Since the large-scale bioethanol industrialization is not fully mature, as one of the main technical steps, the enzymatic hydrolysis process needs to be further studied. In this paper, the three-stage enzymatic hydrolysis method was used to solve the problem of high glucose concentration inhibition during the high substrate concentration hydroly-sis process. The enzymatic hydrolysis rate was improved by the three-stage enzymatic hydrolysis method, which could increase the enzymatic hydrolysis rate and shorten the enzymatic reaction time by removing the reaction products, i.e., cellobiose and glucose, in the hydrolysis process, reducing the product inhibition and increasing the enzyme reaction rate. The hydroxyl radical produced by the fenton reaction caused the loose structure and changed the three-dimensional structure of the cellulose in the biomass, which was propitious to improve the hydrolysis efficiency of the subsequent enzyme by NaOH-Fenton pretreatment method. The results showed that 2% NaOH-Fenton pretreated sample had higher cellulose content and lower hemicellulose content compared with the materials with 2% NaOH pre-treated sample. The enzymatic hydrolysis yield decreased with the increase of substrate concentration, either in one-stage or three-stages enzymatic hydrolysis. The NaOH-Fenton pretreated mulberry at 30% substrate concentration were performed the three-stage hydrolysis, using the enzyme dosage 40 U/g cellulose, and the three-stage hydrolysis could reach much higher hydrolysis yields of 74.16% in 30 h. Compared with the 72 h yield ( 45.61%) of the one-stage hy-drolysis, a 62.60% increase in hydrolysis yield was achieved and 42 h hydrolysis time was reduced. The results have practical significance for improving the yield of cellulase hydrolysis and reducing the cost of making fuel ethanol from biomass resources.%为改善高底物浓度酶水解过程中产物抑制问题,采用三段酶水解方法,通过在水解过程中及时移除反应产物纤维二糖和葡萄糖,降低产物抑制作用,增加酶反应速率,从而提高酶水解得率、缩短酶反应时间.与原料和经NaOH预处理的桑木比较,NaOH-Fenton预处理后的桑木中木聚糖含量明显降低,纤维素含量相对增加,木质素含量变化较小.无论是一段水解还是三段水解,纤维素酶水解得率均随底物质量浓度的升高而下降.在0.30 g/mL(m/V)底物质量浓度下,当酶用量增加为40 U/g(以纤维素质量计)时,三段(10+10+10)h酶水解得率74.16%,比一段水解72 h得率45.61%增长了62.60%,并且水解时间缩短了42 h.该研究结果对提高纤维素酶水解得率、降低纤维资源制取燃料乙醇成本具有指导意义.
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