Creating lactose phosphorylase enzymes by directed evolution of cellobiose phosphorylase

Abstract

Disaccharide phosphorylases are interesting enzymes for the production of sugar phosphates from cheap starting materials and for the synthesis of novel glycosides. Cellobiose phosphorylase (CP) from Cellulomonas uda was subjected to directed evolution in order to create enzyme variants with significantly increased lactose phosphorylase (LP) activity, useful for the production of alpha-D-galactose 1-phosphate. In a first round, random mutagenesis was performed on part of the CP gene and the resultant library was selected on minimal lactose medium. One clone containing six amino acid mutations was found with increased LP activity compared with the wild-type CP enzyme. The negative and neutral mutations were eliminated by site-directed mutagenesis and the resultant enzyme variant containing two amino acid substitutions (T508A/N667T) showed more LP activity than the parent mutant. Saturation mutagenesis of the beneficial sites and screening for improved mutants allowed us to identify the T508I/N667A mutant which has 7.5 times higher specific activity on lactose than the wild-type. The kinetic parameters of the mutants were determined and showed that the increased LP activity was caused by a higher k(cat) value. This is the first report of an engineered CP with modified substrate specificity.

Enzymatic production of β‐D‐glucose‐1‐phosphate from trehalose

“…Glycoside phosphorylases, in contrast, only need inorganic phosphate to produce a glycosylphosphate. In literature, the synthesis of α-glucose-1-phosphate (αGlc1P) from starch [10,11] or sucrose [12] and that of α-galactose-1-phosphate (αGal1P) from lactose [13,14] have previously been reported. A production process for βGlc1P has not yet been described in detail, but is similarly feasible using GH65 disaccharide phosphorylases from cheap substrates [15] briefly mention the production of βGlc1P from trehalose, using typical reaction conditions employed for αGlc1P [16].…”

Phosphoryl Transfer from α-d-Glucose 1-Phosphate Catalyzed by Escherichia coli Sugar-Phosphate Phosphatases of Two Protein Superfamily Types

“…Glycosyl transfer to phosphate is fundamentally limited in application to hexose 1-phosphate products, and the relatively narrow substrate scope of glycoside phosphorylase reactions restricts one to just a few glycosyl phosphates for which effective production has been demonstrated on a preparative scale (e.g., ␣-D-glucose 1-phosphate [␣Glc 1-P] and ␤Glc 1-P [26,27] and ␣-D-galactose 1-phosphate [28]). Phosphotransferase reactions, in contrast, offer convenient access to a large diversity of sugar phosphate products, as shown for nucleoside triphosphate-dependent phosphorylation of various hexose substrates by sugar kinases, for example (29)(30)(31)(32)(33).…”

Structural Basis for Reversible Phosphorolysis and Hydrolysis Reactions of 2-O-α-Glucosylglycerol Phosphorylase

“…Although GGP can efficiently and specifically produce GG via its reverse phosphorolysis activity, a considerably high activity of the side reaction, ␤Glc1P hydrolysis can be an obstacle for industrial applications. The structure determinations of sugar phosphorylases provided solid foundations for endowing altered substrate specificities and increased thermostability to these enzymes (33)(34)(35)(36). Our structural study will also facilitate future protein engineering of GGP to modulate and improve its function.…”