Characterization of a β-D-mannosidase from a marine gastropod, Aplysia kurodai

A β-D-mannosidase (EC 3.2.1.25) with a molecular mass of approximately 100 kDa was purified from the digestive fluid of a marine gastropod Aplysia kurodai by ammonium sulfate fractionation followed by column chromatographies on TOYOPEARL Butyl-650 M, TOYOPEARL DEAE-650 M, and Superdex 200 10/300 GL. This enzyme, named AkMnsd in the present study, showed optimal activities at pH 4.5 and 40 °C and was stable at the acidic pH range from 2.0 to 6.7 and the temperature below 38 °C. The Km and Vmax values for AkMnsd determined at pH 6.0 and 30 °C with p-nitrophenyl β-d-mannopyranoside were 0.10 mM and 3.75 μmol/min/mg, respectively. AkMnsd degraded various polymer mannans as well as mannooligosaccharides liberating mannose as a major degradation product. Linear mannan from green alga Codium fragile was completely depolymerized by AkMnsd in the presence of AkMan, an endolytic β-mannanase, which we previously isolated from the same animal (Zahura et al., Comp. Biochem. Physiol. B 157, 137-148 (2010)). A cDNA encoding AkMnsd was amplified from the Aplysia hepatopancreas cDNA by the PCR using degenerated primers designed on the basis of N-terminal and internal amino-acid sequences of AkMnsd. The cloned AkMnsd cDNA consisted of 2985 bp and encoded an amino-acid sequence of 931 residues with the calculated molecular mass of 101,970 Da. The deduced sequence of AkMnsd showed 20-43% amino-acid identity to those of glycoside-hydrolase-family 2 (GHF2) β-mannosidases. The catalytically important amino-acid residues determined in GHF2 enzymes were completely conserved in AkMnsd. Thus, AkMnsd is regarded as a new member of GHF2 mannosidase from marine gastropod.     

Eudistomidins H-K, new β-carboline alkaloids from the Okinawan marine tunicate Eudistoma glaucus

Four new β-carboline alkaloids, eudistomidins H-K (1-4), were isolated from an Okinawan marine tunicate Eudistoma glaucus and the structures of 1-4 were elucidated on the basis of spectroscopic data. Eudistomidins H (1) and I (2) were new β-carboline alkaloids possessing a unique fused-tetracyclic ring system consisting of a tetrahydro β-carboline ring and a hexahydropyrimidine ring. Eudistomidin J (3) showed relatively potent cytotoxicity against murine leukemia cells P388 and L1210, and human epidermoid carcinoma cells KB in vitro.     

Isolation of β-Tocopherol from the Root Bark of the Mulberry Tree

Kluyvera citrophila KY7844 enzymatically catalyzed N-acylation of 7-amino-desacetoxycephalosporanic acid with 1-(1H)-tetrazolylacetate methylester to produce 7-[1-(1H)-tetrazolylacetamido]-desacetoxycephalosporanic acid. The product was purified and characterized.     

Isolation of β-globin-related genes from a human cosmid library

A human gene library was constructed using an improved cloning technique for cosmid vectors. Human placental DNA was partially digested with restriction endonuclease Mboi, size-fractionated and ligated to BamHI-cut and phosphatase-treated cosmid vector pJB8. After packaging in λ phage particles, the recombinant DNA was transduced into Escherichia coli 1400 or HB101 followed by selection on ampicillin for recombinant E. coli. 150000 recombinant-DNA-containing colonies were screened for the presence of the human β-globin related genes. Five recombinants were isolated containing the human β-globin locus and encompassing approx. 70 kb of human DNA.     

Isolation and characterization of A new β-melanotropin from horse pituitary glands

A new β-melanotropin was isolated from horse pituitaries and its primary structure has been determined. The amino acid sequence of the new peptide differed from that of β-melanotropin in the deletion of the NH₂-terminal aspartic acid. Thus, it is designated as [Des-Asp¹]-β-melanotropin. It possessed higher lipolytic activity when compared with β-melanotropin.     

