Department of Molecular Sciences, Macquarie University, North Ryde, Sydney, NSW, 2109, Australia.
Australian Research Council Industrial Transformation Training Centre for Molecular Technology in the Food Industry, Sydney, NSW, 2109, Australia.
J Ind Microbiol Biotechnol. 2019 Jun;46(6):769-781. doi: 10.1007/s10295-019-02157-7. Epub 2019 Feb 26.
Enzymatic degradation of the β-1,3-glucan paramylon could enable the production of bioactive compounds for healthcare and renewable substrates for biofuels. However, few enzymes have been found to degrade paramylon efficiently and their enzymatic mechanisms remain poorly understood. Thus, the aim of this work was to find paramylon-degrading enzymes and ways to facilitate their identification. Towards this end, a Euglena gracilis-derived cDNA expression library was generated and introduced into Escherichia coli. A flow cytometry-based screening assay was developed to identify E. gracilis enzymes that could hydrolyse the fluorogenic substrate fluorescein di-β-D-glucopyranoside in combination with time-saving auto-induction medium. In parallel, four amino acid sequences of potential E. gracilis β-1,3-glucanases were identified from proteomic data. The open reading frame encoding one of these candidate sequences (light_m.20624) was heterologously expressed in E. coli. Finally, a Congo Red dye plate assay was developed for the screening of enzyme preparations potentially able to degrade paramylon. This assay was validated with enzymes assumed to have paramylon-degrading activity and then used to identify four commercial preparations with previously unknown paramylon degradation ability.
β-1,3-葡聚糖拟南芥的酶促降解可以生产用于医疗保健的生物活性化合物和用于生物燃料的可再生底物。然而，能够有效降解拟南芥的酶很少被发现，其酶促机制也知之甚少。因此，本工作的目的是寻找能够降解拟南芥的酶，并找到促进其鉴定的方法。为此，构建了一株来源于眼虫的 cDNA 表达文库，并将其导入大肠杆菌。建立了基于流式细胞术的筛选方法，以鉴定能够水解荧光底物荧光素二-β-D-吡喃葡萄糖苷的眼虫酶，同时结合省时的自动诱导培养基。此外，从蛋白质组学数据中鉴定了四个潜在的眼虫β-1,3-葡聚糖酶的氨基酸序列。其中一个候选序列（light_m.20624）的开放阅读框在大肠杆菌中异源表达。最后，开发了刚果红染色平板法用于筛选可能具有降解拟南芥能力的酶制剂。该方法通过假定具有降解拟南芥活性的酶进行验证，然后用于鉴定四种具有未知降解拟南芥能力的商业制剂。
