• Journal of Microbiology and Biotechnology
• /
• v.18 no.3
• /
• pp.410-416
• /
• 2008

The gene SfXyn10, which encodes a protease-resistant xylanase, was isolated using colony PCR screening from a genomic library of a feather-degrading bacterial strain Streptomyces fradiae var. k11. The full-length gene consists of 1,437bp and encodes 479 amino acids, which includes 41 residues of a putative signal peptide at its N terminus. The amino acid sequence shares the highest similarity (80%) to the endo-1,4-${\beta}$-xylanase from Streptomyces coelicolor A3, which belongs to the glycoside hydrolase family 10. The gene fragment encoding the mature xylanase was expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to homogeneity by acetone precipitation and anion-exchange chromatography, and subsequently characterized. The optimal pH and temperature for the purified recombinant enzyme were 7.8 and $60^{\circ}C$, respectively. The enzyme showed stability over a pH range of 4.0-10.0. The kinetic values on oat spelt xylan and birchwood xylan substrates were also determined. The enzyme activity was enhanced by $Fe^{2+}$ and strongly inhibited by $Hg^{2+}$ and SDS. The enzyme also showed resistance to neutral and alkaline proteases. Therefore, these characteristics suggest that SfXyn10 could be an important candidate for protease-resistant mechanistic research and has potential applications in the food industry, cotton scouring, and improving animal nutrition.

• 한국생물공학회:학술대회논문집
• /
• 2005.10a
• /
• pp.642-643
• /
• 2005

• Journal of Microbiology and Biotechnology
• /
• v.21 no.12
• /
• pp.1322-1329
• /
• 2011

Filamentous fungi colonizing rice straw were collected from 11 different sites in Korea and were identified based on characterization of their morphology and molecular properties. The fungi were divided into 25 species belonging to 16 genera, including 14 ascomycetes, one zygomycete, and one basidiomycete. Fungal cellulolytic and xylanolytic enzymes were assessed through a two-step process, wherein highly active cellulase- and/or hemicellulase-producing fungi were selected in a first screening step followed by a second step to isolate the best enzyme-producer. Twenty-five fungal species were first screened for the production of total cellulase (TC), endo-${\beta}$-1,4 glucanase (EG), and endo-${\beta}$-1,4 xylanase (XYL) using solid-state fermentation with rice straw as substrate. From this screening, six species, namely, Aspergillus niger KUC5183, A. ochraceus KUC5204, A. versicolor KUC5201, Mucor circinelloides KUC6014, Trichoderma harzianum 1 KUC5182, and an unknown basidiomycete species, KUC8721, were selected. These six species were then incubated in liquid Mandels' media containing cellulose, glucose, rice straw, or xylan as the sole carbon source and the activities of six different enzymes were measured. Enzyme production was highly influenced by media conditions and in some cases significantly increased. Through this screening process, Trichoderma harzianum 1 KUC5182 was selected as the best enzyme producer. Rice straw and xylan were good carbon sources for the screening of cellulolytic and xylanolytic enzymes.

• Animal Bioscience
• /
• v.35 no.11
• /
• pp.1733-1743
• /
• 2022

Objective: This study was aimed to explore the efficacy of combination of endo-xylanase (Xyn) and xylan-debranching enzymes (arabinofuranosidase, Afd and feruloyl esterase, FE) in improving utilization of bran in piglet diet. Methods: In vitro experiments were firstly conducted to examine the enzymological properties of Xyn, Afd, and FE, concurrent with their effect on degradation of arabinoxylan (Abx) in bran. In vivo experiment was then implemented by allocating two hundred and seventy 35-d-old postweaning piglets into 3 groups (6 replicates/group), which received bran-containing diet supplemented with Xyn (1,600 U/kg) or its combination with Afd (0.8 U/kg) and FE (4 U/kg) or without enzyme. Results: Both Xyn, Afd, and FE are relatively stable against the changes in temperature and pH value. Combining Xyn with Afd and FE had a superiority (p<0.05) over Xyn alone and its combination with Afd or FE in promoting (p<0.05) degradation of Abx in different brans. Combined treatment with Xyn, Afd, and FE was more beneficial than Xyn alone to induce increasing trends (p<0.10) of average daily gain, final body weight and feed efficiency of piglets fed bran-containing diet. Moreover, combination of Xyn, Afd, and FE showed advantages (p<0.05) over Xyn alone in causing reductions (p<0.05) in diarrhea rate and cecal pH value, concurrent with increases (p<0.05) in cecal and colonic acetic acid and total volatile fatty acid concentrations, as well as cecal butyric acid concentration of piglets fed bran-containing diet. Conclusion: Combining Xyn with Afd and FE was more beneficial than Xyn alone in promoting degradation of Abx in bran, along with growth performance and intestinal volatile fatty acid profile of piglets received bran-containing diet. Thereby, combination of Xyn, Afd, and FE had a superior efficacy relative to Xyn alone in improving application of cereal bran in piglet diet.