Production and characteristics of some new β-agarases from a marine bacterium,Vibrio sp. strain JT0107

Vibrio sp. strain JT0107 is one of the marine bacteria that secrete β-agarases which catalyze the hydrolysis of agarose. The optimum culture conditions for the production of some β-agarases have been determined. To increase agarase activity, aeration and a sufficient concentration of agarose are needed. One of the enzymes that the bacteria secreted into the culture medium was isolated and purified 39-fold using a combination of ultrafiltration and subsequent anion exchange column chromatography. The purified protein migrated as a single band (72 kDa) on sodium dodecyl sulfate polyacrylamide gel electrophoresis and its isoelectric point was 4.7. Amino acid sequence analysis revealed a single N-terminal sequence that had no sequence identity to other marine bacterial agarases. This novel enzyme was found to be an endo-type β-agarase (EC 3.2.1.81) that catalyzes the hydrolysis of the β-1,4 linkage of agarose to yield neoagarotetraose [O-3,6-anhydro-α-l-galactopyranosyl(1→3)-O-β-d-galactopyranosyl(1→4)-O-3,6-anhydro-α-l-galactopyranosyl(1→3)-d -galactose] and neoagarobiose [O-3,6-anhydro-α-l-galactopyranosyl(1→3)-d-galactose]. The optimum pH and temperature for obtaining high activity of the enzyme were at around 8 and 30°C, respectively. The enzyme did not degrade sodium alginate, λ-carrageenan, ι-carrageenan or κ-carrageenan.     

Isolation and characterization of β-glucosidase from the cytosol of rat kidney cortex

A procedure is described for the preparation of extensively purified β-d-glucosidase (EC 3.2.1.21) from the cytosol fraction of rat kidney. The specific activity of the β-glucosidase in the high speed supernatant (100 000 × g, 90 min) fraction of rat kidney homogenate is 700-fold greater than that in the same fraction from heart, skeletal muscle, lung, spleen, brain or liver. β-Glucosidase activity co-chromatographs with β-d-galactosidase, β-d-fucosidase, α-l-arabinosidase and β-d-xylosidase activities through the last four column steps of the purification and their specific activities are 0.26, 0.39, 0.028 and 0.017 relative to that of β-glucosidase, respectively. The specific activity of the apparently homogeneous β-glucosidase is 115 000 nmol of glucose released from 4-methylumbelliferyl-β-d-glucopyranoside per mg protein per h. All five glycosidase activities possess similar pH dependency (pH optimum, 6–7) and heat lability, and co-migrate on polyacrylamide disc gels at ph 8.9 (R_F, 0.67). β-Glucosidase activity is inhibited competitively by glucono-(1 → 5)-lactone (K_I, 0.61 mM) and non-competitively by a variety of sulfhydryl reagents including N-ethylmaleimide, p-chloromercuribenzoate, 5,5′-dithio-bis(2-nitrobenzoic acid), and iodoacetic acid. Although the enzyme will release glucose from p-nitrophenyl and 4-methylumbelliferyl derivatives of β-d-glucose, it will not hydrolyze xylosyl-O-serine, β-d-glucocerebroside, lactose, galactosylovalbumin or trehalose. The enzyme consists of a single polypeptide chain with a molecular weight of 50 000–58 000, has a sedimentation coefficient of 4.41 S and contains a relatively large number of acidic amino acids. A study of the distribution of β-glucosidase activity in various regions of the dissected rat kidney indicates that the enzyme is probably contained in cells of the proximal convulated tubule. The enzyme is also present in relatively large ammounts in the villus cells, but not crypt cells, of the intestine. the physiological subtrates and function of the enzyme are unknown.     

Isolation and characterization of marine biofilm forming bacteria from a ship’s hull

Background

Diverse aquatic microorganisms are capable of colonizing living and non-living surfaces leading to the formation of biofilms. Commonly visualized as a slimy layer, these biofilms are filled with hundreds of other microorganisms compared to free living planktonic cells. Microbial surface colonization and surface-associated metabolic activities also exert several macroscale deleterious effects, including biofouling, biocorrosion and the persistence and transmission of harmful or pathogenic microorganisms and virulence determinants. The present study deals with the isolation and screening of marine bacteria for biofilm formation. The screened isolates were characterized and identified as Pychrobacter celer, Pychrobacter alimentarius and Kocuria rhizophila by 16S rRNA sequencing.

Methods

Biofilm forming bacteria were isolated by spread plate technique and subjected to screening by microtiter plate assay. The potent biofilm formers were identified by molecular characterization using 16S rRNA gene sequencing.

Results

Twelve bacterial isolates were obtained by pour plate technique and subjected to biofilm assay. Among the 12 isolates three isolates which showed maximum biofilm formation were subjected to molecular characterizationby 16S rRNA gene sequencing method. The isolates were identified as Pychrobacter celer, Pychrobacter alimentarius and Kocuria rhizophila. The EPS produced by the three biofilm forming bacteria was extracted and the protein and carbohydrate content determined.

