nanopores establishes suitable and accessible microenvironment and renders possibility of biological application.     

Simultaneous purification and immobilization of bitter gourd (Momordica charantia) peroxidases on bioaffinity support

This paper demonstrates the construction of an inexpensive bioaffinity adsorbent by simply incubating Sephadex G 50 matrix with jack bean meal extract at room temperature. Sephadex G 50 adsorbed 17 mg Con A (concanavalin A) per g of the matrix. Con A‐adsorbed Sephadex was employed for the immobilization of glycoenzymes directly from ammonium sulfate‐fractionated proteins of bitter gourd. The obtained bioaffinity support was very efficient for high yield immobilization of peroxidases from bitter gourd and it bound nearly 425 enzyme units per g of the matrix. Bitter gourd peroxidase immobilized on lectin–Sephadex support showed a very high effectiveness factor, ‘η,’ of 1.25. Immobilized BGP preparation was quite stable against the denaturation induced by pH, heat, urea, Triton X 100, Tween 20, SDS, Surf Excel and water‐miscible organic solvents: dimethyl sulfoxide and dimethyl formamide. Low concentration of detergents like SDS, Tween 20, and Triton X 100 enhanced the activity of soluble and immobilized bitter gourd peroxidase. Peroxidase bound to the bioaffinity support exhibited very high resistance to proteolysis caused by the trypsin treatment. Con A–Sephadex‐bound bitter gourd peroxidase retained 85% of its initial activity after treatment with 2.5 mg trypsin per cm³ of incubation mixture for 1 h at 37 °C while the soluble enzyme lost nearly 40% of the initial activity under similar incubation conditions. Immobilized bitter gourd peroxidase preparation appeared to be more rigid to proteolysis mediated by trypsin compared with soluble bitter gourd peroxidase. Copyright © 2004 Society of Chemical Industry     

Improvement of lactulose synthesis through optimization of reaction conditions with immobilized β-galactosidase

Kluyveromyces lactis β-galactosidase was immobilized on silica gels using a covalent bonding method. To improve lactulose synthesis using immobilized β-galactosidase, the optimal reaction conditions, such as lactose and fructose concentrations, pH and ionic strength of the buffer, loading amount of the enzyme and temperature, were determined. Lactulose synthesis using the immobilized β-galactosidase was markedly improved after optimization of the reaction conditions. When the lactulose synthesis was carried out at 47 °C using 40% (w/v) lactose, 20% (w/v) fructose and immobilized β-galactosidase of 12 U/ml in 50 mM sodium phosphate buffer at pH 7.5, the lactulose concentration and specific productivity were 15.80 g/l and 1.32 mg/U·h, respectively. In addition, when the immobilized β-galactosidase was reused for lactulose synthesis, its catalytic activity retained 60.5% after 10 reuses.     

大豆脂肪氧合酶(LOX)的固定化及增强稳定性

以活性白土、滑石粉为载体采用吸附法固定化大豆脂肪氧合酶(LOX)的优化条件为:(1)酶液对活性白土的用量比为897U/mg,在浓度为0 05mol/L、pH=7 0的磷酸盐缓冲液中20℃搅拌吸附30min;(2)酶液对滑石粉的用量比为238U/mg,在浓度为0 05mol/L、pH=8 0的磷酸盐缓冲液中10℃搅拌吸附15min。经放大后实验重复性良好。上述固定化酶置于0～4℃保存20d,酶活损失分别为19 2%和17 7%;与游离酶相比,能加快酶促反应的速度并使产率分别提高9 6%和42 5%。以海藻酸钠为载体采用包埋法得到的固定化酶珠的耐热、耐酸碱、耐有机溶剂能力较游离酶有很大提高,在φ(甘油)=75%的水溶液中保存33d,酶活基本保持不变;在50℃热处理60min后酶活仍能保持90%。     

Enhancement of Alkaline Protease Activity and Stability via Covalent Immobilization onto Hollow Core-Mesoporous Shell Silica Nanospheres