• Journal of Microbiology and Biotechnology
• /
• v.21 no.3
• /
• pp.284-292
• /
• 2011

An endocellulase-free multienzyme complex was produced by a thermophilic anaerobic bacterium, Thermoanaerobacterium thermosaccharolyticum strain NOI-1, when grown on xylan. The temperature and pH optima for growth were $60^{\circ}C$ and 6.0, respectively. The bacterial cells were found to adhere to insoluble xylan and Avicel. A scanning electron microscopy analysis showed the adhesion of xylan to the cells. An endocellulase-free multienzyme complex was isolated from the crude enzyme of strain NOI-1 by affinity purification on cellulose and Sephacryl S-300 gel filtration. The molecular mass of the multienzyme complex was estimated to be about 1,200 kDa. The multienzyme complex showed one protein on native PAGE, one xylanase on a native zymogram, 21 proteins on SDS-PAGE, and 5 xylanases on a SDS zymogram. The multienzyme complex consisted of xylanase, ${\beta}$-xylosidase, ${\alpha}$-L-arabinofuranosidase, ${\beta}$-glucosidase, and cellobiohydrolase. The multienzyme complex was effective in hydrolyzing xylan and corn hulls. This is the first report of an endocellulase-free multienzyme complex produced by a thermophilic anaerobic bacterium, T. thermosaccharolyticum strain NOI-1.

• Korean Journal of Microbiology
• /
• v.52 no.4
• /
• pp.463-470
• /
• 2016

The enzymatic degradation of xylans is the most versatile way to obtain the high value-added functional compounds or the fermentable sugars for renewable energy. The endo-${\beta}$-xylanases are the major enzymes which hydrolyze the internal ${\beta}$-1,4-linkages of xylan backbones to produce the mixtures of xylooligosaccharides including xylobiose and xylotriose. Among them, glucuronoxylanase GH30 can exclusively hydrolyze the internal ${\beta}$-1,4-linkages of xylans decorated with methylglucuronic acid branches. In the present study, two xylanolytic enzyme (PaXN_10 and PaGuXN_30) genes were cloned from Paenibacillus amylolyticus KCTC 3005, and expressed in Escherichia coli, respectively. PaXN_10 (38.7 kDa) belongs to the endo-${\beta}$-xylanases GH10 family, while PaGuXN_30 (58.5 kDa) is a member of glucuronoxylanase GH30. They share the same optimal reaction conditions at $50^{\circ}C$ and pH 7.0. Enzymatic characterization proposed that P. amylolyticus can utilize the hardwood glucuronoarabinoxylans via the cooperative actions of xylanases GH10 and GH30. The extracellular PaGuXN_30 is secreted into the medium and hydrolyzes glucuronoarabinoxylans to release a series of aldouronic acid mixtures with a methylglucuronic acid branch. The resultant products being transported into the microbial cell are successively degraded into the smaller xylooligosaccharides by the intracellular PaXN_10, which will be utilized for the cellular metabolism.

• Journal of Life Science
• /
• v.22 no.6
• /
• pp.837-843
• /
• 2012

This study was carried out to investigate the morphological and physiological characteristics of six new cultivars of Hypsizygus marmoreus (Hm) and measure endo-, exo-cellular enzyme-specific activity. The domestic wild stain (Hm3-10) and commercial strain in Japan (Hm1-1) were mated by crossing monokaryon mycelia. We gained 58 strains from one of 400 crosses through the $1^{st}$ cultivation experiment, and selected six strains from one of 58 strains through the $2^{nd}$ cultivation experiment. When six of the selected new strains were grown during several spawn culture periods (60, 70, 80, 90, and 100 days), a spawn culture period of more 80 days was considered to be excellent as being shorter than 19~20 days. Therefore, we determined the period of spawn culture as 80 days. Three strains such as Hm15-3, Hm15-4, and Hm17-5 showed an excellent result. When endo-cellular enzyme activity measured eight strains, we obtained a result of that specific activity of ${\alpha}$-amylase at the highest as 73.9~102.2 unit/mg protein, and chitinase is lower than ${\alpha}$-amylase at 8.1~13.1 unit/mg protein. When exo-cellular enzyme activity measured eight strains, we determined the result of that specific activity of ${\alpha}$-amylase is the highest at 5,292~1,184 unit/mg protein, and CMCase and xylanase were 1,140~245 unit/mg protein, 94~575 unit/mg protein, compared to each other. However, the enzyme activity of ${\beta}$-glucosidase and chitinase is low.

