1 Introduction
L-Ascorbic acid (L-AA) is a water-soluble vitamin, commonly known as vitamin C (VC), which participates in various physiological activities and plays an important role in maintaining and promoting human health[1-3]. VC is an essential nutrient that cannot be synthesized by the human body, and must be obtained from foods and supplements [4-5]. However, the extreme instability in aqueous solutions, especially under specific oxidation conditions, directly reduces its biological activity, resulting in its applications being greatly restricted [6-9]. In order to maintain the same biological activity and improve its stability, various VC derivatives including ascorbyl phosphate, ascorbyl sulfate [10-11], ascorbyl palmitate[12-13] and ascorbyl glucoside[14-15] have been chemically or biologically synthesized, among which 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) is extremely stable and nonreducible, which has attracted considerable attention with its wide range of applications in cosmetics, food, and medicine [16-17].
Enzymatic synthesis is a preferred method for AA-2G production over alternative chemical synthesis owing to the regioselective glucosylation reaction [16]. Up to now, AA-2G has mainly been synthesized by transglucosylation of L-AA catalyzed by several enzymes, including α-glucosidase [18-19], cyclodextrin glycosyltransferase (CGTase) [20-21], amylase[22], sucrose phosphorylase[23-24], and α-isomaltosylglucosaccharide synthase[25]. Among them, the synthesis of AA-2G by α-glucosidase from higher eukaryote such as rice seed and mammals has the advantages of less intermediates and by-products[18, 26, 27]. However, due to the low efficiency and limited source of glycoside hydrolases, there are only a few related reports in recent years [16, 28].
α -Glucosidase (AGL; EC 3.2.1.20), mainly classified into the glycoside hydrolase families GH13 and GH31, represents a group of exoglycosidases widely distributed in microorganisms, plants, and animals, which can react with the α -glucosidic bond at the nonreducing terminal of substrates and release α -glucose[29]. Under high substrate concentration, GH31 AGL can also catalyze transglucosylation to synthesize oligosaccharides or other α-glucosylated compounds [29-30]. The glycosylated hydroxyl group of the receptor of α-glucosidase transglycosylation activity is independent on its hydrolysis specificity, and the substrate specificity of GH 31 AGL hydrolysis activity varies with enzyme source[31]. It is possible to synthesize AA-2G by glycoside hydrolases belonging to family GH31, because these are retaining enzymes with the catalytic mechanism allowing them to act as transferases. The pattern of transglycosylation products of glycoside hydrolase mainly depends on the structure of the enzyme, especially its catalytic center[32-34].
Pichia pastoris is the excellent expression host with well-established genetic tools and cultivation strategies for scale-up producing heterologous proteins, particularly industrial enzymes and biopharmaceuticals [35]. Several AGL fromAspergillus species [28, 36, 37] andApis cerana indica [38] have been successfully expressed in P. pastoris . The inducible alcohol oxidase I promoter (PAOX1) and the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP) are commonly used in genetic engineering of P. pastoris . Unlike PAOX1, PGAP simplifies the cultivation by avoiding the use of toxic methanol as a carbon source and inducer[39]. In the present study, the AGL gene fromOryza sativa was codon-optimized and synthesized for extracellular production of the rAGL in P. pastoris under the regulation of PGAP. The rAGL was characterized, and its potential for AA2G production via transglycosylation of L-AA was investigated.