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 K_m 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.

Keywords:

• Paederia foetida
• thiol β-amylase
• bagasse-cellulose dialdehyde
• raw starch hydrolysis
• gelatinised cereal flour

Disclosure statement

The research work was not funded or supported by any funding agency or grants and this is to acknowledge that no financial interest or benefit has arisen from the direct applications of my research. This Research article has met all the ethical guidelines.