caa a22 4500 605504083 CHVBK 20210128100614.0 cr unu---uuuuu 210128e20150401xx s 000 0 eng 10.1007/s00253-014-6146-0 doi (NATIONALLICENCE)springer-10.1007/s00253-014-6146-0 Biochemical characteristics of an alkaline pectate lyase PelA from Volvariella volvacea [Elektronische Daten] roles of the highly conserved N-glycosylation site in its secretion and activity [Aiqin Shi, Hang Hu, Fei Zheng, Liangkun Long, Shaojun Ding] Alkaline pectate lyases have great application potential in the bioscouring of textiles. They are isolated predominantly from bacteria and a few fungi. Here, we report the biochemical characteristics of a novel alkaline pectate lyase PelA from the basidiomycete Volvariella volvacea. The full-length pelA encodes a 321-amino-acid polypeptide containing a putative 18-residue signal peptide and a pectate lyase family 1 catalytic domain. It contains one conserved and one non-conserved potential N-glycosylation site (N-X-S/T) at the residues N95 and N198, respectively. The enzyme showed optimal activity at 60°C and pH 10, although it was stable between pH 4 and pH 11. Additional Ca2+ was not required to measure PelA activity in vitro, but it could significantly enhance its activity and thermal stability. The V max values using polygalacturonic acid as substrate were increased from 50.71 to 89.96IUmg−1 by the addition of 0.1mM Ca2+, whereas the K m values were decreased from 0.681 to 0.514mgml−1. Site-directed mutagenesis revealed PelA has only one N-glycan attached to the residue N95. This N-glycan is crucial to its efficient secretion and activity possibly due to its role in maintaining the secondary structure of PelA. Amino acid substitution at the residue N198 had no effect on PelA secretion, but resulted in a slight (5.16%) to modest (27.37%) decrease in specific activity and less thermal stability, indicating the amino acid itself is also important for activity due to it being highly conserved and because of its proximity to the catalytic site. Springer-Verlag Berlin Heidelberg, 2014 Alkaline pectate lyase nationallicence Volvariella volvacea nationallicence N-glycosylation site nationallicence Conserved Asn residue nationallicence Site-directed mutagenesis nationallicence Shi Aiqin Department of Biological Engineering, College of Chemical Engineering, Nanjing Forestry University, 210037, Nanjing, Jiangsu, China aut Hu Hang Department of Biological Engineering, College of Chemical Engineering, Nanjing Forestry University, 210037, Nanjing, Jiangsu, China aut Zheng Fei Department of Biological Engineering, College of Chemical Engineering, Nanjing Forestry University, 210037, Nanjing, Jiangsu, China aut Long Liangkun Department of Biological Engineering, College of Chemical Engineering, Nanjing Forestry University, 210037, Nanjing, Jiangsu, China aut Ding Shaojun Department of Biological Engineering, College of Chemical Engineering, Nanjing Forestry University, 210037, Nanjing, Jiangsu, China aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/8(2015-04-01), 3447-3458 0175-7598 99:8<3447 2015 99 253 https://doi.org/10.1007/s00253-014-6146-0 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-6146-0 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Shi Aiqin Department of Biological Engineering, College of Chemical Engineering, Nanjing Forestry University, 210037, Nanjing, Jiangsu, China aut NATIONALLICENCE 700 1- Hu Hang Department of Biological Engineering, College of Chemical Engineering, Nanjing Forestry University, 210037, Nanjing, Jiangsu, China aut NATIONALLICENCE 700 1- Zheng Fei Department of Biological Engineering, College of Chemical Engineering, Nanjing Forestry University, 210037, Nanjing, Jiangsu, China aut NATIONALLICENCE 700 1- Long Liangkun Department of Biological Engineering, College of Chemical Engineering, Nanjing Forestry University, 210037, Nanjing, Jiangsu, China aut NATIONALLICENCE 700 1- Ding Shaojun Department of Biological Engineering, College of Chemical Engineering, Nanjing Forestry University, 210037, Nanjing, Jiangsu, China aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/8(2015-04-01), 3447-3458 0175-7598 99:8<3447 2015 99 253