A note on starch hydrolysis and β-glucuronidase activity among flavobacteria

Most flavobacteria tested with the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide possessed β-glucuronidase (GUD), but when some of the same strains were tested with the API ZYM gallery, all were negative for GUD. Conflicting reports also appear in the literature about starch hydrolysis among flavobacteria. We observed that the results obtained can depend on the medium used and the length of incubation. Our results indicate that GUD activity and starch hydrolysis are more widely distributed in the genus Flavobacterium than previously reported.     

Confirmational identification of Escherichia coli, a comparison of genotypic and phenotypic assays for glutamate decarboxylase and beta-D-glucuronidase.

Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and beta-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E. coli, three major groups of diarrheagenic E. coli, three other non-coli Escherichia species, and various other gram-negative and -positive bacteria found in water. The genotypic assays were performed with hybridization probes generated by PCR amplification of 670- and 623-bp segments of the gadA/B (GAD) and uidA (GUD) genes, respectively. The GAD enzymes catalyze the alpha-decarboxylation of L-glutamic acid to yield gamma-aminobutyric acid and carbon dioxide, which are detected in the phenotypic assay by a pH-sensitive indicator dye. The phenotypic assay for GUD involves the transformation of 4-methylumbelliferyl-beta-D-glucuronide to the fluorogenic compound 4-methylumbelliferone. The GAD phenotypic assay detected the majority of the E. coli strains tested, whereas a number of these strains, including all representatives of the O157:H7 serotype and several nonpathogenic E. coli strains, gave negative results in the GUD assay. Both phenotypic assays detected some but not all strains from each of the four Shigella species. A strain of Citrobacter freundii was also detected by the GUD assay but not by the GAD assay. All E. coli and Shigella strains were detected with both the gadA/B and uidA probes. A few Escherichia fergusonii strains gave weak hybridization signals in response to both probes at 65 degrees C but not at 68 degrees C. None of the other bacterial species tested were detected by either probe. These results were consistent with previous reports which have indicated that the GAD phenotypic assay detects a wider range of E. coli strains than does the GUD assay and is also somewhat more specific for this species. The genotypic assays for the two enzymes were found to be equivalent in both of these respects and superior to both of the phenotypic assays in terms of the range of E. coli strains and isolates detected.     

双酶法水解辐照改性马铃薯淀粉动力学研究

为了解辐照改性马铃薯淀粉的酶解特性,用α-淀粉酶和糖化酶同时作用于马铃薯原淀粉和经400 kGy剂量辐照处理后淀粉,考察了pH值、酶解温度、α-淀粉酶用量、糖化酶用量对反应速率的影响.以米氏方程为基础,用Lineweaver-Burk法求解动力学参数.结果表明,辐照后马铃薯淀粉的酶解反应速率明显高于马铃薯原淀粉.在单一水解体系中,α-淀粉酶和糖化酶对辐照前后马铃薯淀粉的降解都遵循Michaelis-Menten方程,α-淀粉酶的Km分别为11.343 mg· mL-1和9.386 mg· mL-1,Vmax分别为0.406 mg（mL·min）-1和1.079 mg（mL·min）-1;糖化酶的Km分别为10.307 mg· mL-1和8.905 mg·mL-1,Vmax分别为0.338 mg（mL·min）-1和0.821mg（mL·min）-1;水解产物葡萄糖对反应体系具有竞争性抑制剂的作用,其抑制常数Ki分别为1.298 mg·mL-1和0.934 mg·mL-1.研究结果表明辐照有效提高了马铃薯淀粉的酶解反应活性.     

