Heterogeneity and subunit composition of the haemoglobins of five tilapiine species (Teleostei, Cichlidae) of the genera Oreochromis and Sarotherodon

Haemoglobins of five tilapiine species of the genera Oreochromis and Sarotherodon were investigated. By gel filtration chromatography a molecular weight of 67–69 kDa was determined for the tetrameric molecules which remained stable between pH 5.0 and pH 9.1. When subjected to sodium dodecyl sulphate-Urea-polyacrylamide gel electrophoresis (PAGE), haemoglobins of all species each were split into monomers of three different molecular weights ranging between 16.3 kDA and 17.6 kDa. Subsequently, isoelectric focusing separated haemolysates into about 23 differently charged tetrameric haemoglobins that were arranged in species-specific patterns. This diversity was shown to result from the occurrence of different types of globin chains. By acidic urea PAGE a total of seven major α-globins and five major β-globins were detected and species-characteristic chain variants were identified. To determine the globin chain composition of particular haemoglobin tetramers, 26 bands were isolated by isoelectric focusing and analysed by acidic urea PAGE. Tetramers consisted of doublets of identical α- and identical β-chains (α₂β₂, symmetric tetramers), or combinations of three (α₂ββ*; αα*β₂) or four (αα*ββ*) distinct chains (asymmetric tetramers). Finally, globin chains of Oreochromis niloticus were subjected to partial N-terminal amino acid sequencing. Differences in the composition of the three major β-chains could be shown, whereas the α-chains were N-terminally blocked. Accepted: 12 September 1997     

<Emphasis Type="Italic">Poseidonocella pacifica</Emphasis> gen. nov., sp. nov. and <Emphasis Type="Italic">Poseidonocella sedimentorum</Emphasis> sp. nov., novel alphaproteobacteria from the shallow sandy sediments of the Sea of Japan

The taxonomic study of two Gram-negative, aerobic, non-pigmented bacteria KMM 9010^T and KMM 9023^T isolated from a sandy sediment sample collected from the Sea of Japan seashore was performed. On the basis of the nearly complete 16S rRNA gene sequences, strains KMM 9010^T and KMM 9023^T clustered with the Roseobacter lineage (class Alphaproteobacteria) forming a distinct phylogenetic line adjacent to the genus Donghicola. Novel strains shared the highest sequence similarity of 96.4% to each other and lower than 96.1% similarities to other validly named genera of the class Alphaproteobacteria. In both strains, ubiquinone Q-10 was found to be the major respiratory quinone; phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidic acid, and an unknown aminolipid were the major polar lipids and C_18:1ω7c and 11-methyl C_18:1ω7c were predominant fatty acids. The DNA G+C content was 60.5 mol% (KMM 9010^T) and 65.4 mol% (KMM 9023^T). Based on phenotypic properties and phylogenetic evidence, strains KMM 9010^T and KMM 9023^T should be classified as two novel species in a new genus, Poseidonocella gen. nov., with Poseidonocella pacifica sp. nov., the type species with the type strain KMM 9010^T (= NRIC 0794^T = JCM 17310^T), and Poseidonocella sedimentorum sp. nov. as the second species with the type strain KMM 9023^T (= NRIC 0796^T = JCM 17311^T).     

Catalytic properties and amino acid sequence of endo-1→3-β-D-glucanase from the marine mollusk <Emphasis Type="Italic">Tapes literata</Emphasis>

A specific 1→3-β-D-glucanase with molecular mass 37 kDa was isolated in homogeneous state from crystalline style of the commercial marine mollusk Tapes literata. It exhibits maximal activity within the pH range from 4.5 to 7.5 at 45dgC. The 1→3-β-D-glucanase catalyzes hydrolysis of β-1→3 bonds in glucans as an endoenzyme with retention of bond configuration, and it has transglycosylating activity. The K _m for hydrolysis of laminaran is 0.25 mg/ml. The enzyme is classified as a glucan endo-(1→3)-β-D-glucosidase (EC 3.2.1.39). The cDNA encoding this 1→3-β-D-glucanase from T. literata was sequenced, and the amino acid sequence of the enzyme was determined. The endo-1→3-β-D-glucanase from T. literata was assigned to the 16th structural family (GHF 16) of O-glycoside hydrolases.     

