Aqueous two-phase extraction and characterization of thermotolerant alkaliphilic Cladophora hutchinsiae xylanase: biochemical properties and potential applications in fruit juice clarification and fish feed supplementation.

Xylanase finds extensive applications in diverse biotechnological fields such as biofuel production, pulp and paper industry, baking and brewing industry, food and feed industry, and deinking of waste paper. Here, polyethylene glycol (PEG)-phosphate aqueous two-phase system (ATPS) was applied for the purification of an alkaline active and thermotolerant xylanase from a marine source, Cladophora hutchinsiae (C. hutchinsiae). In the purification process, the effects of some experimental factors such as PEG concentration and PEG molar mass, potassium phosphate(K₂HP0₄) concentration, and pH on xylanase distribution were systematically investigated. Relative enzymatic activity and purification factor obtained were 93.21% and 7.18, respectively. A single protein band of 28 kDa was observed on SDS-PAGE. The optimum temperature and pH of xylanase with beechwood xylan were 30 °C and 9.0, respectively. The Lineweaver-Burk graph was utilized to determine the K_m (4.5 ± 0.8 mg/mL), V_max (0.04 ± 0.01 U) and k_cat (0.001 s⁻¹) values of the enzyme. It was observed that the purified xylanase maintained 70% of its activity at 4 °C and was found stable at pH 4.0 by retaining almost all of its activity. Enzymatic activity was slightly enhanced with Na⁺, K⁺, Ca²⁺ and acetone. The highest increase in the reducing sugar amount was 53.6 ± 3.8, for orange juice at 50 U/mL enzyme concentration. [ABSTRACT FROM AUTHOR]