Heterologous co-expression in E. coli of isoamylase genes from cassava Manihot esculenta Crantz 'KU50' achieves enzyme-active heteromeric complex formation.

KEY MESSAGE: Cloning of two isoamylase genes, MeISA1 and MeISA2, from cassava (Manihot esculenta Crantz) tubers, accompanied by their co-expression in E. coli demonstrates a requirement for heteromeric complex formation to achieve debranching activity. Starch debranching enzyme (DBE) or isoamylase (ISA) (EC.3.2.1.68), an important enzyme in starch metabolism, catalyses the hydrolysis of α-1,6 glycosidic linkages of amylopectin. Isoforms of ISAs have been reported in higher plants and algae (Fujita et al. in Planta 208:283-293, 1999; Hussain et al. in Plant Cell 15:133-149, 2003; Ishizaki et al. in Agric Biol Chem 47:771-779, 1983; Mouille et al. in Plant Cell 8:1353-1366, 1996). In the current work, cassava ISA genes were isolated from cDNA generated from total RNA from tubers of Manihot esculanta Crantz cultivar KU50. MeISA1 and MeISA2 were successfully amplified and cloned into a pETDuet1 vector. The putative MeISA1 and MeISA2 proteins comprised 763 and 882 amino acids, with substantial similarity to StISA1 and StISA2 from potato (84.4% and 68.9%, respectively). Recombinant MeISA1 and MeISA2 were co-expressed in Escherichia coli SoluBL21 (DE3). HistrapTM-Purified rMeISA1 and rMeISA2 showed approximate molecular weights of 87 and 99 kDa, respectively, by SDS-PAGE. Debranching activity was only detectable in the column fractions where both recombinant ISA isoforms were present. The heteromeric DBE from crude extracts of 4-5 h induced cultures analysed by gel filtration chromatography and western blot showed combinations of rMeISA1 and rMeISA2 at ratios of 1:1 to 4:1. Pooled fractions with DBE activity were used for enzyme characterisation, which showed that the enzyme was specific for amylopectin, with optimum activity at 37 °C and pH 7.0. Enzyme activity was enhanced by Co2+, Mg2+ and Ca2+, but was strongly inhibited by Cu2+. Debranched amylopectin products showed chain length distributions typical of plant DBE.

Structural Dissection of the Maltodextrin Disproportionation Cycle of the Arabidopsis Plastidial Disproportionating Enzyme 1 (DPE1).

The degradation of transitory starch in the chloroplast to provide fuel for the plant during the night requires a suite of enzymes that generate a series of short chain linear glucans. However, glucans of less than four glucose units are no longer substrates for these enzymes, whereas export from the plastid is only possible in the form of either maltose or glucose. In order to make use of maltotriose, which would otherwise accumulate, disproportionating enzyme 1 (DPE1; a 4-α-glucanotransferase) converts two molecules of maltotriose to a molecule of maltopentaose, which can now be acted on by the degradative enzymes, and one molecule of glucose that can be exported. We have determined the structure of the Arabidopsis plastidial DPE1 (AtDPE1), and, through ligand soaking experiments, we have trapped the enzyme in a variety of conformational states. AtDPE1 forms a homodimer with a deep, long, and open-ended active site canyon contained within each subunit. The canyon is divided into donor and acceptor sites with the catalytic residues at their junction; a number of loops around the active site adopt different conformations dependent on the occupancy of these sites. The "gate" is the most dynamic loop and appears to play a role in substrate capture, in particular in the binding of the acceptor molecule. Subtle changes in the configuration of the active site residues may prevent undesirable reactions or abortive hydrolysis of the covalently bound enzyme-substrate intermediate. Together, these observations allow us to delineate the complete AtDPE1 disproportionation cycle in structural terms.

Purification, characterization and molecular cloning of a novel endo-beta-N-acetylglucosaminidase from the basidiomycete, Flammulina velutipes.

Endo-beta-N-acetylglucosaminidases are thought to be key enzymes in the catabolism of asparagine-linked oligosaccharides. However, little is known about the enzymes of this type in basidiomycetes. We investigated endo-beta-N-acetylglucosaminidases in basidiomycetes using fluorescence-labeled glycoasparagines as substrates. Flammulina velutipes showed high activity and its enzyme was named endo-beta-N-acetylglucosaminidase FV (Endo FV). The enzyme purified from the fruiting bodies of F. velutipes was separated into two forms. Endo FV was specific for high mannose and hybrid-type oligosaccharides. The enzyme was remarkably less active against asparagine-linked oligosaccharides attached to glycoproteins. It transferred an asparagine-linked oligosaccharide to Glc, but not to Gal. cDNA of Endo FV was cloned. It was composed of a 996-bp open reading frame encoding 331 amino acid residues. A recombinant Endo FV expressed in Escherichia coli showed enzymatic activity. The Endo FV gene in the genome of F. velutipes had no introns. The gene encoding Endo FV showed little homology with genes of known endo-beta-N-acetylglucosaminidases. A chitinase active site motif existed in the deduced primary structure, indicating that Endo FV belongs to glycoside hydrolase family 18. The deduced amino acid sequence of Endo FV had regions conserved in class III chitinases from fungi though it showed little homology with the sequence of any other endo-beta-N-acetylglucosaminidases. A folding model of Endo FV indicated it to be homologous with the tertiary structure of Endo H which is quite similar in specificity for asparagine-linked oligosaccharides. This study suggests that Endo FV may become similar to Endo H in substrate specificity as a result of evolutionary convergence.