Subtilisin-like proteinase secreted by the <Emphasis Type="Italic">Bacillus pumilus</Emphasis> KMM 62 strain at different growth stages

Proteinase secreted in the environment by bacilli on different growth stages was isolated by ion chromatography from the culture medium of Bacillus pumilus KMM 62. According to the hydrolysis character of specific chromogenic substrates and inhibition type, the enzyme belongs to subtilisin-like serine proteinases. The isolated proteinase with the molecular mass of 30 kDa displays maximum activity on hydrolysis of the peptide substrate Z-Ala-Ala-Leu-pNA at pH 8.0–8.5 and temperature 30°C. The protein is stable in the range of pH 7.5–10.0. It was shown that subtilisin-like serine proteinase from B. pumilus KMM 62 possessed thrombolytic activity.     

<Emphasis Type="Italic">Bacillus berkeleyi</Emphasis> sp. nov., isolated from the sea urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis>

A bacterial strain, designated KMM 6244^T, was isolated from the sea urchin Strongylocentrotus intermedius and subjected to a polyphasic taxonomic investigation. The bacterium was found to be heterotrophic, aerobic, non-motile and spore-forming. Comparative phylogenetic analysis based on 16S rRNA gene sequencing placed the marine isolate in the genus Bacillus. The nearest neighbor of strain KMM 6244^T was Bacillus decolorationis LMG 19507^T with a 16S rRNA gene sequence similarity of 98.0%. Sequence similarities with the other recognized Bacillus species were less than 96.0%. The results of the DNA–DNA hybridization experiments revealed a low relatedness (37%) of the novel isolate with the type strain of B. decolorationis LMG 19507^T. Strain KMM 6244^T grew at 4–45°C and with 0–12% NaCl. It produced catalase and oxidase and hydrolyzed aesculin, casein, gelatin and DNA. The predominant fatty acids were anteiso-C_15:0, iso-C_15:0, anteiso-C_17:0, C_15:0, iso-C_16:0 and iso-C_14:0. The DNA G + C content was 39.4 mol%. A combination of phylogenetic, genotypic and phenotypic data clearly indicated that strain KMM 6244^T represents a novel species in the genus Bacillus, for which the name Bacillus berkeleyi sp. nov. is proposed. The type strain is KMM 6244^T (KCTC 12718^T = LMG 26357^T).     

Recent trends in the biochemistry of surfactin

The name surfactin refers to a bacterial cyclic lipopeptide, primarily renowned for its exceptional surfactant power since it lowers the surface tension of water from 72 mN m⁻¹ to 27 mN m⁻¹ at a concentration as low as 20 μM. Although surfactin was discovered about 30 years ago, there has been a revival of interest in this compound over the past decade, triggered by an increasing demand for effective biosurfactants for difficult contemporary ecological problems. This simple molecule also looks very promising as an antitumoral, antiviral and anti-Mycoplasma agent. Structural characteristics show the presence of a heptapeptide with an LLDLLDL chiral sequence linked, via a lactone bond, to a β-hydroxy fatty acid with 13–15 C atoms. In solution, the molecule exhibits a characteristic “horse saddle” conformation that accounts for its large spectrum of biological activity, making it very attractive for both industrial applications and academic studies. Surfactin biosynthesis is catalysed non-ribosomally by the action of a large multienzyme complex consisting of four modular building blocks, called the surfactin synthetase. The biosynthetic activity involves the multicarrier thiotemplate mechanism and the enzyme is organized in structural domains that place it in the family of peptide synthetases, a class of enzymes involved in peptidic secondary-metabolite synthesis. The srfA operon, the sfp gene encoding a 4′-phosphopantetheinyltransferase and the comA regulatory gene work together for surfactin biosynthesis, while the gene encoding the acyltransferase remains to be isolated. Concerning surfactin production, there is no indication whether the genetic regulation, involving a quorum-sensing mechanism, overrides other regulation factors promoted by the fermentation conditions. Knowledge of the modular arrangement of the peptide synthetases is of the utmost relevance to combinatorial biosynthetic approaches and has been successfully used at the gene level to modify the surfactin template. Biosynthetic and genetic rationales have been described for building variants. A fine study of the structure/function relationships associated with the three-dimensional structure has led to the recognition of the specific residues required for activity. These studies will assist researchers in the selection of molecules with improved and/or refined properties useful in oil and biomedical industries. Received: 9 October 1998 / Received revision: 29 January 1999 / Accepted: 31 January 1999     

