title: Capacity of thermomonospora alba XylA to impart thermostability in family F/10 chimeric xylanases.
abstract: To reveal structure-function relationships of family F/10 glycanases, an in vitro molecular level shuffling experiment was conducted to accumulate useful amino acid residues from two homologous F/10 xylanases, FXYN of Streptomyces olivaceoviridis E-86 and XylA of Thermomonospora alba ULJB1, into a single chimeric xylanase. The parent genes were shuffled by crossovers at selected module borders using self-priming Polymerase Chain Reaction (PCR)s. The shuffled constructs, designated as FXYN-M3/4-XylA, FXYN-M9/10-XylA, and FXYN-M14/15-XylA were cloned and their nucleotide sequences were confirmed. Two chimera, FXYN-M3/4-XylA and FXYN-M14/15-XylA, demonstrated activity against RBB-xylan and were over-expressed as His-tag fusion proteins under control of T5 promoter of pQE60. The homogeneously pure chimeric proteins, FXYN-M3/4-XylA and FXYN-M14/15-XylA showed improved thermal and pH profiles compared to those of one of the parents, FXYN. This was apparently due to the influence of amino acids inherited from thermophilic XylA. Measured K(m) and kcat values were closer to those of the other parent, XylA. Interestingly, a significant level of heat tolerance up to 60 degrees C, was recorded for FXYN-M3/4-XylA in comparison to only 40 degrees C for FXYN-M14/15-XylA though their temperature optima did not correlates with their thermal stability. These results indicated that the amino acid residues of the larger T. alba XylA DNA fragment present in FXYN-M3/4-XylA were responsible for inducing its thermal stability.
authors: National Food Research Institute, Ministry of Agriculture, Forestry, and Fisheries, 305-8642, Tsukuba, Japan
authors: MM
authors: Ahsan
authors: S
authors: Kaneko
authors: Q
authors: Wang
authors: K
authors: Yura
authors: M
authors: Go
authors: K
authors: Hayash