Mannanase Man23 Mutant Library Construction Based On A Novel Cell-Free Protein Expression System

BACKGROUNDMannanases are important enzymes which are widely used as a tool in agriculture and food industries. To improve the performance of mannanase Man23, a mutant library was created with rational design, and mutations were introduced on loops around the catalytic region. The Brevibacillus brevisB16 cell-free system which was created in this experiment provided the ability to express the mutant library efficiently. The activities of mutants were measured with a multi-volume spectrophotometer.RESULTSThe mutant Man1606 gained from this system is a sextet which has mutations of N146G, S147H, S156P, T157Y, Q206S and T249H simultaneously on loops 6, 8 and 10. Man1606 showed higher activity and stability than Man23. The optimal temperature of Man1606 rose by 5 degrees C (from 55 to 60 degrees C) and the optimal pH increased slightly but its range became broader.CONCLUSIONThis experiment demonstrated the B. brevis cell-free system shortens the expression time and is an efficient tool for mannanase engineering. (c) 2016 Society of Chemical Industry