Protein immobilization to alumina supports: I. characterization of alumina-organophosphate ligand interactions and use in the attachment of papain

The chemical adsorption of organic phosphate compounds to alumina has been used to create surface linkers for protein immobilization. A number of particulate alumina supports were screened for their physical properties and ability to bind organic phosphate compounds. Two aluminas, termed C1 and CPC, were selected based on their suitability for subsequent testing as protein immobilization supports. Papain was successfully immobilized to these supports when derivatized with phosphate compounds containing free terminal carboxyl groups. Protein binding was enhanced when support carboxyl groups were activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The level of papain immobilization was dependent upon the length of the linker used and the mass of protein exposed to the support. (c) 1992 John Wiley & Sons, Inc.     

Purification and in situ immobilization of papain with aqueous two-phase system

Papain was purified from spray-dried Carica papaya latex using aqueous two-phase system (ATPS). Then it was recovered from PEG phase by in situ immobilization or preparing cross-linked enzyme aggregates (CLEAs). The Plackett-Burman design and the central composite design (CCD) together with the response surface methodology (RSM) were used to optimize the APTS processes. The highly purified papain (96-100%) was achieved under the optimized conditions: 40% (w/w) 15 mg/ml enzyme solution, 14.33-17.65% (w/w) PEG 6000, 14.27-14.42% (w/w) NaH2PO4/K2HPO4 and pH 5.77-6.30 at 20°C. An in situ enzyme immobilization approach, carried out by directly dispersing aminated supports and chitosan beads into the PEG phase, was investigated to recover papain, in which a high immobilization yield (>90%) and activity recovery (>40%) was obtained. Moreover, CLEAs were successfully used in recovering papain from PEG phase with a hydrolytic activity hundreds times higher than the carrier-bound immobilized papain.     

Enzyme immobilization on macroreticular polystyrene

Macroreticular polystyrene beads may be converted into suitable supports for covalent binding of enzymes. In many respects the supports are physically similar to controlled pore glass (CPG). Our results for immobilized glucoamylase were very similar to published results using CPG as a carrier. The characteristics of immobilized papain were less satisfactory. The product exhibited a Z-shaped activity-time profile suggestive of the involvement of multiple mechanisms.     

Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics,screening, and stability

The previously uncharacterized Drosophila melanogaster Epsilon-class glutathione transferases E6 and E7 were immobilized on nanoporous alumina. The nanoporous anodized alumina membranes were derivatized with 3-aminopropyl-triethoxysilane, and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes via ε-amino groups. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzyme in solution. A limited kinetic study was also carried out on immobilized human GST S1-1 (also known as hematopoietic prostaglandin D synthase). The stability of the immobilized enzymes was virtually identical to that of enzymes in solution, and no leakage of enzyme from the matrix could be observed.     

Fibrous polymer-grafted chitosan/clay composite beads as a carrier for immobilization of papain and its usability for mercury elimination

Papain, which is an industrially important enzyme, has been immobilized on fibrous polymer-modified composite beads, namely poly(methacrylic acid)-grafted chitosan/clay. Characterization studies have been done using FTIR and SEM analysis. Operating parameters such as pH and initial concentration of papain have been varied to obtain the finest papain immobilized polymer-modified composite beads. The immobilization capacity of composite beads has been determined as 34.47 ± 1.18 (n = 3) mg/g. The proteolytic activity of immobilized papain was operated using bovine serum albumin (BSA) and maximum velocity (V _max) and Michaelis–Menten constant (K_m) values of the free and immobilized enzymes were determined using Lineweaver–Burk and Eadie–Hofstee equations. Usability of papain immobilized polymer-modified composite beads as adsorbents for the elimination of mercury was investigated. The maximum removal capacity of PIPMC beads has been found to be 4.88 ± 0.21 mg Hg/g when the initial metal concentration and weight of polymer-modified composite beads were 50 mg/L and 0.04 g at pH 7, respectively. Mercury removal performance of the papain immobilized polymer-modified composite beads was investigated in conjunction with Cu (II), Zn (II) and Cd (II) ions. The mercury adsorption capacity of papain immobilized polymer-modified composite beads was a slight reduction from 1.15 to 0.89 mg/g in presence of multiple metal salts.     

Preparation and properties of immobilized papain and lipase.

Papain and lipase were immobilized on derivatized Sepharose 4-B. The activated agarose had a binding capacity of 1.2 micronmol amino groups/ml packed agarose or 17 mg proteins/g dry agarose. The immobilized enzyme preparations were tested for the effects of pH of assay, temperature of assay, and substrate concentrations. The effect of 6M urea on the activity of papain was also determined. Soluble forms of the enzymes were used for comparison. Immobilization of the enzymes resulted in slightly different pH and temperature optima for activities. For immobilized papain Km(app) was similar to the one observed with soluble papain. Immobilization of lipase, however, cause a decrease in Km values. The immobilized enzyme preparations were stable when stored at 4 degrees C and pH 7.5 for periods up to eight months. The soluble enzymes lost their activity within 96 hr under similar storage conditions. Immobilized papain did not lose any activity after treatment with 6M urea for 270 min, whereas soluble papain lost 81% of its activity after the urea treatment, indicating that the immobilization of papain imparted structural and conformational stability to this enzyme.     