Conclusion

Among the isolates screened, isolate 8 (Kocuria rhizophila) produced maximum protein and carbohydrate which was also in accordance with the results of microtiter plate assay.

     

Characterization of the type of action of β-1,3-glucanases from marine invertebrates

• 1.1. Ten species of marine invertebrates possessing the highest laminarinase activity have been investigated.
• 2.2. The following approaches were used in determining the type of action of partially purified β-1,3-glucanases: effect on modified substrates, comparative study of enzymolysis products by the Nelson's and glucose oxidase methods and stereochemistry of the enzymolysis products.
• 3.3. Experimental results indicate that the examined marine invertebrates contain β-1,3-glucanases with endo-type activity; exo-enzymes are apparently absent.

     

Isolation and Identification of Two β-Hydroxy-pyridine Derivatives from the Nondialyzable Melanoidin-hydrolyzate

The hydrolyzate of the melanoidin prepared from glucose-ammonia system (kept in pH 5.3~6.0 during the reaction) was fractionated into the two fractions of non-adsorbate and adsorbate on Amberlite IR-120 (H⁺-form). In the present paper, the adsorbed fraction (Fraction B) was examined.

Paper chromatographic examination of the Fraction B indicated the presence of at least eight compounds positive to diazotized sulphanilic acid reagent. The two compounds of them (indicated orange and orange-yellow color) were isolated and identified as 2-methyl-5-hydroxy-pyridine and 2-hydroxymethyl-5-hydroxy-pyridine, respectively.

It is probable that these compounds would loosely be bound as a small moiety in the melanoidin molecule.     

The organization of the β-globin gene of the bivalve mollusc Anadara trapezia and its evolutionary relationship to other invertebrate and vertebrate globin genes

Three pairs of oligonucleotide primers based on partial DNA and amino acid sequences were used in a combination of PCR experiments to amplify the -globin gene of the bivalve mollusc Anadara trapezia. The sequence of 2,139 by presented contains the whole of the -globin gene with the exception of the 5 flanking sequence. This gene possesses the three-exon-and-two-intron gene structure typical of vertebrate globin genes but the lengths of the introns (762 by and 690 bp, respectively) are only approximately half the size of those present in a -variant gene previously characterized from this organism. The encoded amino acid sequence shows two changes when compared to the previously published amino acid sequence. Correspondence to: A.G. Mackinlay     

Cloning, expression, and characterization of a glycoside hydrolase family 50 β-agarase from a marine Agarivorans isolate

The gene for a thermostable β-agarase from Agarivorans sp. JA-1 was cloned and sequenced. It comprised an open reading frame of 2,988 base pairs, which encode a protein of 109,450 daltons consisting of 995 amino acid residues. A comparison of the entire sequence showed that the enzyme has 98.8% sequence similarities to β-agarase from Vibrio sp. JT1070, indicating that it belongs to the family glycoside hydrolase (GH)-50. The gene corresponding to a mature protein of 976 amino acids was inserted and expressed in Escherichia coli. The recombinant β-agarase was purified to homogeneity. It had maximal activity at 40°C and pH 8.0 in the presence of 1 mM NaCl and 1 mM CaCl₂. The enzyme hydrolyzed agarose as well as neoagarohexaose and neoagarotetraose to yield neoagarobiose as the main product. Thus, the enzyme would be useful for the industrial production of neoagarobiose.     

Cloning, expression and characterization of a new ι-carrageenase from marine bacterium, Cellulophaga sp.

Purpose of work

The purpose of this study is to report a ι-carrageenase which degrades ι-carrageenan yielding neo-ι-carratetraose as the main product in the absence of NaCl. The gene for a new ι-carrageenase, CgiB_Ce, from Cellulophaga sp. QY3 was cloned and sequenced. It comprised an ORF of 1,386 bp encoding for a protein of 461 amino acid residues. From its sequence analysis, CgiB_Ce is a new member of GH family 82 and shared the highest identity of 32 % in amino acids with ι-carrageenase CgiA2 from Zobellia galactanovorans indicating that it is a hitherto uncharacterized protein. The recombinant CgiB_Ce had maximum specific activity (1,870 U/mg) at 45 °C and pH 6.5. It was stable between pH 6.0–9.6 and below 40 °C. Although its activity was enhanced by NaCl, the enzyme was active in the absence of NaCl. CgiB_Ce is an endo-type ι-carrageenase that hydrolyzes β-1,4-linkages of ι-carrageenan, yielding neo-ι-carratetraose as the main product (more than 80 % of the total product).