Greener production of low methoxyl pectin via recyclable enzymatic de-esterification using pectin methylesterase cross-linked enzyme aggregates captured from citrus peels

We propose more green and sustainable enzymatic production of low methoxyl (LM) pectin with desired degree of esterification (DE) using a reusable and practically cost-free biocatalyst captured from renewable waste. The pectin methyl esterase (PME) was directly captured as recyclable biocatalyst from the waste citrus peels through cross-linked enzyme aggregates (CLEAs). The optimization of biocatalyst preparation specifically recovered 85% of PME activity available in the waste citrus peels by ammonium sulphate (50%, v/v) precipitation followed by cross-linking for 5 h with 70 mM glutaraldehyde. The PME-CLEAs were characterized by FTIR and SEM and used for batch de-esterification of high methoxyl (HM) pectin obtained from citrus, mango, and pomegranate to LM-pectin. The PME-CLEAs exhibited higher stability in acidic pH (most desired for HM-pectin de-esterification), thermostability, and similar kinetics of de-esterification compared to the native PME. The PME-CLEAs were extremely versatile as they achieved maximum reduction in DE by 75%, 68%, and 66% for pectin derived from citrus, mango, and pomegranate, respectively without changing their molecular weight (MW) and GalA content under the same optimal batch reaction conditions (pectin loading 1% w/v, PME loading 40 U/g of pectin, 35 °C, and pH 6.5, 4 h). De-esterified pectin's strong gelling in presence of Ca²⁺ and decreased methyl ester peak density in FTIR further confirmed production of LM-pectin by PME-CLEAs. Finally, the PME-CLEAs were recycled for 7 batches of LM-pectin production with consistent DE, MW and GalA content of LM-pectin produced in each batch and PME activity which makes this process promising to pectin industry.