Characterization of a highly stable α-galactosidase from thermophilic Rasamsonia emersonii heterologously expressed in a modified Pichia pastoris expression system

Abstract

BackgroundStructurally stable α-galactosidases are of great interest for various biotechnological applications. More thermophilic α-galactosidases with high activity and structural stability have therefore to be mined and characterized. On the other hand, few studies have been performed to prominently enhance the AOX1 promoter activity in the commonly used Pichia pastoris system, in which production of some heterologous proteins are insufficient for further study.ResultsReGal2 encoding a thermoactive α-galactosidase was identified from the thermophilic (hemi)cellulolytic fungus Rasamsonia emersonii. Significantly increased production of ReGal2 was achieved when ReGal2 was expressed in an engineered Pastoris pichia expression system with a modified AOX1 promoter and simultaneous fortified expression of Mxr1 that is involved in transcriptionally activating AOX1. Purified ReGal2 exists as an oligomer and has remarkable thermo-activity and thermo-tolerance, exhibiting maximum activity of 935 U/mg towards pNPGal at 80 °C and retaining full activity after incubation at 70 °C for 60 h. ReGal2 is insensitive to treatments by many metal ions and exhibits superior tolerance to protein denaturants. Moreover, ReGal2 efficiently hydrolyzed stachyose and raffinose in soybeans at 70 °C in 3 h and 24 h, respectively.ConclusionA modified P. pichia expression system with significantly enhanced AOX1 promoter activity has been established, in which ReGal2 production is markedly elevated to facilitate downstream purification and characterization. Purified ReGal2 exhibited prominent features in thermostability, catalytic activity, and resistance to protein denaturants. ReGal2 thus holds great potential in relevant biotechnological applications.

Recent developments in heterologous production of cellulases and xylanases using the Pichia pastoris expression system

“…The transcription of pAOX1, pDAS1, and pDAS2 in the presence of Mxr1 (containing S215A mutant) was decreased up to 60% which indicated that Mxr1 exclusively regulates the transcription of pAOX1, pDAS1, and pDAS2. 56 An et al 51 investigated the regulation of modified pAOX1 and simultaneous fortified expression of Mxr1 that is involved in pAOX1 transcription activation for the production of sufficient amounts of a thermo-active ⊍-galactosidase from Rasamsonia emersonii (ReGal2). The expression of ReGal2 in the engineered P. pastoris enabled the production of suitable www.soci.org H Ullah et al…”

CRISPR/Cas9 mediated gene editing of transcription factor ACE1 for enhanced cellulase production in thermophilic fungus Rasamsonia emersonii

“…Hence, thermophilic fungi which are capable of secreting high yields of thermostable enzymes have gained considerable interest. One such thermophilic fungus R. emersonii has been reported to contain several lignocelluloytic enzymes including endo-1,4-β-glucanase, β-glucosidase, α-galactosidase, xylanase and amylases [3,5,6], thus possessing great industrial and biotechnological potential.…”

Engineering a Trypsin-Resistant Thermophilic α-Galactosidase to Enhance Pepsin Resistance, Acidic Tolerance, Catalytic Performance, and Potential in the Food and Feed Industry

“…α-Galactosidase production and activity have been reported in fungi, bacteria, plants, and insects. − Most known α-galactosidases belong to glycoside hydrolase (GH) families 27 and 36 . Some α-galactosidases are specific for 4-nitrophenyl α-galactopyranoside ( p NPG), 2-nitrophenyl β-D-galactopyranoside, 4-nitrophenyl β-D-glucuronide, melibiose, maltose, raffinose, or stachyose, , and some had high enzyme activity for polymers guar gum, locust bean gum, carob galactomannan, or gum arabic. , …”