Expression Of Aspergilus Fumigatus Z5endo-xylanases And Acetyl Xylan Esterasegenes And The Synergistic Effect Between Them

The world produces about 200 billion tons of agricultural waste each year all over the world,which is one of the most extensive natural sources of lignocelluloses and includes crop straws,rice husk,bagasse,com cob,etc.Lignocellulose mainly includes cellulose,hemicellulose and lignin,and xylan is one of the most abundant type of hemicellulose.At present,the research of hemicellulase biochemistry and molecular biology is focused on xylan degradation enzyme.Xylanase is a type of the degradation enzyme which can hydrolyze xylan into xylose.Due to its biodegradable beta-1,4-glycosidic bond of xylan,xylanase not only can reduce the viscosity and nutritional resistance of xylan,but also can produce low poly xylose with care and regulating gastrointestinal functions.It has been widely used in food,feed,pharmaceutical,paper,textile and other industries.Due to thecomplex structure,xylan degradation needs the synergy of a variety of xylanase,including the enzymes for the hydrolysis of backbone and the enzymes for the hydrolysis of side chains.The mechanism of these xylan degradation enzymes work synergistically to completely hydrolyze xylan is unclear.A cellulose degrading fungal strain Aspergilusfumigatus Z5 has been screened from high temperature compost samples.We analyzed the RT?PCR and mass spectrometry data of A.fumigatus Z5 induced by oat spelt xylan,and got several major xylan degradation related genes,including four endo-xylanase genes?Y699-04481,06333,07611,09486?,two xylosidase genes?Y699-04570,05610?and an acetyl xylan esterase gene?Y699-05457?.This study aimed to achieve efficient heterologous expression of these xylanase genes in Aspergillus niger and Pichia pastoris X33,and to study the synergistic effect between the different endo-xylanases and acetyl xylan esterase.The results were obtained as follows:?1?The condition of protoplast preparation and co-transformant efficiency was optimized to realize the heterologous expression of xylanse gene in filamentous fungi.?2?A GH10 family ?-1,4-endoxylanase gene Y699-09486 and an acetyl xylan esterase gene Y699-05457 with a carbohydrate-binding domain sequence?cbm₁?,were successfully expressed,the corresponding protein are Xyn10C and AXE1.The activity of Xyn10C transformant fermentation was 83.08 IU/mL,the optimal reaction conditions were 70?,pH 5.0-6.0,it can still remain more than 95%activity after 1h in 60?.The activity of Acetyl xylan esterase AXE1 transformant fermentation reached 84.76 IU/mL,the optimal reaction condition is 55-60?,pH7.0-7.5,more than 70%activity still remained after 1h in 60?.?3?A fusion protein Xyn10BaLC was successfully expressed in Pichia pastoris X33,which was engineered from Xyn10B?Y699-06333?with a linker region and CBM1 domain from Y699-04481 added to its C-terminus.It showed similar enzymatic properties to XynlOB,but the enzyme catalytic efficiency?Kcat/Km?decreased from 22.42 s-1 mg-1 ml to 4.57 s-1 mg-1 ml.?4?The reducing sugar production of washed corncob powder?WCCP?hydrolyzed with different endo-xylanase?Xyn10C,Xyn10B and Xyn10BaLC?was compared.It was found that Xyn10C and Xyn10,which belong to GH10 family endoxylanase,showed almost no degradation capacity,while Xyn10BaLC with CBM is capable of degrading WCCP to produce reducing sugar;for WCCP treated with high temperature and pressure,the amount of sugar production increased with Xyn10,Xyn10C and Xyn10B also showed the capacity of producing reducing sugar.These results indicated that CBM1 domain is important in the process of WCCP hydyolysis,and the destruction of the WCCP may release some xylan that can be degraded by endo-xylanase without CBM domain.?5?The synergistic effects between Xyn10C,Xyn10,Xyn10BaLC and AXE1 were compared.XynlOBaLC showed an approximately 2-fold synergistic effect with AXE1 for WCCP degradation,and the other two endo-xylanases without CBM showed no synergistic effect.These results indicate that the localization of CBM1 domain plays the critical role in the synergistic effect of GH10 xylanases and AXE1.?6?For unwashed corncob,CBM 1-lacking GH10 xylanases,including Xyn10B and Xyn10,were demonstrated to be able to degrade the corncob powder and release the reducing sugars.In fact,physical milling could destroy the structure of corncob and result in the internal materials,such as cellulose and xylan,exposed on the surface of the corncob powder.The released soluble and accessible xylan was used as the suitable substrate of CBM1-lacking GH10 xylanases,which could be stopped by the washing.Additionally,Xyn10BaLC was still able to degrade the corncob powder but never show synergy with AXE1 under the interference of the soluble and accessible xylan.If the processing method of corncob was changed,for example manually cutting,the total broken surface would be much less than that of the milling powder.In this condition,the cut corncob could not be degraded by GH10 xylanases without CBMI but could be degraded by Xyn10BaLC.Together,these results suggested the complexity of the corncob structure,and directly shown that the processing methods would result in the different performances of xylan degradation.However,in natural environment,Plant biomass is a solid stable polysaccharide complex and can not be easily destroyed to be the soluble polysaccharides.CBM1-containing GH10 xylanases have the ability to bind and degrade solid plant biomass actually participate in the limiting steps of natural solid xylan degradation by filamentous fungi.