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The transesteriflcation of crude cottonseed oil with methyl acetate to biodiesel catalyzed by combined lipases (Lipozyme TL IM and Novozym 435) was investigated. The significant variables of transesterification (methanol concentration, combined lipases concentration and reaction temperature) were screened from six variables by PlackettBurman (PB) design and were further optimized via response surface methodological approach. A 20-run full factorial central composite design (CCD) was used to construct the statistical model, and the optimal conditions were obtained as follows: temperature 57.36Â°C, reaction time 10 h, 17.7% combined lipases (m (Lipozyme TL IM) : m (Novozym 435)=2:1) based on oil weight, substrate molar ratio (methyl acetate/cottonseed oil) 12:1, 2.96% methanol and 35% n-hexane based on oil weight. The predicted biodiesel yield was 93.61% under the optimal conditions and the subsequent verification experiments with biodiesel yield of 94.92% confirmed the validity of the predicted model.