Mutagenesis Of Rahnella Sp.R3and Purification, Characteirzation Of Its Cold-adapted Lactase

β-Galactosidase （EC3.2.1.23） is commonly known as lactase, has been widely appliedin hydrolysis of lactose in dairy products. The treated products with improved quality aremore easily digested by individuals suffered with lactose intolerance. Cold-adapted lactasehas high catalytic activity at low temperatures（0~4℃）, therefore it is capable of hydrolyzinglactose in dairy during processing and storage with advantages. Firstly, treating foodstuffsunder mild conditions can maintain taste and nutritional values unchanged. Secondly, enzymecatalyzed reaction at low temperatures can suppress the growth of mesophilic microorganismsto improve the dairy safety. In addition, the enzymatic action can be inhibited at a certainpoint by a moderate increase of temperature during processing, then the flavor of food can beunified.In order to obtain an industrial strain with higher cold-adapted lactase production, theoriginal strain of Rahnella sp.R3was mutated by atmospheric and room temperature plasmas.The methods used to screen the mutant strain with higher cold-adapted lactase productionincluded selective medium with X-gal and shaking flask fermentation. The enzymaticproperties of cold-adapted lactase from different mutants were comparatively analyzed byactivity assay. It has been demonstrated that mutants RA300-2, RA300-6and RA300-9maintain good genetic stability. The enzyme activity of three mutants increased by48.9%,36.9%and32.6%comparing with the original strain, respectively.The enzyme from Rahnella sp.R3was purified in sequence by ammonium sulfateprecipitation, hydrophobic chromatography, Q Sepharose High Performance and SephacrylS-200High Resolution and proved by the single band in SDS-PAGE. The enzyme waspurified by354.4folds with a yield of21.3%and Mwof57.3KDa. The cold-adapted lactasefrom mutants RA300-2was purified according to the same method.Enzymatic properties of cold-adapted lactase from Rahnella sp.R3and mutant RA300-2were investigated. The cold-adapted lactase from Rahnella sp.R3was maximally active at45℃, pH6.5～7.5. The cold-adapted lactase from mutant RA300-2was maximally active at35℃, pH6.5～7.5. They were thermal-sensitive and undetectable after incubation at45℃for45min and55℃for15min respectively. Metal ions have similar effects on them. Some metalions such as Ca2+, Cu2+, Al3+and Zn2+reduced enzymes activity, and Al3+strongly inhibitedenzymes activity. Whereas, Mg2+and K+activated enzyme. Kmand Vmaxof Rahnella sp.R3forONPG at25℃was found to be4.64mmol/L and7.19mol/（min·mL）, respectively.