Study On The Thermostable Lactase

To produce low-lactose production with lactase, this can effectively solve the problem of "lactose intolerance". Lactase from thermophilic bacteria is heat-stable and which has forte of high conversion rate, low pollution, et al. This thesis was designed to filter lactase production of thermophilic strains, and then classify and identify it, to study its fermentation conditions for enzyme production, enzyme isolation and enzymatic properties. It is to establish the foundation for the thermostabile lactase gene cloning and expression. The main results were as following:The strain T242was isolated from22samples as the thermostable P-galactosidase producer. Study on cell characteristics, colony morphology, physiological and biochemical identification of bacteria and other traditional identification, as well as the16S rRNA gene sequence similarity comparison and phylogenetic analysis of modern molecular biology to identify, and ultimately determine the strain T242are Bacillus coagulans. Strains T242and Bacillus coagulans have a homology of99%.Thermophilic strain T242produces intracellular thermostable lactase which hydrolyzes lactose to glucose and galactose. The study found that high concentrations of NaCl combined with lysozyme on strain T242broken wall best rules of the worst short-term freeze-thaw, the former activity is8times the latter.Through single-factor analysis of thermophilic strain T242nutritional and fermentation conditions optimize the enzyme production conditions. The best nutritional conditions for enzyme production for:1.5%lactose,1%yeast extract,1.5%peptone,0.7%MgSO4. The best fermentation conditions for enzyme production are as follows:initial pH8.0, inoculation for2%, culture medium volumes for30mL/250mL flask, the seeds age for36h, cultivated at60℃for36h. Then the maximum yield of1.21U/mL was obtained.The thermostable lactase from strain T242was separated and depurated preliminarily. Obtained four protein peaks after separated by DEAE-52(2×10cm, gradient elution:0～0.4mol/L NaCl, flow rate:0.5mL/min,3mL per tube). The enzyme activity of the first peak was4.16U/mL and the second was1.89U/mL. Select peak I for enrichment and dialysis by enzyme activity and protein peak. The sample recovered from the peak I was further purified with Sephadex G-75column chromatography (1.64×70cm,50mmol/L pH6.8buffer phosphate;1mL per tube;0.083mL/min), obtained two protein peaks, and the first peak had a enzyme activity. The sample with enzyme activity after dislysis and enrichment had a clear band on a SDS polyacrylamide gel electrophoresis, and its molecular mass was estimated to be about50kDa.Study on Enzyme property and reaction kinetics,the result is follows:the optimal temperature is60℃; it had good thermostability between40℃to60℃; the optimal pH was6.8and it was stable at pH6.4-7.5; When the metal ions concentration of lmmol/L, Fe2+and Zn2+had inhibition on lactase and Mn2+, Ca2+, Mg2+, K+, Na+had no inhibition or activation. When the metal ions concentration of10mmol/L, Mn2+, Mg2+, Na+had high activation and Ca2+, Zn2+, K+had high inhibition on lactase at high density. The maximum speed of reaction is0.146mmol/L·min and the Km is20.060mmol/L.