A cold-active β-glucosidase (Bgl1C) from a sea bacteria <Emphasis Type="Italic">Exiguobacterium oxidotolerans</Emphasis> A011

By constructing a genomic library, a new gene encoding β-glucosidase (Bgl1C) was cloned from Exiguobacterium oxidotolerans A011, which was isolated from deep sea mud. The putative β-glucosidase gene consisted of an open reading frame (ORF) of 1,347 nucleotides, and encoded a protein of 448 amino acids with a predicted molecular weight of 51.6 kDa. Bgl1C belonged to the glycoside hydrolase family 1, and the deduced amino acid sequence displayed the highest identity (68%) to the β-glucosidase from Bacillus coahuilensis m4-4. Optimal conditions for activity were pH 7 and a temperature of 35°C and Bgl1C was stable in buffers ranging from pH 6.6 to 9. The specific activity, K _m, and V _max for the substrate p-nitrophenyl-β-d-glucopyranoside were 41 U mg⁻¹, 1.72 mg ml⁻¹ and 0.45 μg ml⁻¹ s⁻¹, respectively. Na⁺, Ca²⁺, EDTA and β-mercaptoethanol had no effect on the activity, while Hg²⁺, Cu²⁺, Co²⁺ strongly inhibited it. It is noteworthy that Bgl1C is a cold active enzyme that retains about 61% of its maximum activity at 10°C. Structural model of Bgl1C revealed that some amino acids (glycine, alanine, serine, valine) concerned with plasticity and flexibility were located around the active sites, this may contributed to the cold adaption of Bgl1C. These favorable features make Bgl1C a potential candidate for various industrial applications.     

Characterization of a novel β-glucosidase-like activity from a soil metagenome

We report the cloning of a novel β-glucosidase-like gene by function-based screening of a metagenomic library from uncultured soil microorganisms. The gene was named bgllC and has an open reading frame of 1,443 base pairs. It encodes a 481 amino acid polypeptide with a predicted molecular mass of about 57.8 kDa. The deduced amino acid sequence did not show any homology with known β-glucosidases. The putative β-glucosidase gene was subcloned into the pETBlue-2 vector and overexpressed in E. coli Tuner (DE3) pLacI; the recombinant protein was purified to homogeneity. Functional characterization with a high performance liquid chromatography method demonstrated that the recombinant BgllC protein hydrolyzed d-glucosyl-β-(l–4)-d-glucose to glucose. The maximum activity for BgllC protein occurred at pH 8.0 and 42°C using p-nitrophenyl-β-d-glucoside as the substrate. A CaCl₂ concentration of 1 mM was required for optimal activity. The putative β-glucosidase had an apparent K _m value of 0.19 mM, a V _max value of 4.75 U/mg and a k _cat value of 316.7/min under the optimal reaction conditions. The biochemical characterization of BgllC has enlarged our understanding of the novel enzymes that can be isolated from the soil metagenome.     

