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PubMed2002

Magnetite-alginate beads for purification of some starch degrading enzymes.

Starch degrading enzymes, viz., beta-amylase, glucoamylase, and pullulanase, were purified using magnetite-alginate beads. In each case, the enzyme activity was eluted by using 1.0 M maltose. beta-Amylase (sweet potato), glucoamylase (Aspergillus niger), and pullulanase (Bacillus acidopullulyticus) from their crude preparations were purified 37-, 31-, and 49-fold with 86, 87, and 95% activity recovery, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed single band in each case.

Alginates↗

PubMed1997

Enhancement of cyclodextrin production through use of debranching enzymes.

In the presence of a complexant and a debranching enzyme capable of cleaving alpha-(1-->6) linkages in alpha-D-glucans, Bacillus mascerans cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) converted starch, maltodextrin and glycogen into cyclodextrin (CD) in yields higher than those obtainable in the absence of debranching enzyme. The extent of yield enhancement by pullulanase (EC 3.2.1.41; from Enterobacter aerogenes) was dependent upon temperature, type of substrate (including source of starch) and kind of complexant. Highest yields with pullulanase were attained generally by use of low temperature (15-25 degrees C) and starches of low amylose content. At 25 degrees C and pH 7, with cyclodecanone as complexant, 91-93% yields of beta-CD were obtainable from amylopectin, waxy-maize starch, and tapioca starch. With decan-I-ol as complexant, amylopectin was converted at 15 degrees C into alpha-CD in 84% yield. With cyclotridecanone as complexant, amylopectin was converted at 40 degrees C into gamma-CD in 72% yield. The debranching enzyme isoamylase (EC 3.2.1.68; from Pseudomonas amyloderamosa) was also employed successfully to achieve high beta-CD yields. A 90% yield of beta-CD from amylopectin was obtained by applying isoamylase, CGTase and cyclodecanone at pH 6 and 25 degrees C.

Amylopectin↗

PubMed1977

Production of Schardinger beta-dextrin by soluble and immobilized cyclodextrin glycosyltransferase of an alkalophilic Bacillus sp.

Succinylated cyclodextrin glycosyltransferase (EC 3.2.1.19) of an alkalophilic Bacillus sp. was adsorbed on a vinylpyridine copolymer. The enzyme had about 25% of the activity of soluble enzyme added. No increase of pH or thermal stability of the enzyme was observed by the adsorption, whereas optimum temperature for the enzyme action was shifted from 50 to 55 degrees C. The enzyme converted starch to cyclodextrine without significant loss of activity under the conditions of 4 times reusing of 6 hr conversion by the batch system or 2 weeks continuous reaction by the column system at 55 degrees C and pH 8.0. About 46% of the potato starch solution [15% (w/v)] was converted to cyclodextrins by the enzyme, and 52% was converted by the simultaneous action of the enzyme and alkaline pullulanase of alkalophilic Bacillus sp. (No. 202-1). These values were almost the same as those obtained by the soluble enzyme or enzymes system.

Bacillus↗

PubMed1990

Protein secretion in gram-negative bacteria: transport across the outer membrane involves common mechanisms in different bacteria.

The xcp genes are required for protein secretion by Pseudomonas aeruginosa. They are involved in the second step of the process, i.e. the translocation across the outer membrane, after the exoproteins have reached the periplasm in a signal peptide dependent fashion. The nucleotide sequence of a 2.5 kb DNA fragment containing xcp genes showed at least two complete open reading frames, potentially encoding proteins with molecular weights of 41 and 19 kd. Products with these apparent molecular weights were identified after expression of the DNA fragment in vitro and in vivo. Subcloning and complementation experiments showed that both proteins are required for secretion. The two products are located in the inner membrane and share highly significant homologies with the PulL and PulM proteins which are required for the specific secretion of pullulanase in Klebsiella pneumoniae. These homologies reveal the existence of a common mechanism for protein secretion in Pseudomonas aeruginosa and Klebsiella pneumoniae.

Amino Acid Sequence↗

PubMed1992

Flow injection analysis with immobilized enzymes for process control of pullulan production by fermentation.

A flow injection system is described for the parallel determination of pullulan and glucose during a fermentation of the fungus Aureobasidium pullulans. The polysaccharide was hydrolyzed by pullulanase and amyloglucosidase, immobilized to controlled-pore glass (CPG). The glucose produced was oxidized by glucose dehydrogenase and the NADH formed determined photometrically. The pullulan concentration was calculated from the difference to the response obtained for free glucose. The calibration curves for monomer and polymer were both linear between 2 mg dm-3 and 20 mg dm-3. Analysis of one sample for the determination of glucose and pullulan took about 10 min.