Cloning of the full-length isoamylase3 gene from cassava Manihot esculenta Crantz 'KU50' and its heterologous expression in E. coli.

Isoamylase (EC.3.2.1.68), an essential enzyme in starch metabolism, catalyses the cleavage of α-1,6 glucosidic linkages of branched α-polyglucans such as beta-limit dextrin and amylopectin, but not pullulan. Three different isoamylase isoforms have been reported in plants and algae. We herein report on the first success in preparation of full-length isoamylase3 gene (MeISA3) of cassava Manihot esculenta Crantz 'KU50' from 5' Rapid Amplification of cDNA Ends (5' RACE). The MeISA3 was cloned to pET21b and expressed in E. coli. The HistrapTM-purified rMeISA3 appeared as a single band protein with approximate molecular size of 75 kDa on SDS-PAGE and Western blot, while 80 kDa was shown by gel filtration chromatography. This indicated the existence of a monomeric enzyme. Biochemical characterisation of rMeISA3 showed that the enzyme was specific towards beta-limit dextrin, with optimal activity at 37 °C pH 6.0. Activity of rMeISA3 could be significantly promoted by Mg2+ and Co2+. rMeISA3 debranched glucan chains of amylopectin were confirmed by HPAEC-PAD analysis.

Cloning and expression of cyclodextrin glycosyltransferase gene from Paenibacillus sp. T16 isolated from hot spring soil in northern Thailand.

Gene encoding cyclodextrin glycosyltransferase (CGTase), from thermotolerant Paenibacillus sp. T16 isolated from hot spring area in northern Thailand, was cloned and expressed in E. coli (JM109). The nucleotide sequences of both wild type and transformed CGTases consisted of 2139 bp open reading frame, 713 deduced amino acids residues with difference of 4 amino acid residues. The recombinant cells required 24 h culture time and a neutral pH for culture medium to produce compatible amount of CGTase compared to 72 h culture time and pH 10 for wild type. The recombinant and wild-type CGTases were purified by starch adsorption and phenyl sepharose column chromatography and characterized in parallel. Both enzymes showed molecular weight of 77 kDa and similar optimum pHs and temperatures with recombinant enzyme showing broader range. There were some significant difference in pH, temperature stability and kinetic parameters. The presence of high starch concentration resulted in higher thermostability in recombinant enzyme than the wild type. The recombinant enzyme was more stable at higher temperature and lower pH, with lower K(m) for coupling reaction using cellobiose and cyclodextrins as substrates.

Characterization of a thermostable levansucrase from Bacillus sp. TH4-2 capable of producing high molecular weight levan at high temperature.

A thermoactive and thermostable levansucrase was purified from a newly isolated thermophilic Bacillus sp. from Thailand soil. The purification was achieved by alcohol precipitation, DEAE-Cellulose and gel filtration chromatographies. The enzyme was purified to homogeneity as determined by SDS-PAGE, and had a molecular mass of 56 kDa. This levansucrase has some interesting characteristics regarding its optimum temperature and heat stability. The optimum temperature and pH were 60 degrees C and 6.0, respectively. The enzyme was completely stable after treatment at 50 degrees C for more than 1 h, and its activity increased four folds in the presence of 5 mM Fe(2+). The optimum temperature for levan production was 50 degrees C. Contrary to other levansucrases, the one presented in this study is able to produce high molecular weight levan at 50 degrees C.

New action pattern of a maltose-forming alpha-amylase from Streptomyces sp. and its possible application in bakery.