Biotransformation of β-ionone by engineered cytochrome P450 BM-3

Wild-type cytochrome P450 monooxygenase from Bacillus megaterium (P450 BM-3) has a low hydroxylation activity for β-ionone (<1 min⁻¹). Substitution of phenylalanine by valine at position 87 led to a more than 100-fold increase in β-ionone hydroxylation activity (115 min⁻¹). Enzyme activity could be further increased by both site-directed and random mutagenesis. The mutant R47L Y51F F87V, designed by site-directed mutagenesis, and the mutant A74E F87V P386S, obtained after two rounds of error-prone polymerase chain reaction, exhibited an increase in activity of up to 300-fold compared to the wild-type enzyme. The triple mutant R47 LY51F F87V exhibited moderate enantioselectivity, forming (R)-4-hydroxy-β-ionone with an optical purity of 39%. All mutants regioselectively converted β-ionone into 4-hydroxy-β-ionone. The regioselectivity is determined amongst others by the absolute configuration of the substrate.     

Synthesis and biological activity of oxytocin analogues containing unnatural amino acids in position 9: structure activity study

We report the solid phase synthesis and some pharmacological properties of 24 oxytocin (OT) analogues. Basic modifications at position 9 (introduction of l- or d-β-(2-thienyl)-alanine [L- or D-Thi], or l- or d-3-Pyridylalanine [l- or d-3-Pal]) were combined with d-tyrosine(OEthyl) [d-Tyr(Et)] or d-1-naphthylalanine [d-1-Nal] in position 2 and β-mercaptopropionic acid (Mpa) in position 1 modifications in altogether 14 analogues. Additionally, 8 analogues having α-aminoisobutyric acid [Aib] or d-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (d-Tic) or diethylglycine (Deg) in position 9 and d-Tyr(Et) or d-1-Nal or d-Tic in position 2 and Mpa or Pen (ββ-dimethylcysteine) in position 1 were prepared. Two of these analogues have one more modification in position 6, i.e. Pen. Furthermore, two analogues having Mpa in position 1 and d-Tyr(Et) or d-1-Nal in position 2 were prepared for comparison purposes. The analogues were tested for rat uterotonic activity in vitro, in the rat pressor assay and for binding affinity to human OT receptor. The analogue having the highest anti-oxytocic activity was [Mpa¹, d-Tyr(Et)², Deg⁹]OT (pA₂ = 8.68 ± 0.26); this analogue was also selective.     

Hydroxylation of steroids by <Emphasis Type="Italic">Curvularia lunata</Emphasis> mycelium in the presence of methyl-β-cyclodextrine

Transformation of 16 Δ⁵-3β-hydroxy- and Δ⁴-3-ketosteroids of androstane and pregnane classes was carried out using Curvularia lunata mycelium suspended in phosphate buffer with methyl-β-cyclodextrine (MCD). As the result, 20 monohydroxy- and dihydroxy-metabolites, whose structure was determined using spectra of proton magnetic resonance and mass-spectra, have been isolated. Hydroxylation of Δ⁵-3β-hydroxy-steroids occurred mostly in the C-7α position whereas hydroxylation of Δ⁴-3-ketosteroids was in the C-11β position. Only androst-4-en-3,17-dione, 9α-hydroxy-androstenedione, and androsta-1,4-diene-3,17-dione were hydroxylated at C-14α position. Besides main 11β-derivatives, the 6β- and 7β-hydroxy-derivatives with yield 10 and 30%, respectively, were isolated during transformation of progesterone and hydroxymethyl pregnadienone. The ratio of MCD to transforming steroid was 1: 1 (mol/mol). Hydrocortisone and 7α-hydroxyandrostenolone with the yield 55 and 77%, respectively, were obtained at the maximal concentrations of cortexolone 20 g/l and androstenolone acetate 10 g/l in the presence of MCD. Absorption of steroids on mycelium, lower speed of their transformation, low concentrations of modifying substrates, and low yield of hydroxyderivatives have been observed in the absence of MCD.     

