Cloning and expression of the Bacillus circulans endo-β-1,3-1,4-glucanase gene (bglBC1) in Escherichia coli

A gene coding for the endo--1,3-1,4-glucanase of B. circulans ATCC21367 was cloned into Escherichia coli. The cloned enzyme hydrolyzed lichenan or barley -glucan to produce 3-O--cellobiosyl-d-glucose as a main product but was inactive with carboxymethyl cellulose, laminarin and xylan. The enzyme, M _r=28 kDa, remained within the cytoplasm of E. coli. A 771 bp open reading frame was in the 2 kb PstI fragment of the recombinant plasmid pLL200K. The deduced protein sequence consists of 257 amino acids and has a putative signal peptide of 26 amino acids. The amino acid sequence of the endo--1,3-1,4-glucanase showed 68 and 51% homology to previously reported endo--1,3-1,4-glucanases from Bacillus strain N-137 and B. brevis, respectively.     

Resolution of Clostridium stercorarium cellulase by fast protein liquid chromatography (FPLC)

Summary A fast and efficient separation procedure for the analysis of the cellulase components of the thermophilic anaerobe Clostridium stercorarium was developed. Culture respernatants were concentrated without loss of cellulase activity by tangential flow ultrafiltration. Resolution of the cellulase system was achieved by fast protein liquid chromatography (FPLC) on a Mono Q anion exchange column. Enzyme fractions were assayed for hydrolysis of Avicel, carboxymethylcellulose (CMC), -nitrophenyl--d-cellobioside, and p-nitrophenyl--d-glucoside. Two Avicelases, two -cellobiosidases, and one -glucosidase were identified and characterized by SDS-polyacrylamide electrophoresis and isoelectric focusing. On the basis of their activities towards CMC, Avicelase I was classified as endo--glucanase and Avicelase II as exo--glucanase. Efficient hydrolysis of microcrystalline cellulose was shown to result from the combined action of both Avicelases.     

Beta-1,3-glucanase from unfertilized eggs of the sea urchin Strongylocentrotus intermedius. Comparison with beta-1,3-glucanases of marine and terrestrial mollusks

-1,3-Glucanase (Lu) was isolated from unfertilized eggs of the sea urchin Strongylocentrotus intermedius. A comparative study of some properties of -1,3-glucanase Lu and -1,3-glucanases with different action types—endo--1,3-glucanase from crystalline style of the marine mollusk Spisula sachalinensis (LIV) and exo--1,3-glucanase from the terrestrial snail Eulota maakii (LII)—was performed. It was found that -1,3-glucanase Lu hydrolyzes laminaran with a high yield of glucose in the reaction products. The enzyme hydrolyzes substrates with retention of the glycosidic bond configuration, is able to cleave modified substrates, and exhibits transglycosylating activity. All properties of -1,3-glucanase from S. intermedius were more similar to those of the endo--1,3-glucanase from the marine mollusk (LIV) than exo--1,3-glucanase LII from the terrestrial snail. The differences in the effect of LIV and Lu on laminaran are probably related to the functions of -1,3-glucanase Lu from sea urchin eggs (which, in contrast to LIV, is not a digestive enzyme).     

Peptide des β-Alanins als Ersatz für die freie Aminosäure bei pantothensäurebedürftigen Hefezellen und ihr Verhalten gegenüber dem β-Alanin-Antagonisten Asparagin

Zusammenfassung Pantothensäurebedürftige Hefezellen können ihren Bedarf an diesem Vitamin nicht allein aus -Alanin decken, sondern auch aus Benzoyl--Alanin, -Alanyl-d,l-Norleucin und -Alanyl-l-Histidin. Der Antagonist Asparagin hemmt die Verwertung dieser Peptide genauso wie diejenige der freien Aminosäure. Durch höhere Konzentrationen an -Alanin oder -Alanyl-d,l-Norleucin läßt sich die Hemmwirkung nicht allein kompensieren, es kommt sogar zu einer Förderung des Hefewachstums. Der Antagonist wird dann zum Synergisten.

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Summary The -alanine containing peptides benzoyl--alanine, -alanyl-d,l-norleucine and -alanyl-l-histidine can substitute for the amino acid -alanine in a pantothenic acid requiring yeast. Asparagine, an antagonist of -alanine, affects these peptides in a similar manner. In combination with an overdose of -alanine or -alanyl-d,l-norleucine, asparagine is no longer an antagonist but becomes a synergist.

