Identification and characterization of a novel KG42 xylanase (GH10 family) isolated from the black goat rumen-derived metagenomic library.

This study was conducted to isolate and functionally characterize a novel xylan-degrading enzyme from the microbial metagenomes of black goat rumens. A novel gene, KG42, was isolated from one of the 17 xylan-degrading metagenomic fosmid clones obtained from black goat rumens. The KG42 gene, comprising a 1107 bp open reading frame, encodes a protein composed of 368 amino acids (41 kDa) with a glycosyl hydrolase family 10 (GH10) domain, consisting of a "salad-bowl" shaped tertiary structure (a typical 8-fold α/ß barrel (α/ß)8) and two catalytic residues. KG42 xylanase protein has at best 40% sequence identity with other homologous GH10 xylanase proteins. The enzyme displayed its optimum activity at pH 5.0 and 50 °C. The enzyme was thermally stable at pH and temperature ranges of 5.0-10.0 and 20-60 °C, respectively. Substrate specificity and hydrolytic patterns implied that the KG42 xylanase functions as an endo-ß-1,4-xylanase (EC 3.2.1.8). The KG42 xylanase was also used for the preparation of bifidogenic xylan hydrolysates, demonstrating its potential applications toward preparing prebiotic xylooligosaccharides.

Metagenomic Mining and Functional Characterization of a Novel KG51 Bifunctional Cellulase/Hemicellulase from Black Goat Rumen.

A novel KG51 gene was isolated from a metagenomic library of Korean black goat rumen and its recombinant protein was characterized as a bifunctional enzyme (cellulase/hemicellulase). In silico sequence and domain analyses revealed that the KG51 gene encodes a novel carbohydrate-active enzyme that possesses a salad-bowl-like shaped glycosyl hydrolase family 5 (GH5) catalytic domain but, at best, 41% sequence identity with other homologous GH5 proteins. Enzymatic profiles (optimum pH values and temperatures, as well as pH and thermal stabilities) of the recombinant KG51 bifunctional enzyme were also determined. On the basis of the substrate specificity data, the KG51 enzyme exhibited relatively strong cellulase (endo-ß-1,4-glucanase [EC 3.2.1.4]) and hemicellulase (mannan endo-ß-1,4-mannosidase [EC 3.2.1.78] and endo-ß-1,4-xylanase [EC 3.2.1.8]) activities, but no exo-ß-1,4-glucanase (EC 3.2.1.74), exo-ß-1,4-glucan cellobiohydrolase (EC 3.2.1.91), and exo-1,4-ß-xylosidase (EC 3.2.1.37) activities. Finally, the potential industrial applicability of the KG51 enzyme was tested in the preparation of prebiotic konjac glucomannan hydrolysates.

Isolation and characterization of a novel endo-beta-1, 4-glucanase from a metagenomic library of the black-goat rumen

ABSTRACT The various types of lignocellulosic biomass found in plants comprise the most abundant renewable bioresources on Earth. In this study, the ruminal microbial ecosystem of black goats was explored because of their strong ability to digest lignocellulosic forage. A metagenomic fosmid library containing 115,200 clones was prepared from the black-goat rumen and screened for a novel cellulolytic enzyme. The KG35 gene, containing a novel glycosyl hydrolase family 5 cellulase domain, was isolated and functionally characterized. The novel glycosyl hydrolase family 5 cellulase gene is composed of a 963-bp open reading frame encoding a protein of 320 amino acid residues (35.1 kDa). The deduced amino acid sequence showed the highest sequence identity (58%) for sequences from the glycosyl hydrolase family 5 cellulases. The novel glycosyl hydrolase family 5 cellulase gene was overexpressed in Escherichia coli. Substrate specificity analysis revealed that this recombinant glycosyl hydrolase family 5 cellulase functions as an endo-β-1,4-glucanase. The recombinant KG35 endo-β-1,4-glucanase showed optimal activity within the range of 30-50 °C at a pH of 6-7. The thermostability was retained and the pH was stable in the range of 30-50 °C at a pH of 5-7.

Isolation and characterization of a novel endo-ß-1,4-glucanase from a metagenomic library of the black-goat rumen.