The stability and reusability of soluble enzymes are of major concerns, which limit their industrial applications. Herein, alkaline protease from Bacillus sp. NPST-AK15 was immobilized onto hollow core-mesoporous shell silica (HCMSS) nanospheres. Subsequently, the properties of immobilized proteases were evaluated. Non-, ethane- and amino-functionalized HCMSS nanospheres were synthesized and characterized. NPST-AK15 was immobilized onto the synthesized nano-supports by physical and covalent immobilization approaches. However, protease immobilization by covalent attachment onto the activated HCMSS–NH₂ nanospheres showed highest immobilization yield (75.6%) and loading capacity (88.1 μg protein/mg carrier) and was applied in the further studies. In comparison to free enzyme, the covalently immobilized protease exhibited a slight shift in the optimal pH from 10.5 to 11.0, respectively. The optimum temperature for catalytic activity of both free and immobilized enzyme was seen at 60 °C. However, while the free enzyme was completely inactivated when treated at 60 °C for 1 h the immobilized enzyme still retained 63.6% of its initial activity. The immobilized protease showed higher V_max, k_cat and k_cat/K_m, than soluble enzyme by 1.6-, 1.6- and 2.4-fold, respectively. In addition, the immobilized protease affinity to the substrate increased by about 1.5-fold. Furthermore, the enzyme stability in various organic solvents was significantly enhanced upon immobilization. Interestingly, the immobilized enzyme exhibited much higher stability in several commercial detergents including OMO, Tide, Ariel, Bonux and Xra by up to 5.2-fold. Finally, the immobilized protease maintained significant catalytic efficiency for twelve consecutive reaction cycles. These results suggest the effectiveness of the developed nanobiocatalyst as a candidate for detergent formulation and peptide synthesis in non-aqueous media.     

Synthesis of Vitamin A Esters by Immobilized Candida sp. Lipase in Organic Media

Vitamin A ester was synthesized in organic solvents with immobilized lipase from Candida sp. The types of lipases, influences of solvent, the molar ratio of substrates, the reaction temperature and the water activity in the reaction were studied in detail in order to obtain the optimum conditions for Vitamin A palmitate synthesis. In a system of hexane, 100mg immobilized Candida sp. lipase was used in the presence of 1.2mmol vitamin A acetate and 3.6mmol palmitic acid. The yield of vitamin A palmitate reached 81% in 12h at 25℃. The immobilized Candida sp. lipase was prepared by adsorbing Cand/da sp. fermentation broth on pretreated textile and could be reused for at least six batches.     

Application of Immobilized α-Chymotrypsin to Peptide Synthesis in Frozen Aqueous Systems

In order to combine the high potential of frozen state peptide synthesis and the advantages of the application of immobilized proteases, the capability of carrier-bound α-chymotrypsin (CT, EC 3.4.21.1) to form peptide bonds in frozen aqueous reaction mixtures was investigated. The properties of the support materials strongly influenced the peptide yields. CT bound to rather hydrophobic supports catalysed peptide synthesis as effectively as the soluble enzyme. The immobilized enzyme preparations could be successfully re-used at least 15 times. Peptide synthesis catalysed by immobilized chymotrypsin was also studied in low water organic reaction mixtures at sub-zero temperatures. The advantages and limitations of the different approaches are discussed. © 1997 SCI.     

融合蛋白CR2-GDH固定化及不对称还原制备(S)-4-氯-3-羟基丁酸乙酯

比较介孔分子筛材料SBA-15、MCM-41、海藻酸钙、改性二氧化硅4种载体固定化融合蛋白CR2-GDH其酶固载量和酶活回收率,选择SBA-15为固定化载体。研究固定化条件对固定化融合酶量的影响以及固定化酶的稳定性,固定化酶在双相体系催化不对称还原反应。结果表明,在pH值为5.5、酶浓度为1.4mg/mL、反应1h条件下,固定化酶量为27.7mg/g。加入25mmol/L的Ca²⁺,固定化酶的酶活回收率由58.6%提高到78.1%。与游离酶相比,固定化酶的热稳定性显著提高,40℃条件下酶活回收率提高19.1%。固定化酶水相中反复使用7批次后,剩余活性仍超过30%,具有较好的操作稳定性。与游离酶相比,固定化酶更耐受烷烃类有机溶剂。在水/有机溶剂双相反应体系中,Ca²⁺/SBA-15固定化酶和游离酶催化相比,产物得率提高23.8%。     

