Stability and folding of a thermostable xylanase from humicola lanuginosa

Abstract

Thermostable xylanase from Humicola lanuginosa” submitted by Utpal Tatu for the degree of Doctor of Philosophy, Indian Institute of Science, Bangalore, India. The enzyme xylanase (E.C. 3.2.1.8), elaborated extracellularly by the thermophilic fungus Humicola lanuginosa, has been isolated in a pure state. This highly thermostable enzyme has a single polypeptide chain and only one disulfide bond. The present study on the physicochemical properties of this enzyme primarily focuses on the environmental parameters influencing the reduction and reformation of the disulfide bond. The important features of the study include:

the unusual temperature requirement for reduction of the disulfide bond in the xylanase, the role of the disulfide bond in the stability of this enzyme, and the unique ability of the reduced, unfolded form of the enzyme to refold to its native, compactly folded state in the presence of a denaturant.

Earlier work from this laboratory reported the purification, preliminary characterization, and mode of action of this enzyme. The enzyme is a single-domain protein with a molecular weight of 22,500 daltons. It is a glycoprotein with a carbohydrate content of 2.2%. The protein is rich in acidic amino acids (Asp/Glu), contains no methionine residues, and has two half cystines as shown by amino acid composition. The enzyme hydrolyzes the internal 1,4 linkage of xylan, releasing xylo oligosaccharides of varying chain lengths. The thesis is divided into six chapters. The first chapter discusses information available on the structural and functional properties of xylanases from bacterial and fungal sources and outlines the objectives of the present study. The second chapter describes the experimental methods. The third chapter summarizes purification steps (from earlier work), assessment of starting material purity, and crystallization conditions, and discusses antigenic cross reactivity between xylanases from different thermophilic fungi. The fourth chapter details temperature dependence of disulfide bond reduction and the role of temperature in facilitating reduction. The fifth chapter presents experiments showing the role of the disulfide bond in conferring stability against urea , temperature , and alkali induced unfolding. The final chapter reports findings on refolding of the reduced, unfolded enzyme to its native state. Xylanase was purified from H. lanuginosa culture filtrate using the protocol of Lalitha A., involving ion exchange and gel filtration chromatography. A one step purification method was developed using an affinity column based on antibodies raised against the purified enzyme. Antigenic cross reactivity among xylanases from different thermophilic fungi was tested using antibodies against H. lanuginosa xylanase. The Ouchterlony double diffusion test showed cross reactivity with Paecilomyces varioti, but not with Thermoascus aurantiacus. RIA displacement analysis quantified the extent of cross reactivity. Crystallization conditions were standardized to obtain crystals from concentrated enzyme solution. Amino acid analysis and DTNB oxidation showed that the enzyme has a single disulfide bond with no free –SH groups. Reduction of this disulfide bond was not facile at 30°C, even in the presence of 8 M urea, suggesting that the disulfide is buried in the hydrophobic core. Heating the protein to 60°C for 30 min in the presence of mercaptoethanol was necessary and sufficient to allow reduction. Being thermostable, 60°C does not denature the enzyme. The enzyme retains full functional activity at 60°C for 30 min and is maximally active at 65°C. CD and fluorescence spectra show no detectable structural changes at 60°C. The enzyme apparently undergoes a subtle, reversible conformational shift that renders the buried disulfide accessible. A comparative study of unfolding by urea, heat, and alkali between native and reduced (or reduced–carboxymethylated) enzyme showed that the native disulfide bonded form retains structure and function in 8 M urea. In contrast, the reduced form is functionally inactive and grossly unfolded in urea. Thermal denaturation of the native and carboxymethylated enzyme was studied using circular dichroism. CD based secondary structure analysis demonstrated that cleavage of the disulfide bond results in a ~25% loss of sheet structure. Reversibility experiments indicated that the disulfide bond constrains the protein to fold rather than aggregate during heat perturbation. Alkali induced unfolding was examined by tyrosine ionization using UV absorption. Carboxymethylated xylanase exhibited greater tyrosine exposure, indicating greater unfolding. Thus, the disulfide bond constrains complete alkali induced unfolding. Immunological experiments further showed gross conformational changes upon disulfide cleavage. The reduced, unfolded protein can refold to its native state. This reversible transition-monitored using 4 M urea PAGE-showed that the reduced form migrates more slowly due to increased hydrodynamic volume. Refolding begins as mercaptoethanol is dialyzed out, even in 8 M urea. Refolding does not occur in 6 M guanidine hydrochloride. Unlike unfolding, refolding is temperature independent; however, aggregation increases at elevated temperatures. Functional activity reappeared as confirmed by reducing sugar assay and product release chromatography. The inability to refold in the presence of mercaptoethanol, and the inability of carboxymethylated enzyme to refold, indicate that disulfide bond formation is essential for refolding. The identity of refolded and native protein was confirmed by: a) restoration of enzymatic activity, b) resistance to V8 protease, c) sedimentation coefficient analysis, d) hydrodynamic volume (gel filtration), and e) secondary structure analysis. In conclusion, xylanase from Humicola lanuginosa provides a valuable system for studying the acquisition and maintenance of protein folded states and the expression of catalytic function.