The various types of lignocellulosic biomass found in plants comprise the most abundant renewable bioresources on Earth. In this study, the ruminal microbial ecosystem of black goats was explored because of their strong ability to digest lignocellulosic forage. A metagenomic fosmid library containing 115,200 clones was prepared from the black-goat rumen and screened for a novel cellulolytic enzyme. The KG35 gene, containing a novel glycosyl hydrolase family 5 cellulase domain, was isolated and functionally characterized. The novel glycosyl hydrolase family 5 cellulase gene is composed of a 963-bp open reading frame encoding a protein of 320 amino acid residues (35.1kDa). The deduced amino acid sequence showed the highest sequence identity (58%) for sequences from the glycosyl hydrolase family 5 cellulases. The novel glycosyl hydrolase family 5 cellulase gene was overexpressed in Escherichia coli. Substrate specificity analysis revealed that this recombinant glycosyl hydrolase family 5 cellulase functions as an endo-ß-1,4-glucanase. The recombinant KG35 endo-ß-1,4-glucanase showed optimal activity within the range of 30-50°C at a pH of 6-7. The thermostability was retained and the pH was stable in the range of 30-50°C at a pH of 5-7.

Functional Applications of Lignocellulolytic Enzymes in the Fruit and Vegetable Processing Industries.

Cellulose, hemicellulose, pectin (carbohydrate), and lignin (noncarbohydrate) polymers are the main substrates of lignocellulose-degrading enzymes. They are present in large amounts in the primary cell wall and dietary fibers of major fruits and vegetables. During processing of fruits and vegetables to the corresponding final food products, lignocellulosic substrates are hydrolyzed by different lignocellulolytic enzymes. Currently, lignocellulolytic enzymes such as cellulases, xylanases, pectinases, and laccases are extensively used during the processing of fruits and vegetables, in applications like texturizing and flavoring of products in the food industries. The present article provides an updated overview of functional applications of lignocellulolytic enzymes in the juice processing, oil extraction, and alcoholic beverage processing industries. Extensive use of lignocellulolytic enzymes in different food processing industries not only accelerates the production rates but also improves product quality. It is also possible to ensure the efficient use of fruits and vegetables globally by employing lignocellulolytic enzymes in the corresponding processing industries to convert them into food commodities, which will not only raise their economic value in the global market but also increase food availability, which will help mitigate nutritional problems worldwide.

Isolation and characterization of a novel glycosyl hydrolase family 74 (GH74) cellulase from the black goat rumen metagenomic library.

This study aimed to isolate and characterize a novel cellulolytic enzyme from black goat rumen by using a culture-independent approach. A metagenomic fosmid library was constructed from black goat rumen contents and screened for a novel cellulase. The KG37 gene encoding a protein of 858 amino acid residues (92.7 kDa) was isolated. The deduced protein contained a glycosyl hydrolase family 74 (GH74) domain and showed 77% sequence identity to two endo-1,4-ß-glucanases from Fibrobacter succinogenes. The novel GH74 cellulase gene was overexpressed in Escherichia coli, and its protein product was functionally characterized. The recombinant GH74 cellulase showed a broad substrate spectrum. The enzyme exhibited its optimum activity at pH 5.0 and temperature range of 20-50 °C. The enzyme was thermally stable at pH 5.0 and at a temperature of 20-40 °C. The novel GH74 cellulase can be practically exploited to convert lignocellulosic biomass to value-added products in various industrial applications in future.

Isolation and Characterization of Spore-Forming Bacilli (SFB) from Shepherd's Purse (Capsella bursa-pastoris).

Shepherd's purse (Capsella bursa-pastoris), native to Europe, is commonly consumed fresh and sometimes inadequately washed before consumption in Korea. The objective of this study was to characterize isolates of spore-forming bacilli (SFB) in samples of fresh Shepherd's purse. Three genera were identified: Bacillus (9 species), Paenibacillus (3 species), and Brevibacillus (1 species). None of the genes of the hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE) complexes, or of the emetic toxin, was detected in the 25 SFB isolates, except for 2 Bacillus pseudomycoides isolates, where all 3 genes of the HBL enterotoxin complex were detected. There were significant sequence variations between the 2 species (Bacillus cereus and B. pseudomycoides) in the 3 genes of the HBL enterotoxin complex. These findings may provide insights into the diverse characteristics of the B. pseudomycoides HBL enterotoxin complex. Antibiotic resistance was assessed using 8 antibiotics. Among the 25 SFB isolates, 11 showed resistance to antibiotics, of which 5 were multiresistant. Assessment of the spoilage potential showed that all 25 SFB isolates could produce enzymes that can cause spoilage of foods. In conclusion, our findings may serve as integrative information for food research and industrial sectors.

Various Enterotoxin and Other Virulence Factor Genes Widespread Among Bacillus cereus and Bacillus thuringiensis Strains.

Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen Bacillus thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal diseases are attributed to enterotoxins. Sixteen reference strains of B. cereus and nine commercial and 12 reference strains of B. thuringiensis were screened by PCR for the presence of 10 enterotoxigenic genes (hblA, hblC, hblD, nheA, nheB, nheC, cytK, bceT, entFM, and entS), one emetogenic gene (ces), seven hemolytic genes (hlyA, hlyII, hlyIII, plcA, cerA, cerB, and cerO), and a pleiotropic transcriptional activator gene (plcR). These genes encode various enterotoxins and other virulence factors thought to play a role in infections of mammals. Amplicons were successfully generated from the strains of B. cereus and B. thuringiensis for each of these sequences, except the ces gene. Intriguingly, the majority of these B. cereus enterotoxin genes and other virulence factor genes appeared to be widespread among B. thuringiensis strains as well as B. cereus strains.

Production of volatile phenols by kimchi Lactobacillus plantarum isolates and factors influencing their phenolic acid decarboxylase gene expression profiles.

Potential of kimchi lactic acid bacteria (LAB) isolates to produce volatile phenols and factors affecting their phenolic acid decarboxylase (padA) gene expression profiles were investigated in this study. Twelve percent (12%) of 50 tested LAB isolates were found to decarboxylate hydroxycinnamic acids. All six isolates were identified as Lactobacillus plantarum and possessed the padA gene. The highest padA expression was achieved on the third day of incubation with ferulic acid, with a relative expression of 3.30±0.32. The effects of glucose, substrate, and product concentrations, and the pH of the medium were investigated using response surface methodology for the first time in this study. The expression profiles of the padA gene were diverse in various stress environments. The concentration of p-coumaric acid was the most significant factor being positively correlated with the expression levels of the padA gene, but other factors did not show any significant effects. High concentrations of substrates could confer antibacterial activity. Therefore, decarboxylation reaction was suggested as a bacterial response to overcome the antibacterial activity. The phenolic acid decarboxylase activities of L. plantarum isolates found in this study can provide insights for their potential application in the development of food-grade flavors and additives.

Utilization of barley or wheat bran to bioconvert glutamate to γ-aminobutyric acid (GABA).

This study deals with the utilization of agro-industrial wastes created by barley and wheat bran in the production of a value-added product, γ-aminobutyric acid (GABA). The simple and eco-friendly reaction requires no pretreatment or microbial fermentation steps but uses barley or wheat bran as an enzyme source, glutamate as a substrate, and pyridoxal 5'-phosphate (PLP) as a cofactor. The optimal reaction conditions were determined on the basis of the temperatures and times used for the decarboxylation reactions and the initial concentrations of barley or wheat bran, glutamate, and PLP. The optimal reactions produced 9.2 mM of GABA from 10 mM glutamate, yielding a 92% GABA conversion rate, when barley bran was used and 6.0 mM of GABA from 10 mM glutamate, yielding a 60% GABA conversion rate, when wheat bran was used. The results imply that barley bran is more efficient than wheat bran in the production of GABA.

Single nucleotide polymorphisms are randomly dispersed and mostly synonymous in partial rpoB and cpn60 genes of Campylobacter showae human isolates.

The partial 16S rRNA, rpoB, and cpn60 genes congruently allow this study to identify all the eight isolates as the species Campylobacter showae. To our knowledge, this is the first report to reveal the interspecies and intraspecies sequence variations present in the three genes of the C. showae isolates.

Characterization of Slackia exigua isolated from human wound infections, including abscesses of intestinal origin.

Eleven clinical strains isolated from infected wound specimens were subjected to polyphasic taxonomic analysis. Sequence analysis of the 16S rRNA gene showed that all 11 strains were phylogenetically related to Slackia exigua. Additionally, conventional and biochemical tests of 6 of the 11 strains were performed as supplementary methods to obtain phenotypic identification by comparison with the phenotypes of the relevant type strains. S. exigua has been considered an oral bacterial species in the family Coriobacteriaceae. This organism is fastidious and grows poorly, so it may easily be overlooked. The 16S rRNA gene sequences and the biochemical characteristics of four of the S. exigua strains isolated for this study from various infections indicative of an intestinal source were almost identical to those of the validated S. exigua type strain from an oral source and two of the S. exigua strains from oral sources evaluated in this study. Thus, we show for the first time that S. exigua species can be isolated from extraoral infections as well as from oral infections. The profiles of susceptibility to selected antimicrobials of this species were also investigated for the first time.

Species-specific detection of Listeria monocytogenes using polymerase chain reaction assays targeting the prfA virulence gene cluster.