Photochemical characterization of flavobacterial rhodopsin: The importance of the helix E region for heat stability

Although many microbial rhodopsins have been discovered many of organisms in a variety of habitats, little is known about the property and diversity of rhodopsin in flavobacteria. Recent studies discovered that many proteorhodopsin (PR)-like proteins exist in genomes of flavobacteria. Following the isolation of a flavobacterial rhodopsins (FR) from the flavobacteria IMCC1997 from the East Sea of Korea, we characterized its photochemical features. We confirmed that the FR expression is induced by light in the IMCC1997 cell. Upon receiving light energy in vitro, the proton acceptor (D83) and donor (E94) of the FR translocate protons from intracellular to extracellular regions. Compared with proteorhodopsin (PR), the FR from IMCC 1997 cells is very unstable, which may be explained by their primary sequence differences. The ratio of all trans/13-cis retinal conformation does not influence this stability. To measure the stability of FR, we tested heat endurance at 70 °C and found that the heat endurance time of some FR mutants increased. Based upon these results, we found the helix E of this protein to be critical for the unstability of FR.     

采后芒果的后熟软化与贮藏淀粉变化之间的关系

研究了芒果后熟软化与贮藏淀粉变化之间的关系。结果表明,采后芒果淀粉的水解和消失,是使中果皮细胞壁失去支撑致使果实软化的重要原因之一。杀菌剂普克唑对芒果后熟软化和贮藏淀粉形态学的变化稍有延迟作用;2,4－D处理可以明显推迟后熟软化,减慢果实硬度的降低和淀粉的水解,而乙烯利处理则能催熟芒果,显著加速硬度丧失和淀粉的水解及其含量的减少。     

Enzyme-capture assay for rapid detection of Escherichia coli in oysters.

Enzyme-capture assays (ECAs) for Escherichia coli beta-D-glucuronidase (GUD) were performed directly from 24-h gas-positive lauryl tryptose broth (LTB) fermentation tubes that had been inoculated with oyster homogenate seeded with E. coli. The LTB-ECA method yielded results in 1 day that were equivalent to those obtained in 2 days by an LTB and EC-4-methylumbelliferyl-beta-D-glucuronide (EC-MUG) method. Overall, 62 of 64 (97%) positive EC-MUG broths from which E. coli was isolated were correctly identified by ECA. Of 61 LTB tubes identified as GUD negative by ECA, 59 were confirmed to be free of E. coli by using EC-MUG; thus, the false-negative rate was approximately 3%. Polyclonal antibodies prepared against E. coli GUD reacted only with GUDs of E. coli, Escherichia vulneris, and Shigella sonnei. The antibodies did not react with GUDs from Flavobacterium spp., Staphylococcus spp., Yersinia enterocolitica, shellfish, or bovine liver. The GUD ECA test, when used in conjunction with the most-probable-number technique, was a rapid method for E. coli enumeration in oysters.     

Microbial diversity of genital ulcer disease in men enrolled in a randomized trial of male circumcision in kisumu, kenya

Background

Medical male circumcision (MMC) reduces the risk of genital ulcer disease (GUD) in men by 50%. In Ugandan and Kenyan trials, a sexually transmissible agent was not identified in 50–60% of GUD specimens by polymerase chain reaction (PCR) assay. We sought to better define the etiology of GUD in men participating in the Kenyan trial and examine how MMC affects GUD etiology.

Methods

We defined GUD of unknown etiology as negative for HSV (type 1 and type 2), T. pallidum, and H. ducreyi by PCR, and negative for HSV-2 and T. pallidum by serology. We identified bacterial microbiota in a subset of 59 GUD specimens using multitag pyrosequencing of the 16S rRNA gene, and compared results by unknown vs. STI-associated etiology. Statistical analysis employed Bray-Curtis similarity measure of bacterial community by etiology, hierarchical clustering and logistic regression.

Results

In 59 GUD specimens from 59 men, 23 (39%) had unknown etiology. Bacterial diversity was greater in GUD of unknown than STI etiology (p = 0.01). Fusobacteria (Fusobacterium spp. and Sneathia spp.) were more commonly detected in men with GUD of unknown etiology [adjusted OR = 5.67; 95% CI: 1.63–19.8] as were Oxobacter spp. and Anaerovorax spp. [adjusted OR = 3.12; 95% CI: 0.83–11.7]. Sequences from these four anaerobic bacterial taxa were more often detected in uncircumcised men than circumcised men (p<0.05).

