Purification and characterization of an autolytic enzyme for optimized hydrolysis of microalgae for multiple bioproduct extraction

Abstract. In order to improve the process economics of commercializing microalgae-derived bioproducts, alternative techniques for extracellular matrix and/or organelle membrane lysis must be employed. The application of enzymes for these hydrolysis purposes requires a controlled experimental setup with optimized incubation time, temperature, pH, conductivity, cell concentration, and enzyme dosage. Based on our previous work, gamete autolysin (lytic enzyme) from Chlamydomonas reinhardtii has a demonstrated utility in lysing the C. reinhardtii cell wall and releasing the majority of intracellular proteins. A recognized disadvantage of gamete autolysin is the in-lab production process that leads to batch-to-batch variability in terms of enzyme activity. A study was conducted to purify and characterize gamete autolysin to develop an enzyme standard for use in an enzyme activity assay and to ultimately optimize cell lysis for aqueous enzymatic extraction of microalgae bio-products. Gamete autolysin was first concentrated using membrane filtration (ultrafiltration/diafiltration) and then applied to various chromatographic resins in batching binding purification studies. Binding conditions (buffer pH, salt, resin composition) were identified and used for the scale up of gamete autolysin purification with an ÄKTA low pressure chromatography protein purification system. The final purified gamete autolysin will then be used as an enzyme standard for assignment of an enzyme activity unit to be employed for gamete autolysin activity assays and required for cell lysis.