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Purification and characterization of a new xylanase (xylanase B) produced by Streptomyces lividans 66.

ABSTRACT: A new extracellular xylanase produced by Streptomyces lividans 66 was isolated from a genetically engineered clone of that strain. This enzyme, named xylanase B, has an Mr of 31,000 and acts specifically on xylan as an endo-type xylanase producing short-chain xylo-oligosaccharides. The activity is optimal at pH 6.5 and at a temperature of 55 degrees C, which is similar to that of the previously characterized xylanase A. Xylanase B is glycosylated and has a pI of 8.4; its Km and Vmax. values are 3.71 mg/ml and 1.96 mmol/mg of enzyme respectively. Specific antibodies raised against xylanase A show no cross-reaction with xylanase B; however, the anti-(xylanase B) antibodies react slightly with xylanase A. A comparison of the hydrolysis products obtained from oat-spelts xylan with both enzymes show that xylanase A preferentially degrades short-chain oligo-xylosides, whereas xylanase B acts on the longer, water-insoluble, molecules.

SUBMITTER: Kluepfel D

PROVIDER: S-EPMC1131241 | biostudies-other | 1990 Apr

REPOSITORIES: biostudies-other

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Purification and properties of a xylanase from Streptomyces lividans.

Project description:An extracellular xylanase produced by a cellulase-negative mutant strain of Streptomyces lividans 1326 was purified to homogeneity. The purified enzyme has an apparent Mr of 43,000 and pI of 5.2. The pH and temperature optima for the activity were 6.0 and 60 degrees C respectively, and the Km and Vmax. values, determined with a soluble oat spelts xylan, were 0.78 mg/ml and 0.85 mmol/min per mg of enzyme. The xylanase showed no activity towards CM-cellulose and p-nitrophenyl beta-D-xyloside. The enzyme degraded xylan, producing mainly xylobiose, a mixture of xylo-oligosaccharides and a small amount of xylose as end products. Its pattern of action on beta-1,4-D-xylan indicates that it is a beta-1,4-endoxylanase (EC 3.2.1.8).

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Project description:Xylanases catalyze the hydrolysis of xylan, an abundant carbon and energy source with important commercial ramifications. Despite tremendous efforts devoted to the catalytic improvement of xylanases, success remains limited because of our relatively poor understanding of their molecular properties. Previous reports suggested the potential role of atomic-scale residue dynamics in modulating the catalytic activity of GH11 xylanases; however, dynamics in these studies was probed on time scales orders of magnitude faster than the catalytic time frame. Here, we used nuclear magnetic resonance titration and relaxation dispersion experiments ((15)N-CPMG) in combination with X-ray crystallography and computational simulations to probe conformational motions occurring on the catalytically relevant millisecond time frame in xylanase B2 (XlnB2) and its catalytically impaired mutant E87A from Streptomyces lividans 66. Our results show distinct dynamical properties for the apo and ligand-bound states of the enzymes. The apo form of XlnB2 experiences conformational exchange for residues in the fingers and palm regions of the catalytic cleft, while the catalytically impaired E87A variant displays millisecond dynamics only in the fingers, demonstrating the long-range effect of the mutation on flexibility. Ligand binding induces enhanced conformational exchange of residues interacting with the ligand in the fingers and thumb loop regions, emphasizing the potential role of residue motions in the fingers and thumb loop regions for recognition, positioning, processivity, and/or stabilization of ligands in XlnB2. To the best of our knowledge, this work represents the first experimental characterization of millisecond dynamics in a GH11 xylanase family member. These results offer new insights into the potential role of conformational exchange in GH11 enzymes, providing essential dynamic information to help improve protein engineering and design applications.

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Streptomyces coelicolor A3(2) lacks a genomic island present in the chromosome of Streptomyces lividans 66.

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