Abstract
Rhamnogalacturonan lyase (RGL) cleaves backbone α-1,4 glycosidic bonds between L-rhamnose and D-galacturonic acid residues in type I rhamnogalacturonan (RG-I) by β-elimination to generate RG oligosaccharides with various degrees of polymerization. Here, we cloned, expressed, purified and biochemically characterized two RGLs (Bo3128 and Bo4416) in the PL11 family from Bacteroides ovatus ATCC 8483. Bo3128 and Bo4416 displayed maximal activity at pH 9.5 and pH 6.5, respectively. Whereas the activity of Bo3128 could be increased 1.5 fold in the presence of 5 mM Ca2+, Bo4416 required divalent metal ions to show any enzymatic activity. Both of RGLs showed a substrate preference for RG-I compared to other pectin domains. Bo4416 and Bo3128 primarily yielded unsaturated RG oligosaccharides, with Bo3128 also producing them with short side chains, with yields of 32.4 and 62.4%, respectively. Characterization of both RGLs contribute to the preparation of rhamnogalacturonan oligosaccharides, as well as for the analysis of the fine structure of RG-I pectins.
Highlights
Pectin degrading enzymes are found in a variety of microorganisms (de Vries and Visser, 2001; Masoud and Jespersen, 2006; Martens et al, 2011; Tayi et al, 2016), and are widely used in the food industry to degrade pectin
There are three main classes of pectins: homogalacturonan (HG), type I rhamnogalacturonan (RG-I), and type II rhamnogalacturonan (RG-II) (Willats et al, 2001; Lerouxel et al, 2006; Atmodjo et al, 2013). Both HG and RG-II domains form their backbones via α-1,4-GalA linkages (Ridley et al, 2001; Mohnen, 2008), whereas the RG-I backbone is composed of repeating disaccharides of α-1,2-Rha and α-1,4-GalA, with neutral sugar side chains decorating the polymer at Rha O4 positions
According to the NCBI, Bacteroides ovatus ATCC 8483 contains two putative endo rhamnogalacturonan lyases (RGLs): Bo3128 (GenBank accession Number: ALJ47736.1) and Bo4416 (GenBank accession Number: ALJ49009.1), both belonging to the PL11 family
Summary
Pectin degrading enzymes are found in a variety of microorganisms (de Vries and Visser, 2001; Masoud and Jespersen, 2006; Martens et al, 2011; Tayi et al, 2016), and are widely used in the food industry to degrade pectin. There are three main classes of pectins: homogalacturonan (HG), type I rhamnogalacturonan (RG-I), and type II rhamnogalacturonan (RG-II) (Willats et al, 2001; Lerouxel et al, 2006; Atmodjo et al, 2013) Both HG and RG-II domains form their backbones via α-1,4-GalA linkages (Ridley et al, 2001; Mohnen, 2008), whereas the RG-I backbone is composed of repeating disaccharides of α-1,2-Rha and α-1,4-GalA, with neutral sugar side chains decorating the polymer at Rha O4 positions. Many HG degrading enzymes derived from microorganisms have been extensively studied and used to clarify fruit juices and in other processes (Kashyap et al, 2001; Kc et al, 2020; Wu et al, 2020). Enzymes that degrade the RG-I backbone include rhamnogalacturonan hydrolases (RGHs) and rhamnogalacturonan lyases (RGLs)
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