纳米粒子固定化双酶耦合体系连续催化制备(R)-苯基乙二醇

将活化醇盐水解法制备的SiO2纳米粒子分别与羰基还原酶(CR)和甲酸脱氢酶(FDH)进行共价固定化,固定化CR与FDH耦合,连续催化转化b-羟基苯乙酮制备(R)-苯基乙二醇,考察了NADH的再生与循环利用性. 结果表明,纳米粒子固定化CR和FDH酶载量分别为3.32和5.55 mg/g,催化活性为游离酶的50%~60%,最适反应pH值分别为6.5和8.5,最适反应温度分别为40和45℃. 耦合体系进行12批次反应,产物(R)-苯基乙二醇累积量达35.6 g/L,纳米粒子生产能力达178 g/g. 纳米粒子固定化酶经简单离心收集后可重复利用.     

Pt/C催化加氢3-氯-4-甲基硝基苯制备3-氯-4-甲基苯胺

3-氯-4-甲基苯胺是合成有机颜料、染料和农药的重要有机中间体。以3-氯-4-甲基硝基苯为原料,1%Pt/C为催化剂,低压催化加氢制备3-氯-4-甲基苯胺,考察不同溶剂、反应压力、反应温度和催化剂用量对产物收率的影响。结果表明,在3-氯-4-甲基硝基苯10 g、溶剂甲醇用量30 m L、1%Pt/C催化剂用量0.04 g、反应温度80℃和反应压力1.0 MPa条件下,3-氯-4-甲基苯胺收率99.08%,脱氯率0.2%,催化剂可重复使用5次。     

酶法制备一氯乙酸对硝基苄酯及催化反应动力学模型

研究了固定化脂肪酶Novozym 435催化对硝基苄醇和一氯乙酸制备一氯乙酸对硝基苄酯的过程,并对酶法合成反应条件进行优化,确定最佳反应条件为:甲苯作溶剂,对硝基苄醇与一氯乙酸摩尔比1:2,对硝基苄醇浓度5 g L 1,Novozym 435脂肪酶浓度为3.4 g L 1,反应温度50℃,反应时间10 h,对硝基苄醇转化率为76.7%。最后探索酶催化合成一氯乙酸对硝基苄酯动力学反应,得出该反应动力学模型符合双底物乒乓机理和一氯乙酸底物抑制动力学模型的结论并写出了其动力学方程。     

Immobilization and stabilization of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> SRT9 lipase on tri(4-formyl phenoxy) cyanurate

Lipase was extracted and purified from Pseudomonas aeruginosa SRT9. Culture conditions were optimized and highest lipase production amounting to 147.36 U/ml was obtained after 20 h incubation. The extracellular lipase was purified on Mono QHR5/5 column, resulting in a purification factor of 98-fold with specific activity of 12307.81 U/mg. Lipase was immobilized on tri (4-formyl phenoxy) cyanurate to form Schiff’s base. An immobilization yield of 85% was obtained. The native and immobilized lipases were used for catalyzing the hydrolysis of olive oil in aqueous medium. Comparative study revealed that immobilized lipase exhibited a shift in optimal pH from 6.9 (free lipase) to 7.5 and shift in optimal temperature from 55 °C to 70 °C. The immobilized lipase showed 20–25% increase in thermal stability and retained 75% of its initial activity after 7 cycles. It showed good stability in organic solvents especially in 30% acetone and methanol. Enzyme activity was decreased by ∼60% when incubated with 30% butanol. The kinetic studies revealed increase in K _M value from 0.043 mM (native) to 0.10 mM for immobilized lipase. It showed decrease in the V _max of immobilized enzyme (142.8 μmol min⁻¹ mg⁻¹), suggesting enzyme activity decrease in the course of covalent binding. The immobilized lipase retained its initial activity for more than 30 days when stored at 4 °C in Tris-HCl buffer pH 7.0 without any significant loss in enzyme activity.     

