Preparation,characterization and laboratory-scale application of an immobilized glucoamylase

Glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) has been covalently immobilized on a polyacrylamide-type support containing carboxylic groups activated by water-soluble carbodiimide. The activity was 5.5– 6.0 units g^?1solid. The optimum pH for catalytic activity was pH 3.8. The apparent optimum temperature was found at 60°C. With soluble starch as substrate the K_m value was 14 mg ml^?1. The pH for maximum stability was pH 4.0–4.5. In the presence of 8 m urea the immobilized glucoamylase retained most of its catalytic activity but it was more susceptible to guanidinium hydrochloride than the soluble enzyme. The practical applicability of immobilized glucoamylase was tested in batch process and continuous operation.     

Molecular properties,functions, and potential applications of NAD kinases

NAD kinase catalyzes the phosphorylation of NAD（H） to form NADP（H）, using ATP as phosphoryl donor. It is the only key enzyme leading to the de novo NADP＋/ NADPH biosynthesis. Coenzymes such as NAD（H） and NADP（H） are known for their important functions. Recent studies have partially demonstrated that NAD kinase plays a crucial role in the regulation of NAD（H）/ NADP（H） conversion. Here, the molecular properties, physiologic functions, and potential applications of NAD kinase are discussed.     

嗜热脂肪地芽孢杆菌NAD激酶克隆表达及酶学性质研究

NAD激酶能催化NAD生成NADP。本研究采用PCR技术从嗜热脂肪地芽孢杆菌基因组中获得NAD激酶基因,以pET30a（＋）为表达载体、E．coliBL21（DE3）为宿主菌,实现其在大肠杆菌中异源表达,并进行酶学性质研究。结果显示,嗜热脂肪地芽孢杆菌中NAD激酶编码基因大小为816bp,酶分子量大约为35kD。酶学性质分析表明,来源于嗜热脂肪地芽孢杆菌的NAD激酶最适反应温度和pH分别为35℃、pH7．5,在35qC中保温2h后仍能保持80％左右的活性。Mn2＋、Ca2＋对该酶有较强的激活作用,在最适反应条件下该酶的比活力为4．43U／mg。动力学性质分析结果显示NAD激酶对底物NAD催化的k和圪。,分别为1．46mmol／L和0．25tzmol／（L·min）。NAD激酶在大肠杆菌的异源表达为以NAD为底物生物合成NADP提供了更多生物资源。     

Crystal structures of an NAD kinase from Archaeoglobus fulgidus in complex with ATP, NAD, or NADP

NAD kinase is a ubiquitous enzyme that catalyzes the phosphorylation of NAD to NADP using ATP or inorganic polyphosphate (poly(P)) as phosphate donor, and is regarded as the only enzyme responsible for the synthesis of NADP. We present here the crystal structures of an NAD kinase from the archaeal organism Archaeoglobus fulgidus in complex with its phosphate donor ATP at 1.7 A resolution, with its substrate NAD at 3.05 A resolution, and with the product NADP in two different crystal forms at 2.45 A and 2.0 A resolution, respectively. In the ATP bound structure, the AMP portion of the ATP molecule is found to use the same binding site as the nicotinamide ribose portion of NAD/NADP in the NAD/NADP bound structures. A magnesium ion is found to be coordinated to the phosphate tail of ATP as well as to a pyrophosphate group. The conserved GGDG loop forms hydrogen bonds with the pyrophosphate group in the ATP-bound structure and the 2' phosphate group of the NADP in the NADP-bound structures. A possible phosphate transfer mechanism is proposed on the basis of the structures presented.     

Continuous production of lignin-degrading enzymes by Bjerkandera adusta immobilized on polyurethane foam

Continuous production of lignin-degrading enzymes by Bjerkandera adusta immobilized on polyurethane foam gave maximum activities of 220 U lignin peroxidase ml^–1, 150 U manganese peroxidase ml^–1, 50 U laccase ml^–1 and 6.2 U protease ml^–1 at the retention time of 24 h for 60 days. Protease secretion destabilized the produced lignin peroxidase, manganese peroxidase and laccase.     

