变风补料对里氏木霉产纤维素酶的影响

Effect of fed-batch cultivation with regulated aeration on cellulase production by Trichoderma reesei

为应对资源短缺及环境问题,生物质作为可再生生物资源越来越受到关注。纤维素酶催化纤维素水解为葡萄糖是生物转化的关键环节。为提高纤维素酶的生产能力,在分批补料产酶过程中改变各阶段通风量,控制发酵液溶氧量,促进菌丝体的生长及酶的合成。以微晶纤维素为碳源,里氏木霉(Trichoderma reesei)Rut C30为产酶菌株,分批补料生产纤维素酶,摇瓶滤纸酶活达到14.5 U/m L。在3 L的发酵罐中,恒定通风量,分批补料生产纤维素酶,酶活第10天达到13.7 U/m L。对纤维素酶生产各阶段的通风量进行调整和优化,当通风量第1天为20 L/(L·h)、第2~3天为30 L/(L·h)、第4天及以后为25 L/(L·h)时,发酵液溶氧水平得到明显改善,稳定在15%~30%,菌丝体浓度稳定,滤纸酶活达到18.3 U/m L,纤维素酶产率为93.5 U/(L·h),纤维素酶得率为481.6 U/g微晶纤维素。纤维素酶活提高了33.6%,周期缩短2 d。缩短补料周期至8 h,滤纸酶活达到18.5U/m L,提高35%。通过控制通风量,提高纤维素酶产率,降低纤维素酶的生产成本,促进工业化酶制备。

To cope with the shortage of fossil resources and solve environmental problems,biomass has attracted more and more attention being as the largest renewable resource that can replace fossil on the earth.Lignocellulose is the most abundant biomass on earth with wide distribution and rich in content.The enzymatic hydrolysis of cellulose to glucose by cellulase is one of the key routes of lignocellulose bioconversion.However,the high production cost of cellulase seriously restricts the bioconversion of lignocellulose to fuel and other chemicals.In order to improve the production capacity of cellulase,the ventilation of each stage was changed in the process of batch feeding,the amount of oxygen dissolved in the fermentation broth was controlled,and the growth of mycelium and the synthesis of enzyme were promoted.Using avicel as the carbon source,Trichoderma reesei Rut C30 as an enzyme producing strain,cellulase was produced by fed-batch.The cellulase activity of the shake flask reached 14.5 U/mL,andβ-glucosidase reached 2.33 U/mL with a protein concentration of 3.06 g/L.During the production of cellulose in a 3 L fermentor by fed-batch with constant ventilation,the cellulase activity reached 13.7 U/mL after 10 d,andβ-glucosidase only reached 1.5 U/mL,which was influenced by the deactivation of enzyme caused by shearing force of agitation.The production of cellulase was scaled up in a fermentor.The amount of ventilation was adjusted at all stages of cellulase production.When the ventilation amount was 20,30,and 25 L/(L·h)in the first 3 d,respectively.However,the oxygen content was too low to maintain the metabolism of mycelium in the 4th day,and the cellulase activity reached 17.6 U/mL,andβ-glucosidase was about 1.3 U/mL with a protein concentration of about 5.6 g/L in the 8th day.The cellulase activity increased by 28%and 22%compared with the fermentation under constant ventilation and that in the shake flask,respectively.The optimum condition was as follow:the ventilation amount was 20 L/(L·h)for the first day,30 L/(L·h)in 2nd and 3rd days,and 25 L/(L·h)in the 4th day,the oxygen content of the broth was maintained between 15%and 30%,and the mycelium concentration was stable at the range of 16-18 g/L,the cellulase activity reached 18.3 U/mL andβ-glucosidase about 1.3 U/mL with a protein concentration of 5.6 g/L.The enzyme production cultivation was reduced to 8 d.The productivity was 93.5 U/(L·h),and the yield was 481.6 U/g avicel.The filter paper activity increased by 33.6%,and productivity increased by 67%compared with the fermentation with constant ventilation in fermentor.