Substrate-binding properties of the family 54 module of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> Lic16A laminarinase

Endo-β-1.3–1.4-glucanase Lic16A of the moderate thermophilic anaerobe Clostridium thermocellum has a complex multimodular structure. In addition to the catalytic module, Lic16A contains eight auxiliary modules, five of which are substrate-binding modules. The new family 54 substrate-binding module CBM54 (25.2 kDa), which is at the N terminus of the enzyme, has a cleavage site in its N-terminal part, and its cleavage yields a shortened module CBM54C (17.2 kDa) in vivo and in vitro. CBM54C was cloned in Escherichia coli and purified to electrophoretic homogeneity. The binding constants of CBM54C to xylan, chitin, insoluble yeast cell wall β-glucan, and bacterial crystalline cellulose were of the same order of magnitude as for CBM54. However, CBM54C did not bind pustulan, avicel, and chitosan, in contrast to CBM54. Calcium ions restored the ability of CBM54C to bind these three carbohydrates. CBM54 substrate binding promiscuity suggested multiple binding sites, some of them being Ca²⁺ dependent. The Ca²⁺-independent sites for avicel, pustulan and chitosan were localized to the spontaneously split-off N-terminal part (8 kDa) of CBM54. The arrangement of Ca²⁺-dependent and Ca²⁺-independent binding sites for various substrates was suggested.     

Thermostable xylanase10B from <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> ATCC824

The Clostridium acetobutylicum xylanase gene xyn10B (CAP0116) was cloned from the type strain ATCC 824, whose genome was recently sequenced. The nucleotide sequence of C. acetobutylicum xyn10B encodes a 318-amino acid protein. Xyn10B consists of a single catalytic domain that belongs to family 10 of glycosyl hydrolases. The enzyme was purified from recombinant Escherichia coli. The Xyn10B enzyme was highly active toward birchwood xylan, oat-spelt xylan, and moderately active toward avicel, carboxymethyl cellulose, polygalacturonic acid, lichenan, laminarin, barley--glucan and various p-nitrophenyl monosaccharides. Xyn10B hydrolyzed xylan and xylooligosaccharides to produce xylobiose and xylotriose. The pH optimum of Xyn10B was 5.0, and the optimal temperature was 70°C. The enzyme was stable at 60°C at pH 5.0–6.5 for 1 h without substrate. This is one of a number of xylan-related activities encoded on the large plasmid in C. acetobutylicum ATCC 824.     

Steroid hormone specifically binds to rat kidney plasma membrane

A high-affinity and low-capacity corticosterone specific binding was detected in the purified plasma membrane preparation from rat kidney using anin vitro steroid hormone binding assay. The specific-bound hormone was efficiently distinguished from the irreversible-bound hormone with 10 µM corticosterone. Under standardized conditions of pH 7.4 at 2°C and 30 min incubation time, the binding was saturable and showedK _d=13±3 nM andB _max=616±34 fmol/mg of protein. Competitive binding studies with analogue steroids indicated that corticosterone binding to kidney plasma membrane is hormone-specific. Results indicated that the possible nongenomic effects of steroids could be mediated by their interaction with plasma membrane.     

Effect of temperature and pH on phenol removal using purified Coprinus cinereus peroxidase

The removal of phenol by peroxidase-catalysed polymerization was examined using purified Coprinus cinereus peroxidase. The phenol removal efficiency increased with a decrease in the reaction temperature over the range of 0–70 °C, though only a trace of enzyme activity with 4-aminoantipyrine (4-AAP), phenol and hydrogen peroxide was found at 0 °C. The optimum pH value for phenol removal was 9.0, while the enzyme expressed maximum activity at pH 7.5 in the presence of 4-AAP, phenol and hydrogen peroxide. By measuring residual enzyme activity in the polymerizing reaction mixture, it was shown that enzyme inactivation by free radicals was more suppressed at 0 °C than at 40 °C and that the adsorption of the enzyme on the polymerized precipitate was more suppressed at pH 9.0 than that at pH 7.5.     

