Enhancing Activity of Pleurotus sajor-caju (Fr.) Sing ß-1,3-Glucanoligosaccharide (Ps-GOS) on Proliferation, Differentiation, and Mineralization of MC3T3-E1 Cells through the Involvement of BMP-2/Runx2/MAPK/Wnt/ß-Catenin Signaling Pathway.

Osteoporosis is a leading world health problem that results from an imbalance between bone formation and bone resorption. ß-glucans has been extensively reported to exhibit a wide range of biological activities, including antiosteoporosis both in vitro and in vivo. However, the molecular mechanisms responsible for ß-glucan-mediated bone formation in osteoblasts have not yet been investigated. The oyster mushroom Pleurotus sajor-caju produces abundant amounts of an insoluble ß-glucan, which is rendered soluble by enzymatic degradation using Hevea glucanase to generate low-molecular-weight glucanoligosaccharide (Ps-GOS). This study aimed to investigate the osteogenic enhancing activity and underlining molecular mechanism of Ps-GOS on osteoblastogenesis of pre-osteoblastic MC3T3-E1 cells. In this study, it was demonstrated for the first time that low concentrations of Ps-GOS could promote cell proliferation and division after 48 h of treatment. In addition, Ps-GOS upregulated the mRNA and protein expression level of bone morphogenetic protein-2 (BMP-2) and runt-related transcription factor-2 (Runx2), which are both involved in BMP signaling pathway, accompanied by increased alkaline phosphatase (ALP) activity and mineralization. Ps-GOS also upregulated the expression of osteogenesis related genes including ALP, collagen type 1 (COL1), and osteocalcin (OCN). Moreover, our novel findings suggest that Ps-GOS may exert its effects through the mitogen-activated protein kinase (MAPK) and wingless-type MMTV integration site (Wnt)/ß-catenin signaling pathways.

A chitinolytic endochitinase and ß-N-acetylglucosaminidase-based system from Hevea latex in generating N-acetylglucosamine from chitin.

An endochitinase and ß-N-acetylglucosaminidase (NAGase) were purified and characterised from fresh rubber latex serum. These enzymes were used in a total enzyme-based system to produce pure N-acetylglucosamine (NAG) from chitin. The N-terminal amino acid sequences of both purified endochitinase (KEESRRRRHR) and NAGase (AAVDSDTLEI) lacked homology with other known chitinases, including hevamine from rubber latex lutoids. The apparent kinetic parameters, Km and Vmax, for the endochitinase using 4-MU-ß-(NAG)3 as a substrate were 99.73 µM and 29.49 pkat mg(-1), respectively. For NAGase, using 4-MU-ß-NAG as a substrate, the corresponding Km and Vmax values were 20.4 µM and 25.82 pkat mg(-1). When an enzyme incubation mixture containing a 1:1 (pkat/pkat) activity mixed ratio of endochitinase: NAGase was employed, the maximum yield of N-acetylglucosamine (NAG) obtained was 98% from ß-chitin and 20% from α-chitin. These yields were obtained after 4 days of hydrolysis of equal amounts of ß-chitin and α-chitin in the mixture. Thus, ß-chitin was the preferred substrate compared to α-chitin by a ratio of nearly five to one. Mass spectroscopic analysis, using electrospray ionisation mass spectrometry (ESI-MS), of the product obtained from ß-chitin after digestion (for 24h) depicted one distinct major molecular ion peak m/z 260.1, a small minor ion peak m/z 481.2, a potassium adduct of NAG and a potassium adduct of two NAG molecules. Furthermore, experiments to establish the commercial production of NAG using crude enzymes of Hevea latex serum are currently in progress.