Characterization Of Two Fungal Pectinases With Application In Pectin Oligosaccharide

Pectic oligosaccharides?POS?from pectin are depolymerization functional products with lower molecular weights,Pectic oligosaccharides have different structure and function due to different origins.Presently,POS is mainly prepared from pectin-rich raw materials?including citrus,apple,and sugar beet pomace obtained from food processing industries or pure pectin degradation products?by chemical method,physical method,enzymatic hydrolysis,biological enzymatic method.Food processing by-products are rich in pectin substances,and it is a preferred raw material for obtaining the pectin oligosaccharide product by enzymatic hydrolysis.In this study,a pectate lyase and a pectin methylesterase were cloned from thermophilic cellulolytic fungus Humicola insolens Y1 and acidophilic fungus Bispora sp.MEY-1,respectively.And efficient heterologous expression in Pichia pastoris GS115 was realized and studied the enzymatic properties of the two recombinant proteins.PHLY1 is a basophilic mesophilic pectate lyase from Humicola insolens Y1,The purified PLHY1exhibited maximum activity at 55?,The enzyme was stable at 45? and retained more than 70%of the initial activity after incubation at 45? for 1 h.And retained more than40%of the initial activity after incubation at 55? for 1 h.PLHY1 showed maximum activity at pH 10.0,and was active in between pH 4.0 and 12.0 at 55?.PLHY1 was stable over a comprehensive pH range and retained more than of the maximum activity after incubation at pH 3.0–12.0 for 1 h at 37?.PLHY1 showed no activity in the absence of Ca²⁺and was highly active in the presence of 0.4 mM Ca²⁺.The enzyme activity remained comparable in pectins with different levels of methyl esterification.The apple peel degradation products obtained from the PLHY1 treatment were analyzed by the HPAEC-PAD method,result showed that galacturonic acid-oligosaccharides?GalAOS?with small degrees of polymerization?4–6?were the major hydrolyzates produced by the degradation of apple peel pectin by PLHY1.The corresponding structural formulas are GalA?C₄=C5?-GalA-GalA-GalA,GalA?C₄=C₅?-GalA-GalA-GalA-GalA-GalAand GalA?C₄=C₅?-GalA-GalA?OAc?-GalA-GalA-GalA,respectively.PME8A is a pectin methylesterase belonged to carbohydrate esterase?CE?family 8,PME8A showed maximum activity at 60?,and more than 80%of the maximum activity was retained at 55–80?.Its optimum pH was 3.5,and more than 50%of the maximum activity was retained at pH 2.5–5.0.It was a scarce pectin methylesterase with acidophilic from fungus resource.The significant synergistic effect that demethylate of pectin crude extracts from apple pomace and orange peel was observed by PME8A,which enhanced the polygalacturonase activities of PG8fn from Achaetomium sp.Xz8 to 8.5 times and 1 times,respectively.This study demonstrated that pectin methylesterase can be used in the production of pectin oligosaccharides by food processing by-products,providing a new method for the extraction of pectin oligosaccharides.Enzymatic pectin hydrolysis to obtain POS is a crucial area of research,as this method offers several advantages,namely substrate specificity,mild hydrolysis conditions,low cost,and environmental safety.The structure and degree of POS polymerization are closely related to the hydrolytic properties of the pectinase used.Thus,enzymatic hydrolysis may be the preferred method to achieve the desired degree of polymerization and structure in POS.This study demonstrated that pectate lyase and pectin methylesterase can be used in the production of pectin oligosaccharides by food processing by-products,providing a new method for the extraction of pectin oligosaccharides.Enzymatic can not only effectively control the degree of polymerization and structure of pectin oligosaccharides,but also realize the reuse of waste resources of food processing by-products and create new economic benefits.