Comparative oxygen affinity of fish and mammalian myoglobins

Summary Myoglobins from rat, coho salmon (Oncorhynchus kisutch), buffalo sculpin (Enophrys bison) hearts, and yellowfin tuna (Thunnus albacares) red skeletal muscle were partially purified and their O₂ binding affinities determined. Commercially prepared sperm whale myoglobin was employed as an internal standard. Tested at 20°C, myoglobins from salmon and sculpin bound O₂ with lower affinity than myoglobins from the rat or sperm whale. Oxygen binding studies at 12°C and 37°C suggest that this difference is adaptive, permitting myoglobins from cold-adapted fish to function at physiologically relevant temperatures. Taken together, purification and O₂ binding data obtained in this study reveal a previously unrecognized diversity of myoglobin structure and function.     

Studies on the galactomannan-degrading enzymes produced bySporotrichum cellulophilum

Summary Extracellular mannanase activity produced bySporotrichum cellulophilum was purified into two components using acetone precipitation, SP-Sephadex C50 ion exchange chromatography and preparative polyacrylamide gel electrophoresis. The purified mannanse components, M1 and M2, had molecular weights of 108 000–112 000 and 32 200–36 000 respectively. Component M1 was shown to contain 2 subunits having molecular weights of 62 000 and 50 000. M1 and M2 had similar pH-activity profiles with pH optima of 5.5 and 6.0 respectively. M1 was more thermostable than M2: half lives of the enzymes at 70°C were 30 and 9 min for M1 and M2 respectively.     

Halophilic characterization of starch-binding domain from Kocuria varians α-amylase

The tandem starch-binding domains (KvSBD) located at carboxy-terminal region of halophilic α-amylase from moderate halophile, Kocuria varians, were expressed in E. coli with amino-terminal hexa-His-tag and purified to homogeneity. The recombinant KvSBD showed binding activity to raw starch granules at low to high salt concentrations. The binding activity of KvSBD to starch was fully reversible after heat-treatment at 85 °C. Circular dichroism and thermal scanning experiments indicated that KvSBD showed fully reversible refolding upon cooling after complete melting at 70 °C in the presence of 0.2-2.0 M NaCl. The refolding rate was enhanced with higher salt concentration.     

Effect of temperature on vegetative growth among isolates of Metarhizium anisopliae and M. flavoviride

Effects of temperature on vegetative growth on a semi-synthetic medium of 22 isolates of Metarhizium anisopliae and 14 isolates of M. flavoviride were determined. The majority of isolates of both species grew between 11 and 32°C; several isolates grew at 8 and 37 °C. None of the isolates grew at 40 °C. Relative growth rate, calculated from the maximum growth rate for each isolate, was significantly affected by temperature and isolate, with significant isolate * temperature interactions. The maximum absolute growth rates among the isolates ranged from 2.5 mm to 5.9 mm/day. Optimal temperatures were generally between 25 and 32 °C with several isolates exhibiting optimal growth at temperatures as high as 32 °C. Overall, relative growth rates were greater in isolates of M. anisopliae than M. flavoviride at temperatures of 25 °C or lower; conversely mean relative growth rates were greater in M. flavoviride than M. anisopliae at temperatures higher than 25 °C. However, the two most cold tolerant isolates at 8 °C were M. flavoviride and the three most heat tolerant at 35 °C were M. anisopliae. Since temperature growth responses varied considerably between isolates, strain selection according to thermal tolerance may be warranted when choosing a strain for development as a microbial control agent.This revised version was published online in October 2005 with corrections to the Cover Date.     

Purification of the alkaliphilic xylanases from Myceliophthora sp. IMI 387099 using cellulose-binding domain as an affinity tag

Ten xylanase isoforms produced by Myceliophthora sp. were characterized for their ability to bind to avicel. Three of the xylanases showing differential affinity for avicel were purified by column chromatography. The purified xylanase Xyl IIa, IIb and IIc showed molecular mass of 47, 41 and 30 kDa and pI of ∼3.5, 4.8 and 5.2, respectively. Xyl IIa was optimally active at pH 8.0 and temperature 70 °C, while Xyl IIb and IIc were optimally active at pH 9.0 and 60 °C and 7.0 and 80 °C, respectively. Xyl IIa and Xyl IIb showed higher stability under alkaline conditions (pH 9.0) and retained 80% of the original activity upto 1 h and 3 h respectively, at 50 °C. All three purified iso-xylanases showed enhanced activities in presence of Na⁺, Mg²⁺, Mn²⁺ and K⁺ ions, whereas, Zn²⁺ and Cu²⁺ showed negative effect on Xyl IIa. The activity of Xyl IIa increased in presence of reducing agents DTT and mercaptoethanol, however, SDS showed inhibitory effect. Kinetic studies showed that Xyl IIb and IIc degrade rye arabinoxylan, much more efficiently than oat spelt xylan, whereas, Xyl IIa showed much higher Kcat/Km value for birch wood xylan as compared to oat spelt xylan. The purified xylanases were apparently classified in family 10.     

