Thermostability of soluble and immobilized alpha-amylase from Bacillus licheniformis

In view of a possible application of the alpha-amylase from Bacillus licheniformis as a time-temperature integrator for evaluation of heat processes,(11) thermal inactivation kinetics of the dissolved and covalently immobilized enzyme were studied in the temperature range 90-108 degrees C. The D-values (95 degrees C) for inactivation of alpha-amylase, dissolved in tris-HCl buffer, ranged from 6 to 157 min, depending on pH, ionic strength, and Ca(2+) and enzyme concentration. The z-value fluctuated between 6.2 and 7.6 degrees C. On immobilization of the alpha-amylase by covalent coupling with glutaraldehyde to porous glass beads, the thermoinactivation kinetics became biphasic under certain circumstances. For immobilized enzyme, the D-values (95 degrees C) ranged between 17 and 620 min, depending largely on certain environmental conditions. The z-value fluctuated between 8.1 and 12.9 degrees C. In each case of biphasic inactivation, the z-value of the stable fraction (with the higher D-values) was lower than the z-value of the labile fraction. (c) 1992 John Wiley & Sons, Inc.     

Comparative studies on soluble and immobilized yeast hexokinase

Comparative studies have been carried out on soluble and immobilized yeast hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1). The enzyme was immobilized by covalent attachment to a polyacrylamide type support containing carboxylic functional groups. The effects of immobilization on the catalytic properties and stability of hexokinase were studied. As a result of immobilization, the pH optimum for catalytic activity was shifted in the alkaline direction to ～pH 9.7. The apparent optimum temperature of the immobilized enzyme was higher than that of the soluble enzyme. The apparent K_m value with D-glucose as substrate increased, while that with ATP as substrate decreased, compared with the data for the soluble enzyme. Differences were found in the thermal inactivation processes and stabilities of the soluble and immobilized enzymes. The resistance to urea of the soluble enzyme was higher at alkaline pH values, while that for the immobilized enzyme was greatest at ～pH 6.0.     

Comparison of soluble and immobilized trypsin kinetics: Implications for peptide synthesis

The protease trypsin was immobilized to porous glass in both the presence and absence of acetylated soybean trypsin inhibitor (STI) to determine whether immobilization could alter enzyme activity in favor of aminolysis over hydrolysis. Actiive-site titration with 4-methylumbelliferylguanidinobenzoate (MUGB) showed that only about 10% of immobilized trypsin had catalytic activity. Immobilization in the presence of STI produced a higher yield of active enzyme accessible to the inhibitor but did not increase the total yield of MUGB-active immobilized enzyme. Thus, enzyme inactivation upon immobilization could not be attributed to an inaccessible enzyme orientation, nor did STI prevent inactivation by stabilizing the active-site conformation. Kinetic parameters were determined for soluble and immobilized trypsin for two esters, N-tosyl-L-arginine methyl ester (TAME) and N-benzoyl-L-arginine ethyl ester (BAEE), and two amides, N-benzoyl-L-arginine p-nitroanilide (BAPNA) and N-t-boc-leucylglycylarginine p-nitroanilide (LGRNA). In all cases, immobilization caused a greater decrease in k(cat) for amidase activity than for esterase activity. The ratio [k(cat)/ K(m) (ester)]/[k(cat)/K(m) (amide)] increased slightly or stayed the same (for I.GRNA) or decreased sharply (for BAPNA). Including STI during immobilization had little effect on the active enzyme's intrinsic kinetics. A direct comparison of energy diagrams and free energies of activation for BAEE and BAPNA indicates that immobilization raises the free energy barriers for both amide and ester hydrolysis and lowers the energy barrier for aminolysis. In practice, these effects should lower the amidase activity and increase the aminolysis-hydrolysis ratio, rendering the immobilized enzyme a more efficient catalyst for peptide synthesis. (c) 1993 John Wiley & Sons, Inc.     

Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugates for enzyme immunoassays

The periodate-mediated conjugation of horseradish peroxidase to antibody is one of the most popular methods to prepare conjugates for enzyme immunoassays of antigens or corresponding antibodies. A very simple method to obtain peroxidase, which is both about five times cheaper than the rather expensive commercial preparations and has a significant higher activity, is reported. Moreover, the conjugation method was critically investigated and considerably simplified. Conjugates thus obtained are about three times more active than the best obtained with the original method.     