高分子膜载体固定化木瓜蛋白酶

在浸润条件下,以0.5%(v/v)戊二醛交联的高分子膜尼龙载体固定化木瓜蛋白酶。对固定化条件进行了优化,比较了固定化酶与游离酶的酶学参数。结果表明,4℃、pH6.0条件下,将膜载体浸润于2mg/mL酶液中5h,固定化酶活为303.4U/g。固定化酶最适反应pH为6.0～7.0,最适反应温度为65℃。其pH稳定性、热稳定性均比游离酶高。     

Surface Plasmon Resonance Imaging (SPRI) sensor for cystatin determination based on immobilized papain

A Surface Plasmon Resonance Imaging (SPRI) sensor has been developed for specific determination of cystatin. The sensor contains immobilised papain, which binds cystatin from solution. Papain activated with N-Hydroxysuccinimide (NHS) and N-Ethyl-N'-(3-dimethyl aminopropyl)carbodiimide (EDC) was immobilized on an amine-modified gold surface. Cysteamine was used for modification of the gold surface. Papain concentration and the pH of interaction were optimised. A concentration of papain of 1.5 μg mL(-1) and a pH of 6.5 were selected as optimal. The specificity of interaction was verified by the lack of interaction with human albumin. The sensor's dynamic response range is between 0 and 0.6 mg μL(-1), and the detection limit is 0.09 μg mL(-1). The results were validated by comparison with the PETIA (particle enhanced immunoturbidimetric assay) method showing good agreement. A calibration curve of chicken egg white cystatin or Cystatin C was used. In order to demonstrate the sensor's potential, cystatin C was determined in blood plasma, urine and saliva, showing good agreement with data reported in the literature. The results for cystatin concentration in the blood plasma of people suffering from leukaemia were found to be below the normal level of cystatin.     

壳聚糖固定化木瓜蛋白酶的研究

以壳聚糖为载体,成二醛为交联剂将木瓜蛋白酶固定化。5％戊二醛在4-6℃下处理载体5h,加酶液(3.5mg/mL蛋白,pH7.2)固定12h,活力回收达32％,作用于酪蛋白的半衰期为36天,其表观K_m(酪蛋白)值为0.075％(W/V),溶液酶的K_m值为0.086％;最适pH7.0～7.5,溶液酶为7.0～8.5。固定化酶在pH8.5以下,溶液酶在9.0以下活力稳定。固定化酶在45℃以下,溶液酶在75℃以下稳定。用6mol/L脲洗脱固定化酶4次(5.5h)活力仍有54.5％。用固定化酶处理啤酒浊度比对照下降了1.5-3.7倍,蛋白质含量下降了44％,冷藏(4℃)120天无冷混浊现象发生并保持了啤酒原有风味和理化性状。     

Analysis and optimization of methods using water-soluble carbodiimide for immobilization of biochemicals to porous glass

Two methods employing a water-soluble carbodiimide for carboxyl activation were investigated for the immobilization of biochemicals to succinamidopropyl-porous glass beads. Immobilization using the simultaneous method (simultaneous addition of carbodiimide and nucleophilic ligand to the beads) and large excess of carbodiimide and a small nucleophile should result in covalent binding to all accessible carboxyl groups. Results obtained with glycine methyl ester indicated that 40% of the total surface carboxyl groups were sterically accessible. Using these reaction conditions with the protein, chymotrypsinogen, suggests that a surface monolayer is immobilized. although far fewer sites are required assuming single point attachment. For ligands containing carboxyl groups and several nucleophilic groups (e. g., enzymes), however, biological inactivation may occur using the simultaneous method. Consequently, a sequential method (activation of the surface with carbodiimide followed by washing and addition of the biochemical to be immobilized) was optimized. Using optimal conditions (20 min activation time at pH 4.75 and room temperature; 2 min wash at pH 7 and 0 degrees C) and 0.1M carbodiimide, nearly half of the accessible surface sites remained in the O-acylisourea form and reacted with glycine methyl ester upon its addition. The amount of surface loading as a function of activation time was consistent with a model constructed using rate constants for O-acylisourea formation and hydrolysis previously derived from solution studies with acetic acid [Swaisgood and Natake, J. Biochem 74, 77 (1973)]. Measurement of reaction rates with glycine methyl ester following surface activation suggests that the rate of reaction with amino groups is at least eightfold greater than the hydrolysis rate. Either immobilization procedure gave comparable enzyme loading and specific activities for the case of sulfhydryl oxidase.     