Characterization and possible redox regulation of the purified calmodulin‐dependent NAD+ kinase from Lycopersicon pimpinellifolium

The soluble and calmodulin (CaM)‐dependent NAD⁺ kinase from Lycopersicon pimpinellifolium was previously shown to be largely inactivated in isolated cells exposed to a short‐term NaCl stress (Delumeau, Morère‐Le Paven, Montrichard, Laval‐Martin (2000) Plant Cell & Environment 23, 329–336). Nevertheless, the activity could be restored by adding a high dithiothreitol concentration to the protein extract, suggesting that the salt stress triggers an oxidation of the enzyme which leads to its inactivation. It was then interesting to investigate the effect of thiol‐modifying reagents and disulphide reductants on the activity of L. pimpinellifolium NAD⁺ kinase. A three‐step purification procedure was then established and allowed isolation of the enzyme which exists under two forms: a monomer and a dimer of a 56 kDa subunit, characterized, respectively, by pIs of 6·8 and 7·1. Isolated NAD⁺ kinase had a high affinity for CaM, half saturation being obtained for 7 ng mL^?1 bovine CaM. The activity of NAD⁺ kinase was strongly inhibited by thiol‐modifying reagents and oxidized glutathione. NAD⁺ kinase was also found to be air‐inactivated, the residual activity being stimulated by disulphide reductants. The most efficient of them is reduced thioredoxin from Escherichia coli which induced a five‐fold increase in activity and restored 80% of the initial activity. These results which can be related to those previously observed in vivo suggest that the activity of the L. pimpinellifolium NAD⁺ kinase, besides its dependence on CaM, is also dependent on the reduction state of the protein which could be regulated by the thioredoxin h/NADP‐thioredoxin reductase system.     

Quantitative changes in calmodulin and NAD kinase during early cell development in the root apex of Pisum sativum L.

Calmodulin and NAD kinase were extracted from serial developmental sections of the pea root apex. Highly purified samples of calmodulin were assayed by NAD-kinase activation, and whole-cell extracts were examined by two-dimensional polyacrylamide gel electrophoresis. Calmodulin was found to vary 17-fold in concentration over the apical 2 mm, being high in the region of the root cap and meristem, falling rapidly at the base of the meristem during early stages of rapid cell elongation. The rate of decline was different between stele and cortex. Except for a minor increase in concentration 2.5–5 mm from the apex, which coincides with the region of localised meristematic activity during initiation of lateral root primordia, the concentration of calmodulin remained at the lower level throughout the more basal sections of the apical 10 mm. In-vitro NAD-kinase activity was found to increase 17-fold per cell over the apical 30 mm, almost entirely as the result of an increase in calmodulin-dependent activity. Quantitative estimates of both calmodulin and NAD kinase were found to be highly dependent on extraction procedures.Abbreviation EGTA ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid     

Production of optically pure L-alanine in batch and continuous stirred tank reactors using immobilized Pseudomonas sp.

A process to obtain optically pure l-alanine has been developed using batch and continuous stirred tank reactors with a new l-aminoacylase-producing bacterium Pseudomonas sp. BA2 immobilized in calcium alginate beads coated with glutaraldehyde. The maximum production of l-alanine in a continuous stirred tank reactor was 11.26 g after 2 days of operation which is higher than that previously reported.     

Kinetic locking-on and auxiliary tactics for bioaffinity purification of NADP+-dependent dehydrogenases using N6-linked immobilized NADP+ derivatives: Studies with mammalian and microbial glutamate dehydrogenases

This study is concerned with the development and application of kinetic locking-on and auxiliary tactics for bioaffinity purification of NADP(+)-dependent dehydrogenases, specifically (1) the synthesis and characterization of highly substituted N(6)-linked immobilized NADP(+) derivatives using a rapid solid-phase modular approach; (2) the evaluation of the N(6)-linked immobilized NADP(+) derivatives for use with the kinetic locking-on strategy for bioaffinity purification of NADP(+)-dependent dehydrogenases: Model bioaffinity chromatographic studies with glutamate dehydrogenase from bovine liver (GDH with dual cofactor specificity, EC 1.4.1.3) and glutamate dehydrogenase from Candida utilis (GDH which is NADP(+)-specific, EC 1.4.1.4); (3) the selection of an effective "stripping ligand" for NADP(+)-dehydrogenase bioaffinity purifications using N(6)-linked immobilized NADP(+) derivatives in the locking-on mode; and (4) the application of the developed bioaffinity chromatographic system to the purification of C. utilis GDH from a crude cellular extract.Results confirm that the newly developed N(6)-linked immobilized NADP(+) derivatives are suitable for the one-step bioaffinity purification of NADP(+)-dependent GDH provided that they are used in the locking-on mode, steps are taken to inhibit alkaline phosphatase activity in crude cellular extracts, and 2',5'-ADP is used as the stripping ligand during chromatography. The general principles described here are supported by a specific sample enzyme purification; the purification of C. utilis GDH to electrophoretic homogeneity in a single bioaffinity chromatographic step (specific activity, 9.12 micromol/min/mg; purification factor, 83.7; yield 88%). The potential for development of analogous bioaffinity systems for other NADP(+)-dependent dehydrogenases is also discussed.     

