Purification, cDNA cloning and homology modeling of endo-1,3-beta-D-glucanase from scallop Mizuhopecten yessoensis

The retaining endo-1,3-β-d-glucanase (LV) with molecular mass of 36 kDa was purified to homogeneity from the crystalline styles of scallop Mizuhopecten yessoensis. The purified enzyme catalyzed hydrolysis of laminaran as endo-enzyme forming glucose, laminaribiose and higher oligosaccharides as products (K_m  600 μg/mL). The 1,3-β-d-glucanase effectively catalyzed transglycosylation reaction that is typical of endo-enzymes too. Optima of pH and temperature were at 4.5 and 45 °C, respectively. cDNA encoding the endo-1,3-β-d-glucanase was cloned by PCR-based methods. It contained an open reading frame that encoded 339-amino acids protein. The predicted endo-1,3-β-d-glucanase amino acid sequence included a characteristic domain of the glycosyl hydrolases family 16 and revealed closest homology with 1,3-β-d-glucanases from bivalve Pseudocardium sachalinensis, sea urchin Strongylocentrotus purpuratus and invertebrates lipopolysaccharide and β-1,3-glucan-binding proteins.The fold of the LV was more closely related to κ-carrageenase, agarase and 1,3;1,4-β-d-glucanase from glycosyl hydrolases family 16. Homology model of the endo-1,3-β-d-glucanase from M. yessoensis was obtained with MOE on the base of the crystal structure of κ-carrageenase from P. carrageonovora as template. Putative three-dimensional structures of the LV complexes with substrate laminarihexaose or glucanase inhibitor halistanol sulfate showed that the binding sites of the halistanol sulfate and laminarihexaose are located in the enzyme catalytic site and overlapped.     

Purification and characterization of a new endo-1,4-beta-D-glucanase from Beltraniella portoricensis

A new endoglucanase, designated BCE1, produced by Beltraniella portoricensis, was purified from the culture supernatant. The N-terminal amino acid sequence suggests that BCE1 belongs to family 45 glycoside hydrolase (family 45 endoglucanase). The molecular mass of BCE1 was found to be 40 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for the carboxymethyl cellulase (CMCase) activity of BCE1 was 4.5, and the optimum temperature was 55 degrees C. Among family 45 endoglucanases, RCE1 and RCE2 from Rhizopus oryzae, PCE1 from Phycomyces nitens, and EGL3 and EGL4 from Humicola grisea, BCE1 was most resistant to anionic surfactant and oxidizing agent. These results indicate that BCE1 might prove to be a useful enzyme in the detergent industry.     

Chemical modification of lysine residues of beta-1,3-glucanase from Spisula sachalinensis

The effects of chemical modification of the amino groups of lysine residues on the activity of beta-1.3-glucanase from Spisula sachalinensis were studied. Modification of two lysine residues per molecule did not affect either the enzyme activity with respect to laminarine, nor the Km value. The modified beta-1.3-glucanase retains the ability to catalyze the transglycosylation and cleaves the high molecular weight CM-pachyman at the same rate as does the native enzyme. No significant changes in the enzyme thermal stability were observed. Thus, the modified enzyme groups cannot be involved in the enzyme active center and are exposed on the surface of the protein globule. The chemical modification was shown to have no effect on the enzyme kinetics, which is essential for its immobilization.     

Catalytic properties and mode of action of endo-(1-->3)-beta-D-glucanase and beta-D-glucosidase from the marine mollusk Littorina kurila

A complex of the enzymes from the liver of the marine mollusk Littorina kurila that hydrolyzes laminaran was investigated. Two (1-->3)-beta-d-glucanases (G-I and G-II) were isolated. The molecular mass of G-I as estimated by gel-permeation chromatography and SDS-PAGE analysis was 32 and 40kDa, respectively. The G-II molecular mass according to SDS-PAGE analysis was about 200kDa. The pH optimum for both G-I and G-II was pH 5.4. The G-I had narrow substrate specificity and hydrolyzed only the (1-->3)-beta-d-glucosidic bonds in the mixed (1-->3),(1-->6)- and (1-->3),(1-->4)-beta-d-glucans down to glucose and glucooligosaccharides. This enzyme acted with retention of the anomeric configuration and catalyzed a transglycosylation reaction. G-I was classified as the glucan endo-(1-->3)-beta-d-glucosidase (EC 3.2.1.39). G-II exhibited both exo-glucanase and beta-d-glucoside activities. This enzyme released from the laminaran glucose as a single product, but retained the anomeric center configuration and possessed transglycosylation activity. The hydrolysis rate of glucooligosaccharides by G-I decreased with an increase of the substrate's degree of polymerization. In addition to (1-->3)-beta-d-glucanase activity, the enzyme had the ability to hydrolyze p-nitrophenyl beta-d-glucoside and beta-d-glucobioses: laminaribiose, gentiobiose, and cellobiose, with the rate ratio of 50:12:1. G-II may correspond to beta-d-glucoside glucohydrolase (EC 3.2.1.21).     

