【作者】 杜海英；

【导师】 贾英民；

【作者基本信息】 河北农业大学 ， 微生物学， 2006， 硕士

【摘要】 本论文对产高温乳糖酶的黑曲霉菌株UCo-3的原生质体形成与再生的最佳条件进行了研究,在最佳条件下用紫外线、⁶⁰Co-γ,射线对其原生质体进行复合诱变处理,得到产高温乳糖酶活力较高的菌株黑曲霉DL116,并对该菌株的培养基和发酵条件及产酶机制进行了研究。 对黑曲霉UCo-3原生质体形成与再生条件进行了研究。对菌龄、酶浓度、酶解温度、酶解时间四因素进行正交试验,确定原生质体形成与再生的最佳条件。结果表明:采用1.5%的纤维素酶与0.75%的蜗牛酶的混合酶液,菌龄为16h,酶解温度为30℃,酶解时间为3h,用0.6mol/L KCl溶液作稳渗剂,在此条件下原生质体形成数为3.05×10⁶个/mL,再生率为2.04%。 对诱变程序与步骤进行了研究,确定了2种诱变剂的辐射剂量、致死率与正突变率的相互关系,确定了对黑曲霉菌株UCo-3的原生质体进行诱变的技术参数:使用功率为18W的紫外灯管,照射距离为30cm,照射时间为4min,⁶⁰Co-γ射线辐射剂量为500Gy。 对UCo-3菌株进行1次紫外线和2次⁶⁰Co-γ,射线诱变,获得一株产高温乳糖酶的高产黑曲霉突变株DL116。对其进行菌种传代产酶性能试验,结果证明突变株DL116经传代10次,乳糖酶活力稳定在44.37U/mL左右,证明其遗传性能稳定。 通过对高产乳糖酶突变株DL116进行菌落形态观察,足细胞和分生孢子头显微观察发现,突变株菌落形态较原菌株UCo-3略有改变,菌落呈整齐圆形年轮状,菌落背面呈现明显的辐射性皱褶。将突变株编号为Aspergillus niger DL116。 对产高温乳糖酶的高产突变株DL116进行产酶研究,发现果胶对乳糖酶的产生具有明显的诱导作用,可显著提高酶的表达量。 对黑曲霉菌株DL116的发酵条件进行了研究,确定其最佳培养基成分及最佳培养条件。研究了不同碳源、氮源、无机盐、表面活性剂等因素对黑曲霉DL116产酶的影响,发现果胶、豆粕粉、玉米浆、硫酸铵浓度对产酶影响较大,且无机氮源与有机氮源存在一定的协同效应。对果胶、豆粕粉、玉米浆、硫酸铵四因素进行正交试验,确定黑曲霉DL116产酶的最佳培养基成分为果胶2.5%,豆粕粉6.0%、玉米浆9.0%、硫酸铵0.1%、K₂HPO₄ 1.0%、Tween-80 0.3%。通过摇瓶发酵试验,确定最佳培养条件为:摇床30℃,200 r/min,发酵培养基的起始pH为5.0,接种量为孢子浓度10⁴个/mL,装液量为30 mL/250mL,培养8.5d。在此条件下培养,β-半乳糖苷酶酶活力达98.8 U/mL。是出发菌株UCo-3的6.07倍。更多还原

【Abstract】 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.更多还原