木瓜蛋白酶在飘珠上的固定化和红外活化

用火电厂的废物——飘珠作载体,将木瓜蛋白酶固定化得到成功,比活力为天然酶的26.6％。但发现,采用适当的红外辐射又可使固定化酶活力提高约30％。     

Control of protein immobilization: coupling immobilization and site-directed mutagenesis to improve biocatalyst or biosensor performance

Mutagenesis and immobilization are usually considered to be unrelated techniques with potential applications to improve protein properties. However, there are several reports showing that the use of site-directed mutagenesis to improve enzyme properties directly, but also how enzymes are immobilized on a support, can be a powerful tool to improve the properties of immobilized biomolecules for use as biosensors or biocatalysts. Standard immobilizations are not fully random processes, but the protein orientation may be difficult to alter. Initially, most efforts using this idea were addressed towards controlling the orientation of the enzyme on the immobilization support, in many cases to facilitate electron transfer from the support to the enzyme in redox biosensors. Usually, Cys residues are used to directly immobilize the protein on a support that contains disulfide groups or that is made from gold. There are also some examples using His in the target areas of the protein and using supports modified with immobilized metal chelates and other tags (e.g., using immobilized antibodies). Furthermore, site-directed mutagenesis to control immobilization is useful for improving the activity, the stability and even the selectivity of the immobilized protein, for example, via site-directed rigidification of selected areas of the protein. Initially, only Cys and disulfide supports were employed, but other supports with higher potential to give multipoint covalent attachment are being employed (e.g., glyoxyl or epoxy-disulfide supports). The advances in support design and the deeper knowledge of the mechanisms of enzyme-support interactions have permitted exploration of the possibilities of the coupled use of site-directed mutagenesis and immobilization in a new way. This paper intends to review some of the advances and possibilities that these coupled strategies permit.     

Controlled layer-by-layer immobilization of horseradish peroxidase.

Horseradish peroxidase (HRP) was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester (BcapNHS) in a controlled manner to obtain biotinylated horseradish peroxidase (Bcap-HRP) with two biotin moieties per enzyme molecule. Avidin-mediated immobilization of HRP was achieved by first coupling avidin on carboxy-derivatized polystyrene beads using a carbodiimide, followed by the attachment of the disubstituted biotinylated horseradish peroxidase from one of the two biotin moieties through the avidin-biotin interaction (controlled immobilization). Another layer of avidin can be attached to the second biotin on Bcap-HRP, which can serve as a protein linker with additional Bcap-HRP, leading to a layer-by-layer protein assembly of the enzyme. Horseradish peroxidase was also immobilized directly on carboxy-derivatized polystyrene beads by carbodiimide chemistry (conventional method). The reaction kinetics of the native horseradish peroxidase, immobilized horseradish peroxidase (conventional method), controlled immobilized biotinylated horseradish peroxidase on avidin-coated beads, and biotinylated horseradish peroxidase crosslinked to avidin-coated polystyrene beads were all compared. It was observed that in solution the biotinylated horseradish peroxidase retained 81% of the unconjugated enzyme's activity. Also, in solution, horseradish peroxidase and Bcap-HRP were inhibited by high concentrations of the substrate hydrogen peroxide. The controlled immobilized horseradish peroxidase could tolerate much higher concentrations of hydrogen peroxide and, thus, it demonstrates reduced substrate inhibition. Because of this, the activity of controlled immobilized horseradish peroxidase was higher than the activity of Bcap-HRP in solution. It is shown that a layer-by-layer assembly of the immobilized enzyme yields HRP of higher activity per unit surface area of the immobilization support compared to conventionally immobilized enzyme.     

金属螯合载体定向固定化木瓜蛋白酶的研究

以磁性金属螯合琼脂糖微球为载体,利用金属螯合配体（IDACu²⁺）与蛋白质表面供电子氨基酸相互作用的原理,定向固定了木瓜蛋白酶。固定化最适条件为Cu²⁺1.5×10^-2mol/g载体、固定化时间4h、固定化pH7.0、给酶量30mg/g载体。固定化酶的最适反应温度70℃、最适反应pH8.0,固定化酶的热稳定性明显高于溶液酶,固定化酶活力回收为68.4％,且有较好的操作稳定性,载体重复使用5次后固定化酶酶活为首次固定化酶79.71%。     

Phosphoramidate end labeling of inorganic polyphosphates: facile manipulation of polyphosphate for investigating and modulating its biological activities