Molecular cloning and characterization of an alpha1,3 fucosyltransferase, CEFT-1, from Caenorhabditis elegans

We report on the identification, molecular cloning, and characterization of an alpha1,3 fucosyltransferase (alpha1,3FT) expressed by the nematode, Caenorhabditis elegans . Although C. elegans glycoconjugates do not express the Lewis x antigen Galbeta1-- >4[Fucalpha1-->3]GlcNAcbeta-->R, detergent extracts of adult C.elegans contain an alpha1,3FT that can fucosylate both nonsialylated and sialylated acceptor glycans to generate the Lexand sialyl Lexantigens, as well as the lacdiNAc-containing acceptor GalNAcbeta1-->4GlcNAcbeta1-- >R to generate GalNAcbeta1-->4 [Fucalpha1-->3]GlcNAcbeta1-->R. A search of the C.elegans genome database revealed the existence of a gene with 20-23% overall identity to all five cloned human alpha1,3FTs. The putative cDNA for the C.elegans alpha1,3FT (CEFT-1) was amplified by PCR from a cDNA lambdaZAP library, cloned, and sequenced. COS7 cells transiently transfected with cDNA encoding CEFT-1 express the Lex, but not sLexantigen. The CEFT-1 in the transfected cell extracts can synthesize Lex, but not sialyl Lex, using exogenous acceptors. A second fucosyltransferase activity was detected in extracts of C. elegans that transfers Fuc in alpha1,2 linkage to Gal specifically on type-1 chains. The discovery of alpha-fucosyltransferases in C. elegans opens the possibility of using this well-characterized nematode as a model system for studying the role of fucosylated glycans in the development and survival of C.elegans and possibly other helminths.      

Functional analysis of an endo-1,6-beta-D-glucanase gene (neg-1) from Neurospora crassa

The 1,6-beta-D-glucanase gene (neg1) of Neurospora crassa was disrupted by repeat-induced point mutations. Sequence analysis of the neg1 gene in the R12-1 mutant showed that 9 nucleotides within the coding region of the gene changed from GC to AT. The base transition of C to A at position 662 resulted in a codon. No apparent phenotypic changes were observed in the mutant, but Congo-red, SDS, and cetyltrimethyl ammonium bromide (CTAB), which affect fungal cell walls or membranes, markedly inhibited the hyphal growth of the mutant at a concentration that does not inhibit growth in the wild type.     

Cloning of an endo-1,4-beta-D-glucanase gene from Clostridium josui and its expression in Escherichia coli

The gene for carboxymethyl cellulose-degrading enzyme (endoglucanase) from Clostridium josui (FERM P-9684) was cloned in Escherichia coli HB101 with pBR322. A 5.6-kilobase-pair HindIII fragment encoding an endoglucanase was hybridized with C. josui chromosomal DNA. The size of the cloned DNA fragment was reduced with PvuII, and the resulting active fragment (2 kilobase pairs, with restriction sites of EcoRI and PstI) was ligated into pUC118 at the SmaI sites (pUCJ1). The endoglucanase production by E. coli JM103(pUCJ1) in Luria-Bertani broth was enhanced up to approximately three times by maintaining the pH at 6.5 and using 80 mM NaCl.     

Cloning and expression of an endo-1,6-beta-D-glucanase gene (neg1) from Neurospora crassa

A gene (neg1) encoding an endo-1,6-beta-D-glucanase from Neurospora crassa was cloned. The putative neg1 was 1443-bp long and encoded a mature endo-1,6-beta-D-glucanase protein of 463 amino acids and signal peptide of 17 amino acids. The purified recombinant protein (Neg1) obtained from Escherichia coli showed 1,6-beta-D-glucanase activity. No genes similar in sequence were found in yeasts and fungi.     

