Recombination of thermo-alkalistable, high xylooligosaccharides producing endo-xylanase from Thermobifida fusca and expression in Pichia pastoris.
For xylooligosaccharide (XO) production, endo-xylanase from Thermobifida fusca was modified by error-prone PCR and DNA shuffling. The G4SM1 mutant (S62T, S144C, N198D, and A217V) showed the most improved hydrolytic activity and was two copies expressed in Pichia pastoris under the control of GAP promoter. The maximum xylanase activity in culture supernatants was 165 ± 5.5 U/ml, and the secreted protein concentration reached 493 mg/l in a 2-l baffled shake flask. After 6× His-tagged protein purification, the specific activity of G4SM1 was 2036 ± 45.8 U/mg, 2.12 times greater than that of wild-type enzyme. Additionally, G4SM1 was stable over a wide pH range from 5.0 to 9.0. Meanwhile, half-life of G4SM1 thermal inactivation at 70 °C increased 8.5-fold. Three-dimensional structures suggest that two amino acid substitutions, S62T and S144C, located at catalytic domain may be responsible for the enhanced activity and thermostability of xylanase. Xylobiose was the dominant end product of xylan hydrolysis by G4SM1. Due to its attractive biochemical properties, G4SM1 has potential value in commercial XO production.
Institute of Dairy Science, College of Animal Science, Zhejiang University, Hangzhou, 310058, China.
Qian
Q
Wang
Wen
W
Du
Xiao-Yan
XY
Weng
Ming-Qi
MQ
Liu
Jia-Kun
JK
Wang
Jian-Xin
JX
Liu
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D029662:Betula / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D043364:Endo-1,4-beta Xylanases / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D018628:Gene Dosage; D005798:Genes, Bacterial; D005820:Genetic Testing; D005965:Glucuronates / Q000096:biosynthesis*; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D009844:Oligosaccharides / Q000096:biosynthesis*; D010843:Pichia / Q000235:genetics*; D011995:Recombination, Genetic; D040681:Structural Homology, Protein; D013696:Temperature; D014990:Xylans / Q000378:metabolism
D005965:Glucuronates; D009844:Oligosaccharides; D014990:Xylans; C570991:xylooligosaccharide; D043364:Endo-1,4-beta Xylanases