• Journal of Microbiology and Biotechnology
• /
• v.20 no.5
• /
• pp.893-903
• /
• 2010

A cellulolytic and xylanolytic enzyme complex-producing alkalothermoanaerobacterium strain, Tepidimicrobium xylanilyticum BT14, is described. The cell was Grampositive, rod-shaped, and endospore-forming. Based on 16S rRNA gene analysis and various lines of biochemical and physiological properties, the strain BT14 is a new member of the genus Tepidimicrobium. The strain BT14 cells had the ability to bind to Avicel, xylan, and corn hull. The pH and temperature optima for growth were 9.0 and $60^{\circ}C$, respectively. The strain BT14 was able to use a variety of carbon sources. When the bacterium was grown on corn hulls under an anaerobic condition, a cellulolytic and xylanolytic enzyme complex was produced. Crude enzyme containing cellulase and xylanase of the strain BT14 was active in broad ranges of pH and temperature. The optimum conditions for cellulase and xylanase activities were pH 8.0 and 9.0 at $60^{\circ}C$, respectively. The crude enzyme had the ability to bind to Avicel and xylan. The analysis of native-PAGE and native-zymograms indicated the cellulosebinding protein showing both cellulase and xylanase activities, whereas SDS-PAGE zymograms showed 4 bands of cellulases and 5 bands of xylanases. Evidence of a cohesinlike amino acid sequence seemed to indicate that the protein complex shared a direct relationship with the cellulosome of Clostridium thermocellum. The crude enzyme from the strain BT14 showed effective degradation of plant biomass. When grown on corn hulls at pH 9.0 and $60^{\circ}C$ under anaerobic conditions, the strain BT14 produced ethanol and acetate as the main fermentation products.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.4
• /
• pp.483-488
• /
• 2014

In order to improve the expression of heat-resistant xylanase XYNB from Aspergillus niger SCTCC 400264, XynB has been cloned into Pichia pastoris secretary vector pPIC9K. The XynB production of recombinant P. pastoris was four times that of E. coli, and the $V_{max}$ and specific activity of XynB reached $2,547.7{\mu}mol/mg$ and 4,757 U/mg, respectively. XynB still had 74% residual enzyme activity after 30 min of heat treatment at $80^{\circ}C$. From the van der Waals force analysis of XYNB (ACN89393 and AAS67299), there is one more oxygen radical in AAS67299 in their catalytic site, indicating that the local cavity is much more free, and it is more optimal for substrate binding, affinity reaction, and proton transfer, etc, and eventually increasing enzyme activity. The H-bonds analysis of XYNB indicated that there are two more H-bonds in the 33rd Ser of XYNB (AAS67299) than in the 33rd Ala(ACN89393 ), and two H-bonds between Ser70 and Asp67.

• Mycobiology
• /
• v.48 no.6
• /
• pp.501-511
• /
• 2020

Xylophagous termites are capable of degrading lignocellulose by symbiotic gut microorganisms along with the host's indigenous enzymes. Therefore, the termite gut might be a potential niche to obtain natural yeasts with celluloytic, xylanolytic and ethanologenic traits required for bioethanol production from lignocellulosic biomass. In this study, we cultured 79 yeasts from three different termites viz. Coptotermes heimi, Odontotermes javanicus and Odontotermes obesus. After suitable screening methods, we identified 53 yeasts, which belonged to 10 genera and 16 different species of both ascomycetous and basidiomycetous yeasts. Most yeasts in the present study represent their first-ever isolation from the termite gut. Representative strains of identified yeasts were evaluated for their cellulolytic, xylanolytic, and ethanologenic abilities. None of the isolates showed cellulase activity; 22 showed xylanolytic activity, while six produced substantial quantities of ethanol. Among xylanolytic cultures, Pseudozyma hubeiensis STAG 1.7 and Hannaella pagnoccae STAG 1.14 produced 1.31 and 1.17 IU of xylanase. Among ethanologenic yeasts, the strains belonging to genera Candida and Kodamaea produced high amount of ethanol. Overall, highest ethanol level of 4.42 g/L was produced by Candida tropicalis TS32 using 1% glucose, which increased up to 22.92 g/L at 35 ℃, pH 4.5 with 5% glucose. Fermentation of rice straw hydrolysate gave 8.95 g/l of ethanol with a yield of 0.42 g/g using the strain TS32. Our study highlights the gut of wood-feeding termites as a potential source of diverse yeasts that would be useful in the production of xylanase and bioethanol.

