Glucosylglycerol and glucosylglycerate as enzyme stabilizers

Abstract

Compatible solutes constitute a diverse class of low-molecular-mass organic molecules that are accumulated in high intracellular concentrations in response to the external stress of hyperosmolality or high temperature. Many of these compounds like alpha, alpha-trehalose are well known for their stabilizing effect on protein structure and could lead to development of more stable protein formulations. Negatively charged solutes like mannosylglycerate (R-2-O-alpha-D-mannopyranosyl-glycerate) are widespread among (hyper)thermophilic microorganisms and are thought to be exceptionally potent stabilizers of proteins under high-temperature denaturation conditions. To further inquire into the role of compound charge for protective function, we have compared two naturally occurring and structurally related solutes, glucosylglycerol (2-O-alpha-D-glucopyranosyl-sn-glycerol) and glucosylglycerate (R-2-O-alpha-D-glucopyranosyl-glycerate), as stabilizers of different enzymes undergoing inactivation through elevated temperature or freeze drying, and benchmarked their effects against that of alpha,alpha-trehalose. Glucosylglycerate in concentrations of >/=0.1 M was the most effective in preventing thermally induced loss of enzyme activity of lactate dehydrogenase, mannitol dehydrogenase, starch phosphorylase, and xylose reductase. alpha,alpha-Trehalose could usually be replaced by glucosylglycerol without compromising enzyme stability. Glucosylglycerol and glucosylglycerate afforded substantial (eightfold) protection to mannitol dehydrogenase during freeze drying.

In vitro evaluation of glyceric acid and its glucosyl derivative, α-glucosylglyceric acid, as cell proliferation inducers and protective solutes

“…We preliminarily confirmed that sorbitol, a practical agent for protecting of protein that is used in the food industry at 4-5%, 29) inhibited heat aggregation of egg white at a concentration of 5% (275 mM), whereas 5% GGA-Na (170 mM) had a stronger inhibitory effect on the heat aggregation of egg white than that of 5% sorbitol's (data not shown). The GA structure may therefore contribute a protein-protective property of GGA as an enzyme stabilizer, 30) and GA itself represents a superior protein-protective agent. Such activity will play an important role in vivo, such as protecting biological molecules during adaptation to environmental changes.…”

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

“…GGP shows sequence identities of 28.3 (234/828) and 31.9% (257/805) to LbMP and CsKP, respectively. The GH65 enzymes consist of four distinct regions: an N-terminal ␤ sandwich domain (residues 1-250 in GGP, blue), a linker region (251-286, cyan), a catalytic (␣/␣) 6 barrel domain (290 -687, yellow), and a C-terminal domain (287-289 and 688 -761, red). GGP forms a dimer in solution (11).…”

“…Protein Expression and Purification-The expression vectors for C terminally (His) 6 -tagged wild-type GGP and its E475Q mutant (11) were introduced into Escherichia coli BL21 CodonPlus (DE3)-RIL (Stratagene, La Jolla, CA). The transformants were cultured in Luria-Bertani medium containing 50 mg/liter of kanamycin and 100 mg/liter of chloramphenicol at 37°C for 4 h. Isopropyl 1-thio-␤-D-galactopyranoside was added to a final concentration of 0.1 mM to induce protein expression.…”

Establishing glucosylglycerol synthesis in potato (Solanum tuberosum l. cv. Albatros) by expression of the ggpPS gene from Azotobacter vinelandii

“…Such transgenic crop plants may show sustainable growth under saline or drought conditions. Moreover, significant GG accumulation in potato tubers could be used as raw material for GG purification, which is used under the trade name glycoin in cosmetics or pharmaceuticals as moisture or stabilizer (http://www.bitop.de; [31]). …”

“…GG is effective to protect macromolecules against denaturation has been proven with many in vitro systems [28,29,31]. The improved fitness of salt and drought-stressed transgenic potato plants can also be related to the overall increase of soluble sugars inside leaves.…”