Study On Selected Breeding Of High-Yielding Strains And Mechanism Of Producing The Thermostable Lactase

The protoplast of Aspergillus niger which produces the thermostable β-galactosidase was compoundly treated with UV irradiation and ⁶0Co γ-ray. The mutant strain DL116 with high β-galactosidase production was isolated. The culture medium and fermented condition and mechanism of producing lactase were investigated in this paper.The formation and regeneration of the protoplast from Asperigillus niger UCo-3 was studied. The influence of mycelial age, enzyme system, enzymolysis time and temperature on the preparation and regeneration of Aspergillus niger UCo-3 protoplasts were studied by orthogonal design. The results were as follows: the mycelia were cultured for 16 hours, then enzymelysed for 3 hours at 30℃ with 1.5% cellulase and 0.75% snailase, using 0.6mol/L KC1 as the osmotic pressure stabilizer. Under these conditions, the amount of protoplasts reached 3.05xl0⁶/mL while the regeneration frequency was 2.04%.After studying the conditions of mutagenesis, the relations among the radiation does, death rate and positive mutation rate of two mutagens were obtained. The optimized mutation conditions: the irradiation distance was 30cm, the time of irradiation was 4 min, with 18W ultraviolet lamp, and the dose of ⁶0Co γ-ray was 500 Gy.Aspergillus niger high-yield mutant DL116 producing thermostable β-galactosidase was screened through UV and ⁶0Co γ-ray and the lactase activity of it was 44.37 U/mL. The test on hereditary stability and capability of producing lactase was carried out and the result indicated that the lactase activity of the mutant was still around 44.37 U/mL 10 generations later. This verified that its inheritance was stable.After observation on the colony, foot cell and conidium of mutant strain DL116, it was found that the regularly ring-shaped colony of the mutant strain was somewhat different from that of its parents. The back of colony had the obvious radiating gauffers. It was named Aspergillus niger DL116.The researches on lactase producing conditions showed that pectin had an obvious inductive function on producing lactase.The fermentation conditions of A. niger DL116, including carbon source, nitrogen source, inorganic salt, surface active agent and so on were investigated. It was found that the concentrations of pectin, soybean powder, corn liquor, (NH₄)₂SO₄ had the more obvious influences. The orthogonal test of the four factors showed that the optimized culture medium was: pectin 2.5%, soybean power 6%, corn liquor 9%, (NH₄)₂SO₄ 0.1%, K2HPO4 1%, and tween-80 0.3%. Through shake flask experiments, it was acquired thatthe best culture conditions: pH 5.0, 30°C, 200 r/min, 30 mL/250 mL, and 104 spores per mil inoculum quantity. After optimization, the /?-galactosidase activity reached 98.8 U/mL, which was 6.01 times of the initial strain UCo-3.