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.

Status of the tear film in trachoma

To evaluate the value and usefulness of the clinical and laboratory diagnostic tests in trachoma, forty three patients [86 eyes] with trachoma cicatrized, trachoma trichiasis and corneal opacities in accordance with the WHO-1987 grading system, Were evaluated for Schirmer's test, tear osmolarity and tear lysozyme level. In the 86 eyes [43 patients] of normal eyes, tear fluid osmolarity was 303.4 +/- 9.1 mOsm.IL. In 86 eyes [43 patients] of trachomatous cases it was 311.9 +/- 12.3 mOsm./L. Tear lysozyme level noted in trachomatous cases was 1.68 +/- 0.81 mg/ml. This value was around normal range [1.5-2.5 mg/mi]. The average value of the Schirmer's test in normal cases was 15.1 +/- 8.9 mm/5 mm., whereas in trachomatous cases it was 11.9 +/- 6.8 mm/5 mm. Because of being correlate decreased Schirmer's test and tear osmolarity in trachomatous cases, it is assumed that the status of tear fluid may only be assessed by tear film osmolarity measurements