Site-directed mutagenesis of α-l-rhamnosidase from Alternaria sp. L1 to enhance synthesis yield of reverse hydrolysis based on rational design

Abstract

The α-L-rhamnosidase catalyzes the hydrolytic release of rhamnose from polysaccharides and glycosides and is widely used due to its applications in a variety of industrial processes. Our previous work reported that a wild-type α-L-rhamnosidase (RhaL1) from Alternaria sp. L1 could synthesize rhamnose-containing chemicals (RCCs) though reverse hydrolysis reaction with inexpensive rhamnose as glycosyl donor. To enhance the yield of reverse hydrolysis reaction and to determine the amino acid residues essential for the catalytic activity of RhaL1, site-directed mutagenesis of 11 residues was performed in this study. Through rationally designed mutations, the critical amino acid residues which may form direct or solvent-mediated hydrogen bonds with donor rhamnose (Asp, Asp, Asp, Glu, Arg, His, and Trp) and may form the hydrophobic pocket in stabilizing donor (Trp, Tyr, Tyr, and Trp) in active-site of RhaL1 were analyzed, and three positive mutants (W261Y, Y302F, and Y316F) with improved product yield stood out. From the three positive variants, mutant W261Y accelerated the reverse hydrolysis with a prominent increase (43.7 %) in relative yield compared to the wild-type enzyme. Based on the 3D structural modeling, we supposed that the improved yield of mutant W261Y is due to the adjustment of the spatial position of the putative catalytic acid residue Asp. Mutant W261Y also exhibited a shift in the pH-activity profile in hydrolysis reaction, indicating that introducing of a polar residue in the active site cavity may affect the catalysis behavior of the enzyme.

A novel strategy for efficient disaccharides synthesis from glucose by β-glucosidase

“…In addition to glycosidic bond cleavage, glycoside hydrolases (GHs) can be used for the synthesis of glycoside bonds in vitro via reverse hydrolysis reaction without the need for cofactors, such as uridine diphosphate (Lu et al 2015;Perugino et al 2004;Rosengren et al 2019;Xu et al 2016). Recently, several glycosidases, such as β-glucosidase, α-glucosidase, α-L -rhamnosidase, β-mannosidase and β-galactosidase, have been used to synthesize glycosides via the reverse hydrolysis reaction (Lu et al 2015;Arthornthurasuk et al 2018;Chen et al 2019;Ajisaka et al 2012;Tan et al 2016).…”

“…Honda et al (2008) reported that hydrogen-bonding interaction with catalytic water that reduced the hydrolytic reactivity of an inverting xylanase was dramatically decreased by eliminating the retention of the nucleophilic water molecule at the key amino acid residue. Other studies have focused on improving the hydrophobicity of the entrance to the active site (Xu et al 2016;Qin et al 2019;Frutuoso et al 2013) or acceptor subsite (Lundemo et al 2013). However, the effect of hydrophobicity of amino acid residues in the catalytic site on the catalytic activity needs to be further elucidated.…”

Purification and characterisation of a novel α‐L‐rhamnosidase exhibiting transglycosylating activity from Aspergillus oryzae

“…The reverse hydrolysis product was determined using a TLC plate (Merck, Darmstadt, Germany) and HPLC 1200 system (Agilent). The TLC analysis was carried out using n‐butanol/ethanol/water (5:3:2, v/v/v) as the mobile phase (Xu et al ., ). Spots were visualised by carbonisation using a solution composed of 0.1% (w/v) 3,5‐dihydroxytoluene in H 2 SO 4 /methanol (2:8 v/v).…”

Enhancement of the thermostability of Aspergillus niger α‐ l ‐rhamnosidase based on PoPMuSiC algorithm

“…It has been used in debittering of citrus, manufacturing of prunin from naringin, enhancing of wine aromas, and producing of L-rhamnose (Yadav, Yadav, Yadav, & Yadav, 2010). Moreover, it is a biotechnologically important enzyme in food industry in the preparation of drug and drug precursors, and some α-l-rhamnosidases can catalyze the synthesis of rhamnose-containing chemicals by reverse hydrolysis (RCCs) with the L-rhamnose as a donor (Chin, Murad, Mahadi, Nathan, & Bakar, 2013;Martearena, Daz, & Ellenrieder, 2009;Xu et al, 2016).…”