     

The laminarinase system of Streptomyces sp., ATCC 11238

Summary Extracellular proteins from Streptomyces sp. ATCC 11238 grown on fungal mycelia and chitin as C- and N-sources were concentrated by ultrafiltration and acetone precipitation. The crude preparation containing chitin and laminarin degrading enzymes was fractionated by repeated gel filtrations. Three different types of -1,3-glucanases were found. Besides oligomeric breakdown products laminaritriose is the main product of laminarin hydrolysis by one endo--1,3-glucanase. A second laminarin degrading (exo-splitting) enzyme yields predominantly laminaribiose. Another exo--1,3-glucanase liberates glucose but no, oligosaccharides from the nonreducing end of laminarin.     

Ozone-induced changes of mRNA levels of β-1,3-glucanase,chitinase and ‘pathogenesis-related’ protein 1b in tobacco plants

Treatment of the ozone-sensitive tobacco cultivar Bel W3 with an ozone pulse (0.15 l/l, 5 h) markedly increased the mRNA level of basic -1,3-glucanase and to a lower degree that of basic chitinase. The increase of -1,3-glucanase mRNA level occurred within 1 h and showed a transient maximum. Seventeen hours after ozone treatment, the -1,3-glucanase mRNA level decreased to lower values. The increase of basic chitinase mRNA level was delayed and was less pronounced than that of -1,3-glucanase mRNA. Cultivar Bel B showed only a small increase of -1,3-glucanase mRNA level after the same ozone treatment, whereas its basic chitinase mRNA was more strongly induced. Prolonged ozone treatment for 2 days of tobacco Bel W3 led to a persistent level of -1,3-glucanase and basic chitinase mRNAs, as well as to an increase of acidic chitinase and pathogenesis-related (PR) 1b mRNA levels. The results indicate that genes so far considered to code for PR proteins may also be involved in the plant response to oxidative stress.     

Studies on cellulase production by Clostridium thermocellum

Summary Clostridium thermocellum ATCC 27405 (and its improved cellulase-producing mutant, AS-39) is an anaerobic thermophile that produces endo--glucanase and exo--glucanase when grown on cellobiose or cellulose as major carbon source (Shinmyo et al. 1979). The site of cellulase accumulation was at least 95% extracellular. Optimum conditions for endo--glucanase production in flasks included 1% (w/v) cellobiose, 0.2% (w/v) urea as a nitrogen source, 0.1 M morpholinopropane-sulfonic acid buffer, an initial pH of 7.4, and a yeast extract concentration of 0.6% (w/v). An improved medium (GS medium) was devised for future studies. Xylan was degraded by an extracellular enzyme (s) produced during cultivation on cellobiose, although C. thermocellum does not grow on xylan.     

Molecular characterization of a pea β-1,3-glucanase induced by Fusarium solani and chitosan challenge

-glucanases are prominent proteins in pea endocarp tissue responding to fungal infection. We have cloned and sequenced a partial pea cDNA clone, pPIG312, corresponding to a -1,3-glucanase in pea pods challenged with the incompatible pathogen Fusarium solani f. sp. phaseoli. The insert from the partial pea cDNA was used to probe a genomic library derived from pea leaves of the same cultivar. One of the genomic clones, pPIG4-3, contained the complete coding sequence for a mature -1,3-glucanase protein. The predicted amino acid sequence of the pea -1,3-glucanase has 78% identity to bean -1,3-glucanase, 62% and 60% to two tobacco -1,3-glucanases, 57% to soybean -1,3-glucanase, 51% to barley -1,3-glucanase, and 48% to barley -1,3-1,4-glucanase. Genomic Southern analysis indicates that the pea genome contains only one -1,3-glucanase gene corresponding to the probe used in this study. Accumulation of -1,3-glucanase mRNA homologous with the pPIG312 probe was detected in pea pods within 4 to 8 h after challenge with F. solani f. sp. phaseoli, f. sp. pisi, a compatible strain, or the elicitor, chitosan. In the incompatible reaction, mRNA accumulation remained high for 48h, whereas it rapidly decreased in the compatible reaction. After fungal inoculation of whole pea seedlings, the enhanced mRNA accumulation occurred mainly in the basal region (lower stem and root). This -1,3-glucanase glucanase mRNA was constitutively expressed in the roots of pea seedlings. The sustained levels of -glucanase mRNA expression induced by the incompatible pathogen in the resistance response suggests that the enzyme contributes to the pea plant's general defense.     

