Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization

Laccase has been emerged as a valuable green biocatalyst in cleaner production, which requires vast amount of laccase at low production cost and with good properties. The current work evaluated a wild strain Pycnoporus sp. SYBC-L3 as an efficient microbial cell factory for laccase production. High-cell density cultivation (HCDC) of L3 was developed in a 5-L bioreactor by adjusting the ratio of inoculum to medium, achieving over 4-fold of both laccase activity (240 U/mL) and fungal biomass (20 g/L) than those obtained under routine procedures. Laccase productivity was further assessed at pilot-scale in a 500-L and a 5-ton bioreactor, where the highest activity of around 60 U/mL and 80 U/mL were produced, respectively. Cost percentage analysis for the 5-ton reactor demonstrated that labor was the highest portion (50%) in total cost for industrial production in this case. Using a one-step purification scheme, laccase was purified to homogeneity from the culture broth with yield of 44% and 97% purity. Circular dichroism (CD) analyses suggested that the secondary structure of laccase was mainly composed of β-sheet that played a vital role in maintaining laccase activity, and also revealed the potential mechanisms for the changes in laccase activities under varying conditions. Powdered laccase was efficiently prepared from cell-free culture broth using spray drying technique with above 74% activity retained. This study provides important information for an efficient and scalable production of laccase.