Purification and partial characterization of β-galactosidase from Tritrichomonas foetus

The work presented in this paper describes the purification and properties of a β-galactosidase from the protozoan Tritrichomonas foetus. An inexpensive and straightforward method for extraction of the enzyme involving ammonium sulphate precipitation, ion exchange and affinity chromatography resulted in a high level of purification. After purification β-N-acetylglucosaminidase was the only enzyme present as a contaminant at a significant level. The β-galactosidase isolated had a pH optimum of 5.8. The K_m determined at pH 5.8 was found to be 2.2 mM. Interesting results were obtained when studies were carried out to determine the effect of various metal ions on enzyme activity. Of the metal ions used in this study only manganese ions were found to activate the enzyme. This seems to be a characteristic of trichomonad enzymes, as N-acetyl-β-glucosaminidase, a-galactosidase and N-acetyl-a-galactosaminidase are also activated by manganese ions. The strongest inhibition was recorded with lead and to a lesser extent by zinc. The result with lead is not unexpected as the heavy metal is known to cause irreversible inhibition by binding to the amino-acid backbone of the enzyme. The result with zinc is interesting as high levels of zinc are present and trichomonads are known to be apathogenic in semen. The purified β-galactosidase was found to have the capacity to hydrolyse lactose (Gal β1-4 Glc), lacto-N-biose 1 (Gal β1-3 GlcNAc) and N-acetyllactosamine (Gal β1-4 GlcNAc). When the enzyme was applied to a non-denaturing polyacrylamide gel a single band was observed when stained with Coomassie brilliant blue. This band coincided with that obtained when the gel was stained with p-nitrophenyl β-galactopyranoside. When the same gel was incubated with p-nitrophenyl N-acetyl β-glucopyranoside a band was detected which did not coincide with that of β-galactosidase. Since the β-N-acetylglucosaminidase enzyme does not move to the same position on a non-denaturing gel as the β-galactosidase, we will use this technique to isolate the latter enzyme and determine the N-terminal sequence as a prelude to cloning and further study of the gene. This revised version was published online in November 2006 with corrections to the Cover Date.     

Branched glucan from the fruiting bodies of Piptoporus betulinus (Bull.:Fr) Karst.

A new glucan, namely, piptoporane I, with a molecular mass of 270 kDa was isolated from fruiting bodies of Piptoporus betulinis (Bull.:Fr.) Karst. (Fomitopsidacaeae). Using a combination of physicochemical methods, it was established that piptoporane I was a branched glucan with a backbone consisting of α-(1 → 3)-glucopyranose residues substituted at the C-6 position by single residues of β-D-glucopyranose by 17.3%. A polysaccharide with such a structure was isolated for the first time from the fungus genus Piptoporus.     

Genetic polymorphism and activities of human lung alcohol and aldehyde dehydrogenases: Implications for ethanol metabolism and cytotoxicity

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) exhibit genetic polymorphism and tissue specificity. ADH and ALDH isozyme phenotypes from 39 surgical Chinese lung specimens were identified by agarose isoelectric focusing. The identity of the lung β-ADHs was further demonstrated by their characteristic pH-activity profiles for ethanol oxidation,K _m values for NAD and ethanol, and inhibition by 4-methylpyrazole or 1,10-phenanthroline. The β₂ allele, coding for β₂ polypeptide, was found to be predominant in the lung specimens studied. The ADH activities in the lungs with the homozygous phenotype ADH₂ 2-2 (exhibiting β₂β₂) and ADH₂ 1-1 (exhibiting β₁β₁) and the heterozygous phenotype ADH₂ 2-1 (exhibiting β₂β₂, β₂β₁, and β₁β₁) were determined to be 999±77, 48±17, and 494±61 nmol/min/g tissue, respectively. Fifty-one percent of the specimens studied lacked the ALDH2 activity band on the isoelectric focusing gels. The activities in the lung tissues with the ALDH2-active phenotype and the inactive phenotype were determined to be 30±3 and 17±1 nmol/min/g tissue, respectively. These findings indicate that human pulmonary ethanol-metabolizing activities differ significantly with respect to genetic polymorphism at both theADH ₂ and theALDH ₂ loci. The results suggest that individuals with highV _max β₂-ADH and deficient in low-K _m mitochondrial ALDH2, accounting for approximately 45% of the Chinese population, may end up with acetaldehyde accumulation during alcohol consumption, rendering them vulnerable to tissue injury caused by this highly reactive and toxic metabolite. This work was supported by Grants NSC 77-0412-B016-58 and NSC 80-0412-B016-21 from the National Science Council, Republic of China.     