Purification and properties of an endo-(1 leads to 3)-beta-D-glucanase from malted barley

3,6-Anhydro-α-D-galactopyranose 1,2-(methyl orthoacetate) and its 4-acetate were synthesized from 2,3,4-tri-O-acetyl-6-O-tosyl-α-D-galactopyranosyl bromide. Condensation of the above-mentioned, acetylated ortho ester with 1,2:3,4-di-O-isopropylidene-α-D-galactopyranose gave 6-O-(2,4-di-O-acetyl-3,6-anhydro-β-D-galactopyranosyl)-1,2:3,4-di-O-isopropylidene-α-D-galactopyranose. The same disaccharide derivative was synthesised from 6-O-β-D-galactopyranosyl-1,2:3,4-di-O-isopropylidene-α-D-galactopyranose by mono-O-tosylation followed by treatment with alkali and acetylation.     

Molecular cloning and characterization of an inorganic pyrophosphatase from barley

A cDNA clone with sequence homology to soluble inorganic pyrophosphatase (IPPase) was isolated from a library of developing barley grains. The protein encoded by this clone was produced in transgenic Escherichia coli, and showed IPPase activity. In nondormant barley grains, the gene appeared to be expressed in metabolically active tissue such as root, shoot, embryo and aleurone. During imbibition, a continuous increase of the steady state mRNA level of IPPase was observed in embryos of non-dormant grains. In the embryos of dormant grains its production declined, after an initial increase. With isolated dormant and nondormant embryos, addition of recombinant IPPase, produced by E. coli, enhanced the germination rate. On the other hand, addition of pyrophosphate (PPi), substrate for this enzyme, appeared to reduce the germination rate. A role for this IPPase in germination is discussed.     

Molecular cloning and characterization of the Caenorhabditis elegans alpha1,3-fucosyltransferase family

The genome of Caenorhabditis elegans encodes five genes with homology to known alpha1,3 fucosyltransferases (alpha1,3FTs), but their expression and functions are poorly understood. Here we report the molecular cloning and characterization of these C. elegans alpha1,3FTs (CEFT-1 through -5). The open-reading frame for each enzyme predicts a type II transmembrane protein and multiple potential N-glycosylation sites. We prepared recombinant epitope-tagged forms of each CEFT and found that they had unusual acceptor specificity, cation requirements, and temperature sensitivity. CEFT-1 acted on the N-glycan pentasaccharide core acceptor to generate Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-Asn. In contrast, CEFT-2 did not act on the pentasaccharide acceptor, but instead utilized a LacdiNAc acceptor to generate GalNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc, which is a novel activity. CEFT-3 utilized a LacNAc acceptor to generate Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc without requiring cations. CEFT-4 was similar to CEFT-3, but its activity was enhanced by some divalent cations. Recombinant CEFT-5 was well expressed, but did not act on available acceptors. Each CEFT was optimally active at room temperature and rapidly lost activity at 37 degrees C. Promoter analysis showed that CEFT-1 is expressed in C. elegans eggs and adults, but its expression was restricted to a few neuronal cells at the head and tail. We prepared deletion mutants for each enzyme for phenotypic analysis. While loss of CEFT-1 correlated with loss of pentasaccharide core activity and core alpha1,3-fucosylated glycans in worms, loss of other enzymes did not correlate with any phenotypic changes. These results suggest that each of the alpha1,3FTs in C. elegans has unique specificity and expression patterns.     

Cloning of an endo-1,4-beta-D-glucanase gene from Clostridium josui and its expression in Escherichia coli.

The gene for carboxymethyl cellulose-degrading enzyme (endoglucanase) from Clostridium josui (FERM P-9684) was cloned in Escherichia coli HB101 with pBR322. A 5.6-kilobase-pair HindIII fragment encoding an endoglucanase was hybridized with C. josui chromosomal DNA. The size of the cloned DNA fragment was reduced with PvuII, and the resulting active fragment (2 kilobase pairs, with restriction sites of EcoRI and PstI) was ligated into pUC118 at the SmaI sites (pUCJ1). The endoglucanase production by E. coli JM103(pUCJ1) in Luria-Bertani broth was enhanced up to approximately three times by maintaining the pH at 6.5 and using 80 mM NaCl.     

