title: Effective pH pretreatment and cell disruption method for real-time intracellular enzyme activity assay of a marine fungus covered with pigments.
abstract: 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. Halorosellinia 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 Halorosellinia sp. (No. 1403) and can offer enlightenment for other filamentous fungi with similar problems.
authors.affiliation: a School of Biotechnology, State Key Laboratory of Bioreactor Engineering , East China University of Science and Technology , Shanghai , China.
authors.forename: Xiaoxu
authors.initials: X
authors.lastname: Zhang
authors.affiliation: a School of Biotechnology, State Key Laboratory of Bioreactor Engineering , East China University of Science and Technology , Shanghai , China.
authors.forename: Yanyun
authors.initials: Y
authors.lastname: Gao
authors.affiliation: a School of Biotechnology, State Key Laboratory of Bioreactor Engineering , East China University of Science and Technology , Shanghai , China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Yin
authors.affiliation: a School of Biotechnology, State Key Laboratory of Bioreactor Engineering , East China University of Science and Technology , Shanghai , China.
authors.forename: Menghao
authors.initials: M
authors.lastname: Cai
authors.affiliation: a School of Biotechnology, State Key Laboratory of Bioreactor Engineering , East China University of Science and Technology , Shanghai , China.
authors.forename: Xiangshan
authors.initials: X
authors.lastname: Zhou
authors.affiliation: a School of Biotechnology, State Key Laboratory of Bioreactor Engineering , East China University of Science and Technology , Shanghai , China.|b Shanghai Collaborative Innovation Center for Biomanufacturing , Shanghai , China.
authors.forename: Yuanxing
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D019149:Bioreactors; D004798:Enzymes / Q000378:metabolism; D005658:Fungi / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008386:Marine Biology; D010860:Pigments, Biological / Q000378:metabolism*
chemical_list: D004798:Enzymes; D010860:Pigments, Biological
keywords: Cell disruption; extracellular metabolite; filamentous fungi; intracellular enzyme activity assay; mycelia covered with pigments; pH pretreatment