Substrate specificity of a recombinant chicken <Emphasis Type="Italic">β</Emphasis>-carotene 15,15′-monooxygenase that converts <Emphasis Type="Italic">β</Emphasis>-carotene into retinal

The recombinant β-carotene 15,15′-monooxygenase from chicken liver was purified as a single 60 kDa band by His-Trap HP and Resource Q chromatography. It had a molecular mass of 240 kDa by gel filtration indicating the native form to be tetramer. The enzyme converted β-carotene under maximal conditions (pH 8.0 and 37°C) with a k _cat of 1.65 min⁻¹ and a K _m of 26 μM and its conversion yield of β-carotene to retinal was 120% (mol mol⁻¹). The enzyme displayed catalytic efficiency and conversion yield for β-carotene, β-cryptoxanthin, β-apo-8′-carotenal, β-apo-4′-carotenal, α-carotene and γ-carotene in decreasing order but not for zeaxanthin, lutein, β-apo-12′-carotenal and lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C₃₀ seems to be essential for enzyme activity.     

Biosynthesis of medium-chain-length poly(hydroxyalkanoates) with altered composition by mutant hybrid PHA synthases

Pseudomonas resinovorans harbors two isogenic poly(hydroxyalkanoates) (PHAs) synthase genes (phaC1 _Pre , phaC2 _Pre ) responsible for the production of intracellular medium-chain-length (mcl-)PHAs. Sequence analysis showed that the putative gene-products of these genes contain a conserved α/β-hydrolase fold in the carboxy-terminal half of the proteins. Hybrid genes pha7 and pha8 were constructed by exchanging the α/β-hydrolase-fold coding portions of phaC1 _Pre and phaC2 _Pre at the 3′ terminal. When grown with decanoate as carbon source, the pha7- or pha8-transformed Escherichia coli LS1298 produced PHAs containing 73–75% β-hydroxydecanoate (β-HD) and 25–27% β-hydroxyoctanoate (β-HO). Deletion mutants, Δpha7 and Δpha8, were isolated during the PCR-based construction of pha7 and pha8, respectively. Cells harboring these mutants produced PHAs containing 55–60 mol% β-HD and 40–45 mol% β-HO. These results demonstrate the feasibility of generating active hybrid mcl-PHA synthase genes and their mutants with the potential of producing polymers having a varied repeat-unit composition.     

Leakage and lysis of lipid membranes induced by the lipopeptide surfactin

Surfactin is a lipopeptide produced by Bacillus subtilis which possesses antimicrobial activity. We have studied the leakage and lysis of POPC vesicles induced by surfactin using calcein fluorescence de-quenching, isothermal titration calorimetry and ³¹P solid state NMR. Membrane leakage starts at a surfactin-to-lipid ratio in the membrane, R _b ≈ 0.05, and an aqueous surfactin concentration of C _S^w ≈ 2 μM. The transient, graded nature of leakage and the apparent coupling with surfactin translocation to the inner leaflet of the vesicles, suggests that this low-concentration effect is due to a bilayer-couple mechanism. Different permeabilization behaviour is found at R _b ≈ 0.15 and attributed to surfactin-rich clusters, which can induce leaks and stabilize them by covering their hydrophobic edges. Membrane lysis or solubilization to micellar structures starts at R _b^sat = 0.22 and C _S^w = 9 μM and is completed at R _m^sol = 0.43 and C _S^w = 11 μM. The membrane–water partition coefficient of surfactin is obtained as K = 2 × 10⁴ M⁻¹. These data resolve inconsistencies in the literature and shed light on the variety of effects often referred to as detergent-like effects of antibiotic peptides on membranes. The results are compared with published parameters characterizing the hemolytic and antibacterial activity. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Low-specificity l-threonine aldolase of Pseudomonas sp. NCIMB 10558: purification, characterization and its application to β-hydroxy-α-amino acid synthesis

