Partial Purification and Characterisation of Pectinase Produced by Aspergillus niger LFP-1 Grown on Pomelo Peels as a Substrate

Trop Life Sci Res. 2021 Mar;32(1):1-22. doi: 10.21315/tlsr2021.32.1.1. Epub 2021 Mar 31.

Abstract
in English, Malay (macrolanguage)

In the present study, pectinase was produced by local fungal isolate, Aspergillus niger LFP-1 grown on pomelo peels as a sole carbon source under solid-state fermentation (SSF). The purification process begins with the concentration of crude enzyme using ammonium sulfate precipitation and followed by purification using anion-exchange column chromatography (DEAE-Sephadex) and subsequently using gel filtration column chromatography (Sephadex G-100). On the other hand, the molecular weight of the purified enzyme was determined through SDS-PAGE. The findings revealed the crude enzyme was purified up to 75.89 folds with a specific activity of 61.54 U/mg and the final yield obtained was 0.01%. The molecular mass of the purified pectinase was 48 kDa. The optimum pH and temperature were 3.5 and 50°C, respectively. This enzyme was stable at a range of pH 3.5 to 4.5 and a relatively high temperature (40°C-50°C) for 100 min. The Km and Vmax were found to be 3.89 mg/mL and 1701 U/mg, respectively. Meanwhile, pectin from citrus fruit and the metal ion (Co2+) were the best substrate and inducer to enhance pectinase yield, respectively.

Dalam kajian ini, enzim pektinase telah dihasilkan dari kulat pencilan tempatan, Aspergillus niger LFP-1 yang tumbuh di atas kulit limau bali sebagai satu-satunya sumber karbon melalui kaedah perfermentasian keadaan pepejal. Proses penulenan ini bermula dengan pemekatan enzim kasar menggunakan kaedah pemendapan ammonium sulfate dan diikuti dengan penulenan menggunakan kolum kromatografi pertukaran-ion (DEAE-Sephadex) dan disertai dengan kolum kromatografi penapisan gel (Sephadex G-100). Selain itu, berat molekul enzim yang telah ditulenkan ditentukan menggunakan SDS-PAGE. Keputusan kajian menunjukkan enzim kasar telah ditulenkan sebanyak 75.89 kali ganda dengan aktiviti spesifik sebanyak 61.54 U/mg dan hasil akhir yang diperolehi sebanyak 0.01%. Berat molekul enzim kasar ini adalah 48 kDa. pH dan suhu yang optima masing-masing adalah 3.5 dan 50°C. Enzim ini didapati stabil pada julat pH 3.5 hingga 4.5 dan julat suhu yang tinggi (40°C–50°C) untuk 100 minit. Nilai Km dan Vmax yang diperoleh masing-masing adalah 3.89 mg/mL and 1701 U/mg. Sementara itu, pektin daripada buah-buahan sitrus dan ion besi (Co2+) masing-masing adalah substrat dan pemangkin terbaik untuk meningkatkan hasil pektinase.

Keywords: Aspergillus niger LFP-1; Characterisation; Pectinase; Purification; Solid State Fermentation.