Identification Of Strain Producing Cold-adapted Lactase And Purification, Characterization Of The Lactase

One cold-adapted lactase producing strain 14-1 was isolated from its low temperature environment in XINJINAG province. The strain was identified by studying its characteristics of morphology, cultivation, physiology and biochemistry and 16S rDNA sequence alignment. The results showed that the strain belongs to Rahnella aquatilis.Shake-flask culture conditions of cold-adapted lactase were studied with psychrotrophic strain14-1. The fermentation conditions were optimized: lactose concentration 2.5%, initial pH 6.5, seed inoculation volume 3%, 500 mL flask contained 100mL medium. The maximumβ-Galactosidase activity, about 4.51 U·mL^-1 was obtained at 20℃when cultured for 48 hours.The lactase was intracellular enzyme,so the cell pellets were disrupted with a sonfier sonicator.The crude enzyme was purified by column chromatography and a single band was obtained on SDS-PAGE. Apparent molecular weight of subunits of the purified enzyme estimated by SDS-PAGE were about 60 KDa.The specific activity of crude enzyme was increase up to 321.7 U·mg^-1 with purifying fold of 4.19 and the yield was 25%.Research about the properties of the enzyme showed that the optimal temperature was 35℃, and the optimal pH value was 6.5 to 7.0 for the enzyme with ONPG as a substrate. The enzyme activity was sensitive to temperatures above 40℃and was undetectable after 2h incubation at 45℃. The metal ions Mn²⁺, K⁺ activated the enzyme activity while Cu²⁺, Zn²⁺ , Fe³⁺ and Al³⁺ inhibited activity.When milk was treated with the lactase at 35℃for 2 hours, the degree of hydrolysis for lactose can reached 80%.