Enzymes, Immobilized↗

PubMed2004

Production of D-lactic acid by bacterial fermentation of rice starch.

D-Lactic acid was synthesized by the fermentation of rice starch using microorganisms. Two species: Lactobacillus delbrueckii and Sporolactobacillus inulinus were found to be active in producing D-lactic acid of high optical purity after an intensive screening test for D-lactic acid bacteria using glucose as substrate. Rice powder used as the starch source was hydrolyzed with a combination of enzymes: alpha-amylase, beta-amylase, and pullulanase to obtain rice saccharificate consisting of maltose as the main component. Its average gross yield was 82.5%. Of the discovered D-lactic acid bacteria, only Lactobacillus delbrueckii could ferment both maltose and the rice saccharificate. After optimizing the fermentation of the rice saccharificate using this bacterium, pilot scale fermentation was conducted to convert the rice saccharificate into D-lactic acid with a D-content higher than 97.5% in a yield of 70%. With this yield, the total yield of D-lactic acid from brown rice was estimated to be 47%, which is almost equal to the L-lactic acid yield from corn. The efficient synthesis of D-lactic acid can open a way to the large scale application of high-melting poly(lactic acid) that is a stereocomplex of poly(L-lactide) and poly(D-lactide). Schematic representation of the production of D-lactic acid starting from brown rice as described here.

Fermentation↗

PubMed1997

Module swaps between related translocator proteins pIV(f1), pIV(IKe) and PulD: identification of a specificity domain.

In Gram-negative bacteria, type II and type III secretion and filamentous phage assembly systems use related outer membrane proteins for substrate-specific transport across the outer membrane. We show here that the specificity domain of the phage f1 outer membrane protein pIV is contained within the 149 N-terminal amino acid residues. When the pIV(f1) specificity domain is fused to the translocator domain of the related pIV of phage IKe, the chimeric construct supports f1 but not IKe assembly. Functional coupling between the two domains in this chimeric construct is poor and is improved by a single amino acid change in the translocator domain of the pIV(IKe). In native pIV(IKe), two amino acid changes within its specificity domain are both necessary and sufficient to change the specificity from IKe to f1 assembly. Analysis of 39 chimeric constructs between pIV(f1) and the outer membrane protein PulD of the pullulanase secretion system failed to identify a comparable exchangeable specificity domain. These results indicate that the two domains may not function autonomously, and suggest that tertiary and quarternary changes of the entire translocator component rather than of an autonomous functional domain are required for specific translocation across the outer membrane.

Amino Acid Sequence↗

PubMed1998

Macromolecular assembly and secretion across the bacterial cell envelope: type II protein secretion systems.

A decade ago, Pugsley and colleagues reported the existence of a large region of Klebsiella DNA, distinct from the Klebsiella gene encoding pullulanase, which was necessary for secretion of this enzyme to the cell surface in Escherichia coli (d'Enfert et al., 1987a,b). The pul genes it contained proved to be the tip of an iceberg. The sequences reported before 1992 (d'Enfert et al., 1987a,b; d'Enfert & Pugsley, 1989; Pugsley & Reyss, 1990; Reyss & Pugsley, 1990) included only one gene (pulD) that matched any sequence in the data base; a 220 amino acid residue segment of PulD was 32% identical with a portion of the filamentous phage-encoded protein, pIV. But by the time the sequence of the 18.8 kb DNA fragment that contained the pul genes had been completed (Possot et al., 1992), reports of sets of homologous genes in several species of Gram-negative plant and animal pathogens had appeared. For the most part, these gene clusters were cloned by their ability to complement mutants that produced, but failed to secrete, proteins normally found in the extracellular milieu; when tested, the mutants showed reduced pathogenicity or were totally avirulent. The secreted proteins included hydrolytic enzymes such as cellulase and pectinase from plant pathogens, and proteases and toxins from animal pathogens. The multi-gene family necessary for secretion of these enzymes is now known as the type II system or the main terminal branch (MTB) of the general secretion pathway (GSP). As summarized by Pugsley et al. (1997), the current tally includes type II systems from Klebsiella oxytoca (pul), Erwinia chrysanthemi and carotovora (out), Xanthomonas campestris (xps), Pseudomonas aeruginosa (xcp), Aeromonas hydrophila (exe), and Vibrio cholerae (eps). A second type II system (sps) necessary for deposition of the S-layer on the cell surface in A. hydrophila is more similar to the X. campestris than A. hydrophila genes (Thomas & Trust, 1995). The biggest surprise has been the discovery of a complete set of type II secretion genes in E. coli K12. The E. coli genes are not expressed under normal growth conditions, and a search is underway to find inducing conditions and secretion substrates (Francetic & Pugsley, 1996). Impressive progress has already been made in defining components of the pathway. What remains to be understood in mechanistic detail is how this protein secretion system functions.

