Effective pH pretreatment and cell disruption method for real-time intracellular enzyme activity assay of a marine fungus covered with pigments

<p>Filamentous fungi are capable producers of many bioactive compounds, and real-time intracellular enzyme activity assay is an essential guidance for their bioprocess developments. However, there are many difficulties in preparing homogenate for enzyme activity assay, such as disrupting fungal cell with complicated cellular structure and solid cell wall, removing abundant extracellular metabolites accumulating on mycelia, and so on. <i>Halorosellinia</i> sp. (No. 1403) was a marine-derived filamentous fungus producing a potential antitumor compound 1403C, and the deep red pigments (with main component of 1403C) covering on its mycelia showed strong absorption in a wide range, which critically affected the measurement of many enzyme activities. In this study, we developed an effective pH pretreatment and cell disruption method to prepare homogenate for enzyme activity assay. When mycelia were washed by the solution with pH 5.0 for 3 min, most pigments could be removed without severe loss on enzyme activities. Afterward, grinding with mini bead for 15 min with alternating cooling could effectively disrupt both cell wall and mitochondrial membrane. These methods have been successfully applied on real-time intracellular enzyme activity assay of <i>Halorosellinia</i> sp. (No. 1403) and can offer enlightenment for other filamentous fungi with similar problems.</p

Peroxisome-targeted and tandem repeat multimer expressions of human antimicrobial peptide LL37 in <i>Pichia pastoris</i>

<p>Although the human antimicrobial peptide LL37 has a broad spectrum of antimicrobial activities, it easily damages host cells following heterologous expressions. This study attempted two strategies to alleviate its damage to host cells when expressed in <i>Pichia pastoris</i> using the <i>AOX1</i> promoter. Tandem repeat multimers of LL37 were first designed, and secretion expression strains GS115-9K-(DPLL37DP)_<i>n</i> (<i>n</i> = 2, 4, 6 and 8) containing different copies of the LL37 gene were constructed. However, LL37 tandems still killed the cells after 96 hr of induction. Subsequently, peroxisome-targeted expression was performed by adding a peroxisomal targeting signal 1 (SKL) at the C-terminus of LL37. The LL37 expression strain GS115-3.5K-LL37-SKL showed no significant inhibition in the cells after induction. Antibacterial activity assays showed that the recombinant LL37 expressed in peroxisomes had good antimicrobial activities. Then, a strain GS115-3.5K-LL37-GFP-SKL producing LL37, green fluorescent protein, and SKL fusion proteins was constructed, and the fusion protein was confirmed to be targeting the peroxisomes. However, protein extraction analysis indicated that most of the fusion proteins were still located in the cell debris after cell disruption, and further studies are required to extract more proteins from the peroxisome membrane.</p