SOP for determination of pectin Methylesterase (PME) activity in sweetpotato roots

Pectin methylesterase (PME) activity correlates well with texture in potatoes (Solanum tuberosum); higher PME activity is associated with increased firmness as the PME rapidly demethylates pectin in the cell walls and middle lamellae, allowing for hardening through cross linking with divalent cations. In order to understand cooked sweet potato root texture, an assay was developed for PME activity PME removes methyl groups from pectin producing methanol and free acids. A simple and sensitive method for determination of methanol is important in the study of pectin methyl esterase (PME) in plant material. A determination of the amount of methanol produced by a particular plant tissue can be used to assess the level of PME activity or, indeed, determine the degree of methylation of purified pectins. An approach to assay methanol is to oxidize the methanol to formaldehyde and then colorimetrically determine the formaldehyde. The oxidation is carried out by alcohol oxidase. This enzymatic method improves selectivity and also eliminates the use of hazardous chemicals. The formaldehyde produced is condensed with N-methylbenzothiazolinone-2-hydrazone (MBTH) under neutral conditions. When the medium is acidified and an oxidant (i‧e., Fe3+) is added, this adduct then forms a blue formazan dye that can be determined colorimetrically. Assess PME activity in roots of sweet potato genotypes known to have different textural properties. Correlate PME activity against measurements of textural differences upon cooking to assess role of pectin modification in tissue softening.