• Journal of Life Science
• /
• v.25 no.6
• /
• pp.680-685
• /
• 2015

Previously, cellulase and xylanase producing microorganism, Bacillus subtilis NC1, was isolated from soil. Based on the 16S rRNA gene sequence and API 50 CHL test the strain was identified as Bacillus subtilis, and named as B. subtilis NC1. We cloned and sequenced the genes for cellulase and xylanase. Plus, the deduced amino acid sequences from the genes of cellulase and xylanase were determined and were also identified as glycosyl hydrolases family (GH) 5 and 30, respectively. In this study to optimize the medium parameters for cellulase production by B. subtilis NC1 the RSM (response surface methodology) based on CCD (central composite design) model was performed. Three factors, tryptone, yeast extract, and NaCl, for N or C source were investigated. The cellulase activity was measured with a carboxylmethyl cellulose (CMC) plate and the 3,5-dinitrosalicylic acid (DNS) methods. The coefficient of determination (R²) for the model was 0.960, and the probability value (p=0.0001) of the regression model was highly significant. Based on the RSM, the optimum conditions for cellulase production by B. subtilis NC1 were predicted to be tryptone of 2.5%, yeast extract of 0.5%, and NaCl of 1.0%. Through the model verification, cellulase activity of Bacillus subtilis NC1 increased from 0.5 to 0.62 U/ml (24%) compared to the original medium.

• Asian-Australasian Journal of Animal Sciences
• /
• v.16 no.2
• /
• pp.248-256
• /
• 2003

Two experiments were conducted to determine the effects of expander processing and enzyme supplementation of wheat-based diets on growth performance and nutrient digestibility in finishing pigs. For Exp. 1, 60 finishing pigs (average initial BW of 49.5 kg) were fed meal, standard pellets and expanded pellets in a 70 d growth assay. From 49.5 to 79.0 kg, 79.0 to 111.8 kg, and overall (49.5 to 111.8 kg), ADG and ADFI were not affected by pelleting or standard vs expander conditioning (p>0.22). However, from 49.5 to 79.0 kg, pigs fed pellets have greater gain/feed than pigs fed mash (p<0.04), and pigs fed expanded pellets tended to have greater (p<0.10) gain/feed than pigs fed standard pellets. Overall (i.e. from 49.5 to 111.8 kg), gain/feed (p<0.02) and apparent fecal digestibilities of DM (p<0.001) and N (p<0.02) were improved by pelleting the diets. Also, expander processing further improved gain/feed (p<0.06) and digestibility of DM (p<0.04) compared to standard steam conditioning. Scores for keratinization (p<0.002) and ulceration (p<0.003) of the stomach were increased by pelleting, but the mean scores for the various treatments ranged only from 0.05 to 1.08 (i.e., low to mild keratosis and ulceration). For Exp. 2, 80 pigs (average initial BW of 54.1 kg) were fed mash and pellets (standard or expander) without and with xylanase. The enzyme was added to supply 4,000 units of xylanase activity/kg of diet. Adding xylanase to the mash diet improved gain/feed from 90.7 to 115.9 kg (p<0.04) of the growth assay and digestibility of DM (p<0.05) on d 39. However, in pelleted diets, adding the enzyme did not improve growth performance or digestibility of nutrients. Pelleting tended to increase scores for ulceration (p<0.06), and enzyme supplementation decreased stomach keratinization scores for pigs fed the standard pellets (p<0.01). However, as in Exp. 1, the mean scores for all treatment groups were quiet low (i.e., ranging from normal to mild). In conclusion, pelleting improved efficiency of growth, but additional benefits from expander conditioning were observed only in Exp. 1. Finally, xylanase tended to improve growth performance and nutrient digestibility, only in pigs fed mash diets but not in pigs fed pellets.

• Journal of Animal Science and Technology
• /
• v.50 no.5
• /
• pp.713-720
• /
• 2008

This study was conducted to isolate and identify xylanase- and cellulase-producing thermophilic bacteria from stacked spent mushroom substrates and to determine the optimal medium conditions for their growth. Bacteria with the highest xylanase and CMCase activities were strain 3 and 201-7. Both of them were identified as Bacillus spp. and named Bacillus subtilis KU3 and Bacillus subtilis KU201-7. The optimal medium condition of Bacillus subtilis KU3 was obtained when 3%(w/v) of yeast extract and 1%(w/v) of maltose were used as nitrogen and carbon sources, respectively. That of Bacillus subtilis KU201-7 was obtained when 0.5%(w/v) of yeast extract and 0.5%(w/v) of CMC were used as nitrogen and carbon sources, respectively.

