Purification and Characterization of β-Mannanase Derived from Rhizopus microsporus var. rhizopodiformis Expressed in Komagataella phaffii

The demand for food-grade β-mannanases, ideal for high-temperature baking, is increasing. Using the <i>Komagataella phaffii</i> (<i>P. pastoris</i>) expression system for β-mannanase production, this study aimed to enhance purification methods. We evaluated better conditions for production and purification of β-mannanase (<i>PpRm</i>Man134A) from recombinant <i>P. pastoris</i> X-33, focusing on a higher purity and reducing the production of endogenous secretory proteins in fermentation. By adjusting carbon and nitrogen sources, culture time, and temperature, we controlled cell growth to reduce the production of endogenous secretory proteins. The better-evaluated conditions involved culturing recombinant <i>P. pastoris</i> in 70 mL buffered glycerol complex medium for 24 h at 30 °C, then in modified buffered methanol-complex medium with 0.91% (<i>w</i>/<i>v</i>) methanol, 0.56% (<i>w</i>/<i>v</i>) sorbitol, and 0.48% (<i>w</i>/<i>v</i>) mannitol for another 24 h, which improved the <i>PpRm</i>Man134A yield and reduced endogenous secretory proteins, shortening the fermentation time by 72 h. An affordable purification method using ultrafiltration and salt-out precipitation was utilized. <i>PpRm</i>Man134A showed thermostability up to 100 °C and effectively degraded locust bean gum into smaller fragments, mainly producing mannotriose. In conclusion, with its enhanced purity due to reduced levels of endogenous secretory proteins, purified <i>PpRm</i>Man134A emerges as a promising enzyme for high-temperature baking applications.