Effect of temperature on the adsorption and one-step growth of the Nostoc virus N-1

This study was an attempt to observe the effects of temperature on adsorption and one-step growth of the virus N-1 infecting the nitrogen-fixing cyanobacterium Nostoc muscorum. Adsorption rate was found to be maximum at 40° C whereas no adsorption occurred at 10° C. The Q ₁₀ value was about 2.03 and the energy of activation, Ea was 16.3 kcal/ mole for the adsorption process. The development cycle of the virus was temperature sensitive. With increase in temperature, a gradual increase in inhibition of virus yield i.e. 8.33% at 30° C, 35.3% at 35° C and complete inhibition at 40° C was observed. Out of 7 h latent period, the early 4 h were temperature sensitive and heat treatment had a reversible inhibitory effect on virus development. The temperature treatment did not affect the rise period but burst-size was reduced.List of Abbreviattions PFU plaque-forming units - IM input multiplicity     

Application of adsorption isotherms to chromium adsorption onZ.ramigera

Summary Adsorption isotherms for the adsorption of chromium onZoogloea ramigera are developed. The rates were affected by the pH and temperature of adsorption medium. The biomass ofZ. ramigera at pH 2.0 where the optimum pH for biosorption lies exhibited the highest chromium adsorptive uptake capacity. In general, higher adsorptive uptake was observed at 25°C than 35°C and 45°C.     

Fast and efficient adsorption/desorption of protein by a novel magnetic nano-adsorbent

A novel magnetic nano-adsorbent was prepared by covalently binding polyacrylic acid (PAA) on Fe₃O₄ superparamagnetic nanoparticles (13.2 nm) via carbodiimide activation. The maximum weight ratio of PAA to Fe₃O₄ was 0.12 (i.e., average of two PAA molecules on a magnetic nanoparticle). The magnetic nano-adsorbent possessed a high ionic exchange capacity of 1.64 meq g^–1 and was efficient for the recovery of lysozyme. The lysozyme could be completely adsorbed in 0.1 M phosphate buffer at pH 3–5 and completely desorbed in NaSCN solution (>1 M) within 1 min, and retained 95% activity after adsorption/desorption. In addition, the adsorption behavior followed the Langmuir adsorption isotherm with a maximum adsorption amount of 0.224 mg mg^–1 and a Langmuir adsorption equilibrium constant of 10 ml mg^–1 at 25 °C. The change of enthalpy at 15–35 °C was –4.2 kJ ml mol^–1 mg^–1.     

Purification, characterization, and primary structure of a chitinase from Pseudomonas sp. YHS-A2

A chitinase gene (chiA) from Pseudomonas sp. YHS-A2 was cloned into Escherichia coli using pUC19. The nucleotide sequence determination revealed a single open reading frame of chiA comprised of 1902 nucleotide base pairs and 633 deduced amino acids with a molecular weight of 67,452 Da. Amino acid sequence alignment showed that ChiA contains two putative chitin-binding domains and a single catalytic domain. Two proline-threonine repeat regions, which are linkers between catalytic and substrate-binding domains in some cellulases and xylanases, were also found. From E. coli, ChiA was purified 12.8-fold relative to the periplasmic fraction. The Michaelis constant and maximum initial velocity for p-nitrophenyl-N,N′-diacetylchitobiose were 1.06 mM and 44.4 μmol/h per mg protein, respectively. The purified ChiA binds not only to colloidal chitin but also to other substrates (avicel, chitosan, and xylan), but the binding affinity of avicel, chitosan, and xylan is around 10 times lower than that of colloidal chitin. The reaction of ChiA with colloidal chitin and chitooligosaccharides (trimer-hexamer) produced an end product of N,N′-diacetylchitobiose, indicating that ChiA is a chitobiosidase. Received: 29 October 1999 / Received revision: 16 March 2000 / Accepted: 24 March 2000     

Purification and characterization of membrane-bound aldehyde dehydrogenase from Acetobacter polyoxogenes sp. nov.

