ABSTRACT
The model 3-D structure of xylanase KRICT PX3 (JF320814) identified by DNA sequence analysis revealed a catalytic domain and CBM4-9 which functions as a xylan binding domain (XBD). To identify its role in xylan hydrolysis, six expression plasmids were constructed encoding the N-terminal CBM plus the catalytic domain or different glycosyl hydrolases, and the biochemical properties of the recombinant enzymes were compared to the original structure of PX3 xylanase. All six of the recombinant xylanases with the addition of CBM in the pIVEX-GST expression vector showed no improved PX3 hydrolytic activity. However, the absence of the CBM domain resulted in a decrement of 40% in thermostability, movement of the optimal temperature from 55°C to 45°C, alteration of the optimal pH range from 5-10 to 6-8, and reduction of the enzymatic activity to one-second under the same condition, respectively. The putative XBD in PX3 comprises a new N-terminal domain homologous to the catalytic thermostabilizing domains from other xylanases. Analysis of the main products released from xylan indicate that the recombinant enzymes act as endo-1,4-ß-xylanases but differ in their hydrolysis of xylan from beech wood, birch wood, and oat spelt.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Endo-1,4-beta Xylanases/chemistry , Endo-1,4-beta Xylanases/metabolism , Paenibacillus/enzymology , Bacterial Proteins/genetics , Catalytic Domain , Cellulose/metabolism , Endo-1,4-beta Xylanases/genetics , Enzyme Stability , Hot Temperature , Hydrogen-Ion Concentration , Kinetics , Paenibacillus/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Xylans/metabolismABSTRACT
A new bi-modular, wide pH spectrum and highly active xylanase KRICT PX3 (JF320814) isolated from Paenibacillus terrae HPL-003 (KCTC11987BP) has been cloned and expressed in Escherichia coli. Purified recombinant xylanase KRICT PX-3 (1,620 bp, 540aa, NCBI accession number JF320814) showed highly active at 55°C in pH 4.0-11.0, and stability for at least 24h at 50°C, and exhibited Km and Vmax of 0.2mg/mL and 153.8 U/mg on birchwood xylan. Most common ions did not affect the enzyme activity at 1mM concentration. This enzyme could belong to glycoside hydrolase family 10 because hydrolyzed glucuronoxylan and arabinoxylan substrate to xylobiose, xylotriose, and some traces of xylose as hydrolysis products. Model 3-D structure was composed of two domains, the catalytic domain of a (ß/α)8 barrel fold while the small domain probably functions as a xylan binding domain, and the two domains are connected by a flexible linker peptide (PPLAIEKDIPSL). However, sequence alignment between xylan-binding module in this xylanase KRICT PX3 and CBM22 showed 21% of identity and 35% of similarity. This xylanase structure showed a distinctive group of enzyme cluster separately from the rest of GH10 xylanases, and seems to constitute a new type of xylanases.
Subject(s)
Bacterial Proteins/genetics , Endo-1,4-beta Xylanases/genetics , Paenibacillus/enzymology , Paenibacillus/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Base Sequence , Cloning, Molecular , DNA, Bacterial/genetics , Endo-1,4-beta Xylanases/chemistry , Endo-1,4-beta Xylanases/metabolism , Enzyme Stability , Genome, Bacterial , Kinetics , Models, Molecular , Molecular Sequence Data , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
This article reports on the full genome sequence of Paenibacillus terrae HPL-003, which is a gram-positive, endospore-forming, xylanase-producing bacterium isolated from soil found in forest residue on Gara Mountain. The strain HPL-003 contains 6,083,395 bp with a G+C content of 46.77 mol%, 2,633 protein-coding genes, and 117 structural RNAs.
