The worldwide contamination of cereals, oilseeds, and other crops by mycotoxin-producing moulds is a significant problem. Mycotoxins have adverse effects on humans and animals that result in illnesses and economic losses. Reduction or elimination of mycotoxin contamination in food and feed is an important issue. This study aimed to screen soil bacteria for degradation of zearalenone (ZEN). A pure culture of strain CK1 isolated from soil samples showed most capable of degradation of ZEN. Using physiological, biochemical, and 16S rRNA gene sequence analysis methods, CK1 was identified as Bacillus licheniformis. Addition of 2 ppm of ZEN in Luria-Bertani (LB) medium, B. licheniformis CK1 decreased 95.8% of ZEN after 36 h of incubation. In ZEN-contaminated corn meal medium, B. licheniformis CK1 decreased more than 98% of ZEN after 36 h of incubation. In addition, B. licheniformis CK1 was non-hemolytic, non-enterotoxin producing, and displayed high levels of extracellular xylanase, cellulase, and protease activities. These findings suggest that B. licheniformis CK1 could be used to reduce the concentrations of ZEN and improve the digestibility of nutrients in feedstuffs simultaneously.
A cDNA encoding a bifunctional acetylxylan esterase/xylanase, XynS20E, was cloned from the ruminal fungus Neocallimastix patriciarum. A putative conserved domain of carbohydrate esterase family 1 was observed at the N-terminus and a putative conserved domain of glycosyl hydrolase family 11 was detected at the C-terminus of XynS20E. To examine the enzyme activities, XynS20E was expressed in Escherichia coli as a recombinant His(6) fusion protein and purified by immobilized metal ion-affinity chromatography. Response surface modeling combined with central composite design and regression analysis was then applied to determine the optimal temperature and pH conditions of the recombinant XynS20E. The optimal conditions for the highest xylanase activity of the recombinant XynS20E were observed at a temperature of 49 degrees C and a pH of 5.8, while those for the highest carbohydrate esterase activity were observed at a temperature of 58 degrees C and a pH of 8.2. Under the optimal conditions for the enzyme activity, the xylanase and acetylxylan esterase specific activities of the recombinant XynS20E toward birchwood xylan were 128.7 and 873.1 U mg(-1), respectively. To our knowledge, this is the first report of a bifunctional xylanolytic enzyme with acetylxylan esterase and xylanase activities from rumen fungus.
A gene encoding a xylanase, named xynS20, was cloned from the ruminal fungus Neocallimastix patriciarum. The DNA sequence of xynS20 revealed that the gene was 1,008 bp in size and encoded amino acid sequences with a predicted molecular weight of 36 kDa. The amino acid sequence alignment showed that the highest sequence identity (28.4%) is with insect gut xylanase XYL6805. According to the sequence-based classification, a putative conserved domain of glycosyl hydrolase family 11 was detected at the N-terminus of XynS20 and a putative conserved domain of family 1 carbohydrate-binding module (CBM) was observed at the C-terminus of XynS20. An Asn-rich linker sequence was found between the N-terminal catalytic domain and the C-terminal CBM of XynS20. To examine the activity of the gene product, xynS20 gene was cloned as an oleosin-fused protein, expressed in Escherichia coli, affinity-purified by formation of artificial oil bodies, released from oleosin by intein-mediated peptide cleavage, and finally harvested by concentration of the supernatant. The specific activity of purified XynS20 toward oat spelt xylan was 1,982.8 U mg(-1). The recombinant XynS20 was stable in the mild acid pH range from 5.0 to 6.0, and the optimum pH was 6.0. The optimal reaction temperature of XynS20 was 45 degrees C; at temperatures below 30 and above 55 degrees C, enzyme activity was less than 50% of that at the optimal temperature.
BACKGROUND: Xylan is the second most abundant renewable polysaccharide in nature and also represents an important industrial substrate. The complete degradation of xylan requires the combination of several types of xylanolytic enzymes, including endo-β-1,4-xylanases, β-xylosidases, and acetylxylan esterases. As a biocatalyst, xylanolytic enzymes with good thermal stability are of great interest, therefore, a thermo-tolerant acetylxylan esterase, AxeS20E, was investigated.
Four chimeric xylanolytic enzymes were formed by fusion of a thermally stable xylanase XynCDBFV either to the N-terminus or C-terminus of a thermally stable acetylxylan esterase AxeS20E, with or without a Gly-rich flexible linker (S2). The three-dimensional (3D) structures of the chimeric enzymes were predicted using the I-TASSER server, and the results indicated that the structures of Axe-S2-Xyn and Xyn-S2-Axe were more similar to the native structures than were those of Axe-Xyn and Xyn-Axe. Axe-S2-Xyn and Xyn-S2-Axe were expressed in Escherichia coli and purified by means of affinity chromatography. Response surface modeling (RSM), combined with central composite design (CCD) and regression analysis, was then employed to optimize the xylanase activities of the chimeric enzymes. Under the optimal conditions, Xyn-S2-Axe had greater hydrolytic activities on natural xylans and rice straw than did the parental enzymes. These results suggested that the chimeric enzyme Xyn-S2-Axe could be effective at hydrolyzing xylan in biomass and that it has potential to be used in a range of biotechnological applications.
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