Developmental and hormonal regulation of β-1,3-glucanase in tobacco

A highly sensitive and specific rocket immunoassay was used to measure the content of an endo-type -1,3-glucanase (EC 3.2.1.39) in tissues of Nicotiana tabacum L. cv. Havana 425. We show that the accumulation of -1,3-glucanase in cultured pith-parenchyma tissue is blocked by combinations of the auxin, -naphthaleneacetic acid (NAA), and the cytokinin, kinetin. When tissues pre-incubated for 7 d on complete medium containing 2.0 mg·l^-1 NAA and 0.3 mg·l^-1 kinetin are transferred onto medium without hormones or with either hormone added separately, the -1,3-glucanase content expressed per mg soluble protein increases approx. ten fold over a 7-d period. Under these inductive conditions, up to approx. 5% of the soluble protein is -1,3-glucanase. The induction is inhibited by >90% when tissues are cultured over the same period on medium containing both hormones. This -1,3-glucanase is developmentally regulated in the intact plant. It is a major component of the soluble protien in the lower leaves and roots but is not detectable in leaves near the top of the plant.Abbreviation NAA -naphthaleneacetic acid     

The Morphogenetic Potential of Transgenic Tobacco Plants Expressing Genes of Bacterial Glucanases with Distinct Substrate Specificity

The expression of the bacterial gene for thermostable -1,3-glucanase in transgenic tobacco plants was shown to induce substantial changes in plant morphogenetic potential, whereas the expression of -1,3; 1,4-glucanase did not affect essentially plant morphogenesis. Our results permit the suggestion that the expression of bacterial -1,3-glucanase in plants elevated the level of endogenous auxin.     

Genes encoding acidic and basic class III β-1,3-glucanases are expressed in tomato plants upon viroid infection

-1,3-glucanases are hydrolytic enzymes considered to constitute part of the general array of defense genes induced by pathogen infection in higher plants. We have isolated and characterized two complementary DNA clones, corresponding to new -1,3-glucanases from tomato plants (Lycopersicon esculentum) which are expressed upon challenge with citrus exocortis viroid. Amino acid sequence comparison revealed that they are most similar to -1,3-glucanases from tobacco, particularly to PR-Q, the unique component of the class III -1,3-glucanase. The deduced amino acid sequences of the two tomato -1,3-glucanases indicate that, although being highly similar in amino acid sequence, they have different isoelectric points: pI 10.5 for the basic isoform (Tom PR-Q b) and pI 5.2 for the acidic one (Tom PR-Q a). The expression of these two -1,3-glucanase messenger RNAs (mRNAs) in response to viroid infection and ethephon treatments was examined. mRNAs for these two isoforms are coordinately expressed and induced similarly to mRNAs for other PR proteins, indicating that they are part of a general and coordinate mechanism of response of tomato plants susceptible to viroid infection.     

β-1,3-Glucanase and dimorphism in Paracoccidioides brasiliensis

Mycelial and yeast forms of P. brasiliensis were tested for several glucohydrolases. In addition to high levels of -blucanases, low amounts of -glucanase, chitinase and maltase were found. Tests for invertase, amylase and lactase were negative. The levels of -1,3-glucanase were higher in the mycelial form. The shift to the mycelial phase correlated with an increase in the levels of -1,3-glucanase. The enzyme was present in the cytoplasm, cell wall and culture medium. The extracellular enzyme was purified 42 fold by ammonium sulphate precipitation and gel filtration. Maximal activity was obtained at 60°C and pH of 5.0 acetate buffer or pH 6.0 (phosphate buffer). Its K _m was 0.205 mg/ml. The cell wall-bound enzyme showed a higher temperature optimum. Optimum pH and K _m were also slightly different. Following treatment of the cell walls with chitinase, -1,3-glucanase was released into the medium.     

β-Glucanases in developing cotton (Gossypium hirsutum L.) fibres

Cotton fibres possess several -glucanase activities which appear to be associated with the cell wall, but which can be partially solubilised in buffers. The main activity detected was that of an exo-(13)--d-glucanase (EC 3.2.1.58) but which also had the characteristics of a -glucosidase (EC 3.2.1.21). Endo-(13)--d-glucanase activity (EC 3.2.1.39) and much lower levels of (14)--d-glucanase activity were also detected. The exo-(13)--glucanase showed a maximum late on (40 days post-anthesis) in the development of the fibres, whereas the endo-(13)--glucanase activity remained constant throughout fibre development. The -glucanase complex associated with the cotton-fibre cell wall also functions as a transglucosylase introducing, inter alia, (16)--glucosyl linkages into the disaccharide cellobiose to give the trisaccharide 4-O--gentiobiosylglucose.Abbreviations CMC carboxymethylcellulose - ONPG o-nitrophenyl--d-glucopyranoside - TLC thin-layer chromatography Presented at the Third Cell Wall Meeting held in Fribourg in 1984     

Primary structure and expression of mRNAs encoding basic chitinase and 1,3-β-glucanase in potato