Removal of phenols from wastewater by soluble and immobilized tyrosinase

An enzymatic method for removal of phenols from industrial wastewater was investigated. Phenols in an aqueous solution were removed after treatment with mushroom tyrosinase. The reduction order of substituted phenols is catechol > p-cresol > p-chlorophenol > phenol > p-methoxyphenol. In the treatment of tyrosinase alone, no precipitate was formed but a color change from colorless to dark-brown was observed. The colored products were removed by chitin and chitosan which are available abundantly as shellfish waste. In addition, the reduction rate of phenols was observed to be accelerated in the presence of chitosan. Tyrosinase, immobilized by using amino groups in the enzyme on cation exchange resins, can be used repeatedly. By treatment with immobilized tyrosinase, 100% of phenol was removed after 2 h, and the activity was reduced very little even after 10 repeat treatments. (c) 1993 John Wiley & Sons, Inc.     

Design and characterization of a prototype enzyme microreactor: Quantification of immobilized transketolase kinetics

In this work, we describe the design of an immobilized enzyme microreactor (IEMR) for use in transketolase (TK) bioconversion process characterization. The prototype microreactor is based on a 200‐μm ID fused silica capillary for quantitative kinetic analysis. The concept is based on the reversible immobilization of His₆‐tagged enzymes via Ni‐NTA linkage to surface derivatized silica. For the initial microreactor design, the mode of operation is a stop‐flow analysis which promotes higher degrees of conversion. Kinetics for the immobilized TK‐catalysed synthesis of L ‐erythrulose from substrates glycolaldehyde (GA) and hydroxypyruvate (HPA) were evaluated based on a Michaelis–Menten model. Results show that the TK kinetic parameters in the IEMR (V_max(app) = 0.1 ± 0.02 mmol min^–1, K_m(app) = 26 ± 4 mM) are comparable with those measured in free solution. Furthermore, the k_cat for the microreactor of 4.1 × 10⁵ s^?1 was close to the value for the bioconversion in free solution. This is attributed to the controlled orientation and monolayer surface coverage of the His₆‐immobilized TK. Furthermore, we show quantitative elution of the immobilized TK and the regeneration and reuse of the derivatized capillary over five cycles. The ability to quantify kinetic parameters of engineered enzymes at this scale has benefits for the rapid and parallel evaluation of evolved enzyme libraries for synthetic biology applications and for the generation of kinetic models to aid bioconversion process design and bioreactor selection as a more efficient alternative to previously established microwell‐based systems for TK bioprocess characterization. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

褐藻降解菌群筛选及其复合酶固定化研究

以大连地区的褐藻为材料,筛选了褐藻降解菌群,其在30℃、pH7．5条件下培养74h时,酶活达1．883IU／mL。利用硅藻土吸附法经冷冻干燥制备了固定化复合酶,分析了复合酶系的酶学性质,其最适反应温度为45℃,并在40℃～55℃范围内具有良好的热稳定性;最适pH为7．5,并在pH7．0～8．5之间pH稳定性良好。利用固定化酶进行褐藻酸钠制备,提取率达48．3％,粘度为2．9Pa·s,与传统方法相比均有显著提高,为工程化生产褐藻酸钠提供一定基础。     

Evolution of beetle bioluminescence: the origin of beetle luciferin

Bioluminescence, the conversion of chemical energy into light in living organisms, is dependent on two principal components, an enzyme luciferase and the substrate luciferin. In beetles, the enzyme luciferase has been extensively studied, with signi?cant enzymological, sequence and structural data now available. Furthermore, the enzyme has been employed in a remarkable number of important applications, from microbial detection and medical imaging to GM gene expression studies. However, there is little information regarding the biosynthesis of beetle luciferin, and here we review the literature and speculate as to its evolutionary origins. Luciferin consists of a benzothiazole moiety attached to a thiazole carboxylic acid moiety, the former being rarely observed in nature but the latter being observed in a broad range of biologically derived molecules. Benzothiazoles are, however, observed in melanogenesis and we speculate as to whether this may be relevant to the understanding of luciferin biosynthesis in beetles. This review examines recent novel insights into beetle luciferin recycling and we assess a range of possible biosynthetic mechanisms. Copyright © 2004 John Wiley & Sons, Ltd.     

