Hristozova Tsonka, Michailova Lilia, Tuneva Dimka, Gotcheva Velitchka, Angelov Angel, Roshkova Zlatka
Institute of Microbiology, Bulgarian Academy of Sciences, Plovdiv.
Z Naturforsch C J Biosci. 2002 Sep-Oct;57(9-10):858-62. doi: 10.1515/znc-2002-9-1017.
A mutant of the methylotrophic yeast Hansenula polymorpha with constitutive alcohol oxidase (AOX) and peroxisome biosynthesis was obtained after UV treatment followed by cell plating on a medium containing methanol and 2-deoxy-D-glucose (DOG). DOG-resistant colonies of mutants were insensitive to catabolic repression by glucose and methanol. A selection procedure is described that allows the isolation of a mutant exhibiting a constitutive phenotype of AOX involved in methanol utilization. Furthermore, additional features of the constitutive presence of peroxisomes are demonstrated. 562 DOG-resistant colonies were tested, 24 of them demonstrating constitutive AOX formation. Based on quantitative analysis, one of the strains--DOG-13 was selected and its growth, biochemical and ultrastructural characteristics were examined. Its specific enzyme activity when cultivated on a yeast nitrogen base + 1% glucose (YNB + 1% Glucose) was found to reach 145 nmol x min(-1) x mg(-1) protein (compared to zero of the parent strain) after he 20th hour of cultivation. This was confirmed by fine-structure analysis, showing typical peroxisomes, which number and size increased with the enzyme activity. This study demonstrates a constitutive AOX and peroxisome biosynthesis by the mutant strain H. polymorpha DOG-13 obtained.
在紫外线处理后，将细胞接种在含有甲醇和2-脱氧-D-葡萄糖（DOG）的培养基上，获得了多形汉逊酵母的一种突变体，该突变体具有组成型乙醇氧化酶（AOX）和过氧化物酶体生物合成能力。突变体的DOG抗性菌落对葡萄糖和甲醇的分解代谢阻遏不敏感。本文描述了一种筛选方法，该方法可分离出表现出参与甲醇利用的AOX组成型表型的突变体。此外，还证明了过氧化物酶体组成型存在的其他特征。对562个DOG抗性菌落进行了测试，其中24个显示出组成型AOX形成。基于定量分析，选择了其中一个菌株——DOG-13，并对其生长、生化和超微结构特征进行了研究。在酵母氮源+1%葡萄糖（YNB+1%葡萄糖）培养基上培养时，发现其在培养第20小时后比酶活达到145 nmol·min⁻¹·mg⁻¹蛋白质（亲本菌株为零）。精细结构分析证实了这一点，显示出典型的过氧化物酶体，其数量和大小随酶活性增加。本研究证明了所获得的多形汉逊酵母突变体菌株DOG-13具有组成型AOX和过氧化物酶体生物合成能力。