• Microbiology and Biotechnology Letters
• /
• v.45 no.1
• /
• pp.43-50
• /
• 2017

A xylan-degrading bacterium (strain WJ-1) was isolated from soil collected from Jeju Island, Republic of Korea. Strain WJ-1 was characterized as a gram-positive, aerobic, and spore-forming bacterium. The predominant fatty acid in this bacterium was anteiso-$C_{15:0}$ (42.99%). A similarity search based on 16S rRNA gene sequences suggested that the strain belonged to the genus Streptomyces. Further, strain WJ-1 shared the highest sequence similarity with the type strains Streptomyces spinoveruucosus NBRC 14228, S. minutiscleroticus NBRC 13000, and S. glaucescens NBRC 12774. Together, they formed a coherent cluster in a phylogenetic tree based on the neighbor-joining algorithm. The DNA G+C content of strain WJ-1 was 74.7 mol%. The level of DNA-DNA relatedness between strain WJ-1 and the closest related species S. glaucescens NBRC 12774 was 85.7%. DNA-DNA hybridization, 16S rRNA gene sequence similarity, and the phenotypic and chemotaxonomic characteristics suggest that strain WJ-1 constitutes a novel subspecies of S. glaucescens. Thus, the strain was designated as S. glaucescens subsp. WJ-1 (Korean Agricultural Culture Collection [KACC] accession number 92086). Additionally, strain WJ-1 secreted thermostable endo-type xylanases that converted xylan to xylooligosaccharides such as xylotriose and xylotetraose. The enzymes exhibited optimal activity at pH 7.0 and $55^{\circ}C$.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.12
• /
• pp.1636-1643
• /
• 2012

Enzymatic pre-bleaching by modification of pulp fibers with xylanases is an attractive approach to reduce the consumption of toxic bleaching chemicals in the paper industry. In this study, an alkaliphilic endoxylanase gene was isolated from metagenomic DNA of a structurally stable thermophilic lignocellulose-degrading microbial consortium using amplification with conserved glycosyl hydrolase family 10 primers and subsequent genome walking. The full-length xylanase showed 78% sequence identity to an endo-${\beta}$-1,4-xylanase of Clostridium phytofermentans and was expressed in a mature form with an N-terminal His6 tag fusion in Escherichia coli. The recombinant xylanase Xyn3F was thermotolerant and alkaliphilic, working optimally at $65-70^{\circ}C$ with an optimal pH at 9-10 and retaining >80% activity at pH 9, $60^{\circ}C$ for 1 h. Xyn3F showed a $V_{max}$ of 2,327 IU/mg and $K_m$ of 3.5 mg/ml on birchwood xylan. Pre-bleaching of industrial eucalyptus pulp with no prior pH adjustment (pH 9) using Xyn3F at 50 IU/g dried pulp led to 4.5-5.1% increase in final pulp brightness and 90.4-102.4% increase in whiteness after a single-step hypochlorite bleaching over the untreated pulp, which allowed at least 20% decrease in hypochlorite consumption to achieve the same final bleaching indices. The alkaliphilic xylanase is promising for application in an environmentally friendly bleaching step of kraft and soda pulps with no requirement for pH adjustment, leading to improved economic feasibility of the process.

• Asian-Australasian Journal of Animal Sciences
• /
• v.23 no.12
• /
• pp.1632-1638
• /
• 2010

Two experiments with growing pigs were conducted to investigate the effects of two distinct multienzyme preparations on nutrient digestibility, growth performance and blood profiles. In Exp. 1, a total of 96 pigs ($29.7{\pm}0.69\;kg$) were utilized in a 42-day performance and digestibility trial using four dietary treatments: CON (control diet), ENDO (control+0.10% Endopower), NSPase1 (control+0.10% NSPase) and NSPase2 (control+0.20% NSPase). Endopower was a commercial multienzyme preparation which contained ${\alpha}$-galactosidase, galactomannase, xylanase and ${\beta}$-glucanase. NSPase mainly contained ${\alpha}$-1,6-${\beta}$-galactosidase, ${\beta}$-1,4-mannanase and ${\beta}$-1,4-mannosidase. There were six replication pens per treatment with four pigs per pen. Pigs fed NSPase1 diet had a higher ADG (p<0.05) and G:F (p<0.05) than those fed the control diet. There were no significant differences in growth performance among the multienzyme treatments (p>0.05). Compared with CON, apparent digestibility of DM was increased (p<0.05) by ENDO treatment. N digestibility was improved (p<0.05) in response to multienzyme treatments during the experimental period. Blood urea nitrogen (BUN) was higher (p<0.05) in ENDO treatment than in CON and NSPase1 treatments at the end of the experiment, while the glucose level improved (p<0.05) due to ENDO and NSPase2 treatments. In Exp. 2, four ileal-cannulated, growing barrows ($20.17{\pm}1.31\;kg$) were housed in individual metabolism crates and randomly assigned to 1of 4 treatments (same as Exp. 1) within a $4{\times}4$ Latin square design. Enzyme supplementations improved the majority of apparent ileal amino acid digestibilities (p<0.05). It is concluded that the supplementation of NSPase1 improved growth performance as well as N digestibility and partially improved apparent ileal amino acid digestibility in growing pigs fed a diet based on corn and soybean meal.