Studies On Gene Cloning, Expression And Enzymatic Characterization Of Acetyl Xylan Esterase From Aspergillus Usamii

Acetyl xylan esterase(AXE, EC 3.1.1.72), discovered in the 1990 s, is one of the key enzymes in the xylanolytic enzyme system, and can hydrolyze O-acetyl located at the C2 and/or C3 positions of the D- xylose in acetylated xylan. Due to its low expression in microbes and the low and non-specific enzyme activity, the indusctrial application of natural AXE is limited. In this paper, the acetyl xylan esterase of Aspergillus usamii E001 was expressed in Pichia pastoris expression system, and its enzymatic properties were analyzed.The Auaxe c DNA was obtained by reverse transcriptom PCR using the Aspergillus usamii E001 total RNA as template, and its primers Auaxe-F and Auaxe-R were designed by the software of O ligo 7.0. The amilified fragment was inse rted into p UC m-T vector and sequenced. The Auaxe c DNA was a 828 bp fragment encoding 275 amino acids.The Auaxe gene was inserted into the expression vector p PIC9 KM, generating a recombinant expression plasmid p PIC9KM-Auaxe. The recombinant plasmid was then linearized with Sal I and transformed into Pichia pastoris GS115 by electrotransformation. After G418 screening, the recombinant transformant GS115/Auaxe was selected for AXE expression, which was induced by 1% methanol for 72 h. The crude enzyme was extracted from culture supernatant for enzyme activity analysis using p- nitrophenyl acetate as substrate at 50℃, p H 6.0 was 35.6 U/m L by spectrophotometry. In addition, the expression conditions of GS115/Auaxe were optimized, and the activity of re Au Axe reached 98.5 U/m L, which was 2.11 times higher than that before optimization.The purification of recombinant Au Axe(re Au Axe) was performed by a combination of(NH4)2SO4 precipitation, DEAE-52 Sepharose Fast Flow ion-exchange chromatography and dialysis. As a result, re Au Axe was purified by 1.7- fold with a recovery of 63.1%. SDS-PAGE analysis showed that the apparent molecular mass of purified re Au Axe was about 34.0 k Da. The specific activity of purified re Au Axe was 390.5 U/mg. The purified re Au Axe was optimally active at 50℃ and p H 6.0. It was stable at 45℃ or below, and at a p H range of 4.5~7.0. Its enzyme acvitity was not signficantly affected by an array of tested metal ions or EDTA. The Km and Vmax of re Au Axe at 50℃, p H 6.0, towards p- nitrophenyl acetate, were 0.72 mmol/L and 555.6 U/mg, respectively.The synergistic effect of the recombinant acetyl xylan esterase with the recombinant xylanase, the recombinant cellulase and enzymes compound on the wheat bran was investigated, respectively. As a result, when the ratio of re Au Axe and re Au Xyn11 A was 5:1, the released reduce sugar was increased by 46% compared to re Au Xyn11 A alone. The ratio of re Au Axe and re Au Xyn10 A was 5:1, the released reduce sugar was increased by 16%. The ratio of re Au Axe and re Au Cel12 A was 5:1, the released reduce sugar was increased by 28.4% compared to re Au Cel12 A alone. The ratio of re Au Axe and enzymes compound was 5:1, the released reduce sugar was increased by 33.6%.