de Roubin M R, Bastien L, Shen S H, Groleau D
Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec.
J Ind Microbiol. 1991 Oct;8(3):147-56. doi: 10.1007/BF01575847.
Various physico-chemical parameters have been studied in order to improve the production of hepatitis B virus pre-S2 antigen (middle surface antigen) by the methylotrophic yeast Hansenula polymorpha. Antigen production was done in two steps: first, production of cells on glycerol (Phase 1), followed by induction of antigen expression with methanol (Phase 2). Dense cultures of H. polymorpha, equivalent to 35-40 g/l (dry weight), were readily obtained in small fermenters using minimal medium containing glycerol as carbon source. Antigen expression in this minimal medium, after induction with methanol, was however, low and never exceeded 1.6 mg/l of culture. Antigen production was greatly enhanced by adding complex organic nitrogen sources along with methanol at induction time; yeast extract was the best of all the sources tested. In shake flasks, antigen production was proportional to yeast extract concentration up to 7% (w/v) yeast extract, it became clear the the nutritional conditions for good antigen expression were different from those for good biomass production. The effects of yeast extract were reproduced in small fermenters: antigen levels reached 8-9 mg/l in medium containing 6% (w/v) yeast extract during induction with methanol. The mechanisms of yeast extract's effects are still unknown but are probably nutritional. The recombinant H. polymorpha strain produced both periplasmic and intracellular antigen. The periplasmic antigen was shown to be present as 20-22-nm particles and was therefore immunogenic. Immunoblotting indicated that part of the pre-S2 antigen was present as a 24-kDa degradation product. These studies have led to a 140-fold increase in volumetric productivity of antigen and to a 4.6-fold increase in specific production.
为了提高多形汉逊酵母生产乙型肝炎病毒前S2抗原（中表面抗原）的产量，研究了各种物理化学参数。抗原生产分两步进行：首先，在甘油上培养细胞（阶段1），然后用甲醇诱导抗原表达（阶段2）。使用含有甘油作为碳源的基本培养基，在小型发酵罐中很容易获得相当于35-40 g/l（干重）的多形汉逊酵母高密度培养物。然而，在用甲醇诱导后，这种基本培养基中的抗原表达很低，从未超过1.6 mg/l培养物。在诱导时添加复合有机氮源和甲醇可大大提高抗原产量；酵母提取物是所有测试来源中最好的。在摇瓶中，抗原产量与酵母提取物浓度成正比，直至酵母提取物浓度达到7%（w/v），很明显，良好抗原表达的营养条件与良好生物量生产的营养条件不同。酵母提取物的作用在小型发酵罐中得到了重现：在用甲醇诱导期间，含有6%（w/v）酵母提取物的培养基中抗原水平达到8-9 mg/l。酵母提取物作用的机制仍然未知，但可能是营养方面的。重组多形汉逊酵母菌株产生周质和细胞内抗原。周质抗原显示以20-22纳米颗粒形式存在，因此具有免疫原性。免疫印迹表明，部分前S2抗原以24 kDa降解产物形式存在。这些研究使抗原的体积生产力提高了140倍，比生产率提高了4.6倍。
