Screening of ?-Rhamnosidase High-yield Strain from Aspergillus niger / 微生物学通报

In this study, plate transparent circle by Davis method was introduce firstly screening ?-Rhamnosidase high-yield strain. The spore-sprouted Aspergillus niger 8-hour were mutagenized by ethyl methane sulphonate and pre-screened via transparent circle. 11% mutants yield 40% higher of ?-rhamnosidase than the original strain. A high-yield strain, T-226 with the highest ?-rhamnosidase activity of 373.4 U/mL was finally selected from these potential high-yield mutants after rescreened by shake flask fermentation twice. When the T-226 strain was fermented in 5 L bioreactor, the enzyme activity could reach to 631.9 U/mL after 84 h. Thus, the established screening method is highly efficient to isolate ?-rhamnosidase high-yield mutant of A. niger.

Detection of intracellular reactive oxygen species by flow cytometry in Pichia pastoris fermentation / 生物工程学报

In Pichia pastoris fermentation, methanol was oxidized into carbon oxide and produced a byproduct H2 O2, one of the partially reduced forms of molecular oxygen known as reactive oxygen species (ROS) . ROS are highly damaging towards cellular constituents. Flow cytometry (FCM) is an excellent method that permits the rapid, optical analysis of individual cells and has many advantages over conventional cytometry. However, its use in detecting intracellular ROS levels during Pichia fermentation was rarely reported. In our work, by means of flow cytometry, two fluorescent dye 2', 7'-dichlorofluorescin diacetate (DCFH-DA) and propidium iodide (PI) were used to detect ROS. The effect of intracellular ROS on Pichia pastoris cells during fermentation was studied through the comparison between DCFH-DA/PI double-stained cells and PI single-stained cells. In this study, the loss of cell viability during fermentation was correlated with the accumulation of ROS. At the glycerol batch and fed-batch phase, little ROS was accumulated intracellularly and cell viability reached almost 100%. At the early methanol fed-batch phase, intracellular ROS accumulation took place but 98.5% cells still kept viable. At the later methanol fed-batch phase, 94.0% cells accumulated high ROS. As a result, some cells lost their viability because of the damage of ROS. 25.4% dead cells accumulated high ROS in the total 29.1% dead cells.

Application of Flow Cytometry in Viability Detection of Pichia pastoris Cells / 微生物学通报

Cell viability of Pichia pastoris was detected by flow cytometry (FCM) with two reagents fluorescein diacetate (FDA) and propidium iodide (PI). Compared with FDA/PI double-stained dot plots and PI single-stained dot plots,the latter could divide dead and living cells into two separate zones,and get the correct proportion. Then PI single-stained method was used to detect the change of cell viability in Pichia patoris fermentation. At glycerol batch and fed-batch phase,little dead cells were detected. At methanol fed-batch phase,cell viability decreased when cell weight increased,and was only 73.8% at 88 h.