Bacterial Proteins↗

PubMed1975

Comparative biochemistry of alpha-glucan-utilization in Pseudomonas amyloderamosa and Pseudomonas saccharophila: physiological significance of variations in the pathway.

Growth patterns on and utilization of various alpha-glucans were investigated in Pseudomonas amyloderamosa and P. saccharophila. Maltose, maltodextrins (average chain length 7 glycosyl units) and glycogen supported excellent growth of both organisms and were extensively metabolized, although glycogen utilization in P. saccharophila was preceded by a prolonged lag phase. P. amyloderamosa produced limited growth on amylopectin and the carbohydrate was only partly degraded. It seemed likely that many of the unit chains liberated from amylopectin had a length exceeding the substrate range accepted by the maltodextrin permease (transport) system. A correlation was established between the pH of the medium and the utilization of glycogen and amylopectin for growth in P. amyloderamosa. The carbohydrates were at least partly utilizable at pH 6.0, whereas they could not support any growth at pH 6.5. Most likely, the lack of growth at the higher pH reflected the low activity of isolamylase at this pH. The enzyme patterns of maltodextrin catabolism in the two bacteria were established. Intracellularly, maltodextrin phosphorylase and 4-alpha-glucanotransferase occurred in both. Degradation of extracellular alpha-glucans was mediated by a mainly intracellular isoamylase in P. amyloderamosa, whereas P. saccharophila possessed an extracellular alpha-amylase and a firmly cell-bound pullulanase.

Amylases↗

PubMed1996

Characterization of genes from Thermoanaerobacterium thermosulfurigenes EM1 that encode two glycosyl hydrolases with conserved S-layer-like domains.

Two genes from Thermoanaerobacterium thermosulfurigenes EM1 were identified which are predicted to encode a xylanase (XynA) and a polygalacturonate hydrolase (Pg1A). The xynA gene has the potential to encode a 1234-amino acid product consisting of a signal peptide followed by a repeated domain, a xylanase family F domain, two cellulose-binding domains and a triplicated sequence at its C-terminus. The gene pglA is predicted to encode a product of 1148 amino acids consisting of a signal sequence followed by a fibronectin type III-like domain (Fn3 domain), the catalytic domain, a Gly/Thr/Ser/Asn-rich segment and a triplicated domain. The triplicated segments at the C-termini of deduced XynA and Pg1A are about 95% identical to each other and to the S-layer-like domains of the previously characterized pullulanase (AmyB) from the same organism. In contrast, sequence comparisons revealed only distant amino acid sequence similarities between the fibronectin type III-like domains of Pg1A and AmyB from T. thermosulfurigenes EM1.

Amino Acid Sequence↗

PubMed2001

Production of polysaccharide hydrolases in the genus Rhizopus.

Polysaccharide hydrolase activity was assayed in a group of 28 selected Rhizopus strains. The production of lichenases, mannanases, cellulases, xylanases, amylases and pullulanases was demonstrated using the gel-testing method during growth of the strains on suitably meshed polysaccharide gels.

Fermentation↗

PubMed1996

Molecular mimicry: the geographical distribution of immune responses to Klebsiella in ankylosing spondylitis and its relevance to therapy.

The discovery that HLA-B27 is linked to ankylosing spondylitis (AS) and HLA-DR1/DR4 to rheumatoid arthritis (RA) has provided new approaches to the study of the possible causation of these diseases. Several theories have been proposed to explain these associations but only one, namely "molecular mimicry", has provided a specific aetiological agent for each of these diseases. Molecular mimicry between HLA-B27 and two molecules in Klebsiella microbes: nitrogenase and pullulanase D has been reported whilst in Proteus microbes, the haemolysin molecule shows sterochemical similarity to HLA-DR1/DR4. Elevated immune responses to Klebsiella microbes have been demonstrated in AS patients from 10 different countries and this wide geographical distribution suggests that the same aetiological agent is probably acting in producing this condition. Furthermore RA patients show similar immune responses to Proteus microbes. Whether AS or RA are caused by these bacteria can only be resolved by tissue typing all rheumatological patients early, in the course of their disease and then assessing their response to antibiotic chemotherapy in longitudinal studies involving double-blind crossover trials. It is possible that in the future, the course of AS or even RA could be modified by adequate antibiotic chemotherapy or even diets which affect the substrates on which these bacteria grow.

