小分子封闭提高固定化青霉素酰化酶的稳定性

为提高青霉素酰化酶的催化性能和热稳定性,在酶组装过程中添加小分子试剂对介孔泡沫硅载体表面过量的活化位点进行封闭。详细考察了小分子添加质量分数和种类对青霉素酰化酶负载率、催化活力及热稳定性的影响。实验结果得到:经精氨酸封闭的固定化酶活力提高至1.92倍;甘氨酸封闭的固定化酶5 h的50℃热稳定性提高至2.9倍,甘氨酸和谷氨酸封闭的固定化酶50℃热处理25 h仍保持87.9%和82.2%的残余活力;甘氨酸和谷氨酸封闭的固定化酶最适催化pH值向中性偏移且对pH值的耐受性增强。结果表明,在青霉素酰化酶共价组装过程中添加合适的小分子封闭能显著提高酶的催化性能和热稳定性。     

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.     

修饰固定化青霉素酰化酶在非水相中的催化

为提高酶的催化水解活力和稳定性,将青霉素酰化酶组装于介孔泡沫二氧化硅(MCFs)中,并应用于水/有机混合体系催化水解。分别考察了有机介质种类和体积分数、葡聚糖(Dex10k)修饰对固定化酶活力的影响,研究了不同条件下固定化酶的稳定性。实验结果显示:体积分数20%石油醚中,添加Dex10k的介孔泡沫硅固定化酶比活力达209.5U/mg,是缓冲液中MCFs固定化酶活力的196.2%。20%石油醚中,经25次连续操作,固定化酶保持初始活力的71.5%。结果表明:石油醚等烷烃形成的水/有机体系是适合青霉素酰化酶催化的二相体系,且添加Dex10k能提高固定化酶在二相体系中的催化活力及稳定性。     

介孔纳米孔道中单维交联酶策略固定青霉素酰化酶

为解决酶固定化过程中稳定性较差的问题,将单维交联酶策略引入青霉素酰化酶的固定化中,通过在介孔泡沫硅载体(MCFs)表面初步吸附作用将酶固定,再用对本醌对酶分子进行交联,从而制得单维交联酶聚体。实验结果显示:单维交联酶聚体(CLEA)的稳定性有明显提高,最适温度从游离酶的45℃上升到55℃,催化温度的适用范围更宽,在50℃下热处理25 h仍保持74.4%活力,在测试操作稳定性方面,连续操作15次,其酶活保持在61.9%。     

Freeze‐drying of lignin peroxidase: influence of lyoprotectants on enzyme activity and stability

The effect of different lyoprotectants (sucrose, dimethyl‐succinate buffer (DMS), bovine serum albumin (BSA), mannitol and dextran, mw 60 kDa) on the stability of the enzyme lignin peroxidase (LiP, EC number: 1.11.1.‐), both during the freeze‐drying process and storage were investigated. The shelf stability tests were performed at 4 °C and 27 °C. Both DMS buffer and sucrose showed a good protective action: the former was particularly effective during the process, while the latter improved the stability during storage. In contrast, mannitol and dextran had negative effects, reducing the activity also in the lignin peroxidase solution. BSA was discarded because, in the range of compatible concentrations with LiP, it does not confer a consistent structure to the freeze‐dried product. © 2002 Society of Chemical Industry     

Characterization of a chemically modified β-amylase immobilized on porous silica

β-Amylase was coupled to a periodate oxidized dextran by reductive alkylation in the presence of sodium cyanoborohydride. The loss of activity (57%) during the cross-linking of the enzyme was the result of steric hindrance near the catalytic site. In order to verify this hypothesis, the residual activity was determined with substrates of variable molecular size. The residual activity was inversely proportional to the particulate size of the substrate. Increases in residual activity, of up to 53% were obtained using an orientated chemical modification in the presence of a substrate which protects the catalytic site. Native and dextran-conjugated β-amylase were immobilized on an amino activated silica by a classical method using glutaraldehyde for the native enzyme and by reductive alkylation for the modified enzyme. The relative activity of the enzymes obtained after this insolubilization was very high for the modified amylase, 45% for a medium enzyme concentration, compared with 4% at the same concentration for the native enzyme. This difference can be attributed to the variation of the length of the space arm between the silica and the enzyme. The soluble β-amylase dextran conjugate had a superior thermoresistance to that of the native enzyme; its optimal temperature of activity was 5°C higher than that of the native enzyme. This stabilization may be attributable to a rigidification of the protein structure. Immobilization of both native and modified enzymes on a amino silica resulted in thermostabilization of the enzymes. The optimal temperature of activity was 70°C for the native immobilized β-amylase (some 10°C higher than that of the native enzyme) and 75°C for the chemically modified, and subsequently immobilized, β-amylase. The immobilized forms of the enzymes were used for 14 days at 55°C in continuous substrate processing. The greater eflciency of the chemically modified β-amylase was demonstrated by a four-fold increase in maltose production compared to the classical method of immobilization. A kinetic study confirmed the stabilization of the β-amylase by a reduction of the rate constant of inactivation of the different modified enzymes.     