Continuous enzymatic synthesis of Z-kyotorphin amide in an enzyme-immobilized fixed-bed reactor

α-Chymotrypsin immobilized on natural and inexpensive supports such as diatomaceous earth was used as catalyst for Z-Tyr-Arg-NH₂ (Z-kyotorphin amide) synthesis. In order to obtain the optimal reaction conditions, a 2² factorial experimental design was used. The factors considered were cosolvent (dimethylformamide) concentration and temperature; optimal product yield was achieved at 40% (v/v) dimethylformamide and 25°C. A sequential kinetic model was considered which generally gave good agreement between experimental and theoretical data for continuous synthesis of Z-kyotorphin amide in a packed-bed immobilized reactor system. The activation energy for the synthesis was determined to be 48.0 ± 2.3 kJ mol^?1.     

Carbonaceous–siliceous composite materials as immobilization support for lipase from Alcaligenes sp.: Application to the synthesis of antioxidants

Two carbonaceous–siliceous composite materials, produced by hydrothermal and carbonization processes, were evaluated as immobilization support for lipase from Alcaligenes sp. These materials exhibited similar chemical characteristics but their carbon content and porous characteristics were different, which explain the catalytic behavior and stability of the biocatalysts immobilized on them. Higher activity and immobilization selectivity was achieved with the microporous material that had higher carbon content. The lipase immobilized on the mesoporous material had a higher thermal stability at 55 °C, pH 7.0 or at 40 °C in tert-butanol, simulating the reaction conditions required for organic synthesis. Both biocatalysts were tested in the synthesis of palmitoyl ascorbate and they were compared with the commercial biocatalyst QLC. The synthesis conversions with the lipase immobilized in mesoporous materials and with the biocatalyst QLC were similar (50%), but only the former could be reused. These are promising biocatalysts for industrial applications.     

Entrapment of porous and stable concanavalin A–peroxidase complex into hybrid calcium alginate–pectin gel

Ammonium sulfate‐fractionated proteins of turnip (Brassica rapa) were used for the simultaneous purification and immobilization of peroxidase by using crude jack bean extract. The concanavalin A–turnip peroxidase complex retained nearly 70% of the original activity. Calcium alginate–pectin‐entrapped soluble turnip peroxidase and the concanavalin A complex of peroxidase retained 63% and 52% of the original activity, respectively. The concanavalin A–peroxidase complex, the alginate–pectin‐entrapped soluble peroxidase and the alginate–pectin‐entrapped concanavalin A–peroxidase complex showed very high stability against denaturation mediated by heat, pH, urea, organic solvents and detergents. The exposure of soluble and immobilized turnip peroxidase to trypsin resulted in an enhancement of the peroxidase activity. The concanavalin A–peroxidase complex entrapped preparation was markedly more stable as compared with the directly entrapped soluble enzyme preparation. The results suggested that such preparations have great potential in the construction of bioreactors to be used for the remediation of aromatic compounds present in polluted wastewater/industrial effluents. Copyright © 2006 Society of Chemical Industry     

A novel method to prepare chitosan powder and its application in cellulase immobilization

In this study, chitosan microspheres and sponges with uniform spherical and porous morphologies were prepared by coiling the stretched chains of chitosan with addition of salt and choosing different kinds of organic solvents as evaporation solvents. Cellulase was immobilized to the support by a covalent method. The enzyme exhibited a considerable affinity to the support, and the protein loading of 145.5 mg g^?1 support was fairly high. The immobilized cellulase had a higher K_m than free cellulase and had better stability with respect to pH, thermal stability, reuses and storage stability than free cellulase. Copyright © 2005 Society of Chemical Industry     