Conclusions

Anaerobic bacteria are more common in genital ulcers of uncircumcised men. The specific anaerobic bacteria associated with GUD of unknown etiology have cytotoxic properties that can exacerbate epithelial disruptions leading to ulcer-like appearance. MMC may reduce GUD through a reduction in these anaerobic bacteria.     

The enzymatic hydrolysis of starch-based PVOH and polyol plasticised blends

Thermoplastic starch (TPS) materials present several advantages to the plastic industry and when blended with other materials they can exhibit improved mechanical and moisture sensitivity properties compared to pure TPS materials. However, the biodegradability of these blends, through such processes as enzymatic degradation, needs to be characterised to ensure the beneficial properties of TPS are not compromised. The aims of the study were to investigate the effect of varying polyvinyl alcohol (PVOH) content and polyol type within the TPS blends on the rate and extent of starch enzymatic hydrolysis using enzymes α-amylase and amyloglucosidase. The results of this study have revealed that TPS:PVOH blends with a PVOH content at 50 wt% exhibited a significantly reduced rate and extent of starch hydrolysis. The results suggest that this may have been attributed to interactions between starch and PVOH that further prevented enzymatic attack on the remaining starch phases within the blend. The extent of starch hydrolysis was not significantly affected by polyol type, however, the rate of starch hydrolysis from the maltitol blend was significantly reduced compared to sorbitol and glycerol substrates.     

Hydrolysis of starches by the action of an α-amylase from Bacillus subtilis

A moderately thermophilic Bacillus subtilis strain, isolated from fresh sheep’s milk, produced extracellular thermostable α-amylase. Maximum amylase production was obtained at 40 °C in a medium containing low starch concentrations. The enzyme displayed maximal activity at 135 °C and pH 6.5 and its thermostability was enhanced in the presence of either calcium or starch. This thermostable α-amylase was used for the hydrolysis of various starches. An ammonium sulphate crude enzyme preparation as well as the cell-free supernatant efficiently degraded the starches tested. The use of the clear supernatant as enzyme source is highly advantageous mainly because it decreases the cost of the hydrolysis. Upon increase of reaction temperature to 70 °C, all substrates exhibited higher hydrolysis rates. Potato starch hydrolysis resulted in a higher yield of reducing sugars in comparison to the other starches at all temperatures tested. Soluble and rice starch took, respectively, the second and third position regarding reducing sugars liberation, while the α-amylase studied showed slightly lower affinity for corn starch and oat starch.     

Enzyme-capture assay for rapid detection of Escherichia coli in oysters

Enzyme-capture assays (ECAs) for Escherichia coli beta-D-glucuronidase (GUD) were performed directly from 24-h gas-positive lauryl tryptose broth (LTB) fermentation tubes that had been inoculated with oyster homogenate seeded with E. coli. The LTB-ECA method yielded results in 1 day that were equivalent to those obtained in 2 days by an LTB and EC-4-methylumbelliferyl-beta-D-glucuronide (EC-MUG) method. Overall, 62 of 64 (97%) positive EC-MUG broths from which E. coli was isolated were correctly identified by ECA. Of 61 LTB tubes identified as GUD negative by ECA, 59 were confirmed to be free of E. coli by using EC-MUG; thus, the false-negative rate was approximately 3%. Polyclonal antibodies prepared against E. coli GUD reacted only with GUDs of E. coli, Escherichia vulneris, and Shigella sonnei. The antibodies did not react with GUDs from Flavobacterium spp., Staphylococcus spp., Yersinia enterocolitica, shellfish, or bovine liver. The GUD ECA test, when used in conjunction with the most-probable-number technique, was a rapid method for E. coli enumeration in oysters.     