Urease immobilized on chitosan membrane: Preparation and properties

Urease was covalently immobilized on glutaraldehyde-pretreated chitosan membranes. The optimum immobilization conditions were determined with respect to glutaraldehyde pretreatment of membranes and to reaction of glutaraldehyde-pretreated membranes with urease. The immobilized enzyme retained 94% of its original activity. The properties of free and immobilized urease were compared. The Michaelis constant was about five times higher for immobilized urease than for the free enzyme, while the maximum reaction rate was lower for the immobilized enzyme. The stability of urease at low pH values was improved by immobilization; temperature stability was also improved. The optimum temperature was determined to be 65°C for the free urease and 75°C for the immobilized form. The immobilized enzyme had good storage and operational stability and good reusability, properties that offer potential for practical application.     

Immobilization of proteolytic enzyme on highly porous activated carbon derived from rice bran

Highly porous activated carbon (HPAC) was used as carrier matrix for immobilization of acid protease (AP). Immobilization of acid protease on mesoporous activated carbon (AP-HPAC) performs as best enzyme carrier. At pH 6.0, 250 mg acid protease g⁻¹ HPAC was immobilized. The optimum temperature for both free and immobilized AP activities were 50 °C. After incubation at 50 °C, the immobilized AP maintained about 50% of its initial activity, while the free enzyme was completely inactivated. When testing the reusability of AP-HPAC combination immobilized system, a significant catalytic efficiency was maintained along more than five consecutive reaction cycles. The highly porous nature of the carbon permits significant higher loadings of enzyme, which results in a higher enzyme-support strength and increased stability. The changes in the AP, HPAC and AP-HPAC were confirmed by Fourier Transform Infrared spectroscopy (FT-IR). Furthermore, scanning electron microscopy (SEM) allowed us to observe that the morphology of the surface of HPAC and the AP-HPAC.     

Purification and properties of β-amylase produced by bacillus polymyxa

1,4-α-D -glucan maltohydrolase (β-amylase, EC.3.2.1.2.) produced by Bacillus polymyxa was isolated and purified. Some of its properties were examined and compared with those of a typical plant β-amylase. Hydrolysis of periodate oxidised amylose demonstrated an exo mechanism of substrate attack similar to that of sweet potato β-amylase. The effect of sulphydryl reagents on enzyme activity was similar to that reported for plant β-amylases. Consistent with the observation that the enzyme has an exo mechanism of action, it also failed to degrade Schardinger cyclodextrins. These latter compounds acted as inhibitors of the enzyme. The optimum temperature for activity was 37 °C. The enzyme was quite stable at temperatures up to and including 37 °C; 90% of the original activity remained after storage at 37 °C for 6 days. However, the stability decreased rapidly when exposed to temperatures above 37 °C; only 20% of the activity remained after 1 h at 45 °C. The hydrolase exhibited a rather sharp optimum at pH 6.8 for stability at 37 °C. However, the enzyme was quite stable in the pH range 6.4–7.2 at 20 °C but it was shown to be less stable in acidic conditions than the corresponding plant enzymes.     

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     

Immobilization of β-glucosidase and its aroma-increasing effect on tea beverage

β-Glucosidase was effectively immobilized on alginate by the method of crosslinking–entrapment–crosslinking. After optimization of the immobilized conditions, the activity recovery of immobilized β-glucosidase achieved to 46.0%. The properties of immobilized β-glucosidase were investigated. Its optimum temperature was determined to be 45 °C, decreasing 10 °C compared with that of free enzyme, whereas the optimum pH did not change. The thermal and pH stabilities of immobilized β-glucosidase increased to some degree. The K_m value for immobilized β-glucosidase was estimated to be 1.97 × 10^?3 mol/L. The immobilized β-glucosidase was also applied to treat the tea beverage to investigate its aroma-increasing effect. The results showed that after treated with immobilized β-glucosidase, the total amount of essential oil in green tea, oolong tea and black tea increased by 20.69%, 10.30% and 6.79%, respectively. The storage stability and reusability of the immobilized β-glucosidase were improved significantly, with 73.3% activity retention after stored for 42 days and 93.6% residual activity after repeatedly used for 50 times.     