• Journal of Microbiology and Biotechnology
• /
• v.16 no.8
• /
• pp.1269-1275
• /
• 2006

The selection of multienzyme complex-producing bacteria under aerobic condition was conducted for improving the degradation of lignocellulosic substances. The criteria for selection were cellulase and xylanase enzyme production, the presence of cellulose-binding domains and/or xylan-binding domains in enzymes to bind to insoluble substances, the adhesion of bacterial cells to insoluble substances, and the production of multiple cellulases and xylanases in a form of a high molecular weight complex. Among the six Bacillus strains, isolated from various sources and deposited in our laboratory, Paenibacillus curdlanolyticus B-6 strain was the best producer of cellulase and xylanase enzymes, which have both cellulose-binding factors (CBFs) and xylan-binding factors (XBFs). Moreover, multiple carboxymethyl cellulases (CMCases) and xylanases were produced by the strain B-6. The zymograms analysis showed at least 9 types of xylanases and 6 types of CMCases associated in a protein band of xylanase and cellulase with high molecular weight. These cells also enabled to adhere to both avicel and insoluble xylan, which were analyzed by scanning electron microscopy. The results indicated that the strain B-6 produced the multienzyme complex, which may be cellulosome or xylanosome. Thus, P. curdlanolyticus B-6 was selected to study the role and interaction between the enzymes and their substrates and the cooperation of multiple enzymes to enhance the hydrolysis due to the complex structure for efficient cellulases and xylanases degradation of insoluble polysaccharides.

• KSBB Journal
• /
• v.17 no.2
• /
• pp.195-202
• /
• 2002

The production conditions of cellulolytic enzymes by a fungus, strain FJ1, were optimized using response surface analysis. The culture factors which largely affected the production of enzymes such as cultivation time, carbon source concentration, nitrogen source concentration, and composition ratio of carbon sources were employed. Optimizedconditions of the factors above corresponding to each cellulolytic enzyme production were as fellowing: CMCase production was obtained in the conditions of cultivation time of 5.4 days, carbon source concentration of 3.5%, nitrogen source concentration of 0.6%, and composition ratio of carbon sources of 52:48 (avicel:CMC), xylanase appeared in the conditions of 5.3 days, 3.5%, 0.8%, and 54:46, respectively, and $\beta$-glucosidase were 7.0 days, 5.0%, 1.0%, and 83:17, respectively, and avicelase were 6.5 days, 4.0%, 0.9%, and 64:36, respectively. The activities of CMCase, xylanase, p-glucosidase, and avicelase predicted by the response surface methodology were 33.5, 52.6, 2.88, and 1.84 U/mL, respectively, and $\beta$-glucosidase activity was enhanced up to 74% when compared to that obtained in the experimental conditions.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.46 no.9
• /
• pp.1114-1121
• /
• 2017

Bacillus strains not producing harmful components were isolated from Korean traditional soybean products. Extracellular enzyme activities (amylase, protease, cellulase, and xylanase) of isolated Bacillus strains were measured, and Bacillus strains with high protease activity were selected. The selected 15 strains were identified as Bacillus amyloliquefaciens (10), Bacillus methylotrophicus (1), Bacillus velezensis (1), and Bacillus subtilis (3). Among them, B. subtilis JBG17019, B. amyloliquefaciens JBD17076, and B. amyloliquefaciens JBD17109 showed antimicrobial activities against food-borne microorganisms. The production abilities of glutamate, glutamine, and poly-${\gamma}$-glutamic acid (${\gamma}$-PGA) of the selected Bacillus strains were measured to analyze fermentation characteristics related to glutamic acid metabolism. The factor for multivariate was analyzed by the principal components analysis (PCA) method between fermentation characteristics and ${\gamma}$-PGA production. The three principal components were classified according to the PCA method: PC1 [enzyme activity (amylase, cellulase, and xylanase)], PC2 (${\gamma}$-PGA), and PC3 (protease, glutamate, and glutamine). As a result, B. amyloliquefaciens JBD17076 and B. subtilis JBG17019 strains were evaluated as having excellent enzyme activity and ${\gamma}$-PGA production.

• Journal of The Korean Society of Grassland and Forage Science
• /
• v.33 no.1
• /
• pp.58-66
• /
• 2013

This study was conducted in order to develop slaughterhouse rumen content (SRC) as a potential feed additive. The moisture content of SRC can reach 80%, and therefore an appropriate dewatering process is required before it can be used. In this study, the effects of heating temperature, heating time, and filler content during the dewatering process on the activity of various enzymes in SRC were investigated. The Box-Behnken experimental design was employed, involving a total of 45 experimental runs, consisting of three variables (heating time, heating temperature, and filler content) with three levels per variable (12, 30 and 48 hr; 60, 75 and $90^{\circ}C$; 12, 22.5 and 33% for heating time, heating temperature, and filler content, respectively). For enzyme activities, xylanase, cellulase, and amylase were examined, and the results were subjected to an analysis of variance. Heating time, heating temperature and filler content had significant effects on the activity of each enzyme (p<0.05). Cellulase and amylase activities decreased (p<0.05) at elevated heating temperatures, whereas xylanase was reasonably stable around $90^{\circ}C$. The activities of all enzymes decreased (p<0.05) with increased heating time. Optimum filler contents for xylanase, cellulase, and amylase activities were 22.5, 12 and 33%, respectively. However, optimum conditions for all variables that simultaneously maximize the activity of all three enzymes could not be ascertained in this study. Nevertheless, the results from the current study can be useful as basic information for the development of SRC as a feed additive enriched with improved major enzymes for livestock feed digestion.