σ-Amylase inactivation during corn starch hydrolysis process

The effects of operating conditions on the enzymatic hydrolysis of corn starch were investigated. A commercial α-amylase produced by Bacillus sp. was used for the hydrolysis experiments. The degree of starch hydrolysis (%) and residual α-amylase activity (%) was investigated versus process variables, including pH, temperature, viscosity, impeller speed, processing time and some materials added such as hydrolysate, maltose, glucose, ethanol and CaCl₂ using a stirred batch reactor. The mathematical models depending on the operating conditions were also derived using the experimental data of residual starch concentration. Some inactivation models were tested to determine the relationship between process variables and enzyme stability during the hydrolysis process.     

Complete DNA sequence analysis of enterohemorrhagic Escherichia coli plasmid pO157_2 in β-glucuronidase-positive E. coli O157:H7 reveals a novel evolutionary path

Strains of enterohemorragic Escherichia coli (EHEC) O157:H7 that are non-sorbitol fermenting (NSF) and β-glucuronidase negative (GUD(-)) carry a large virulence plasmid, pO157 (>90,000 bp), whereas closely related sorbitol-fermenting (SF) E. coli O157:H(-) strains carry plasmid pSFO157 (>120,000 bp). GUD(+) NSF O157:H7 strains are presumed to be precursors of GUD(-) NSF O157:H7 strains that also carry pO157. In this study, we report the complete sequence of a novel virulence plasmid, pO157-2 (89,762 bp), isolated from GUD(+) NSF O157:H7 strain G5101. PCR analysis confirmed the presence of pO157-2 in six other strains of GUD(+) NSF O157:H7. pO157-2 carries genes associated with virulence (e.g., hemolysin genes) and conjugation (tra and trb genes) but lacks katP and espP present in pO157. Comparative analysis of the three EHEC plasmids shows that pO157-2 is highly related to pO157 and pSFO157 but not ancestral to pO157. These results indicated that GUD(+) NSF O157:H7 strains might not be direct precursors to GUD(-) NSF O157:H7 as previously proposed but rather have evolved independently from a common ancestor.     

A quantitative assessment of the importance of barley seed alpha-amylase, beta-amylase, debranching enzyme, and alpha-glucosidase in starch degradation.

Extracts of germinated barley (Hordeum vulgare L.) seeds of 41 different genotypes were analyzed for their activities of alpha-amylase, beta-amylase, alpha-glucosidase, and debranching enzyme and for their abilities to hydrolyze boiled soluble starch, nonboiled soluble starch, and starch granules extracted from barley seeds with water. Linear correlation analysis, used to quantitate the interactions between the seven parameters, revealed that boiled soluble starch was not a good substrate for predicting activities of enzymes functioning in in vivo starch hydrolysis as the extracts' abilities to hydrolyze boiled soluble starch was not correlated with their abilities to hydrolyze native starch granules. Activities of alpha-amylase and alpha-glucosidase were positively and significantly correlated with the seed extracts' abilities to hydrolyze all three starches. beta-Amylase was only significantly correlated with hydrolysis of boiled soluble starch. No significant correlations existed between debranching enzyme activity and hydrolysis of any of the three starches. Interactions between the four enzymes as they functioned together to hydrolyze the three types of starch were evaluated by path coefficient analysis. alpha-Amylase contributed to hydrolyses of all three starches primarily by its direct effect (noninteractive component). This direct contribution increased as the substrate progressed from the completely artificial boiled soluble starch, to the most physiologically significant substrate, native starch granules. alpha-Glucosidase contributed to the hydrolysis of boiled soluble starch primarily by its direct effect (noninteractive) yet contributed to starch granule hydrolysis primarily via its interaction with alpha-amylase (indirect effect). The contribution of beta-amylase to hydrolysis of boiled soluble starch was direct and it did not contribute significantly to hydrolysis of native starch granules.     