Summary Membrane-bound aldehyde dehydrogenase (ALDH) was purified from the membrane fraction of an industrial-vinegar-producing strain, Acetobacter polyoxogenes sp. nov. NBI1028 by solubilization with Triton X-100 and sodium N-lauroyl sarcosinate and subsequent column chromatography on DEAE-Sepharose CL-6B and hydroxyapatite. The purified enzyme was homogeneos on polyacrylamide disc gel electrophoresis. Upon sodium dodecyl sulphate-polyacrylamide gelelectrophoresis, the enzyme showed the presence of two subunits with a molecular mass of 75 000 daltons and 19 000 daltons, respectively. From the absorption and fluorescence spectra, the absence of cytochrome c and the presence of pyrroloquinoline quinone in the purified enzyme were demonstrated. The ALDH preferentially oxidized aliphatic aldehyde with a straight carbon chain except for formaldehyde. The apparent K _m for acetaldehyde was 12 mM. The optimum pH and temperature were 7.0 and 50°–60°C, respectively. The enzyme remained active after storage at 4°C for 20 days. p-Chloromercuribenzoic acid and heavy metal salts such as CuSO₄ were inhibitory to the enzyme. Ferricyanide was effective as an electron acceptor.Offprint requests to: M. Fukaya     

Purification and properties of dipeptidase from Escherichia coli AJ005

Summary A Co²⁺-dependent dipeptidase from E. coli strain AJ005, a peptidase-deficient mutant, was purified with streptomycin sulfate, ammonium sulfate and DEAE-cellulose. The purified dipeptidase increased by about 106-fold in specific activity, with dilysine as a substrate. The dipeptidase cleaved dilysine to two lysines among the lysine homopolymers, the possibility remaining that it is active toward peptides other than dilysine, since it was investigated in the present study only for activity toward lysine homopolymers. Activity was inhibited 54% by 10^–3 M KCN and completely by 10^–3 M PCMB, EDTA and benzethonium chloride, but not at all by soybean trypsin inhibitors. 78% and 95% of its activity was lost with 30 minutes' treatment at 45°C and 50°C, respectively. The apparent Km value was 6.7 × 10^–4 M for dilysine. It is probable that the dipeptidase differs from dipeptidase DP.Abbreviations EDTA Ethylenediaminetetraacetate - PCMB pchloromercuribenzoate     

Adsorption of a xylanase purified from Pulpzyme HC onto alkali-lignin and crystalline cellulose

Adsorption of a xylanase purified from a commercial xylanase, Pulpzyme HC, onto two model components of kraft pulp, crystalline cellulose (Avicel) and alkali-lignin (Indulin AT), was studied at 40°C. A considerable amount of the purified xylanase was adsorbed onto alkali-lignin in alkaline solutions. The adsorption of the purified xylanase onto crystalline cellulose was not significant and could be described by the Langmuir-type adsorption isotherm. The adsorption of the purified xylanase onto alkali-lignin was assumed to be caused by physical or van der Waals interaction based on the result that NaCl did not change the adsorption isotherm. © Rapid Science Ltd. 1998     

Coliphage 434 tofprotein: NH2-terminal amino acid sequence and kinetic and equilibrium measurements of DNA binding

Summary Coliphage 434 tof protein was purified to a substantially pure state from imm ⁴³⁴ cI dv carrier cells. The minimum molecular weight is 7,500±500 as estimated by polyacrylamide gel electrophoresis. The amino acid sequence of the nine NH₂-terminal residues was determined, by manual Edman degradation of the intact protein, to be Met-Gln-Thr-Leu-Ser-Glu-Arg-Leu-(Lys)-.The purified protein at low concentrations binds specifically to imm ⁴³⁴dv DNA and at high concentrations also binds to imm ²¹dv and dv DNA. The curve of the specific binding is of Michaelis type, while that of the nonspecific binding is sigmoidal. The specific binding does not show marked temperature dependency at 4°–37°C. We have analyzed the equilibrium and kinetic data of specific binding. The equilibrium dissociation constant is 1.9x10^-11M at 0°C. The association rate constant and the dissociation rate constant are 1.1–2.9x10⁸M^-1s^-1 and 2.7x10^-3s^-1, respectively, at 0°C. The half life of the tof protein-operator DNA complex is 260s. These results suggest that the tof protein-operator interaction is much weaker than the interaction between the cI repressor and the operator reported by other workers.     

