The Heterologous Expression Of Cellulase Accessory Enzyme AA9 In Pichia Pastoris And Preliminary Application

Lignocellulosic biomass is abundant renewable resource.It is widely recognized that the production of enzyme-based biofuel is of a great significant potential in solving the present energy problem.However,the low efficiency of the cellulase enzyme hydrolysis in the biofuel process has become a big impedence to industrial biofuel production.Some increasing studies have demonstrated that lytic polysaccharide monooxygenates Auxiliary Activity 9（AA9）can act on cellulose crystallinity and work in synergy with cellulase to hydrolyze the lignocellulosic substrate.Accordingly,this thesis attempted to construct recombinant that was capable to efficiently express AA9 and further explore its active role in the cellulase enzymatic hydrolysis.The experiment first constructed the recombinant strain capable of expressing AA9.The Podospora anserina AA9 gene was firstly optimized.And two recombinants P.pastoris/pPIC9K-gh61 and P.pastoris/pPICZαA-gh61 containing pPIC9K and pPICZαA,respectively,were constructed with resistance screening and shake flask fermentation.Further,a double-plasmid recombinant P.pastoris GS115-D-gh61 with a high enzyme activity was constructed.The optimal reaction temperature of recombinant AA9 was 60℃and its optimum reaction was pH 6.0.The addition of Co²⁺,Ba²⁺and Cu²⁺(5 mmol·L^-1)played their respective active role on the recombinant AA9 enzyme activity（increased by 39%,51%and19%respectively）.Then,some key variables of recombinant incubation were selected at the flask level for high AA9 yield.With a single-factor and orthogonal design,the fermentation condition of recombinants was optimized as follows:pH 6.0,1%of methanol concentration,3%of inoculation size,and 2 g·L^-1 of PEG 4000 addition.Under optimized condition,the recombinant enzyme activity was 0.58 U·L^-1.In a 5-L fermentation bioreactor,the recombinant yielded 3.4 g·L^-1 of AA9 protein at 188 h with 2.28 U·L^-1 of enzyme activity.Finally,the recombinant AA9 was used preliminarily for cellulase enzymatic hydrolysis of lignocellulosic substrate.Result showed that the AA9 addition had no obvious role on the enzymatic hydrolysis of CMC,but its addition of 0.75 mg·g^-1 enabled the enzymatic hydrolysis of filter paper to increase 40%.And the enzymatic hydrolysis of Avicel with the AA9 addition of 1 mg·g^-1 was twice that of no addition.Further,the recombinant AA9 was used in lignocellulosic enzymatic hydrolysis.It was shown that the hydrolysis rate increased21.3%and 27.3%,respectively,when the AA9(1.0 mg·g^-1 and 0.50 mg·g^-1,respectively)was added in the hydrolysis of alkali-/acid-catalyzed atmospheric glycerol（AGO）pretreated sugarcane bagasse.The active role of AA9 on the hydrolysis of alkali catalyzed AGO pretreated substrate can be strengthened by Co²⁺(2 mmol·L^-1)and L-ascorbic acid(1 mg·g^-1),contributing to the increase by 41.7%and 41.5%,respectively.With the addition of 0.5mmol·L^-1 Cu²⁺and L-ascorbic acid(1 mg·g^-1),AA9 enabled the hydrolysis of acid catalyzed AGO pretreated substrate to increase by 45%and 40.5%,respectively.When the AA9 was used in enzymatic hydrolysis of the alkaline catalyzed AGO pretreated substrate,the enzyme loading reduced by 30%as the hydrolysis level at 3 FPU·g^-1 was the same to that at 4 FPU·g^-1.With the AA9 use in the hydrolysis of acid catalyzed AGO pretreated substrate,the cellulase enzyme loading saved by 17%.In conclusion,the accessory enzyme AA9 expressed heterologously herein is applicable and robust in the cellulase hydrolysis of lignocellulosic biomass,which is of great promise in development of the ongoing enzyme-based lignocellulosic refining industry.