Purification,characterization and cloning of an endo-1,4-B-glucanase fromRuminococcus sp.

Endoglucanase ofRuminococcus sp. is composed of seven active protein components when chromatographed on an ion exchange column (Q-Sepharose). Component I (endoglucanase A) did not bind to the column and was purified to homogeneity by molecular sieve chromatography. It had a mol. wt. of 22 000. Component II was fractionated into two active protein peaks (endoglucanase B and C) having mol. wt. of 225 000 and 10 000. The endoglucanase A had high affinity for CMC (Km 8 mg/ml). The temperature optimum of all three endoglucanase was between 40–45°C. The gene encoding for endolucanase activity was cloned inE. coli HB101 with pBR322. A 4.3 kilobaseBamH1 fragment encoding endoglucanase was hybridized toRuminococcus chromosomal DNA.     

Biochemical characterization and antifungal activity of an endo-1,3-beta-glucanase of Paenibacillus sp. isolated from garden soil

A 44-kDa 1,3-beta-glucanase was purified from the culture medium of a Paenibacillus strain with a 28-fold increase in specific activity with 31% recovery. The purified enzyme preferentially catalyzes the hydrolysis of glucans with 1,3-beta-linkage and has an endolytic mode of action. The enzyme also showed binding activity to various insoluble polysaccharides including unhydrolyzable substrates such as xylan and cellulose. The antifungal activity of this Paenibacillus enzyme and a previously purified 1,3-beta-glucanase from Streptomyces sioyaensis were examined in this study. Both enzymes had the ability to damage the cell-wall structures of the growing mycelia of phytopathogenic fungi Pythium aphanidermatum and Rhizoctonic solani AG-4. Nonetheless, the Paenibacillus enzyme had a much stronger effect on inhibiting the growth of fungi tested.     

Molecular cloning and nucleotide sequence of the gene encoding an endo-1,4-beta-glucanase from Bacillus sp. KSM-330.

The gene encoding an acid endo-1,4-beta-glucanase from Bacillus sp. KSM-330 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The recombinant plasmid contained a 3.1 kb HindIII insert, 1.8 kb of which was sufficient for the expression of endoglucanase activity in E. coli HB101. Nucleotide sequencing of this region (1816 bp) revealed an open reading frame of 1389 bp. The protein deduced from this sequence was composed of 463 amino acids with an Mr of 51882. The deduced amino acid sequence from amino acids 56 through 75 coincided with the amino-terminal sequence of the endoglucanase, Endo-K, purified from culture of Bacillus sp. KSM-330. The deduced amino acid sequence of Endo-K had 30% homology with that of the celA enzyme from Clostridium thermocellum NCIB 10682 and 25% homology with that of the enzyme from Cellulomonas uda CB4. However, the Endo-K protein exhibited no homology with respect to either the nucleotide or the amino acid sequences of other endoglucanases from Bacillus that had been previously characterized. These results indicate that the gene for Endo-K in Bacillus sp. KSM-330 has evolved from an ancestral gene distinct from that of other Bacillus endoglucanases.     

Molecular cloning and characterization of an NADPH quinone oxidoreductase from Kluyveromyces marxianus

NAD(P)H quinone oxidoreductase is a ubiquitous enzyme that is known to directly reduce quinone substrates to hydroquinones by a two-electron reaction. We report the identification of NADPH quinone oxidoreductase from Kluyveromyces marxianus (KmQOR), which reduces quinone substrates directly to hydroquinones. The KmQOR gene was sequenced, expressed in Escherichia coli, purified, and characterized. The open-reading frame of the KmQOR gene consists of 1143 nucleotides, encoding a 380 amino acid polypeptide. The nucleotide sequence of the KmQOR gene was assigned to EMBL under accession number AY040868. The M(r) that was determined by SDS-PAGE for the protein subunit was about 42 kDa, and the molecular mass of the native KmQOR was 84 kDa, as determined by column calibration, indicating that the native protein is a homodimer. The KmQOR protein efficiently reduced 1,4-benzoquinone, whereas no activities were found for menadiones and methoxyquinones. These observations, and the result of an extended sequence analysis of known NADPH quinone oxidoreductase, suggest that KmQOR possesses a different action mechanism.