METHOD OF IMMOBILISED BETA-FRUCTOFURANOZIDASE Russian patent published in 2008 - IPC C12N11/00 C12N11/04 C12N11/12 C12N11/14 

FIELD: microbiology.

SUBSTANCE: method includes revival of pure culture Klyuveromyces marxianus Y-303 by Koch's method, seeding in Petri dish with 2% wort agar at temperature 48-50°C. Dishes are turned upside-down and kept for 2-3 days in thermostat at temperature 28-30°C. Grown colonies are moved to Petri dish with wort agar for biomass growth, then to bevels with wort agar. Submerged yeast cultivation is carried out in flask of capacity 500 cm, each containing 50 cm of beer wort during 47.5-48.5 h at temperature 30°C. Grown biomass is mixed with distilled water in ratio 1:5, autolysis is carried out at temperature 38-42°C during 59.5-60.5 h. Produced enzyme-containing mixture is deposited with ethyl alcohol at pH 5.0 or with acetone at pH 5,5. β-fructofuranozidase is purified within 3 steps: removal of low-molecular additives by gel filtration on column with sephadex G-25, increasing enzyme preparation purity by ion-exchange chromatography on column with DEAE-cellulose, fractionation on column with sephadex G-150. Concentration on membrane Amicon PM-30 is carried out up to 5 cm³, followed with lyophilisation at t=-30°C. β-ructofuranozidase is immobilised on polymer fibre sorbent (anionite). Before immobilisation, sorbent is processed with 5% hydrochloric acid, distilled water, 5% caustic soda solution and water again. Each process step takes 12 h. Washed sorbent is additionally processed with 95% ethyl alcohol and water again. Aqueous solution of β-fructofuranosidase is made of content 0.018-0.098 mole/dm³, processed sorbent is incubated. Method enables to produce immobilised enzyme in amount 0.005-0.025 mg/g.

EFFECT: provided purity of end product.

2 tbl, 3 ex