Gene Cloning, Expression And Characterization Of Acetyl Xylan Esterase From Streptomyces Albus

Xylan is the main component of plant hemicelluloses, and its main chain and side chain contain a variety of different substituents. Due to its complexity, complete hydrolysis of xylan requires cooperation of a large variety of enzymes. Among them, xylanase plays an important role in the xylanolytic enzyme system by hydrolyzing the β-1,4-glycoside linkages in xylan backbone. Acetyl xylan esterase (Axe) can hydrolyze O-acetyl in the acetylated xylan, through which it can eliminate O-acetyl’s steric hindrance effects to xylanase hydrolysis, and enhance capacity of affinity and degradation to xylan. So far, Axe is rarely reported over the world. Therefore, obtaining good enzymes with superior properties is significance.In this study, Streptomyces albus was used as the experimental material to verify the ability to produce xylanolytic enzymes by Congo red staining. By using bioinformatics methods, the known amino acid sequences were aligned and analyzed, and designed a set of degenerate primers. Then obtained Axe genes of Streptomyces albus by degenerate PCR amplified, the size of Axe gene fragment is312bp. By conducting TAIL-PCR between SP1, SP2, SP3designed from the gene fragment and and the known degenerate primers AD1, AD2, AD3, AD4, the size of949bp gene was found. The open reading frame consists of741bp and encodes247amino acids. The peptide fragment has high similarity with the acetyl xylan esterase gene sequences of Streptomyces sp. ACTE (ZP06273846.1) and Streptomyces avermitilis MA-4680(NP822477.1),78%and58%, respectively. And the peptide fragment has conserved regions of AXE1family proteins. The region has similar secondary and tertiary structure with known acetyl xylan esterase protein C-terminal region, suggesting that this peptide fragment is the C-terminal region of acetyl xylan esterase from Streptomyces albus.The gene Axe was inserted into the expression vector pET-28a (+) with correct reading frame and transformed into E. coli BL21(DE3) to express the recombinant protein. After Ni-NTA purifying, SDS-PAGE show it has molecular masses of26.5kDa, whose weight is as same as the theoretical molecular. The isoelectric point is6.0. As determined by a2min assay with4-methylumbelliferyl acetate as the aubstrate, the optimal activity of Axe occurres at7.0and65℃. By analyzing the effects to activity in different metal ions and chemical reagents situation, Hg2+completely inhibited the enzyme activity, Zn2+and Cu2+showed highly inhibition on them which inhibition of the enzyme activity53.2%and46.9%respectively, Mn2+and Ca2+could slightly activate them, no dramatic effects were found in Mg2+or Co2+.