Sagiroglu Ayten, Yavuz M Onder
Department of Chemistry, Trakya University, Faculty of Science and Letters, Edirne, Turkey.
Artif Cells Blood Substit Immobil Biotechnol. 2005;33(3):343-55. doi: 10.1081/bio-200066640.
Modified methylotrophic yeast Hansenula polymorpha (HP A16) was used in this study, which was obtained with recrossing a leucine oxotrophic yeast and a wild type Hansenula polymorpha CB4732 and was discovered by Ogata et al. The yeast is grown with methanol as a sole carbon source in which methanol oxidase (MOX) is a key enzyme of methanol metabolism. Because of its stability and low substrate specificity, alcohol oxidase is of considerable interest for a range of biotechnological processes. Various methanol feeding regimes were evaluated in an effort to increase the biomass concentration and productivity that could be achieved from fermentations using the other Hansenula polymorpha species. This yeast was grown by batch fermentation. The effects of conditions of inoculation media for increasing amount of MOX enzyme in peroksizomes of yeast with MOX activity were observed. The highest MOX activity of yeast was found within optic density of grown media of OD600 1.5, at 0.35 microM of methanol as used an oxotrophic substrate, at 35 degrees C temperature, at pH 7.0 of 0.1 M potassium phosphate buffer (KPB), at 40 microL of buffered yeast cell volume and at the incubation time of 50 minutes. Whole yeast cells were cultivated at above optimized incubation conditions. The cells were immobilized within the polyacyrlamide gels by entrapment method. Free whole cells and immobilized whole cells were compared using bioconversion percentages of methanol to formaldehyde.
本研究使用了经改良的多形汉逊酵母(HP A16),它是通过将亮氨酸营养缺陷型酵母与野生型多形汉逊酵母CB4732杂交获得的,由Ogata等人发现。该酵母以甲醇作为唯一碳源生长,其中甲醇氧化酶(MOX)是甲醇代谢的关键酶。由于其稳定性和低底物特异性,醇氧化酶在一系列生物技术过程中备受关注。为了提高使用其他多形汉逊酵母物种进行发酵所能达到的生物量浓度和生产力,对各种甲醇进料方式进行了评估。该酵母通过分批发酵培养。观察了接种培养基条件对具有MOX活性的酵母过氧化物酶体中MOX酶量增加的影响。在生长培养基的光密度OD600为1.5、以0.35 microM甲醇作为营养缺陷型底物、温度为35℃、pH值为7.0的0.1 M磷酸钾缓冲液(KPB)、缓冲酵母细胞体积为40微升以及孵育时间为50分钟的条件下,发现酵母的MOX活性最高。在上述优化的孵育条件下培养全酵母细胞。通过包埋法将细胞固定在聚丙烯酰胺凝胶中。使用甲醇转化为甲醛的生物转化百分比对游离全细胞和固定化全细胞进行了比较。