Low-specificity l-threonine aldolase, catalyzing the reversible cleavage/condensation reaction between l-threonine/l-allo-threonine and glycine plus acetaldehyde, was purified to homogeneity from Pseudomonas sp. NCIMB 10558. The enzyme has an apparent molecular mass of approximately 145 kDa and consists of four identical subunits with a molecular mass of 38 kDa. The enzyme, requiring pyridoxal- 5′-phosphate as a coenzyme, is strictly l-specific at the α position, whereas it can not distinguish between threo and erythro forms at the β position. Besides the reversible cleavage/condensation of threonine, the enzyme also catalyzes the reversible interconversion between glycine plus various aldehydes and l-β-hydroxy-α-amino acids, including l-β-(3,4-dihydroxyphenyl)serine, l-β-(3,4-met‐hylenedioxyphenyl)serine and l-β-phenylserine, providing a new route for the industrial production of these important amino acids. Received: 10 November 1997 / Received revision: 7 January 1998 / Accepted 30 January 1998     

Fluorescence study of neurohypophyseal hormones and their analogues

Analogues of arginine-vasopressin (AVP) in which substitution of the proline residue in position 7 (by either sarcosine or N-methylalanine) combined with replacement of the cysteine residue in position 1 were the subject of a fluorescence and molecular mechanics study. We obtained two groups of analogues: selective antidiuretic agonists (cysteine or β-mercaptopropionic acid in position 1) and pressor and uterotonic antagonists (deaminopenicillamine or β-mercapto-β,β-cyclopentamethylene- propionic acid in position 1). Using frequency-domain measurements of fluorescence resonance energy transfer (FRET) we estimated the distance distribution between the phenolic ring of Tyr² and the disulphide bridge Cys¹–Cys⁶. We also analyzed acrylamide quenching of tyrosyl fluorescence to determine the exposure of the tyrosyl ring to the solvent. Results from fluorescence experiments were compared with those from Monte Carlo simulation (ECEPP/3 force-field). Received: 5 August 1996 / Accepted: 8 December 1996     

Biochemical Characterization of a β-1,3-Glucanase from Trichoderma koningii Induced by Cell Wall of Rhizoctonia solani

Trichoderma species are readily isolated from Brazilian cerrado soil by conventional methods and some of them were characterized as Trichoderma koningii. The effect of carbon source on the production of β-1,3-glucanases in the culture filtrates of a specific Trichoderma koningii strain (ALL 13) was investigated. Enzyme activity was detected in all carbon sources tested and only one band of β-1,3-glucanase was detected in non-denaturing PAGE. This enzyme was purified by Sephacryl S-200 gel filtration and Phenyl Sepharose CL 4B chromatography. A typical procedure provided 105-fold purification with 13.4% yield. The molecular weight of the purified enzyme was 75 kDa as estimated by SDS-PAGE. The enzyme hydrolyzed laminarin in an endo-like fashion to form small oligosaccharides and glucose. The K_m and V_max values for β-1,3-glucanase, using laminarin as substrate, were 0.148 mg.mL⁻¹ and 0.159 U.min⁻¹, respectively. The pH optimum for the enzyme was pH 4.6 and maximum activity was obtained at 50°C. Hg²⁺ inhibited the purified enzyme.