Cloning And Expression Of Cellulase Genes From Penicillum Crustosum

Cellulose is the most abundant renewable biomass resources on earth. Due to its wide range ofsources and low cost, it plays a very important role for the sustainable development of human. Thecellulase mediated conversion from cellulose to bioenergy has a broad prospect. With the continuousdevelopment of biotechnology, to use genetic engineering methods to obtain a high activity cellulaserecombinant strain is one of the major measure to facilitate cellulose conversion.In nature, fungi were regarded as the main decomposer of cellulose. Although the microorganismsthat can degradate cellulose are widely present in nature, only a few representatives of them weresystematically characterred in the decomposition mechanism and the enzymology at present. AlthoughPenicillium is the major cellulase production microorganism, its cellulose genetic resources mining isstill limited, especially Penicillium crustosum cellulase genes have not been cloned yet. In this study,we use cloned Penicillium crustosum strain601endo cellulase gene (Cel I), and expressed it inEscherichia coli and Pichia pastoris. We detected the enzymatic properties of the expression products,and make a preliminary study on the function of cellulose binding domain (CBD). The main works arelisted following:1) Based on the ITS rDNA sequence, we identified a strain sceened in our laboratory as Penicilliumcrustosum strains601.2) Using RT-PCR and3’-RACE technics, we cloned its full-length cDNA of endo cellulose.3) We separately cloned the full length cellulose gene Cel I and fragment contains theβ-glucosidasedomain Cel-PG in to pPICZα and then transferred them into Pichia X-33. After methanol inducedfermentation, their enzyme characteristics were checked as: For Cel I, the most suitabletemperature is40℃, the most suitable pH is7, and the highest enzyme activity is6.95U/mL; ForCel I-PG, the most suitable temperature is40℃, the most suitable pH is7, the highest enzymeactivity is7.65U/mL.min.4) We successfully expressed Cel I and Cel-PG in E.coli DE3. After Ni-column purification, the sizeof the target band is50.3kDa and36.1kDa, respectively.For expression product Cel I, the most suitable temperature is40℃, the most suitable pH is7, the highest activity is29.43U/mL; ForCel I-PG, the most suitable temperature is40℃, the most suitable pH is7, the highest enzymeactivity is30.97U/mL.5) We realized the fusion expression of CBD with green fluorescent protein GFP in E. coli DE3.After Ni-purification, the size of the GFP and GFP-CBD were28.4kDa and46.8kDa,respectively.6) We conducted the cellulose binding assay of GFP-CBD and GFP. Checked by the fluorescencemicroscopy, we observed the binding of the CBD-GFP protein on the filter paper fiber, and provedthat cellulase CBD domain even if separated from the cellulase, are still having a biologicalactivity.