Polyphosphates, linear polymers of inorganic phosphates linked by phosphoanhydride bonds, are widely present among organisms and play diverse roles in biology, including functioning as potent natural modulators of the human blood clotting system. However, studies of protein-polyphosphate interactions are hampered by a dearth of methods for derivatizing polyphosphate or immobilizing it onto solid supports. We now report that EDAC (1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide) efficiently promotes the covalent attachment of a variety of primary amine-containing labels and probes to the terminal phosphates of polyphosphates via stable phosphoramidate linkages. Using (31)P NMR, we confirmed that EDAC-mediated reactions between primary amines and polyphosphate result in phosphoramidate linkages with the terminal phosphate groups. We show that polyphosphate can be biotinylated, labeled with fluorophores, and immobilized onto solid supports, that immobilized polyphosphate can be readily used to quantify protein binding affinities, that covalently derivatized or immobilized polyphosphate retains its ability to trigger blood clotting, and that derivatizing the ends of polyphosphate with spermidine protects it from exopolyphosphatase degradation. Our findings open up essentially the entire armamentarium of protein chemistry to modifying polyphosphate, which should greatly facilitate studies of its biological roles.     

Functional polymeric supports for immobilization of cholesterol oxidase

Here, we have reported the useful functional polymeric supports for possible application of enzyme immobilization. Functional polymers were prepared by free radical polymerization from different monomers (i.e., methylmetacrylate, glycidylmethacrylate, acrylamide, etc.) and N,N-methylenebis(acrylamide) (MBAAm) crosslinker. Cholesterol oxidase (ChOx) [EC.1.1.3.6] was then covalently immobilized onto these functional supports via epichlorohydrin (ECH) and carbodiimide (EDAC) as the activating agents. It was observed that, after 60th use in 5 days, the retained activities for immobilized enzymes onto poly(methyl methacrylate-co-glycidyl methacrylate) [P(MMA-co-GMA)] and poly(acrylamide-co-acrylic acid)/polyethyleneimine [P(AAm-co-AA)/PEI] supports were found as 56% and 83%, respectively.     

Hydrolysis of soyabean by papain immobilized on wood by radiation polymerization

Summary Papain was immobilized on wood chips by radiation polymerization without substantial loss of enzyme activity. The immobilized papain was used to hydrolyse soyabean meal and found to be stable upto 6 cycles of operation. Maximum hydrolysis occurred with 15% (W/V) immobilized matrix.     

Substituted polymethylglutamate membrane for immobilization of urease

Poly-γ-methyl-l-glutamate (PMG) was modified and used for enzyme immobilization. Trichloroethyl ester (TCE) and three kinds of amino groups (ethylenediamine, ED; 1,8-diamino-4-amino-methyloctane, TA; polyethyleneimine, PEI) were introduced into the pendant group of PMG. A membrane was prepared from these polymers for enzyme immobilization. Urease was immobilized on each membrane using glutaraldehyde or water-soluble carbodiimide. Urease was very stable when it was immobilized with water-soluble carbodiimide on the membrane having PEI in the pendant group. The characteristics of immobilized urease were also discussed.     

Design of a Papain Immobilized Antimicrobial Food Package with Curcumin as a Crosslinker

Contamination of food products by spoilage and pathogenic microorganisms during post process handling is one of the major causes for food spoilage and food borne illnesses. The present green sustainable approach describes the covalent immobilization of papain to LDPE (low density polyethylene), HDPE (high density polyethylene), LLDPE (linear low density polyethylene) and PCL (polycaprolactam) with curcumin as the photocrosslinker. About 50% of curcumin and 82-92% of papain were successfully immobilized on these polymers. After 30 days, the free enzyme retained 87% of its original activity, while the immobilized enzyme retained more than 90% of its activity on these polymers. Papain crosslinked to LLDPE exhibited the best antibiofilm properties against Acinetobacter sp. KC119137.1 and Staphylococcus aureus NCIM 5021 when compared to the other three polymers, because of the highest amount of enzyme immobilized on this surface. Papain acts by damaging the cell membrane. The enzyme is able to reduce the amount of carbohydrate and protein contents in the biofilms formed by these organisms. Meat wrapped with the modified LDPE and stored at 4°C showed 9 log reduction of these organisms at the end of the seventh day when compared to samples wrapped with the bare polymer. This method of crosslinking can be used on polymers with or without functional groups and can be adopted to bind any type of antimicrobial agent.     

Properties of papain, immobilized on organosilica

Papain, a proteolytic enzyme, is used in the reactions of organic synthesis for preparing peptides. The use of immobilized papain with this aim is very promising. Preparations of papain immobilized by organosilica have been studied for their physicochemical properties as well kinetics of the papain immobilization by amino-organosilica activated by cyanuric chloride. Retention of the enzyme activity of immobilized papain reached 40% and depended on the amount of enzyme bound with the carrier. Kmobs of the immobilized enzyme did not differ significantly from that of the soluble enzyme. After immobilization the pH-optimum an pH-profile of the catalytical activity of papain remained unchangeable. For the period of 20 days immobilized papain has lost 20-50% activity.