Biohydrogen production by immobilized Chlorella sp. using cycles of oxygenic photosynthesis and anaerobiosis

Hydrogen production was studied using immobilized green alga Chlorella sp. through a two-stage cyclic process where immobilized cells were first incubated in oxygenic photosynthesis followed by anaerobic incubation for H₂ production in the absence of sulfur. Chlorella sp. used in this study was capable of generating H₂ under immobilized state in agar. The externally added glucose enhanced H₂ production rates and total produced volume while shortened the lag time required for cell adaptation prior to H₂ evolution. The rate of hydrogen evolution was increased as temperature increased, and the maximum evolution rate under 30 mM glucose was 183 mL/h/L and 238 mL/h/L at 37 °C and 40 °C, respectively. In order to continue repeated cycles of H₂ production, at least two days of photosynthesis stage should be allowed for cells to recover H₂ production potential and cell viability before returning to H₂ production stage again.     

Tween-20 Activates and Solubilizes the Mitochondrial Membrane-bound, Calmodulin Dependent NAD+ Kinase of Avena sativa L.

Among different treatments assayed, a mix of a nonionic detergent (5% Tween-20) with 0.5 m NaCl was found to solubilize a large part of the calmodulin-dependent NAD⁺ kinase bound to the inner mitochondrial membrane. It also stimulated its activity by increasing 7 times the maximal velocity. Activity stimulation was also observed with phosphatidylcholine, phosphatidylethanolamine and with reductants (HSO₃ and DTT). This solubilized NAD⁺ kinase and the calmodulin-dependent cytosoluble isoform displayed distinct molecular masses, as well as different kinetic parameters. We propose that solubilization of membrane-bound NAD⁺ kinase could occur in vivo in Avena sativa and could generate a soluble isoform. Received: 6 November 2000/Revised: 18 April 2001     

Cloning,characterization and expression of two alternatively splicing isoforms of Ca2+/calmodulin-dependent protein kinase I gamma in the rat brain

Ca2+/calmodulin-dependent protein kinase I (CaMKI), originally identified as a protein kinase phosphorylating synapsin I, has been shown to constitute a family of closely related isoforms (alpha, beta and gamma). Here, we have isolated and determined the complete primary structures of two alternatively splicing isoforms of CaMKI termed CaMKI gamma 1 and -gamma 2. CaMKI gamma 1 and -gamma 2 contain an identical N-terminal catalytic domain with different C-terminal regions due to the deletion of the 425-bp nucleotide sequence of CaMKI gamma 1 in CaMKI gamma 2. In vitro kinase assay has demonstrated the marked enhancement of the Ca2+/CaM-dependent activity of CaMKI gamma 1 by the preincubation with Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), but no significant activation of CaMKI gamma 2. Northern blot analysis has demonstrated the predominant expression of CaMKI gamma in the brain. RT-PCR analysis has revealed similar expression patterns between CaMKI gamma 1 and CaMKI gamma 2 in various brain regions. In situ hybridization analysis has demonstrated that CaMKI gamma mRNA is expressed in a distinct pattern from other isoforms of CaMKI with predominant expression in some restricted brain regions such as the olfactory bulb, hippocampal pyramidal cell layer of CA3, central amygdaloid nuclei, ventromedial hypothalamic nucleus and pineal gland. In the primary hippocampal neurons and NG108-15 cells, transfected CaMKI gamma 1 and -gamma 2 are localized primarily in the cytoplasm and neurites but not in the nucleus. These findings suggest that both isoforms of CaMKI gamma may be involved in Ca2+ signal transduction in the cytoplasmic compartment of certain neuronal population.     

