Gene Cloning, Heterologous Expression And Characterization Of Three Polygalacturonases From Thermophilic Fungus Neosartorya Fischeri P1

Pectic substance （pectin） is an important constituent of non-starch polysaccharides. It is composed of D-galacturonic acid residues linked together by a-1,4 glycosidic linkages and mainly exist in the middle lamella of plant cell wall. Although pectin cannot provide energy to animals directly, it has probiotic effect and can influence the metabolism of animals. Moreover, pectin is another renewable resource besides cellulose and xylan, whose degradation releases galacturonic oligosaccharides of high values.Pectinase is a group of pectin-degrading enzymes. According to the substrate specificity and mode of action, pectinase can be classified into three types: polygalacturonase that degrades polygalacturonic acid by cleaving the a-1,4 glycosidic linkages, pectate lyase that breaks the glycosidic bonds via trans-elimination reaction, and pectin esterase that degrades polygalacturonic acid with esterification. Pectinase has a broad application prospect in many industries, such as food, textile, feed, and papermaking. It is of significance to explore more pectinases with excellent properties.In this study, one endo-polygalacturonase gene （Nfpg5） and two exo-polygalacturonase genes （Nfpg4 and Nfpg6） were cloned from thermophilic fungus Neosartorya fischeri P1. To verify their biological functions, the genes were subcloned into the expression vector pPIC9y and then transformed into Pichia pastoris GS115. All genes were successfully expressed and their properties were determined. Deduced NfPG4shares 97.6% amino acid sequence identity with the protein XM₀01257488.1 from N. fischeri NRRL 181 and 79% identity with the closest characterized homolog from Aspergillus tubingensis. Purified recombinant NfPG4 showed the optimal activity at pH 3.5 and 65 ℃. It was stable at pH 3.0-8.5 when incubated at 37 ℃ for 1 h and at 55 ℃ after incubation for 1 h. The Km and kcat values of NfPG4 were 2.9 mg/ml and 720 s-1, respectively. Its specific activity was 700.9 U/mg. The deduced amino acid sequence of NfPG5 shares 100% identity with that of XM₀01264034.1 from N. fischeri NRRL 181 and 58% identity with its closest characterized homolog from Achaetomium sp.Xz-8. Purified recombinant NfPG5 showed the optimal activity at pH 4.5 and 70 ℃. It was stable at pH 2.0-11.0 when incubated at 37 ℃ for 1 h and at 55 ℃ after incubation for 1 h. The Km, kcat and specific activity values of NfPG5 were 1.0 mg/ml,2854 s-1, and 3630.4 U/mg, respectively. Deduced NfPG6 shares 100% amino acid sequence identity with that of XM₀01263571.1 from N. fischeri NRRL 181. Purified recombinant NfPG6 showed the optimal activity at pH 4.5 and 55 ℃. It was stable at pH 2.0-11.0 when incubated at 37 ℃ for 1 h and retained almost all activity after incubation at 50 ℃ for 1 h. The Km, at and specific activity values of NfPG6 were 3.4 mg/ml, 724 s-1, and53.9 U/mg, respectively.In summary, this study identified three polygalacturonase genes with excellent properties from N. fischeri P1. Among them, NfPG4 and NfPG5 are particularly interesting due to their high temperature optima, excellent thermostability and high catalytic efficiency. These superior properties make them valuable candidates for industrial applications with a preference for thermophilic pectinases.