Characterization of Two Novel Rumen-Derived Exo-Polygalacturonases: Catalysis and Molecular Simulations

Qian Deng; Xiaobao Sun; Deying Gao; Yuting Wang; Yu Liu; Nuo Li; Zhengguang Wang; Mingqi Liu; Jiakun Wang; Qian Wang

doi:10.3390/microorganisms11030760

Characterization of Two Novel Rumen-Derived Exo-Polygalacturonases: Catalysis and Molecular Simulations

Microorganisms. 2023 Mar 16;11(3):760. doi: 10.3390/microorganisms11030760.

Authors

Qian Deng^{1

2}, Xiaobao Sun^{1

2}, Deying Gao^{1

2}, Yuting Wang³, Yu Liu⁴, Nuo Li^{1

2}, Zhengguang Wang², Mingqi Liu³, Jiakun Wang^{1

2}, Qian Wang^{1

2}

Affiliations

¹ Key Laboratory of Molecular Animal Nutrition, Ministry of Education, Zhejiang University, Hangzhou 310058, China.
² Institute of Dairy Science, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.
³ Key Laboratory of Marine Food Quality and Hazard Controlling Technology of Zhejiang Province, College of Life Sciences, China Jiliang University, Hangzhou 310018, China.
⁴ College of Life Sciences, Zhejiang University, Hangzhou 310058, China.

Abstract

Pectinases are a series of enzymes that degrade pectin and have been used extensively in the food, feed, and textile industries. The ruminant animal microbiome is an excellent source for mining novel pectinases. Two polygalacturonase genes, IDSPga28-4 and IDSPga28-16, from rumen fluid cDNA, were cloned and heterologously expressed. Recombinant IDSPGA28-4 and IDSPGA28-16 were stable from pH 4.0 to 6.0, with activities of 31.2 ± 1.5 and 330.4 ± 12.4 U/mg, respectively, against polygalacturonic acid. Hydrolysis product analysis and molecular dynamics simulation revealed that IDSPGA28-4 was a typical processive exo-polygalacturonase and cleaved galacturonic acid monomers from polygalacturonic acid. IDSPGA28-16 cleaved galacturonic acid only from substrates with a degree of polymerization greater than two, suggesting a unique mode of action. IDSPGA28-4 increased the light transmittance of grape juice from 1.6 to 36.3%, and IDSPGA28-16 increased the light transmittance of apple juice from 1.9 to 60.6%, indicating potential application in the beverage industry, particularly for fruit juice clarification.

Keywords: exo-polygalacturonase; expression; hydrolytic pattern; molecular dynamics stimulation; rumen microbe.

Grants and funding

This work was supported by the Key R&D Program of Zhejiang Province (2022C02043) and the National Key Research and Development Program of China (2018YFD0501903).