Folding of the firefly luciferase polypeptide chain with the immobilized C terminus

Refolding of firefly Photinus pyralis luciferase from a denatured state is a slow process; its rate and productivity depend on molecular chaperones of the Hsp70 family. In contrast, cotranslational folding of luciferase is fast and productive in the absence of chaperones. During cotranslational folding, the C termini of polypeptides are associated with ribosomes, massive particles. The question arises as to whether C-terminal immobilization on a massive particle promotes folding. To study this problem experimentally, luciferase was C-tagged with hexahistidine to allow its C-terminal immobilization of chelating Sepharose. Both immobilized and free chains of the urea-denatured enzyme refolded at the same rate. At the same time, immobilization led to a higher refolding yield owing to the prevention of intermolecular aggregation. Chaperones of the Hsp70 family promoted folding of both immobilized and free luciferase polypeptides. It was assumed that the high rate of cotranslational folding is not ensured by mere immobilization of the C terminus of the polypeptide, but is rather due to a favorable start conformation of the growing peptide in the peptidyltransferase center of the ribosome and/or the vectorial character of the folding, proceeding from the N to the C end during polypeptide synthesis.     

Potential application of immobilized streptokinase extracted from Streptococcus equinus VIT_VB2

Streptokinase purified from Streptococcus equinus VIT_VB2 isolated from bovine milk sample was immobilized in various solid supports namely entrapment in agarose gel, calcium alginate beads and gelatin gel by cross-linking with formaldehyde. Immobilization of streptokinase in calcium alginate beads showed maximum efficiency (81.8?±?1.06%) when compared with entrapment with agarose gel (55.6?±?2.17%) and cross-linked gelatin formaldehyde gel (71.0?±?1.54%). The purified SK activity was expressed maximum in calcium alginate (1%) and gelatin gel (0.25%) with 1292.68?±?1.33 and 1121.9?±?1.2?U?mL^?1, respectively. Similarly, SK entrapped in gelatin gel and calcium alginate showed maximum in vitro blood clot lysis activity with 77.67?±?2.64% and 76.16?±?2.72%, respectively. The immobilized SK in gelatin gel showed complete clot lysis within 15?min; hence, this application of the study could be used in the treatment of superficial thrombophlebitis, phlebitis, and venous thrombosis. These beads were used for three repeated cycles to check the conversion of substrates into their products, and we concluded that SK can be immobilized in the suitable matrices. Therefore, this helps in the drug-delivery strategies in highly efficient way, moreover, economically competent process in the pharmaceutics.     

Application of immobilized enzyme technologies for the textile industry: a review

Abstract

A number of enzymes have applications in the textile sector, as several natural fibres (e.g. cotton, wool and flax) can be subjected to processing using such natural biocatalysts. In the latest two decades, demand for industrial enzymes has increased considerably; however, the textile industry requires highly stable enzymes, and good performance at extreme values of pH and temperature. New strategies are continuously emerging for the immobilization of enzymes with superior efficiency and usage. Enzymatic immobilization can stabilize enzymes and extend their useful life. Additionally, reduction in effluent treatment costs and improvement of efficiency is possible. This paper briefly reviews the most recent research efforts pertaining to immobilization of enzymes with potential application in the textile industry.     

Equine marker genes: Polymorphism for soluble erythrocyte malic enzyme

Polymorphism for equine erythrocyte malic enzyme is detectable on starch gel electrophoresis. The frequency of MEI^S was 0.06 in 667 Standardbred and 0.09 in 85 Thoroughbred horses. No genetically determined electrophoretic variation in soluble malate dehydrogenase was detected.     

Comparison of properties of commercially available crystalline native and recombinant firefly luciferases

Commercially available crystalline native and recombinant firefly luciferases were compared. The two types of luciferase had indistinguishable responses to variation in ATP and luciferin concentrations and to omission of reaction components. The time courses of light production, the responses to nucleotide analogues, and the stability of the enzymes under several storage conditions were identical. The native enzyme had a slightly greater specific activity and was more sensitive to trypsin degradation. These differeces are probably attributable to differences in conformation.     

Hydrolysis of oils by using immobilized lipase enzyme: A review

This review focuses on the use of immobilized lipase technology for the hydrolysis of oils. The importance of lipase catalyzed fat splitting process, the various immobilization procedures, kinetics, deactivation kinetics, New immobilized lipases for chiral resolution, reactor configurations, and process considerations are all reviewed and discussed.     