Preparation of an immobilized two-enzyme system, beta-amylase-pullulanase, to an acrylic copolymer for the conversion of starch to copolymer for the conversion of starch to maltose. I. Preparation and stability of immobilized beta-amylase

β-Amylase (EC 3.2.1.2), obtained from barley, was chemically attached to a crosslinked copolymer of acrylamide-acrylic acid using a water-soluble carbodiimide. The derivative showed 23% β-amylase activity in relation to that of free enzyme with a coupling yield of 40% based on the amount of added β-amylase. In order to find optimal coupling conditions, the effect of pH and different carbodiimide concentrations was investigated. The enzymic activity associated with different β-amylase concentrations was further outlined. A slightly increased operational stability for the enzyme upon immobilization was observed. Markedly improved operational stability has been obtained by coupling in the presence of reduced glutathione of bovine serum albumin.     

Preparation and characterization, using high-performance liquid chromatography, of an enzyme forming glucosides of cytokinins.

Cytokinins can occur naturally as glycosides with beta-D-glucose as the sugar substituent. From radish (Raphanus sativus) cotyledons, an enzyme has been partly purified which synthesizes the 7-glucopyranoside of zeatin [6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine], a compound known to occur in this species. High-performance reverse-phase liquid chromatography was uniquely useful as the analytical procedure for quantitative study of the minute amounts of enzyme available. The enzyme uses UDPglucose as the source of the sugar residue. A large number of derivatives of purine are glucosylated, but adenine derivatives with an alkyl side chain at least three carbon atoms in length at position N6 are preferentially glucosylated. This corresponds to the structural features required for high cytokinin activity. The 7-glucoside of zeatin is known to be very weakly active in cytokinin bioassays. Hence, this enzyme, and others catalyzing the same reaction, have a role in the regulation of cytokinin activity.     

Natural killer cell cytolytic granule-associated enzymes. I. Purification, characterization, and analysis of function of an enzyme with sulfatase activity

An enzyme with sulfatase activity has been isolated from the granules of a rat NK leukemia cell line, CRNK-16. The enzyme has been purified from crude preparation, with a specific activity of 52 nmol/min/mg of protein, by DEAE ion exchange and Con A-Sepharose affinity chromatography, resulting in a specific activity of 230 nmol/min/mg of protein. The molecular mass of the purified enzyme was estimated to be 40 kDa by gel filtration chromatography at pH 7.4, but the enzyme had the ability to complex to molecular masses of greater than 300 kDa at low pH when crude granule extract was used as the starting sample, suggesting that it associates with other granule components. The enzyme was determined to be an arylsulfatase by its ability to (a) hydrolyze p-nitrophenyl sulfate (Km = 26.0 mM) and p-nitrocatechol sulfate (pNC sulfate) (Km = 1.1 mM) and (b) be inhibited by sulfite (Ki = 6.0 x 10(-7) M), sulfate (Ki = 1 x 10(-3) M), and phosphate (Ki = 4 x 10(-5) M) in a competitive manner. The pH optimum for enzymatic activity was determined to be 5.6. The role of this enzyme in cytolytic function was investigated by examining the effect of its substrates and inhibitors on granule- and cell-mediated lysis. pNC sulfate was shown to cause a dose-dependent inhibition of target cell lysis by isolated cytolytic granules (complete inhibition at 12.5 mM). Sulfite induced an incomplete inhibition (50% at 1 mM), whereas phosphate was essentially without inhibitory effect. Sulfate, on the other hand, altered lytic activity in a biphasic manner, inasmuch as it induced an inhibition of lysis at high concentrations and an increase of lysis at low concentrations. Cell-mediated lysis was inhibited by pNC sulfate in a dose-dependent fashion at concentrations greater than 2.5 mM, with nearly complete inhibition at 50 mM. Sulfate also altered the lytic activity by intact cells in a biphasic manner, although the effect was much less pronounced. Sulfite and phosphate caused only a 30% inhibition of lytic activity. These results suggest that the sulfatase enzyme is involved in NK cytolytic function, presumably at the lethal hit stage.     

