| The enzyme beta~D-galactosidase(beta-D-galactoside galactohydrolase, EC 3.2.1.23, also referred to as lactase)hydrolysis the disaccharide lactose to glucose and galactose. It is widely distributed various microorganisms such as bacteria, fungi, and also in various plants and animals intestine. Lactase can be used for treatment of "lactose intolerance", and production of low-lactose milk, whey and other dairy products. Although lactase has important applications in the fields of medicine, dairy industry, some problems obstructs large-scale industrial production of lactase. Main problems ate: (l)Low production and Enzymatic activity of lactase; (2)Commercial lactase mainly derived from yeasts(Kluyveromyces lactis <. Kluyveromyces fragilis) with low thermostability and narrow pH range; (3)An intracellular lactase resulting in the high cost due to extraction and purification processes.The purposes of our research are: (1) Screening lactase-yielded strain with superior enzymatic characteristics, identifying and isolating the lactase gene from the microorganism. (2) Enhancing production of lactase and reducing production cost via constructing high effective bioreactor of Pichia pastoris which can express and secrete lactase effectively.The results obtained as follows:1. A lactase-yielded strain Aspergillus candidus is screened. Genomic DNA and cDNA sequences of lactase from Aspergillus candidus are cloned. Sequences analysis reveals that the genomic DNA with the length of 345 8bp containing eight introns, cDNA with the length of 3015bp encoding a polypeptide of 1005 amino acid residues. Signal peptide is consisted of 19 amino acid residues. 11 potential N-glycosylation sites are assumed. Comparing the gene cDNA and the deduced amino acid sequences with the lactase sequences from various organism sources shows a very low homology, and the highest homology of cDN A and the deduced amino acid sequence are observed between the lactase of Aspergillus candidus and from Aspergillus oryzae ATCC 20423 which used as control strain. Homology of cDNA sequence is 99%, and homology of the deduced amino acid sequence is 99.5%. Although only 3 amino acid residues aredifferent in the lactase from A. candidus2, 1(A. oryzae P (A. candidus); 420, l(A. ory~ae)^M(A. candidus); 989, N(^. oryzae-℃D(A. candidus)}, the characteristics of the enzyme exhibits obvious difference from that of A. oryzae. The lactase gene from Aspergillus candidus is a new gene for food and diary industry.2. The lactase from Aspergillus candidus shows optimum pH of 5.2, optimum temperature of 60℃, and the stable pH ranging from 3.0 to 9.0. Specific activity is 706.54U/mg. The Km is 1.67mmol/L, Fraax is 3.33umol/L with ONPG as a substrate. The enzymatic characteristics of Aspergillus candidus is superior in many aspects to that of Aspergillus oryzae ATCC 20423 including the thermostability, specific activity, metal ion stability, Km, and pH stable range.3. Lactase gene from Aspergillus candidus is expressed in E. coil and Pichia pastoris. By SDS-PAGE, the molecular weight of lactase expressed in E. coil is estimated to be about 11 OkD according with calculated molecular weight. The molecular weight of lactase protein expressed in Pichia pastoris is about 120-13 OkD probably due to glycosylation. Lactase from Aspergillus candidus is expressed in Pichia pastoris efficiently. High-level secreted lactase is about 6g/L with enzymatic activity of 3600U/mL. Therefore, the research open a new and convenient avenue for high-yielded lactase production.4. 3000U and 1500U of lactase is added separately in lOmL milk and the lactose content in milk by HPLC at the condition of various temperature (12℃ > 37 ℃ > 50 ℃ .. 60 ℃) and various incubated times is determined. The result shows, (1) lactose hydrolysis rate under the condition of 3000U lactase is higher than that of 1500U at the same temperature and incubated tune. (2) The lactose hydrolysis rate is higher at the higher temperature than that of lower temperature at the same incubated time. The...
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