Sequence comparison of individual genes in the prfA virulence gene cluster (pVGC), a central virulence gene cluster, from different Listeria species indicated that priming sites within the genes appeared to be specific for Listeria monocytogenes exclusively. Therefore, the pVGC was targeted for polymerase chain reaction (PCR) assays to detect and specifically identify L. monocytogenes. Each gene of the pVGC was specifically amplified in L. monocytogenes but not in other Listeria species.

Enhanced rapidity for qualitative detection of Listeria monocytogenes using an enzyme-linked immunosorbent assay and immunochromatography strip test combined with immunomagnetic bead separation.

An enzyme-linked immunosorbent assay (ELISA), immunochromatography (ICG) strip test, and immunomagnetic bead separation (IMBS) system based on a monoclonal antibody were individually developed for the detection and isolation of Listeria monocytogenes in meat samples. The three methods showed a strong reaction with Listeria species and a weak reaction with Staphylococcus aureus. To increase the rapidity of L. monocytogenes detection, combinations of the ELISA and ICG strip test with the IMBS system (ELISA-IMBS and ICG-IMBS) were investigated. In comparative analyses of artificially inoculated meat and samples of processed meat, the ELISA and ICG strip test required 24 h of enrichment time to detect the inoculated meat samples with > or =1 X 10(2) CFU/10 g, whereas the ELISA-IMBS and ICG-IMBS required only 14 h of enrichment. Analyses of naturally contaminated meat samples (30 pork samples, 20 beef samples, 26 chicken samples, 20 fish samples, and 20 processed meat samples) performed by ELISA-IMBS, ICG-IMBS, and API kit produced similar results. The ELISA-IMBS and ICG-IMBS provide a more rapid assay than the individual ELISA and the ICG strip test and are appropriate for rapid and qualitative detection of L. monocytogenes (or Listeria species) in meat samples. With the ICG-IMBS, L. monocytogenes could be detected in meat samples within 15 h and the method has potential as a rapid, cost-effective on-site screening tool for the detection of L. monocytogenes in food samples and agricultural products at a minimum detection level of approximately 100 CFU/10 g.

Development of predictive mathematical model for the growth kinetics of Staphylococcus aureus by response surface model.

A response surface model was developed for predicting the growth rates of Staphylococcus aureus in tryptic soy broth (TSB) medium as a function of combined effects of temperature, pH, and NaCl. The TSB containing six different concentrations of NaCl (0, 2, 4, 6, 8, and 10%) was adjusted to an initial of six different pH levels (pH 4, 5, 6, 7, 8, 9, and 10) and incubated at 10, 20, 30, and 40 degrees C. In all experimental variables, the primary growth curves were well (r2=0.9000 to 0.9975) fitted to a Gompertz equation to obtain growth rates. The secondary response surface model for natural logarithm transformations of growth rates as a function of combined effects of temperature, pH, and NaCl was obtained by SAS's general linear analysis. The predicted growth rates of the S. aureus were generally decreased by basic (pH 9-10) or acidic (pH 5-6) conditions and higher NaCl concentrations. The response surface model was identified as an appropriate secondary model for growth rates on the basis of correlation coefficient (r=0.9703), determination coefficient (r2=0.9415), mean square error (MSE=0.0185), bias factor (B(f)=1.0216), and accuracy factor (A(f)=1.2583). Therefore, the developed secondary model proved reliable for predictions of the combined effect of temperature, NaCl, and pH on growth rates for S. aureus in TSB medium.

Production and characterization of monoclonal and recombinant antibodies against antimicrobial sulfamethazine.

A monoclonal antibody (mab) against the antimicrobial sulfamethazine was prepared and characterized by an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA). Sulfamethazine in the range of 0.2 and 45 ng/ml could be determined with the mab by IC-ELISA. cDNAs encoding a variable heavy chain and variable light chain of the mab were cloned to produce recombinant antibodies using phage display technology. Following phage rescue and three rounds of panning, a single-chain variable fragment (scFv) antibody with high sulfamethazine-binding affinity was obtained. ELISA analysis revealed that scFv antibody and parent mab showed similar, but not identical, characteristics. The IC50 value by IC-ELISA with scFv antibody was 4.8 ng/ml, compared with 1.6 ng/ml with the parent mab. Performances of the assays in the presence of milk matrix were compared; the mab-based assay was less affected than the scFv-based assay. Sixty milk samples were analyzed by mab-based IC-ELISA, and four samples were sulfamethazine positive; these results were favorably correlated with those obtained by HPLC.

