Screening And Mutation Breeding Of A Strain Producing Cold-adapted Lactase And The Study On Optimization Of Fermentation Conditions And Its Crude Enzymatic Properties

Lactase, named β-galactosidase （EC3.2.1.23） which not only can hydrolyze the β-D-1,4-galactosidic linkage but also has transglycosylation activity, plays an important role in food,medicine, analytical chemistry and other fields. Cryogenic lactase has high activity at lowtemperatures. Lactose is the main carbohydrate in milk and its content is about4.5%to5%[1].However, some people are in lack of lactase. Intake of milk or dairy products causes bowel,abdominal pain and diarrhea, which names lactose intolerance[2]. Using the lactase to producelow-lactose products can solve this problem effectively which is also the one of the largestpurpose of lactase in the food industry. Most of the dairy processing operations are carried outat low temperature （4°C）[5]. Cold-adapted lactase does hydrolyze lactose of milk significantlyat low tempreture and has little influence on products. A strain producing cold-adapted lactasehas been isolated and identified from the frozen soil of Tianshan Mountains. The yield ofcold-adapted lactase was enhanced by UV and HNO2treatment. The conditions that producingof cold-adapted lactase were optimized. The properties of enzyme was researched. The resultswere reported as follows:With three-stage screening, one cold-adapted lactase producing strain R3was isolatedfrom Tiansan mount permafrost in the Xin jiang uygur Autonomous Region. The lactaseproduced by strain R3was intracellular enzyme. The strain was identified by studying itscharacteristics of morphology, cultivation, physiology and biochemistry and16S rDNAsequence alignment. The results showed that the strain belongs to Rahnella sp. and namedRahnella sp.R3.By UV radiation mutation and nitrous acid mutagenesis methods to the parental strainRahnella sp.R3, the results showed that optimal conditions for complex mutation was asfollows: using30W uviolized100s, then treated with0.10mol/L nitrite for2minutes. A highyield mutant was obtained and named UN-9. The enzyme activity of UN-9was4.06U/mL,which increased32%compared with the original strain. Genetic stability test of strain UN-9showed that the relative activity was more than98%in10generations.Medium composition and fermentation conditions were optimized by single factor andorthogonal experiments. The optimum medium composition （g/L） was lactose40g/L,peptone20g/L, yeast extract12g/L and MgSO42g/L. The optimum conditions wereinoculation time24h, inoculation volume4%, liquid volume50mL/250mL, initial pH7.0±0.2, incubation time32h under15°C. Under the optimum conditions, the enzymeactivity reached4.51U/mL.The lactase was purified by ammonium sulfate precipitation step by step. Research onthe enzymatic properties showed that the lactase was maximally active at25°C and pH7.0,and it had higher enzyme activity in the low temperature. The enzyme recovery was92%at15°C and31%at4°C, respectively. The enzyme activity was thermal-sensitive and wasundetectable after1h incubated at65°C．Some metal ions had shown inhibiting effect onenzyme activity. Ca²⁺and Na⁺slightly reduced enzyme activity while heavy metal ions,particularly Cu²⁺、Al³⁺and Zn²⁺strongly inhibited enzyme activity. When milk was treated with the cold-adapted lactase of the strain UN-9, the resultsshowed that the hydrolysis rate of the lactase reached82%.