An a-Amylase (EC 3.2.1.1) was purified that catalyses the production of a high level of maltose from starch without the attendant production of glucose. The enzyme was produced extracellularly by thermophilic Streptomyces sp. that was isolated from Thailand's soil. Purification was achieved by alcohol precipitation, DEAE-Cellulose, and Gel filtration chromatographies. The purified enzyme exhibited maximum activity at pH 6-7 and 60 degrees C. It had a relative molecular mass of 45 kDa, as determined by SDS-PAGE. The hydrolysis products from starch had alpha-anomeric forms, as determined by 1H-NMR. This maltose-forming alpha-Amylase completely hydrolyzed the soluble starch to produce a high level of maltose, representing up to 90%. It hydrolyzed maltotetrose and maltotriose to primarily produce maltose (82% and 62% respectively) without the attendant production of glucose. The high maltose level as a final end-product from starch and maltooligosaccharides, and the unique action pattern of this enzyme, indicate an unusual maltose-forming system. After the addition of the enzyme in the bread-baking process, the bread's volume increased and kept its softness longer than when the bread had no enzyme.

Expression analysis of calmodulin and calmodulin-like genes from rice, Oryza sativa L.

BACKGROUND: In plants, a large family of calmodulin (CaM) and CaM-like (CML) proteins transduce the increase in cytosolic Ca2+ concentrations by binding to and altering the activities of target proteins, and thereby affecting the physiological responses to a vast array of stimuli. Here, transcript expression analysis of Cam and CML gene family members in rice (Oryza sativa L.) was extensively examined. RESULTS: Cam and CML genes in rice exhibited differential expression patterns in tissues/organs. Under osmotic stress and salt stress, expression of OsCam1-1, OsCML4, 5, 8, and 11 was induced with different kinetics and magnitude. OsCML4 and 8 mRNA levels significantly increased by 3 h after treatment and remained elevated for at least 24 h while expression of OsCam1-1, OsCML5 and 11 was up-regulated as early as 1-3 h before rapidly returning to normal levels. Several cis-acting elements in response to abiotic stresses, including DREs (important promoter elements responsive to drought, high salt, and cold stress), were detected in the 5' upstream regions of these genes. The observed induction of the GUS activity of transgenic rice plants via the OsCam1-1 promoter appeared to be biphasic and dependent on the severity of salt stress. CONCLUSIONS: Large OsCam and OsCML gene family members likely play differential roles as signal transducers in regulating various developmental processes and represent important nodes in the signal transduction and transcriptional regulation networks in abiotic stresss responses mediated by the complex Ca2+ signals in plants, which are rich in both spatial and temporal information.

An expedient enzymatic route to isomeric 2-, 3- and 6-monodeoxy-monofluoro-maltose derivatives.

2-Deoxy-2-fluoro-d-glucose, 3-deoxy-3-fluoro-D-glucose and 6-deoxy-6-fluoro-D-glucose were converted into the corresponding maltose derivatives using Arabidopsis thaliana DPE2-mediated trans-glycosylation reaction with glycogen acting as a glucosyl donor. (19)F NMR spectroscopy proved to be a valuable tool for monitoring the progress of these reactions and to assess the nature of resulting oligomeric products.

Structure and expression analysis of the OsCam1-1 calmodulin gene from Oryza sativa L.

Calmodulin (CaM) proteins, members of the EF-hand family of Ca(2+)- binding proteins, represent important relays in plant calcium signals. Here, OsCam1-1 was isolated by PCR amplification from the rice genome. The gene contains an ORF of 450 base pairs with a single intron at the same position found in other plant Cam genes. A promoter region with a TATA box at position-26 was predicted and fused to a gus reporter gene, and this construct was used to produce transgenic rice by Agrobacterium-mediated transformation. GUS activity was observed in all organs examined and throughout tissues in cross-sections, but activity was strongest in the vascular bundles of leaves and the vascular cylinders of roots. To examine the properties of OsCaM1-1, the encoding cDNA was expressed in Escherichia coli. The electrophoretic mobility shift when incubated with Ca(2+) indicates that recombinant OsCaM1-1 is a functional Ca(2+)-binding protein. In addition, OsCaM1-1 bound the CaMKII target peptide confirming its likely functionality as a calmodulin.

Purification and characterization of two alkaline, thermotolerant alpha-amylases from Bacillus halodurans 38C-2-1 and expression of the cloned gene in Escherichia coli.

A newly isolated strain, 38C-2-1, produced alkaline and thermotolerant alpha-amylases and was identified as Bacillus halodurans. The enzymes were purified to homogeneity and named alpha-amylase I and II. These showed molecular masses of 105 and 75 kDa respectively and showed maximal activities at 50-60 degrees C and pH 10-11, and 42 and 38% relative activities at 30 degrees C. These results indicate that the enzymes are thermotolerant. The enzyme activity was not inhibited by a surfactant or a bleaching reagent used in detergents. A gene encoding alpha-amylase I was cloned and named amyI. Production of AmyI with a signal peptide repressed the growth of an Escherichia coli transformant. When enzyme production was induced by the addition of isopropyl beta-D(-)-thiogalactopyranoside in the late exponential growth phase, the highest enzyme yield was observed. It was 45-fold that of the parent strain 38C-2-1.