Detection of methicillin resistance in Staphylococcus aureus isolates using two-step triplex PCR and conventional methods.

A two-step triplex PCR assay targeting the mecA, femA, and nuc genes was developed for the detection of methicillin resistance genes harbored by some Staphylococcus aureus isolates and for the simultaneous identification of such isolates at the species level. The triplex PCR revealed the presence of the femA and nuc genes in all the S. aureus isolates examined (n=105). Forty-four clinical isolates were mecA positive and no foodborne isolates were mecA positive. The PCR results had a 98 or 99% correlation with the results of PBP2a latex agglutination tests or oxacillin susceptibility tests, respectively.

Production of monoclonal antibody against Listeria monocytogenes and its application to immunochromatography strip test.

An immunochromatography (ICG) strip test based on a monoclonal antibody for the rapid detection of L. monocytogenes in meat and processed-meat samples was developed in this study. A monoclonal antibody (MAb) specific to L. monocytogenes was produced from cloned hybridoma cells (FKLM-3B12-37) and used to develop an ICG strip test. The antibody showed a stronger binding to L. monocytogenes than other Listeria species, and a weak cross-reaction to S. aureus based on an ELISA. The detection limit of the ICG strip test was 10(5) cell/ml. In total, 116 meat and processed-meat samples were collected and analyzed using both the ICG strip test and a PCR. The ICG strip test and PCR indicated L. monocytogenes contamination in 34 and 27 meat samples, respectively. The 7 meat samples not identified as L. monocytogenes positive by the PCR were also tested using an API kit and found to be contaminated by Listeria species. In conclusion, the ICG strip test results agreed well with those obtained using the PCR and API kit. Thus, the developed ICG has potential use as a primary screening tool for L. monocytogenes in various foods and agricultural products, generating results within 20 min without complicated steps.

Morphology and adhesion of Campylobacter jejuni to chicken skin under varying conditions.

The adhesion of Campylobacter jejuni to chicken skin, along with the associated morphological changes under aerobic conditions at 4, 25, and 37 degrees C and microaerobic (O2 5%, CO2 10%, N2 85%) conditions, were investigated using confocal laser scanning microscopy (CLSM), flow cytometry, and plate counting. The morphological change of C. jejuni from a spiral shape to a coccoid form or VBNC form (viable but nonculturable form) progressed rapidly under aerobic conditions at 25, 37, and 4 degrees C. As regards adhesion, the C. jejuni cells were mostly located in the crevices and feather follicles of the chicken skin, where the cells in the feather follicles floated freely in the entrapped water, even after the skin was rinsed quite thoroughly. CLSM also revealed the penetration of some spiral-shaped C. jejuni cells into the chicken skin. Even after changing their shape at various temperatures, coccoid-form C. jejuni cells were still found in the crevices and feather follicles of the chicken skin.

Molecular cloning and characterization of a large subunit of Salmonella typhimurium glutamate synthase (GOGAT) gene in Escherichia coli.

Two pathways of ammonium assimilation and glutamate biosynthesis have been identified in microorganisms. One pathway involves the NADP-linked glutamate dehydrogenase, which catalyzes the amination of 2-oxoglutarate to form glutamate. An alternative pathway involves the combined activities of glutamine synthetase, which aminates glutamate to form glutamine, and glutamate synthase, which transfers the amide group of glutamine to 2-oxoglutarate to yield two molecules of glutamate. We have cloned the large subunit of the glutamate synthase (GOGAT) from Salmonella typhimurium by screening the expression of GOGAT and complementing the gene in E. coli GOGAT large subunit-deficient mutants. Three positive clones (named pUC19C12, pUC19C13 and pUC19C15) contained identical Sau3AI fragments, as determined by restriction mapping and Southern hybridization, and expressed GOGAT efficiently and constitutively using its own promoter in the heterologous host. The coding region expressed in Escherichia coli was about 170 kDa on SDS-PAGE. This gene spans 4,732 bases, contains an open reading frame of 4,458 nucleotides, and encodes a mature protein of 1,486 amino acid residues (Mr = 166,208). The FMN-binding domain of GOGAT contains 12 glycine residues, and the 3Fe-4S cluster has 3 cysteine residues. The comparison of the translated amino acid sequence of the Salmonella GOGAT with sequences from other bacteria such as Escherichia coli, Salmonella enterica, Shigella flexneri, Yersinia pestis, Vibrio vulnificus and Pseudomonas aeruginosa shows sequence identity between 87 and 95%.