高产木聚糖酶菌株的诱变选育及酶学性质研究
摘要
用紫外线照射的方法对出发菌株Aspergillus niger M1进行处理后，从中筛选到了一株木聚糖酶活力比较高的突变株A. niger J506，传代10次后产酶能力仍然稳定性良好。
经过正交试验设计，得出突变株A. niger J506产木聚糖酶最佳的工艺条件为：主碳源浓度4％、麸皮与玉米芯的比例为5：5、辅加碳源葡萄糖的浓度是0.1％、辅加氮源草酸铵的浓度是2.0％，培养基初始pH为5.0，250ml三角瓶的装液量为100ml。
聚丙烯酰胺凝胶电泳研究发现，突变株和出发菌株的蛋白质种类有较大不同。尤其是在木聚糖酶谱带检测中发现，突变株发酵液中有三种类型的木聚糖酶，而出发菌株中只有两种类型的木聚糖酶，并且通过考马斯亮蓝G250染色和琼脂糖板上的透明圈发现，突变株中第三种类型的木聚糖酶不仅表达量很大，活力也很高。
出发菌株木聚糖酶最适作用pH是5.4；突变株木聚糖酶的最适作用pH是6.0。出发菌株在pH7.0到10.0之间木聚糖酶的稳定性较好，而突变株在pH6.0到10.0之间木聚糖酶的稳定性较好
突变株和出发菌株的木聚糖酶最适作用温度均为52℃；在20℃到50℃之间突变株和出发菌株木聚糖酶稳定性比较好，二者的半失活温度都在55℃左右。
The spores of A. niger M1, the initial strain, was treated by U.V. at morality of 90% . A mutant with high xylanase activity was screened ,naming A. niger J 506. The xylanase activity of the mutant kept stable after 10 generations.
After orthogonal designing experiment, the optimum fermentation conditions of A. niger J 506 were obtained, which is as followed: concentration of the major carbon resource 4 %, ratio between bran and corncob 5:5, concentration of glucose 0.1%, concentration of ammonium oxalate as supplemental nitrogen resource 2.0%, the initial pH of liquid medium 5.0, 100ml/250ml flask.
The PAGE revealed the culture supernatant of the initial strain and the mutant contained different protein bands, which exactly demonstrated at protein level that A. niger J 506 was surely a mutant of A. niger M1. Zymogram stained with xylan-Remazol brilliant blue for detecting xylanase showed there are three different xylanases in the mutant culture, while two xylanases in initial strain. What is important, the third xylanase in A. niger J 506 have higher activity and more production levels from PAGE and zymogram of xylanase.
Xylanases of the initial strain and the mutant have their optimum activity at pH 5.4 and pH 6.0 , respectively. They have better stability in the range pH 7.0-10.0 when incubated at various pHs at 45℃ for 2 hr. The two xylanases have maximal activity at 52℃ and have better stability up to 50℃ when incubated at various temperatures in pH 6.0 for 30min. Their temperatures for loss half activity in 30mm are 55℃ or so.
经过正交试验设计，得出突变株A. niger J506产木聚糖酶最佳的工艺条件为：主碳源浓度4％、麸皮与玉米芯的比例为5：5、辅加碳源葡萄糖的浓度是0.1％、辅加氮源草酸铵的浓度是2.0％，培养基初始pH为5.0，250ml三角瓶的装液量为100ml。
聚丙烯酰胺凝胶电泳研究发现，突变株和出发菌株的蛋白质种类有较大不同。尤其是在木聚糖酶谱带检测中发现，突变株发酵液中有三种类型的木聚糖酶，而出发菌株中只有两种类型的木聚糖酶，并且通过考马斯亮蓝G250染色和琼脂糖板上的透明圈发现，突变株中第三种类型的木聚糖酶不仅表达量很大，活力也很高。
出发菌株木聚糖酶最适作用pH是5.4；突变株木聚糖酶的最适作用pH是6.0。出发菌株在pH7.0到10.0之间木聚糖酶的稳定性较好，而突变株在pH6.0到10.0之间木聚糖酶的稳定性较好
突变株和出发菌株的木聚糖酶最适作用温度均为52℃；在20℃到50℃之间突变株和出发菌株木聚糖酶稳定性比较好，二者的半失活温度都在55℃左右。
The spores of A. niger M1, the initial strain, was treated by U.V. at morality of 90% . A mutant with high xylanase activity was screened ,naming A. niger J 506. The xylanase activity of the mutant kept stable after 10 generations.
After orthogonal designing experiment, the optimum fermentation conditions of A. niger J 506 were obtained, which is as followed: concentration of the major carbon resource 4 %, ratio between bran and corncob 5:5, concentration of glucose 0.1%, concentration of ammonium oxalate as supplemental nitrogen resource 2.0%, the initial pH of liquid medium 5.0, 100ml/250ml flask.
The PAGE revealed the culture supernatant of the initial strain and the mutant contained different protein bands, which exactly demonstrated at protein level that A. niger J 506 was surely a mutant of A. niger M1. Zymogram stained with xylan-Remazol brilliant blue for detecting xylanase showed there are three different xylanases in the mutant culture, while two xylanases in initial strain. What is important, the third xylanase in A. niger J 506 have higher activity and more production levels from PAGE and zymogram of xylanase.
Xylanases of the initial strain and the mutant have their optimum activity at pH 5.4 and pH 6.0 , respectively. They have better stability in the range pH 7.0-10.0 when incubated at various pHs at 45℃ for 2 hr. The two xylanases have maximal activity at 52℃ and have better stability up to 50℃ when incubated at various temperatures in pH 6.0 for 30min. Their temperatures for loss half activity in 30mm are 55℃ or so.
引文
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