Infection of potato leaves (Solanum tuberosum L. cv. Datura) by the late blight fungus Phytophthora infestans, or treatment with fungal elicitor leads to a strong increase in chitinase and 1,3--glucanase activities. Both enzymes have been implicated in the plant's defence against potential pathogens. In an effort to characterize the corresponding genes, we isolated complementary DNAs encoding the basic forms (class I) of both chitinase and 1,3--glucanase, which are the most abundant isoforms in infected leaves. Sequence analysis revealed that at least four genes each are expressed in elicitor-treated leaves. The structural features of the potato chitinases include a hydrophobic signal peptide at the N-terminus, a hevein domain which is characteristic of class I chitinases, a proline- and glycine-rich linker region which varies among all potato chitinases, a catalytic domain, and a C-terminal extension. The potato 1,3--glucanases also contain a N-terminal hydrophobic signal peptide and a C-terminal extension, the latter comprising a potential glycosylation site. RNA blot hybridization experiments showed that basic chitinase and 1,3--glucanase are strongly and coordinately induced in leaves in response to infection, elicitor treatment, ethylene treatment, or wounding. In addition to their activation by stress, both types of genes are regulated by endogenous factors in a developmental and organ-specific manner. Appreciable amounts of chitinase and 1,3--glucanase mRNAs were found in old leaves, stems, and roots, as well as in sepals of healthy, untreated plants, whereas tubers, root tips, and all other flower organs (petals, stamen, carpels) contained very low levels of both mRNAs. In young leaves and stems, chitinase and 1,3--glucanase were differentially expressed. While chitinase mRNA was abundant in these parts of the plant, 1,3--glucanase mRNA was absent. DNA blot analysis indicated that in potato, chitinase and 1,3--glucanase are encoded by gene families of considerable complexity.     

Nucleotide sequence of an endo-β-1,4-glucanase gene fromBacillus subtilis CK-2

The gene encoding endo--1,4-glucanase inBacillus subtilis CK-2 was cloned intoEscherichia coli DH5, and the nucleotide sequence determined. The 1500 bp gene encodes a protein of 499 amino-acid residues with a calculated molecular mass of 55 261, and is equipped with a typicalB. subtilis signal peptide. Nucleotide sequence comparison revealed only 2 basepairs deviation between this gene and the endo--1,4-glucanase gene ofB. subtilis PAP115, and 93% to 95% homology was found between the amino acid sequences of these enzymes and otherB. subtilis endo--1,4-glucanases. Regions of similarity were also observed between the carboxy-terminal part of these enzymes and the part of theB. lautus PL236celA enzyme constituting the cellulose-binding domain.     

Synthesis of selectively radiolabeled hexasaccharides for the determination of enzymatic regioselectivity

Poly-N-acetyllactosamines provide backbone structures for functional modifications such as sialyl Lewis X. To understand how the biosynthesis of poly-N-acetyllactosamines is regulated, two branched oligosaccharides of the structure Gal1,4GlcNAc1, 6(Gal1,4GlcNAc1,2)-Man1,6Man-octyl 1 and 2 were synthesized in which one of the terminal galactose units was selectively radiolabeled. Hexasaccharides 1 and 2 were assembled from the chemically synthesized pentasaccharide precursors GlcNAc1,6(Gal1,4GlcNAc1,2)-Man1,6Man-octyl3 and Gal1,4GlcNAc1,6(GlcNAc1, 2) - Man1,6 Man-octyl 4 respectively, through treatment with UDP-1-[³H]-Gal and 1,4 galactosyltransferase. Compounds 1 and 2 were subsequently incubated with UDP-GlcNAc and the UDP-GlcNAc: Gal1-4Glc(NAc)1,3-N-acetylglucosaminyltransferase (i-GlcNAc transferase) resulting in a partial conversion to a mixture of heptasaccharides which were purified by HPLC. The branch selectivity of the addition of N-acetylglucosamine to compounds 1 and 2 was then characterized by endo--galactosidase digestion of the heptasaccharides, followed by isolation of the resultant pentasaccharides on C18 reverse-phase silica cartridges. Comparison of the amount of radiolabel to a control reaction lacking endo--galactosidase indicated the favored site of GlcNAc addition to be the lower 1,2-branch over the 1,6-branch by a 3:1 ratio.     

