Construction of mutant variants of gayal rumen cellulase CMX-1 and study on its enzymatic characterization

LAO Fengjuan; HUO Dengliang; LAI Jiewei; JIU Mei; BA Sangzhuzha; DAN Zengluosang; YANG Shuli

doi:10.13557/j.cnki.issn1002-2813.2025.24.015

Abstract：The experiment aims to analyze the basic enzymatic properties of the highly expressed cellulase CMX-1 screened from the gayal rumen Fosmid library.To further improve the catalytic efficiency of the enzyme,the CMX-1 encoding gene was mutated and modified using error-prone polymerase chain reaction(ep-PCR).The results showed that the mutant gene EMX-1 was successfully obtained.Sequence analysis revealed that EMX-1 encodes 305 amino acids(aa),with a theoretical molecular weight of 34.29 kDa and an isoelectric point(pI)of 5.70.It belongs to the glycoside hydrolase family 5(GH5)and shares a maximum sequence similarity of 80%with homologous sequences in the GenBank database.The mutant gene was efficiently expressed in Escherichia coli BL21 and exhibited cellulase activity.Enzymatic characterization results showed that the optimal temperature of the mutant enzyme EMX-1 was the same as that of the original enzyme CMX-1,both at 50℃.At this temperature,the optimal pH value of EMX-1 increased from 5.0 of CMX-1 to 5.5.At their respective optimal pH value and 50℃conditions,the cellulase activity of EMX-1 was 247.76 U/mL,which was about 1.4 times higher than that of CMX-1(178.92 U/mL).EMX-1 exhibited stronger stability than CMX-1 within the temperature range of 40 to 50℃and pH value of 3.0 to 9.0.The study shows that this study successfully obtained the mutated enzyme EMX-1 with significantly improved catalytic performance through ep-PCR technology,providing new enzyme resources for the development of ruminant feed additives.