Bioprocessing of laccase produced by submerged culture of Ganoderma sp. WR-1

The current study carried out, developed a protocol to efficiently process laccase from fermentation broths of Ganoderma sp. WR-1. Selection of a downstream protocol based on required yield and purity was achieved. Purification using fractional ammonium sulphate precipitation, three phase partitioning, ultrafiltration, diafiltration, and chromatography techniques were investigated. Ammonium sulphate and three phase partitioning resulted in a loss of 20% and 40% laccase activity. Successful ultrafiltration required separation of the extracellular polymeric substances (EPS) which was formed during fermentation. Freeze-thawing of the broth post-fermentation, followed by depth and microfiltration separated the EPS successfully. Operating parameters of pH, temperature and transmembrane pressure during ultrafiltration were optimized as 7, 21 °C and 25 psi respectively. Yields of 97% and ease of scalability made ultrafiltration the operation of choice for laccase concentration. Further product improvement by diafiltration in different operational modes, mode 1 (pre-diafiltration followed by post-concentration) and mode 2 (pre-concentration followed by post-diafiltration) using 10 and 30 kDa membranes was carried out. Diafiltration, using a 30 kDa membrane in mode 1 resulted in 3.7-fold purification with 90% yields. Anion, cation and hydrophobic interaction chromatography of the diafiltered broth to increase fold purity was performed. Anion exchange chromatography was the most suited technique as a substantially purified laccase was obtained with overall yields of 81%.

Research highlights▶ The paper provides a detailed study of downstream operations for laccase recovery. ▶ The separation of polymeric substances by freeze-thawing is not reported elsewhere. ▶ Tangential flow filtration was optimized to concentrate laccase effectively. ▶ The research focused on scalability and high yields of laccase, unlike most reports. ▶ This work helps select recovery protocols with a trade off between purity and yield.