title: Analytical method development for directed enzyme evolution research: a high throughput high-performance liquid chromatography method for analysis of ribose and ribitol and a capillary electrophoresis method for the separation of ribose enantiomers.
abstract: Analytical methods were developed for a directed enzyme evolution research programme, which pursued high performance enzymes to produce high quality L-ribose using large scale biocatalytic reaction. A high throughput HPLC method with evaporative light-scattering detection was developed to test ribose and ribitol in the enzymatic reaction, a β-cyclobond 2000 analytical column separated ribose and ribitol in 2.3 min, a C(18) guard column was used as an on-line filter to clean up the enzyme sample matrix and a short gradient was applied to wash the column, the enzymatic reaction solution can be directly injected after quenching. Total run time of each sample was approx. 4 min which provided capability of screening 4×96-well plates/day/instrument. Meanwhile, a capillary electrophoresis method was developed for the separation of ribose enantiomers, while 7-aminonaphthalene-1,3-disulfonic acid was used as derivatisation reagent and 25 mM tetraborate with 5 mM β-cyclodextrin was used as electrolyte. 0.35%of D-ribose in L-ribose can be detected which can be translated into 99.3% ee of L-ribose. Derivatisation reagent and sample matrix did not interfere with the measurement.
authors.affiliation: Codexis Laboratories Singapore, 61 Science Park Road, Science Park II, Singapore 117525, Singapore. barry.sun@codexis.com
authors.forename: Baoguo
authors.initials: B
authors.lastname: Sun
authors.forename: Gregory
authors.initials: G
authors.lastname: Miller
authors.forename: Wan Yee
authors.initials: WY
authors.lastname: Lee
authors.forename: Kelvin
authors.initials: K
authors.lastname: Ho
authors.forename: Michael A
authors.initials: MA
authors.lastname: Crowe
authors.forename: Leslie
authors.initials: L
authors.lastname: Partridge
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D008354:Mannitol Dehydrogenases / Q000737:chemistry* / Q000378:metabolism; D012255:Ribitol / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012266:Ribose / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013237:Stereoisomerism
chemical_list: D012255:Ribitol; D012266:Ribose; D008354:Mannitol Dehydrogenases; D007770:L-Lactate Dehydrogenase