Among the possible delivery routes, the oral administration of a protein is simple and achieves high patient compliance without pain. However, the low bioavailability of a protein drug in the intestine due to the physical barriers of the intestinal epithelia is the most critical problem that needs to be solved. To overcome the low bioavailability of a protein drug in the intestine, we aimed to construct a recombinant Pichia pastoris expressing a human growth hormone (hGH) fusion protein conjugated with a transcytotic peptide (TP) that was screened through peroral phage display to target goblet cells in the intestinal epithelia. The TP-conjugated hGH was successfully produced in P. pastoris in a secreted form at concentrations of up to 0.79 g/l. The function of the TP-conjugated hGH was validated by in vitro and in vivo assays. The transcytotic function of the TP through the intestinal epithelia was verified only in the C terminus conjugated hGH, which demonstrated the induction of IGF-1 in a HepG2 cell culture assay, a higher translocation of recombinant hGH into the ileal villi after oral administration in rats and both IGF-1 induction and higher body weight gain in rats after oral administration. The present study introduces the possibility for the development of an effective oral protein delivery system in the pharmaceutical and animal industries through the introduction of an effective TP into hGH.

IntroductionL-Arabinose is a pentose sugar that can be obtained by the hydrolysis of various plant cell wall hemicellulosic polymers such as arabinoxylans and arabinans [1]. Due to the health-beneficial functions, L-arabinose and arabino-oligosaccharides (AOS) have been considered as promising candidates for low-calorie sweeteners and prebiotic materials [2][3][4]. For the enzymatic production of L-arabinose, α-L-arabinofuranosidase (ABF; E.C. 3.2.1.55) is the key player which catalyzes the exo-type hydrolysis of L-arabinose-containing polymers [5,6]. Especially, its synergistic actions with endo-α-(1,5)-L-arabinanase (ABN; E.C. 3.2.1.99) were suggested for the cost-effective L-arabinose production from sugar beet arabinan [7,8].The ABFs possess versatile hydrolyzing activities towards the terminal non-reducing α-(1,2)-, α-(1,3)-, and/or α-(1,5)-L-arabinofuranosidic linkages in various polymeric and oligomeric substrates [5,6]. Depending on their structural features, the common microbial ABFs can be mainly categorized to the members of glycoside hydrolase (GH) families 51 and 43. To date, various ABFs have been reported from mainly bacteria, as well as a few fungi and plants [5,6,9]. Based on the genome analyses, the probable gene clusters for the utilization of L-arabinose and arabinans were found from Bacillus [10, 11], Geobacillus [12], and Corynebacterium spp. [13]. Bacillus subtilis produces two intracellular exo-ABFs GH51 and two extracellular endo-ABNs GH43, which can synergistically degrade the arabinan polymers to L-arabinose [11]. On the contrary, only a limited number of eukaryotic ABFs were genetically and enzymatically characterized from the fungal microorganisms such as Penicillium [14], Aspergillus [15], Chrysosporium [16], and Aureobasidium spp. [17]. As the eukaryotic ABFs share relatively low Two genes encoding probable α-L-arabinofuranosidase (E.C. 3.2.1.55) isozymes (ABFs) with 92.3% amino acid sequence identity, ABF51A and ABF51B, were found from chromosomes 3 and 5 of Saccharomycopsis fibuligera KJJ81, an amylolytic yeast isolated from Korean wheat-based nuruk, respectively. Each open reading frame consists of 1,551 nucleotides and encodes a protein of 517 amino acids with the molecular mass of approximately 59 kDa. These isozymes share approximately 49% amino acid sequence identity with eukaryotic ABFs from filamentous fungi. The corresponding genes were cloned, functionally expressed, and purified from Escherichia coli. SfABF51 A and SfABF51 B showed the highest activities on p-nitrophenyl arabinofuranoside at 40~45 o C and pH 7.0 in sodium phosphate buffer and at 50 o C and pH 6.0 in sodium acetate buffer, respectively. These exoacting enzymes belonging to the glycoside hydrolase (GH) family 51 could hydrolyze arabinoxylooligosaccharides (AXOS) and arabino-oligosaccharides (AOS) to produce only L-arabinose, whereas they could hardly degrade any polymeric substrates including arabinans and arabinoxylans. The detailed product analyses revealed that both SfABF51 isozymes can catalyze the ver...

The gene encoding an α-L-arabinofuranosidase (BvAF) GH51 from Bacillus velezensis FZB42 was cloned and expressed in Escherichia coli. The corresponding open reading frame consists of 1,491 nucleotides which encode 496 amino acids with the molecular mass of 56.9 kDa. BvAF showed the highest activity against sugar beet (branched) arabinan in 50 mM sodium acetate buffer (pH 6.0) at 45 o C. However, it could hardly hydrolyze debranched arabinan and arabinoxylans. The time-course hydrolyses of branched arabinan and arabinooligosaccharides (AOS) revealed that BvAF is a unique exo-hydrolase producing exclusively L-arabinose. BvAF could cleave α-(1,2)-and/or α-(1,3)-L-arabinofuranosidic linkages of the branched substrates to produce the debranched forms of arabinan and AOS. Although the excessive amount of BvAF could liberate L-arabinose from linear AOS, it was extremely lower than that on branched AOS. In conclusion, BvAF is the arabinan-specific exo-acting α-L-arabinofuranosidase possessing high debranching activity towards α-(1,2)-and/or α-(1,3)-linked branches of arabinan, which can facilitate the successive degradation of arabinan by endo-α-(1,5)-Larabinanase.

Trait-based functional studies are widely used to elucidate the relationships between ecological indicators and environmental parameters as well as to predict functional change in aquatic biota in response to various types of human disturbance. Clarifying how functional traits of aquatic organisms depend on environmental conditions can facilitate aquatic conservation and management, but determining the importance of these traits to ecological river health requires further investigation. As fish play a key role in the assessment of ecological conditions, we examined the relevance of the functional diversity of lotic fish to the river health assessment using multi-metric models of water pollution (mWPI) and fish-based biological integrity (mIBI). Twelve fish traits related to food acquisition, environmental stability, and mobility were used for the functional analyses. Chemical river health was highly sensitive to downstream organic matter and nutrient pollution according to mWPI. Based on the present gradient of chemical health and water chemical variables, we identified three water quality groups (G-I, G-II, and G-III). G-I, G-II, and G-III showed low, intermediate, and high levels of water quality degradation, respectively. Spatially significant differences among these groups were observed for both the taxonomic and functional structures of lotic fish as well as ecological river health based on mIBI. The dominance of sensitive species was high in G-I, whereas tolerant and exotic species contributed strongly to the species compositions of G-II and G-III. Functional richness and dispersal were significantly reduced in G-III, and their decreases correlated with ecological health and the loss of species that are insectivorous, rheophilic, and sensitive to water pollution. Regarding redundancy analyses, both the models of functional trait metrics (F = 8.06, p < 0.001) and mIBI metrics (F = 4.88, p < 0.01) indicated good performance in terms of the variation in water quality and chemical river health parameters. Overall, the functional trait-based diversity of lotic fish is significant to the assessment of ecological river health and reflects water chemical quality. This association arises because niche occupation in functional space by all species, along with their abundance distribution, is highly responsive to the loss of species with sensitive traits due to water pollution.