Purification, characterisation and immobilisation of an acid-stable, raw-starch hydrolysing thiol β-amylase, over produced in the stem of Paederia foetida

Abstract

A 200 KDa acid-stable thiol β-amylase, appreciably present in the stem of Paederia foetida (48,000 ± 5,500 Units (U)/100 g fresh wt.) was purified by (NH4)2SO4 precipitation, ion exchange chromatography, size exclusion chromatography and HPLC. The enzyme was optimally active in pH 6.0 at 60 °C, showed a specific activity of 3466 U/mg protein and displayed a Km of 3.5 ± 0.1 mg/ml (soluble starch). Activity was stable in the pH range of 3.0–8.0, retaining 94 ± 1% activity at pH 3. The enzyme was stable up to 55–57 °C, beyond which the activity fell sharply. Hg2+ and Ag+ (1 mM concentration) completely inhibited the enzyme activity. Enzyme was strongly inhibited by DTNB, PCMB, N-ethylmaleimide, iodoacetic acid, iodoacetamide, and was experimentally determined to be a thiol amylase (3 SH group/mole); the DTNB inhibition of activity being released by cysteine. The enzyme efficiently hydrolysed potato starch (DE = 51), corn starch (DE= 46), gelatinised cereal flours (wheat, rice and gram), amylopectin, raw starch and raw cereal flours. The enzyme was determined to be a β-amylase (maltose as the only product) by end product analysis and its inability to hydrolyse β-limit dextrin. Immobilisation of the enzyme (crude) on oxidised bagasse (dialdehyde cellulose) increased the temperature optima (by 10 °C) and thermo-stability (retaining 48 ± 2% and 38 ± 1% activity at 70 and 80 °C respectively). The immobilised enzyme system efficiently produced maltose from cereal and tuber starches, remaining 85 ± 1% and 80 ± 1% active after 10th and 20th cycles respectively.