Antibodies, Bacterial↗

PubMed1999

Cell wall of Thermoanaerobacterium thermosulfurigenes EM1: isolation of its components and attachment of the xylanase XynA.

Thermoanaerobacterium thermosulfurigenes EM1 has a gram-positive type cell wall completely covered by a surface layer (S-layer) with hexagonal lattice symmetry. The components of the cell envelope were isolated, and the S-layer protein was purified and characterized. S-layer monomers assembled in vitro into sheets with the same hexagonal symmetry as in vivo. Monosaccharide analysis revealed that the S-layer is associated with fucose, rhamnose, mannosamine, glucosamine, galactose, and glucose. The N-terminal 31 amino acid residues of the S-layer protein showed significant similarity to SLH (S-layer homology) domains found in S-layer proteins of different bacteria and in the exocellular enzymes pullulanase, polygalacturonate hydrolase, and xylanase of T. thermosulfurigenes EM1. The xylanase from T. thermosulfurigenes EM1 was copurified with the S-layer protein during isolation of cell wall components. Since SLH domains of some structural proteins have been shown to anchor these proteins noncovalently to the cell envelope, we propose a common anchoring mechanism for the S-layer protein and exocellular enzymes via their SLH domains in the peptidoglycan-containing layer of T. thermosulfurigenes EM1.

Amino Acid Sequence↗

PubMed1999

Extremophiles as a source of novel enzymes for industrial application.

Extremophilic microorganisms are adapted to survive in ecological niches such as at high temperatures, extremes of pH, high salt concentrations and high pressure. These microorganisms produce unique biocatalysts that function under extreme conditions comparable to those prevailing in various industrial processes. Some of the enzymes from extremophiles have already been purified and their genes successfully cloned in mesophilic hosts. In this review we will briefly discuss the biotechnological significance of extreme thermophilic (optimal growth 70-80 degrees C) and hyperthermophilic (optimal growth 85-100 degrees C) archaea and bacteria. In particular, we will focus on selected extracellular-polymer-degrading enzymes, such as amylases, pullulanases, cyclodextrin glycosyltransferases, cellulases, xylanases, chitinases, proteinases and other enzymes such as esterases, glucose isomerases, alcohol dehydrogenases and DNA-modifying enzymes with potential use in food, chemical and pharmaceutical industries and in environmental biotechnology.

Archaea↗

PubMed1997

Starch utilization by Bacteroides ovatus isolated from the human large intestine.

Starch supported growth of continuous cultures of Bacteroides ovatus when this carbohydrate provided the sole source of carbon and energy. Inducible amylase and alpha-glucosidase activities were inversely related to dilution rate in starch-limited and starch-excess chemostats over the dilution rate (D) range D = 0.03/h to D =0.20/h, and were partly repressed during growth under conditions of starch-excess. Preparative isoelectric focusing of B. ovatus cytoplasmic extracts indicated the existence of three distinct starch-hydrolyzing enzymes. Incubation of active fractions from the isoelectric focusing cell with maltose and a variety of low-molecular-weight oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose) identified a single amylase activity, an enzyme with combined beta-amylase and glucoamylase/alpha-glucosidase properties, and also a possible pullulanase. The ability of B. ovatus to synthesize several starch-hydrolyzing enzymes with different specificities and activities may confer a significant competitive advantage to this organism in the colonic ecosystem.

Bacteroides↗

PubMed1999

Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice.