Covalent immobilization of penicillin G acylase onto amine-functionalized PVC membranes for 6-APA production from penicillin hydrolysis process. II. Enzyme immobilization and characterization

This article describes the covalent immobilization of penicillin G acylase (PGA) onto glutaraldehyde-activated NH₂-PVC membranes. The immobilized enzyme was used for 6-aminopenicillanic acid production from penicillin hydrolysis. Parameters affecting the immobilization process, which affecting the catalytic activity of the immobilized enzyme, such as enzyme concentration, immobilization's time and temperature were investigated. Enzyme concentration and immobilization's time were found of determine effect. Higher activity was obtained through performing enzyme immobilization at room temperature. Both optimum temperature (35°C) and pH (8.0) of immobilized enzyme have not been altered upon immobilization. However, immobilized enzyme acquires stability against changes in the substrate's pH and temperature values especially in the higher temperature region and lower pH region. The residual relative activities after incubation at 60°C were more than 75% compared to 45% for free enzyme and above 50% compared to 20% for free enzyme after incubation at pH 4.5. The apparent kinetic parameters K_M and V_M were determined. K_M of the immobilized PGA (125.8 mM) was higher than that of the free enzyme (5.4 mM), indicating a lower substrate affinity of the immobilized PGA. Operational stability for immobilized PGA was monitored over 21 repeated cycles. The catalytic membranes were retained up to 40% of its initial activity after 10.5 working h. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

固定化L-天门冬酰胺酶的性质及其填充床反应器的研究

利用海藻酸钙包埋、戊二醛交联的方法对L-天门冬酰胺酶进行固定化。研究了固定化L-天门冬酰胺酶的最适pH、最适温度、米氏常数、半衰期等酶学性质,并考察了影响固定化酶柱式填充床反应器转化率的因素。结果表明:固定化酶最适pH值为8.5,最适温度为67°C,固定化酶的米氏常数Km增大,固定化酶半衰期随着温度的增加而逐渐减小;温度、反应柱内径、底物溶液浓度、流速等因素对填充床反应器转化率均有显著影响。     

Study of cyclodextrin production using cyclodextrin glycosyltransferase immobilized on chitosan

Cyclodextrin glycosyltransferase (CGTase EC.3.2.1.19) was immobilized on chitosan through covalent bonding using glutaratdehyde. Various characteristics of immobilized CGTase (ICGTase) such as the pH—activity curve, the temperature—activity curve, pH and thermal stability, batch re-usability and continuous operation stability were evaluated. Among them, the pH optimum and temperature optimum of CGTase were shifted from pH 7.5 and 55°C to pH 8.5 and 60°C after immobilization. No increase of pH stability was observed, whereas the thermal stability of ICGTase was superior to that of free CGTase. ICGTase retained 83 % of relative activity in converting liquefied starch (5% (w/v)) to cyclodextrins (CDs) under the conditions of four times of repeated use of 23 h conversion by the batch system at 55°C and pH 8.5. The half-life of ICGTase under the same conditions by the continuous operation system was about 6 days. About 46 % of the liquefied potato starch solution (5 % (w/v)) was converted to CDs by ICGTase. Addition of ethanol in starch solution was found to increase the formation of β-CD, and 58-3 % yield of CDs was obtained by the addition of 16 % (v/v) ethanol in starch. The kinetic constants K_m and V_max of ICGTase and free CGTase were also examined.     

Triglyceride interesterification by lipases. 1. Cocoa butter equivalents from a fraction of palm oil

Twelve commercially available triacylglycerol lipase preparations were screened for their suitability as catalysts in the interesterification of palm oil mid fraction and ethyl stearate to form a cocoa butter equivalent. Five fungal lipase preparations were found to be suitable. The hydrolytic activity of the commercial lipase preparations was tested with sunflower seed oil and was independent of their interesterification activity. The operational stability of three of the preparations most suited for production of cocoa butter equivalents was examined. The amount of a commercial lipase preparation loaded onto a support was surveyed for optimum short-term catalytic activity. The influence of solvent concentration on the reaction rate and the purity of the product was examined at two temperatures. The optimum solvent concentration at 40°C was 1–1.5 grams of solvent/gram of substrate; at 60°C, the rate of interesterification diminished and the purity of the product decreased with increasing amounts of solvent. Four of the commercial lipase preparations found to be suitable interesterification catalysts were immobilized on five supports and their ability to catalyze the interesterification of a triglyceride and palmitic acid or ethyl palmitate was measured. The choice of support and substrate form (esterified or free fatty acid) greatly affected the catalytic activity. Some preparations were more affected by the choice of support, others by the form of the substrate. No preparation yielded maximum activity on all supports, and no support was found which produced an immobilized enzyme preparation of high activity with every commercial lipase preparation. Caution is advised in transferring observations about the suitability of a support from tests on one commerical enzyme preparation to others; individual testing is required.     

