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.