Purification and characterization of bovine brain calmodulin-dependent protein kinase II. The significance of autophosphorylation in the regulation of 63 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme

Bovine brain contains two calmodulin-dependent phosphodiesterase kinases which are separated on Sephacryl S-300 column. One of these kinases has been purified to homogeneity and shown to belong to the calmodulin-dependent protein kinase II family. Phosphorylation of the 63 kDa phosphodiesterase by this purified protein kinase results in the incorporation of 1.0 mol phosphate per mol subunit and an accompanying increase in Ca²⁺ concentrations required for the phosphodiesterase activation by calmodulin. The protein kinase undergoes autophosphorylation to incorporate 1.0 mol phosphate per mol of subunit of the enzyme and the autophosphorylated enzyme is active, independent of the presence of Ca²⁺. The autophosphorylation reaction as well as the protein kinase reaction are rendered Ca²⁺ independent in less than 15 seconds when approximately one mol phosphate per mol protein kinase is incorporated. The result suggests that activation of phosphodiesterase phosphorylation reaction may occur prior to the activation of phosphodiesterase and phosphatase during a cell Ca²⁺ flux via the protein kinase autophosphorylation mechanism.Abbreviations SDS sodium dodecyl sulfate - EGTA ethylene glycol bis (-aminoethyl ether) - N,N,N,N tetra acetic acid - EDTA ethylenediamine-tetraacetic acid - cAMP cyclic adenosine 35 monophosphate This work is supported by grants from the Medical Research Council of Canada (JHW), the Heart and Stroke Foundation of Alberta (JHW and RKS) and the Heart and Stroke Foundation of Saskatchewan (RKS)     

Expression, purification from inclusion bodies, and crystal characterization of a transition state analog complex of arginine kinase: a model for studying phosphagen kinases.

Phosphagen kinases catalyze the reversible transfer of a phosphoryl group between guanidino phosphate compounds and ADP, thereby regenerating ATP during bursts of cellular activity. Large quantities of highly pure arginine kinase (EC 2.7.3.3), the phosphagen kinase present in arthropods, have been isolated from E. coli, into which the cDNA for the horseshoe crab enzyme had been cloned. Purification involves size exclusion and anion exchange chromatographies applied in the denatured and refolded states. The recombinant enzyme has been crystallized as a transition state analog complex. Near complete native diffraction data have been collected to 1.86 A resolution. Substitution of a recombinant source for a natural one, improvement in the purification, and data collection at cryo temperatures have all yielded significant improvements in diffraction.     

Expression,purification, and characterization of bovine chymosin enzyme using an inducible pTOLT system

In recent years, various studies in the field of industrial enzymes of biotechnology have gained importance due to increasing development in enzyme technology. The different areas where enzymes are used and their economic value of biotechnological products further increases their importance. There are hundreds of different types of cheese but each is made by coagulating milk using rennet to give curds. Today, researchers have begun to develop alternative systems in the cheese industry related to milk-clotting enzymes. In this study, the nucleic acid sequence encoding the optimized chymosin enzyme was used and cloned by Not I and Mlu I restriction enzymes into pTOLT vector system. Then using this construct, the enzyme as a fusion with Tol-A-III protein was produced in Escherichia coli BL21 (DE3) cells. After disrupting the E. coli cell and separating from the constituents by high speed centrifugation, the enzyme was purified by affinity chromatography and fractions were analyzed by SDS–PAGE. Purified enzyme has shown its activity. Optimum temperature and pH of CHY-Tol-A-III protein were 40°C and 6.5, respectively.     

Subchloroplastic localization of NAD kinase activity: evidence for a Ca2+, calmodulin-dependent activity at the envelope and for a Ca2+, calmodulin-independent activity in the stroma of pea chloroplasts

Chloroplasts were prepared from pea seedlings and tested for NAD kinase activity. More than half of a Ca²⁺, calmodulin-dependent activity and most of a Ca²⁺, calmodulin-independent activity of the homogenate were associated with chloroplasts. The Ca²⁺, calmodulin-dependent activity could be detected by adding Ca²⁺ and calmodulin to the incubation medium containing intact chloroplasts. This activity could not be separated from the chloroplasts by successive washes or by phase partition in aqueous two-polymer phase systems. After chloroplasts fractionation, the Ca²⁺, calmodulin-dependent NAD kinase activity was localized at the envelope, and the Ca²⁺, calmodulin-independent activity was recovered from the stroma. In view of these results and of a previous report [Simon (1982) Plant Cell Rep. 1, 119–122] the occurrence and presumed role of calmodulin in the chloroplast are discussed.