High-yield production of the human lysozyme by Pichia pastoris SMD1168 using response surface methodology and high-cell-density fermentation

Background Lysozyme plays a crucial role in innate immunity with its well-recognized bacteriolytic activity. In this study, the influence of expression parameters (inoculation volume, culture volume, growth time, induction temperature and time, initial pH and methanol concentration) on human lysozyme (HLZ) production in recombinant P. pastoris SMD1168 was investigated through Plackett-Burman (PB) design and response surface methodology (RSM). Results It was revealed that induction temperature, induction time and culture volume had significant influence (P < 0.01) on HLZ expression level, which were elected for further optimization with three-dimensional response surface designs for enhanced HLZ production. The highest lysozyme activity reached 3301 U/mL under optimized conditions (at 23.5°C for 90 h with culture volume of 48 mL) in shake flask, which increased 2.2 fold compared with that achieved with the standard protocol (Invitrogen). When high-cell-density fermentation of the recombinant Pichia pastoris was performed in a 15 L fermenter under optimized conditions, the extracellular lysozyme activity reached 47,680 U/mL. SDS-PAGE analysis of the product demonstrated that HLZ was produced as a single major protein with a molecular weight of approximately 14.7 kDa, consistent with its expected size. Conclusions The results indicated that the optimized culture conditions using PB design and RSM significantly enhanced the expression level of HLZ, and the Pichia expression system for HLZ production was successful and industrially promising.

Functional analysis of the GAF domain of NifA in Azospirillum brasilense: effects of Tyr-->Phe mutations on NifA and its interaction with GlnB.

Regulation of NifA activity in Azospirillum brasilense depends on GlnB (a PII protein), and it was previously reported that the target of GlnB activity is the N-terminal domain of NifA. Furthermore, mutation of the Tyr residue at position 18 in the N-terminal domain resulted in a NifA protein that did not require GlnB for activity under nitrogen fixation conditions. We report here that a NifA double mutant in which the Tyr residues at positions 18 and 53 of NifA N-were simultaneously replaced by Phe (NifA-Y1853F) displays high nitrogenase activity, which is still regulatable by ammonia, but not by GlnB. The yeast two-hybrid technique was used to investigate whether GlnB can physically interact with wild-type and mutant NifA proteins. GlnB was found to interact directly with the N-terminal GAF domain of wild-type NifA, but not with its central or C-terminal domain. GlnB could still bind to the single NifA mutants Y18F and Y53F. In contrast, no interaction was detected between GlnB and the double mutant NifA-Y18/53F or between GlnB and NifA-Y43.