【作者】 赵明明；

【导师】 陈珊；

【作者基本信息】 东北师范大学 ， 微生物学， 2005， 硕士

【摘要】 β-羟基丁酸与β-羟基戊酸的共聚酯（PHBV）是某些细菌在非平衡生长阶段体内碳源和能源的贮藏物质,它作为一种新型的可生物降解材料,继聚β-羟基丁酸酯（PHB）后越来越引起人们的关注。 PHBV 的降解依赖于许多细菌和真菌分泌的一种胞外酶,它能水解聚合物生成水溶性的产物。本论文工作的主要目的是通过紫外诱变筛选出能高效降解 PHBV 的突变菌株,分离纯化 PHBV 解聚酶,并对 PHBV 解聚酶的酶学性质进行比较研究。主要结果如下: 1. 以青霉（Penicillium sp.）DS9702-D10 为出发菌株,通过紫外线诱变分生孢子,采用透明圈初筛和摇瓶培养复筛的方法获得 5 株能稳定遗传的分泌 PHBV 解聚酶的高产菌株,其中 06 PHBV 解聚酶的酶活是原始菌株 PHBV 的 5.6 倍,H2 PHBV 解聚酶是原始菌株的 5.38 倍。 2. 测定 5 株突变株每日 PHBV 解聚酶酶活和蛋白含量,其中 11、19 突变株的酶活最高峰出现在 72h,H2 突变株出现在 96h,05、06 突变株出现在 120h。11、19、H2、05和 06 突变株 PHBV 解聚酶酶活分别是原始菌株 PHBV 解聚酶的 2.75、1.55、5.38、4.37和 5.6 倍。 3. 对原始菌株、06 突变株和 H2 突变株的 PHBV 解聚酶粗酶液的基本性质进行了对照研究。06 突变株和 H2 突变株的 PHBV 解聚酶粗酶液为 40℃和 50℃,分别相对于原始菌株 PHBV 解聚酶粗酶液最适反应温度提高了 20℃和 10℃,06 突变株 PHBV 解聚酶粗酶液在 40℃～60℃范围内稳定,H2 突变株 PHBV 解聚酶粗酶液在 30℃～60℃稳定,较原始菌株 PHBV 解聚酶粗酶液的温度稳定范围有所提高。原始菌株 PHBV 解聚酶粗酶液的最适 pH 值为 7.0,经紫外诱变所产生的 06 突变株 PHBV 解聚酶粗酶液最适 pH 值为 7.38;H2 突变株 PHBV 解聚酶粗酶液的最适 pH 值为 6.98 和 8.04 两个峰,酶活在此条件下较原始菌株 PHBV 解聚酶的酶活力有所提高。 4. 06 突变株 PHBV 解聚酶纯化倍数为 33.81,酶活回收率为 13.57%,酶蛋白相对分子质量为 28KD,最适反应温度为 60℃,最适反应 pH 值为 7.38,在 40℃～50℃,pH 为6.98～7.15 范围内较稳定;H2 突变株 PHBV 解聚酶纯化倍数为 39.28,酶活回收率为3.27%,酶蛋白相对分子质量为 44KD,最适反应温度为 50℃,最适反应 pH 值为 8.04,在 40℃～50℃,pH 为 7.6～8.4 范围内较稳定。Na⁺、Ca²⁺对上述两种酶的影响较弱,而 Fe²⁺,Zn²⁺,,Cu²⁺对两酶影响较大,使酶失活。质谱仪测得酶降解 PHBV 的产物均为单体,即 β-羟基丁酸。更多还原

【Abstract】 Poly(β-Hydroxybutyrate-co-β-Hydroxyvalerate) (PHBV) are biodegradable polysters that are synthesized and accumulated intracellularly during unbalanced growth by some bacteria. It has attracted much commercial and academic interest as a kind of new biodegradable material. The ability to d egrade PHBV is widely distributed among b acteria and fungi, and depends o n the secretion of specific extracellular PHBV depolymerase which can hydrolyse the polymer to water-soluble products. The aim of this work is to select the mutant with the treatment of UV, which can degrade PHBV efficiently, to purify PHBV depolymerase and to study the fundamental characteristics of PHBV depolymerase. The main results obtained from this work are as follows: 1. The pinicillium sp. DS9702-D10, a strain of degrading PHBV, was mutagenized by UV treatment. Through screening a lot of mutants with the method of transparent zones and culture filtrate, best five ones were obtained with high –yield of stable PHBV depolymerase, named as 05,06,11,19 and H2. The enzyme activity of the06 and H2 were 5.6 and 5.38 times of the original one. 2. The enzyme activity and the protein of the five muntants were surveyed with their crude enzyme, the highest enzyme yielding of them were found in 72h of 11, 19 ; 96h of H2 and 120h of 05,06. The activity of 11、19、H2、05、06 were 2.75;1.55;5.38;4.37;5.6 times of the original one. 3. Comparative study among the crude extracts of 06、H2 mutants and the original one。The optimum temperature of 06 was 20℃ higher and H2 was 10℃ higher than the original one, the range of temperature stability of 06 was from 40℃ to 60℃ and that of H2 was from 30℃ to 60℃, but the original one was from 30℃ to 50℃.The optimum pH of 06 was 7.38 and that of H2 was 6.98 and 8.04. It was different from the original one which was 7.0. Moreover, the range of pH stability of 06 and H2 was also much better. 4. The extracellular PHBV depolymerase was selected and purified from 06 and H2 mutants by using filtration、C2H5OH precipitation、gel filtration technique in Sephrouse CL-6B.The activity of the purified enzyme was increased by 33.81 folds and the recovery yield was 13.57% of 06. The molecular mass of 06 was 28KD and the optimum activity of enzyme was observed at the temperature 60℃ and at pH 7.38. Its range of temperature stability was 40℃~50℃ at pH 6.24~6.64; The activity of the purified enzyme was increased by 39.28 folds and the recovery yield was 3.27% of H2. The molecular mass of H2 was 44KD and the optimum activity of enzyme was observed at the temperature 50℃ and at pH 6.98 or 8.04. Its range of temperature stability was 40℃~50℃ at pH 7.6-8.4. some metal ions could activate or inhibit the two PHBV depolymerase activity and the hydroxyl-products with PHBV depolymerase were both monomer 3-hydroxybutyrate acid, analyzed by using mass spectrometer.更多还原