Improvement of catalytic efficiency of chloroperoxidase by its covalent immobilization on SBA-15 for azo dye oxidation

Chloroperoxidase from the fungus Caldariomyces fumago was covalently immobilized on SBA-15 mesoporous material and assayed for the enzymatic oxidation of four azo dyes. All dyes were oxidized by the free and the immobilized enzyme to different extent. Acid Blue 120 and Direct Blue 85 dyes were decolorized almost completely. The catalytic efficiency, k _cat/K _M, of the immobilized enzyme was 27, 2.9, 137 and 28 times higher than the free enzyme for Basic Blue 41, Disperse Blue 85, Acid Blue 120 and Direct Black 22 oxidation, respectively. The immobilized enzyme displayed improved thermostability and a similar pH profile compared to the free enzyme. The immobilized chloroperoxidase showed excellent storage stability and maintained 100 % catalytic activity after 90 days at both 4 and 25 °C.     

Immobilization of polyphenol oxidase on carboxymethylcellulose hydrogel beads: preparation and characterization

Carboxymethylcellulose (CMC) beads were prepared by a liquid curing method in the presence of trivalent ferric ions, and epicholorohydrin was covalently attached to the CMC beads. Polyphenol oxidase (PPO) was then covalently immobilized onto CMC beads. The enzyme loading was 603 µg g⁻¹ bead and the retained activity of the immobilized enzyme was found to be 44%. The K_m values were 0.65 and 0.87 mM for the free and the immobilized enzyme, and the V_max values were found to be 1890 and 760 U mg⁻¹ for the free and the immobilized enzyme, respectively. The optimum pH was 6.5 for the free and 7.0 for the immobilized enzyme. The optimum reaction temperature for the free enzyme was 40 °C and for the immobilized enzyme was 45 °C. Immobilization onto CMC hydrogel beads made PPO more stable to heat and storage, implying that the covalent immobilization imparted higher conformational stability to the enzyme. © 2000 Society of Chemical Industry     

Immobilization of α-Amylase to Temperature-Responsive Polymers by Single or Multiple Point Attachments

Temperature-responsive N-isopropylacrylamide (NIPAAm) polymer (PNIPAAm) with a free carboxyl functional end group and a copolymer (NIPNAS) of NIPAAm and N-acryloxysuccinimide (NAS) were synthesized and used for immobilization of α-amylase. The enzyme forms covalent bonds with the former polymer by single point attachment and with the latter polymer by multiple point attachment. Such a difference influences the enzyme activity and properties of the immobilized enzymes. The polymers are temperature-sensitive with lower critical solution temperatures (LCST) of 34·7 and 36·0°C for NIPNAS and PNIPAAm, respectively. The immobilized enzyme exhibited an LCST of 35·5°C for NIPNAS-amylase and 37·1°C for PNIPAAm-amylase. They precipitated and flocculated in aqueous solution above the LCST and redissolved when cooled below that temperature. The activity of the immobilized enzyme depended on the pH of the coupling buffer, with 8·0 being the optimum value. The specific activities of the immobilized enzymes were 87% and 108% compared with that of free enzyme with soluble starch as the substrate for NIPNAS-amylase and PNIPAAm-amylase, respectively. By characterizing the properties of the immobilized enzymes and comparing with those of free enzyme, no diffusion limitation of substrate was found for the immobilized enzymes and they are more thermal stable than the free enzyme. Within the two immobilized enzymes, NIPNAS-amylase showed better thermal stability and reusability. Repeated batch hydrolysis of soluble starch can be carried out efficiently with the immobilized enzymes by intermittent thermal precipitation and recycle of the enzyme. © 1997 SCI.     

Efficient Immobilization of Lecitase in Gelatin Hydrogel and Degumming of Rice Bran Oil Using a Spinning Basket Reactor

Immobilization of Lecitase (Phospholipase A1) in gelatin hydrogel and its stability is studied with a view to utilizing the immobilized enzyme for degumming rice bran oil. Excellent retention of enzyme activity (>80%) is observed in hydrogel containing 43.5% gelatin crosslinked with glutaraldehyde. Compared to the free enzyme which has a broad pH-activity profile (6.5–8.0), the activity of the immobilized enzyme is strongly dependent on pH and has a pH-optimum of pH 7.5. The optimum temperature of enzyme activity increases from 37 to 50 °C. Compared to the free enzyme which loses all its activity in 72 h at 50 °C, the immobilized enzyme retains its activity in full. The immobilized enzyme has been used efficiently in a spinning basket bioreactor for the degumming of rice bran oil with 6 recycles without loss of enzyme activity. The phosphorus content of the oil decreases from 400 ppm to 50–70 ppm in each cycle. After charcoal treatment and dewaxing, a second enzymatic treatment brings down the phosphorus content to <5 ppm.     

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.