Pd/C催化剂催化邻硝基苯胺加氢制备邻苯二胺

卤代芳胺是重要的有机中间体,广泛应用于合成染料、农药、医药、香料及橡胶助剂等。卤代芳香硝基化合物通过液相催化加氢制备卤代芳胺的技术以其环境友好、产品质量稳定和工艺先进而受到重视。用负载型贵金属催化剂催化芳香硝基化合物选择加氢制备相应的芳胺有广泛的应用价值。采用邻硝基苯胺为原料,Pd/C为催化剂,低压催化加氢还原合成邻苯二胺,考察不同溶剂、反应压力、反应温度和反应时间对产物收率的影响。结果表明,在甲醇为溶剂、反应温度100 ℃、反应压力0.8 MPa和反应时间100 min条件下,邻苯二胺平均收率为97%。与传统硫化碱还原或铁粉化学法还原工艺相比,以甲醇为溶剂,Pd/C催化剂催化加氢法在减少废水和降低成本等方面有较大优势。     

Liver alcohol dehydrogenase immobilized on polyvinylidene difluoride

A physical method for immobilization of liver alcohol dehydrogenase (ADH) by hydrophobic adsorption onto a supporting membrane of polyvinylidene difluoride (PVDF) was performed. Simultaneously, a physicochemical characterization of the immobilized enzyme regarding its kinetic behaviour was performed. The activity/pH profile observed points to an effect of pH on activity that is completely different from the case of ADH in solution. The disturbance in the typical bell-shaped profile owing to the fact that the enzyme was immobilized is explained on the basis of a potent limitation to the diffusion of the protons in the support. The findings of the present work also reveal the existence of an effect that limits free external diffusion of the substrate towards and/or the product from the support; this effect seems to be the determinant of the overall rate of the enzymatic reaction and is thus of great importance in the effective kinetic behaviour (v([S])) of immobilized ADH, whose kinetic behaviour is complex (non-Michaelian), as may be seen from the lack of linearity observed in the corresponding double reciprocal and Eadie-Hofstee plots. By non-linear regression numerical analysis of the v([S]) data and application of the F-test for model discrimination, the minimum rate equation necessary to describe the intrinsic kinetic behaviour ofPVDF-immobilized ADH proved to be one of the polynomial quotient type of degree 2:2 (in substrate concentration).     

Covalent immobilization of naringinase for the transformation of a flavonoid

Naringinase (EC 3.2.1.40) from Penicillium sp was immobilized by covalent binding to woodchips to improve its catalytic activity. The immobilization of naringinase on glutaraldehyde‐coated woodchips (600 mg woodchips, 10 U naringinase, 45 °C, pH 4.0 and 12h) through 1% glutaraldehyde cross‐linking was optimized. The pH–activity curve of the immobilized enzyme shifted toward a lower pH compared with that of the soluble enzyme. The immobilization caused a marked increase in thermal stability of the enzyme. The immobilized naringinase was stable during storage at 4 °C. No loss of activity was observed when the immobilized enzyme was used for seven consecutive cycles of operations. The efficiency of immobilization was 120%, while soluble naringinase afforded 82% efficacy for the hydrolysis of standard naringin under optimal conditions. Its applicability for debittering kinnow mandarin juice afforded 76% debittering efficiency. Copyright © 2005 Society of Chemical Industry     

Kinetics of synthesizing 2,4,6-tribromophenyl benzyl ether in a triphase catalysis

The reaction of benzyl bromide with 2,4,6-tribromophenol in an organic solvent/aqueous alkaline solution of KOH to synthesize 2,4,6-tribromophenyl benzyl ether by triphase catalysis was investigated. The macroporous and microporous polymer pellets that served to support the catalyst were prepared by reacting styrene monomers, chloromethylstyrene monomers, and divinylbenzene in a suspension polymerization. Tributylamine was then immobilized on the surface of the polymer pellet to form the three-phase phase-transfer catalysts. The effects of the rate of agitation, degree of cross-linking of the polymer, solvents, inorganic salts, and temperature on the conversion of benzyl bromide and the reaction rate in the three-phase catalytic reaction were investigated in detail. We recommend that a macroporous polymer pellet with a small degree of cross-linking be used to react in a highly polar solvent to obtain high conversion of benzyl bromide and a rapid reaction. © 1994 John Wiley & Sons, Inc.