Expression of a secretory α-glucosidase II from Apis cerana indica in Pichia pastoris and its characterization

Abstract

Backgroundα–glucosidase (HBGase) plays a key role in hydrolyzing α-glucosidic linkages. In Apis mellifera, three isoforms of HBGase (I, II and III) have been reported, which differ in their nucleotide composition, encoding amino acid sequences and enzyme kinetics. Recombinant (r)HBGase II from A. cerana indica (rAciHBGase II) was focused upon here due to the fact it is a native and economic honeybee species in Thailand. The data is compared to the two other isoforms, AciHBGase I and III from the same bee species and to the three isoforms (HBGase I, II and III) in different bee species where available.ResultsThe highest transcript expression level of AciHBGase II was found in larvae and pupae, with lower levels in the eggs of A. cerana indica but it was not found in foragers. The full-length AciHBGase II cDNA, and the predicted amino acid sequence it encodes were 1,740 bp and 579 residues, respectively. The cDNA sequence was 90% identical to that from the HBGase II from the closely related A. cerana japonica (GenBank accession # NM_FJ752630.1). The full length cDNA was directionally cloned into the pPICZαA expression vector in frame with a (His)6 encoding C terminal tag using EcoRI and KpnI compatible ends, and transformed into Pichia pastoris. Maximal expression of the rAciHBGase II–(His)6 protein was induced by 0.5% (v/v) methanol for 96 h and secreted into the culture media. The partially purified enzyme was found to have optimal α-glucosidase activity at pH 3.5 and 45°C, with > 80% activity between pH 3.5–5.0 and 40–55°C, and was stabile (> 80% activity) at pH 4–8 and at < 25–65°C. The optimal substrate was sucrose.ConclusionsLike in A. mellifera, there are three isoforms of AciHBGase (I, II and III) that differ in their transcript expression pattern, nucleotide sequences and optimal enzyme conditions and kinetics.

Comprehensive enzymatic analysis of the amylolytic system in the digestive fluid of the sea hare, Aplysia kurodai: Unique properties of two α‐amylases and two α‐glucosidases

“…Compared with bacterial and fungal α-glucosidase reports, there are very few reports concerning enzymatic characterization of α-glucosidase purified from invertebrates. To date, invertebrate α-glucosidase has been purified from honeybee ( Apis cerana ) [39] , ( A. mellifera ) [25,29] , fruit fly ( Drosophila melanogaster ) [40] , abalone ( Haliotis discus hannai ) [31] , mottled sea hare ( A. fasciata ) [41] , and shrimp ( Penaeus vannamei and P. japonicus ) [12,42] . These enzymes belong to GHF 13 but differ in substrate specificity and molecular mass.…”

Transcriptomic analysis of the honey bee (Apis mellifera) queen spermathecae reveals genes that may be involved in sperm storage after mating

“…The α Glucosidase 2 protein is predominant in the ventriculus and hemolymph of honey bee workers [ 65 ], and it cleaves glucose residues in N-linked oligosaccharides. Moreover, real time semi-quantitative PCR data has previously shown that α Glucosidase 1 mRNA is more highly expressed in adult Apis cerana foragers than α Glucosidase 2 mRNA, while the levels of α Glucosidase 2 mRNA are greater in eggs, larvae and pupae [ 67 ]. Our RT-qPCR results confirmed that α Glucosidase 2 mRNA levels were greater in spermathecae from virgin honey bee queens compared to those in mated queens.…”

Biocatalytic synthesis of 2‐O‐α‐D‐glucopyranosyl‐L‐ascorbic acid using an extracellular expressed α‐glucosidase from Oryza sativa

“…[ 44 ] The thermal stability of AGLs among different species showed obvious differences. A secretory AGL from A. cerana that was expressed in P. pastoris , was stable (> 90% residual activity) to a 15‐min exposure of < 45°C, [ 29 ] and a thermostable AGL from Geobacillus sp. that was expressed in E. coli , maintained 90% of activity after 30 h of incubation at 60°C.…”

“…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. [ 27 ] Several AGLs from Aspergillus species [ 21,28 ] and Apis cerana indica [ 29 ] have been successfully expressed in P. pastoris . The inducible alcohol oxidase I promoter (P AOX1 ) and the constitutive glyceraldehyde‐3‐phosphate dehydrogenase promoter (P GAP ) are commonly used in genetic engineering of P. pastoris .…”