Effect of phytoalexins on hyphal growth and β-glucanases of Phytophthora infestans

Phytophthora infestans excretes an endo--1,3-, an endo--1,4-, and a-1,3-glucanase (laminarinase), a-1,6-glucosidase and possibly small amounts of a-1,4-glucosidase. Ether extracts from the infected resistant cultivar Eba but not from the susceptible Bintje inhibited growth of the parasite. Solavetivone and rishitin, two phytoalexins, and the steroid glycoalkaloid tomatine inhibited growth of the fungus and also activities of some of the fungal glucanases, whereas phytuberin, another phytoalexin, and the two phenolic compounds scopoletin and chlorogenic acid inhibited neither fungal growth nor fungal glucanases. The phytoalexin lubimin strongly reduced fungal growth but did not reduce the activities of any of the fungal glucanases tested. A potential role for host derived fungal glucanase inhibitors as factors of resistance in thePhytophthora-potato system is discussed.     

Structural studies of the glycopeptides of B-chain of cinnamomin – a type II ribosome-inactivating protein by nuclear magnetic resonance

Cinnamomin is a plant type II ribosome-inactivating protein (RIP) isolated from the seeds of Cinnamomum camphora. It consists of two nonidentical polypeptide chains (A- and B-chain) held together through one disulfide linkage. Its A- and B-chain contain 0.3% and 3.9% sugars respectively. The B-chain of cinnamomin was digested by pronase E and then the liberated glycopeptides were separated from non-glycopeptides by gel filtration chromatography on a Bio-Gel P-4 column. Three crude glycopeptides were obtained by continuing chromatography over anion-exchange resin (AG1-X2) in the buffer of 2% pyridine-acetic acid (pH 8.3) with a polygradient elution system. Through further purification by the gel filtration chromatography and HPLC, three major glycopeptides, GP1, GP2 and GP3 were obtained. Mainly by two-dimensional Nuclear Magnetic Resonance (NMR) including TOCSY, DQF-COSY, NOESY, HMQC and HMBC, their primary structures were analyzed as: Man1,3Man1,6(Man1,3)(Xyl1,2)Man1,4GlcNAc1,4GlcNAc1-(Gly-)Asn-Asn-Thr(GP1), Man1,6(Man1,3)(Xyl1,2)Man1,4GlcNAc1,4(Fuc1,3)GlcNAc1-Asn-Ala-Thr(GP2),Man1,6(Man1,3)Man1,6(Man1,2 Man1,3)Man1,4GlcNAc1,4GlcNAc1-(Ala-)Asn-Gly-Thr(GP3).     

β-1,3-Glucanase is highly-expressed in laticifers of Hevea brasiliensis

Clones encoding -1,3-glucanase have been isolated from a Hevea cDNA library prepared from the latex of Hevea brasiliensis using a probe Nicotiana plumbaginifolia cDNA encoding -1,3-glucanase, gnl. Nucleotide sequence analysis showed that a 1.2 kb Hevea cDNA encoding a basic -1,3-glucanase showed 68% nucleotide homology to gnl cDNA. Northern blot analysis using the Hevea cDNA as probe detected a mRNA of 1.3 kb which was expressed at higher levels in latex than in leaf. In situ hybridization analysis using petiole sections from Hevea localized the -1,3-glucanase mRNA to the laticifer cells. Genomic Southern analysis suggested the presence of a low-copy gene family encoding -1,3-glucanases in H. brasiliensis.     

Evidence for a third structural class of β-1,3-glucanase in tobacco

Glucan endo-1,3--glucosidases (-1,3-glucanases) have been implicated in several developmental processes and they may also play a direct role in the plant's defense against fungal pathogens. In an effort to characterize the glucanase gene family, complementary DNA clones encoding an acidic form of -1,3-glucanase have been isolated from tobacco. The cDNA was expressed in E. coli and shown to encode a -1,3-glucanase activity. The protein sequence encoded by the cDNA was found to match the partial protein sequence of PR-35, a previously characterized -1,3-glucanase [29]. The protein encoded by the cDNA was purified from the extracellular fluid of TMV-infected tobacco leaves and found by immunological methods to correspond to glucanase PR-Q' [10]. From a detailed analysis of the cDNA it is clear that this glucanase represents a third structural class of enzyme which differs substantially from both the basic, vacuolar glucanase and the acidic, extracellular forms (PR-2, PR-N and PR-O). It has previously been demonstrated that the basic form of -1,3-glucanase is synthesized as a pre-pro-enzyme and upon maturation the 21 amino acid signal peptide and a 22 amino acid carboxy-terminal peptide are removed. This processing event has been proposed to be involved with the vacuolar localization of the enzyme. By comparing the deduced protein structure of PR-Q' to that of the basic form it is evident that this extracellular enzyme is missing the carboxy-terminal 22 amino acids. The role of a conserved phenylalanine-glycine dipeptide in the processing of glucanases and other pathogenesis-related proteins from tobacco is discussed.