High Production of Trametes cinnabarina Laccase (lac1) by Suspended and Immobilized Cells of Recombinant Pichia pastoris from Crude Glycerol

Laccases are green catalysts widely employed in various industrial, environmental, clinical and biotechnological applications. This study was aimed to develop the cost effective and efficient process for high level of fungal laccase production from crude glycerol. Initially, laccase gene (lac1) of Trametes cinnabarina was chemically synthesized, and integrated into Pichia pastoris strains (BG10, 11 and 16) by using pD915 vector which can facilitate the constitutive secretory expression of laccase (lac1). Among the recombinant P. pastoris strains developed, st.BG10 (wild type) was supported the high level of laccase production (2851 +/- 72 U/1) from pure glycerol based media. While replacing the pure glycerol by crude glycerol (60%), the media supported the cellular growth of st.BG10 (lac1), and produced 2343 +/- 60 U/1 of laccase after 48 h. The free cell biomass of st.BG10 (lac1) was recycled for 6times and produced > 8800U of laccase by using pure/crude glycerol media. Simultaneously, different matrices (agar-agar, calcium alginate, and polyacrylamide-gel) were tested to immobilize the st.BG10 (lac1) for laccase production, in which 6% calcium alginate immobilized cells to the bead size of 2.98 +0.07 mm were found to be stable, and recycled for 18 batches with 27,681-33926 U of laccase production from pure/crude glycerol based media. These findings would significantly assist to develop cost effective bioprocess for laccase production from crude glycerol (a waste product of biodiesel industry), and this is the first report on fungal laccase production by recyclable recombinant yeast using crude glycerol as substrate. [GRAPHICS] .

Automated summary

In this study, a cost-effective and efficient process for high-level fungal laccase production from crude glycerol was developed. The laccase gene was chemically synthesized and integrated into Pichia pastoris strains to facilitate constitutive secretory expression of laccase. The recombinant strains showed high levels of laccase production from pure glycerol and crude glycerol media. Additionally, immobilization of the cells using different matrices improved laccase production and stability.