Studies on enzymatic continuous production of cyclodextrins in an ultrafiltration membrane bioreactor

Studies on simultaneous hydrolysis of starch and synthesis of cyclodextrins by Thermo-aerobacter cyclodextrin glucosyltransferase were conducted in an ultrafiltration membrane bioreactor, allowing enzyme recovery and reduction of product inhibition. The influence of various reaction parameters like starch concentration, enzyme dosage and residence time on cyclodextrin composition was tested. A comparison of batch and continuous cyclodextrin production indicates that employing an ultrafiltration membrane bioreactor increases process efficiency.     

Fluorogenic selective and differential medium for isolation of fecal streptococci

Of 44 fluorogenic substrates tested for their ability to differentiate species of fecal streptococci, four yielded species-differentiating reactions. The remaining substrates either yielded uniformly positive, negative, or variable strain-dependent reactions. One substrate, 4-methylumbelliferone-alpha-D-galactoside, was hydrolyzed by Streptococcus bovis and S. faecium and its biotypes. 4-Methylumbelliferone-alpha-D-galactoside and a colorimetric starch substrate were incorporated into the fecal streptococcal selective medium of Donnelly and Hartman (Appl. Environ. Microbiol. 35:576-581, 1978). Three phenotypic groups were identifiable on the new fluorescent gentamicin-thallous-carbonate agar: (i) starch hydrolysis and fluorescence (S. bovis), (ii) no starch hydrolysis but fluorescence (S. faecium and its biotypes), and (iii) no starch hydrolysis or fluorescence (S. faecalis, S. avium, S. equinus, S. mitis, and S. salivarius). Of the presumptive identifications from sewage, swine, and bovine samples, 86% were confirmed as being correct. The new medium has potential application in water, food, environmental, and possibly clinical microbiology.     

Fluorogenic selective and differential medium for isolation of fecal streptococci.

Of 44 fluorogenic substrates tested for their ability to differentiate species of fecal streptococci, four yielded species-differentiating reactions. The remaining substrates either yielded uniformly positive, negative, or variable strain-dependent reactions. One substrate, 4-methylumbelliferone-alpha-D-galactoside, was hydrolyzed by Streptococcus bovis and S. faecium and its biotypes. 4-Methylumbelliferone-alpha-D-galactoside and a colorimetric starch substrate were incorporated into the fecal streptococcal selective medium of Donnelly and Hartman (Appl. Environ. Microbiol. 35:576-581, 1978). Three phenotypic groups were identifiable on the new fluorescent gentamicin-thallous-carbonate agar: (i) starch hydrolysis and fluorescence (S. bovis), (ii) no starch hydrolysis but fluorescence (S. faecium and its biotypes), and (iii) no starch hydrolysis or fluorescence (S. faecalis, S. avium, S. equinus, S. mitis, and S. salivarius). Of the presumptive identifications from sewage, swine, and bovine samples, 86% were confirmed as being correct. The new medium has potential application in water, food, environmental, and possibly clinical microbiology.     

The raw starch-degrading alkaline amylase ofBacillus sp IMD 370

The amylase ofBacillus sp IMD 370 is the first report of an alkaline amylase with the ability to digest raw starch. The amylase could degrade raw corn and rice starches more effectively than raw potato starch. It showed no adsorb-ability to any type of raw starch at any pH value tested. The enzyme digested raw corn starch to glucose, maltose, maltotriose and maltotetraose. The maximum pH for raw starch hydrolysis was pH 8.0 compared to pH 10.0 for soluble starch hydrolysis. The metal chelator, ethylenediaminetetraacetic acid, strongly inhibited raw starch-digestion and its effect was reversed by the addition of divalent cations. Degradation of raw starch was stimulated six-fold in the presence of -cyclodextrin (17.5 mM).     

