Proteomic profiling of amylases secreted by Penicillium crustosum UEM-45 and characterization of two major enzymes
摘要
Despite the industrial relevance of fungal amylases, those from Penicillium crustosum remain poorly characterized. This study aimed to use a proteomics-based approach to identify and characterize extracellular amylases from P. crustosum UEM-45. The extracellular proteome of P. crustosum UEM-45 was analyzed by LC–MS/MS, resulting in the identification of seven amylases among 1,641 extracellular proteins. An α-amylase (CAZy GH13_1) and a glucoamylase (CAZy GH15) were partially purified and characterized. Both enzymes exhibited molecular masses of approximately 67 kDa, were predicted to be monomeric, and to contain a CBM20 module in addition to their catalytic domains. The α-amylase exhibited optimal activity at pH 6.5 and 40 °C, whereas the glucoamylase showed optimal activity at 45 °C. The melting temperatures (Tm) of the α-amylase and glucoamylase were 50.16 °C and 47.8 °C, respectively. Both enzymes were stable at 4 °C. The KM values for starch were 0.98 mg/mL for the α-amylase and 4.51 mg/mL for the glucoamylase, whereas the Vmax was 0.12 mg/min of starch consumed by the α-amylase and 0.3 µmol/min of glucose released by the glucoamylase. Regulatory and substrate specificity assays revealed distinct profiles. When combined, the enzymes efficiently converted starch into glucose and degraded raw starch. Overall, this study demonstrates that proteomics is effective for discovering novel amylolytic enzymes in P. crustosum. Notably, the characterization of a glucoamylase from this fungus contributes to the limited body of knowledge on this enzyme in Penicillium. The liquefaction and saccharification capabilities of the combined enzymes highlight their potential for industrial starch processing.