Properties of two endoglucanases from a mutant strain Trichoderma sp. M7

Two endoglucanases (1,4--d-glucan-4-glucanohydrolase, EC 3.2.1.4) were purified to electrophoretic homogeneity from the culture filtrate of a mutant strain Trichoderma sp. M₇. The purified endoglucanases had Mr of 57.4 and 55 kDa, and estimated pI values of 4.1 and 3.95/3.75, respectively. Optimal activity for the first cellulase was at pH 4.5 and 50 °C, and at pH 5.5 and 60 °C for the other. Carbodiimide inactivated the one of the purified endoglucanases, while the other was inhibited by iodoacetamide, indicating the involvement of carboxylic or thiol groups in the catalysis. N-Bromsuccinimide strongly inhibited both endoglucanases, suggesting that tryptophan residues are essential for the activity and binding to the substrate.     

Resistance of Rosa microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin

The inhibition of the polymerization of tubulin from cultured cells of rose (Rosa. sp. cv. Paul's scarlet) by colchicine and the binding of colchicine to tubulin were examined in vitro and compared with data obtained in parallel experiments with bovine brain tubulin. Turbidimetric measurements of taxol-induced polymerization of rose microtubules were found to be sensitive and semiquantitative at low tubulin concentrations, and to conform to some of the characteristics of a nucleation and condensation-polymerization mechanism for assembly of filamentous helical polymers. Colchicine inhibited the rapid phase of polymerization at 24°C with an apparent inhibition constant (K _i) of 1.4·10^-4 M for rose tubulin and an apparent K _i=8.8·10^-7 M for brain tubulin. The binding of [³H]colchicine to rose tubulin to form tubulin-colchicine complex was mildly temperature-dependent and slow, taking 2–3 h to reach equilibrium at 24°C, and was not affected by vinblastine sulfate. The binding of [³H]colchicine to rose tubulin was saturable and Scatchard analysis indicated a single class of low-affinity binding sites having an apparent affinity constant (K) of 9.7·10² M^-1 and an estimated molar binding stoichiometry (r) of 0.47 at 24°C. The values for brain tubulin were K=2.46·10⁶ M^-1 and r=0.45 at 37°C. The binding of [³H]colchicine to rose tubulin was inhibited by excess unlabeled colchicine, but not by podophyllotoxin or tropolone. The data demonstrate divergence of the colchicine-binding sites on plant and animal tubulins and indicate that the relative resistance of plant microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin.Abbreviations MT microtubule - TC tubulin-colchicine complex     

Isolation and partial characterization of bacteriocins from<Emphasis Type="Italic"> Pediococcus</Emphasis> species

Lactic acid bacteria have received increased attention as a potential food preservative due to their strong antagonistic activity against many food-spoilage and pathogenic organisms. Three Pediococcus species, P. acidilactici NCIM 2292, P. pentosaceous. NCIM 2296 and P. cervisiae NCIM 2171, were evaluated for bacteriocin production. Inhibitory substance were produced during the late growth phase and maximum production occurred at 37 °C after 36–48 h of incubation. Bacteriocins partially purified from these species by cold-acetone precipitation at 0 °C and cell adsorption desorption techniques have a broad inhibitory spectrum against microorganisms, including gram-negative bacteria such as Escherichia coli and Pseudomonas. Proteolytic enzymes inactivated these peptides, but amylase and lipase did not show any effect. The bacteriocins were stable over a wide pH range (3–8) and apparently most active at pH 4.0–5.0. They were heat-stable (1 h at ~80 °C and autoclaving) at pH 5.0. No loss in activity was observed when stored under refrigeration (4–8 °C). Tris-Tricine SDS-PAGE revealed the molecular masses of these peptides to be between 3.5 and 5.0 kDa.