Isoamylase (EC 3.2.1.68) in rice (Oryza sativa L.) was efficiently purified within a day to homogeneity, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), from developing endosperm by sequential use of Q Sepharose HP anion-exchange chromatography, ammonium sulfate fractionation, and TSKgel G4000SWXL and G3000SWXL gel filtration chromatography. Although the protein exhibited a molecular size of ca. 83 kDa on SDS-PAGE, the apparent size of the native enzyme was approximately 340 and 490 kDa on TSKgel G3000SWXL and G4000SWXL gel filtration chromatograms, respectively, suggesting that rice isoamylase exists in a homo-tetramer to homo-hexamer form in developing endosperm. The purified rice isoamylase was able to debranch glycogen, phytoglycogen and amylopectin but could not attack pullulan. The optimum pH and temperature for isoamylase activity were found to be pH 6.5 to 7.0 and 30 degrees C, respectively. The enzyme activity was completely inhibited by HgCl2 and p-chloromercuribenzoate at 1 mM. These results indicate that rice isoamylase possesses properties which are distinct from those reported for bacterial isoamylase. Complementary-DNA clones for rice endosperm isoamylase were isolated with a polymerase-chain-reaction product as probe which was generated by primers designed from nucleotides conserved in cDNA for maize Sugary-1 isoamylase (M.G. James et al., 1995. Plant Cell 7: 417-429) and a Pseudomonas amyloderamosa gene encoding isoamylase (A. Amemura et al. 1988, J Biol Chem 263: 9271-9275). The nucleotide sequence and deduced amino acid sequence of the longest clone showed a high similarity to those of maize Surgary-1 isoamylase, but a lesser similarity to those of Pseudomonas amyloderamosa isoamylase. Southern blot analysis and gene mapping analysis indicated that the isoamylase gene exists as a single copy in the rice genome and is located on chromosome 8 of cv. Nipponbare which belongs to the Japonica rice group. Phylogenetic analysis indicated that isoamylases from maize and rice are more closely related to a number of glgX gene products of the blue green alga Synechocystis and various bacteria than to isoamylases from Pseudomonas and Flavobacterium. Hence, it is proposed that glgX proteins are classified as isoamylase-type debranching enzymes. Our tree also showed that all starch- and glycogen-debranching enzymes from plants and bacteria tested can be classified into two distinct types, an isoamylase-type and a pullulanase-type.

Amino Acid Sequence↗

PubMed2001

Starch-hydrolyzing bacteria from Ethiopian soda lakes.

Alkaliphilic bacteria were isolated from soil and water samples obtained from Ethiopian soda lakes in the Rift Valley area--Lake Shala, Lake Abijata, and Lake Arenguadi. Starch-hydrolyzing isolates were selected on the basis of their activity on starch agar plate assay. Sixteen isolates were chosen, characterized, and subjected to 16S rRNA gene sequence analysis. All the isolates were gram positive and catalase- and beta-galactosidase positive. All isolates except one were motile endospore-forming rods and were found to be closely related to the Bacillus cluster, being grouped with Bacillus pseudofirmus, Bacillus cohnii, Bacillus vedderi, and Bacillus agaradhaerens. The one exception had nonmotile coccoid cells and was closely related to Nesterenkonia halobia. The majority of the isolates showed optimal growth at 37 degrees C and tolerated salinity up to 10% (w/v) NaCl. Both extracellular and cell-bound amylase activity was detected among the isolates. The amylase activity of two isolates, related to B. vedderi and B. cohnii, was stimulated by ethylenediaminetetraacetic acid (EDTA) and inhibited in the presence of calcium ions. Pullulanase activity was expressed by isolates grouped with B. vedderi and also most of the isolates clustered with B. cohnii; cyclodextrin glycosyltransferase was expressed by most of the B. agaradhaerens-related strains. Minor levels of alpha-glucosidase activity were detected in all the strains.

Amylose↗

PubMed2006

A possible link between Crohn's disease and ankylosing spondylitis via Klebsiella infections.

Crohn's disease (CD) is an immune-mediated gastrointestinal inflammatory disease, which could arise from an interplay between genetic and environmental factors. Klebsiella microbes were suggested to have a vital role in the initiation and perpetuation of the disease through the mechanism of molecular mimicry. This proposition is based on the results of various studies where significantly elevated levels of antibodies against the whole bacteria or preparations from Klebsiella microbes and antibodies to collagen types I, III, IV, and V were detected in patients with CD and patients with ankylosing spondylitis (AS). Molecular similarities were found between Klebsiella nitrogenase and HLA-B27 genetic markers and between Klebsiella pullulanase and collagen fibers types I, III, and IV. Furthermore, significantly positive correlations and cross-reactivity binding activities were observed between anti-Klebsiella and anticollagen antibodies among patients with CD and AS. Early treatment of CD patients with anti-Klebsiella measures is proposed, which may involve the use of antibiotics and low starch diet together with other traditionally used immunomodulatory, immunosuppressive, or biologic agents.

Anti-Bacterial Agents↗