在大肠杆菌中合成的萤火虫荧光素酶的分离和性质

用生物工程技术将萤火虫荧光素酶基因转移到大肠杆菌,在大肠杆菌中合成荧光素酶。这种工程菌已可通过发酵大量培养,并从菌体分离得到接近纯化的荧光素酶。这种酶的分子量是103kD;巯基试剂5,5’-巯基-2(2-硝基苯甲酸)“DTNB”能抑制酶的活性;对于底物荧光素的K_m为1.2μmol/L;酶反应最适pH为7.77;酶催化的生物发光峰在560nm。     

Preparations of immobilized lysozyme with reversibly soluble polymer for hydrolysis of microbial cells

Hen egg white lysozyme was immobilized by carbodiimide method to form amide bonds with a polymer (AS-L) showing reversibly soluble-insoluble characteristics with pH change. The immobilized enzyme (LY-AS) was soluble above pH 6 and precipitate below pH 4.5, offering advantages in that it can carry out hydrolysis of microbial cells in a soluble form yet be recovered after precipitation at low pH. The maximum specific activity of LY-AS was 66% of that of free lysozyme with M. lysodeikticus cells as substrate, which is much higher than the values reported in the literature using water-insoluble materials as carriers. The effects of pH and temperature on the activity of LY-AS were studied and compared with those of free lysozyme. With repeated pH cycles between 6.6 and 4.5, the operation half-life of immobilized enzyme activity was nine cycles. Repeated batch lysis of microbial cells could be carried out with intermittent enzyme precipitation and recovery steps. In such an operation the insoluble residual cells should be recovered together with the immobilized enzyme to minimize enzyme loss arising from adsorption to cells.     

Common structural features of the luxF protein and the subunits of bacterial luciferase: evidence for a (beta alpha)8 fold in luciferase.

The amino acid sequence identity and potential structural similarity between the subunits of bacterial luciferase and the recently determined structure of the luxF molecule are examined. The unique beta/alpha barrel fold found in luxF appears to be conserved in part in the luciferase subunits. From secondary structural predictions of both luciferase subunits, and from structural comparisons between the protein product of the luxF gene, NFP, and glycolate oxidase, we propose that it is feasible for both luciferase subunits to adopt a (beta alpha)8 barrel fold with at least 2 excursions from the (beta alpha)8 topology. Amino acids conserved between NFP and the luciferase subunits cluster together in 3 distinct "pockets" of NFP, which are located at hydrophobic interfaces between the beta-strands and alpha-helices. Several tight turns joining the C-termini of beta-strands and the N-termini of alpha-helices are found as key components of these conserved regions. Helix start and end points are easily demarcated in the luciferase subunit protein sequences; the N-cap residues are the most strongly conserved structural features. A partial model of the luciferase beta subunit from Photobacterium leiognathi has been built based on our crystallographically determined structure of luxF at 1.6 A resolution.     

Methodological problems of direct bioluminescent ATP assay in platelets and erythrocytes

Direct bioluminescent ATP determination in platelets and erythrocytes involves the study of different parameters which are discussed here. Some parameters are linked to the bioluminescent reaction and to the analyte (ATP); others have regard to the biological matrix. The composition of bioluminescent reagents and the preparation and conservation of the ATP standard, also in the presence of excipients, are among the first given. Matrix problems involve cell characteristics related to age and form, lysis resistance and the possible formation of aggregates (platelets) that may inhibit the complete release of ATP. For these reasons we used the most efficient ATP release agent with the lowest inhibitory effect on luciferase. The data obtained correlate well with a bioluminescent method requiring extraction with ethanol/EDTA, and therefore more time, for ATP determination in platelets and erythrocytes.     

Comparative study of soluble and immobilized phenol oxidase from the fungus Mycelia sterilia IBR 35219/2

Phenol oxidase (EC 1.14.18.1) from the microscopic fungus Mycelia sterilia IBR 35219/2 was immobilized using glutaraldehyde on macroporous silica carriers. The enzyme immobilized on amino-Silochrome SKh-2 or aminopropyl-Silochrome 350/80 exhibited maximum activity. Soluble and immobilized phenol oxidases were compared. Compared to the soluble enzyme, the activity of which was optimum at pH 5.5, immobilized phenol oxidase exhibited optimum activity under slightly more acidic conditions (pH 5.2). Immobilization considerably increased the enzyme stability. Both soluble and immobilized forms of phenol oxidase from M. sterilia IBR 35219/2 catalyze oxidative conversion of phenolic compounds of the green tea extract.