Starch-binding domain shuffling in Aspergillus niger glucoamylase

Aspergillus niger glucoamylase (GA) consists mainly of two forms, GAI [from the N-terminus, catalytic domain + linker + starch-binding domain (SBD)] and GAII (catalytic domain + linker). These domains were shuffled to make RGAI (SBD + linker + catalytic domain), RGAIDeltaL (SBD + catalytic domain) and RGAII (linker + catalytic domain), with domains defined by function rather than by tertiary structure. In addition, Paenibacillus macerans cyclomaltodextrin glucanotransferase SBD replaced the closely related A.niger GA SBD to give GAE. Soluble starch hydrolysis rates decreased as RGAII approximately GAII approximately GAI > RGAIDeltaL approximately RGAI approximately GAE. Insoluble starch hydrolysis rates were GAI > RGAIDeltaL > RGAI > GAE approximately RGAII > GAII, while insoluble starch-binding capacities were GAI > RGAI > RGAIDeltaL > RGAII > GAII > GAE. These results indicate that: (i) moving the SBD to the N-terminus or replacing the native SBD somewhat affects soluble starch hydrolysis; (ii) SBD location significantly affects insoluble starch binding and hydrolysis; (iii) insoluble starch hydrolysis is imperfectly correlated with its binding by the SBD; and (iv) placing the P.macerans cyclomaltodextrin glucanotransferase SBD at the end of a linker, instead of closely associated with the rest of the enzyme, severely reduces its ability to bind and hydrolyze insoluble starch.     

Amylase partitioning and extractive bioconversion of starch using thermoseparating aqueous two-phase systems.

The effectiveness of thermoseparating polymer-based aqueous two-phase systems (ATPS) in the enzymatic hydrolysis of starch was investigated. In this work, the phase diagrams of PEO-PPO-2500/ammonium sulfate and PEO-PPO-2500/magnesium sulfate systems were determined at 25 degrees C. The partition behavior of pure alpha-amylase and amyloglucosidase in four ATPS, namely, PEO-PPO/(NH(4))(2)SO(4), PEO-PPO/MgSO(4), polyethylene glycol (PEG)/(NH(4))(2)SO(4), and PEG/MgSO(4), was evaluated. The effects of phase-forming component concentrations on the enzyme activity and partitioning were assessed. Partitioning of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii was also investigated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. The PEO-PPO-2500/MgSO(4) system was extremely attractive for starch hydrolysis. Polymer-based starch hydrolysis experiments containing PEO-PPO-2500/MgSO(4) indicated that the use of ATPS had a significant effect on soluble starch hydrolysis. Batch starch hydrolysis experiments with PEO-PPO/salt two-phase systems resulted in higher production of maltose or glucose and exhibited remarkably faster hydrolysis. A 22% gain in maltose yield was obtained as a result of the increased productivity. This work is the first reported application of thermoseparating polymer ATPS in the processing of starches. These results reveal the potential for thermoseparating polymer-enhanced extractive bioconversion of starch as a practical technology.     

Process development of starch hydrolysis using mixing characteristics of Taylor vortices

In food industries, enzymatic starch hydrolysis is an important process that consists of two steps: gelatinization and saccharification. One of the major difficulties in designing the starch hydrolysis process is the sharp change in its rheological properties. In this study, Taylor–Couette flow reactor was applied to continuous starch hydrolysis process. The concentration of reducing sugar produced via enzymatic hydrolysis was evaluated by varying operational variables: rotational speed of the inner cylinder, axial velocity (reaction time), amount of enzyme, and initial starch content in the slurry. When Taylor vortices were formed in the annular space, efficient hydrolysis occurred because Taylor vortices improved the mixing of gelatinized starch with enzyme. Furthermore, a modified inner cylinder was proposed, and its mixing performance was numerically investigated. The modified inner cylinder showed higher potential for enhanced mixing of gelatinized starch and the enzyme than the conventional cylinder.