Cloning And Expression Of Neurospora Crassa CBH? Gene

Cellulose is a kind of abundant organic compound which is widely distributed in nature.But cellulose is difficult to be decomposed because of its structure constraint.There are a lot of methods,such as:physical,chemical and microbiological.Using biodegradable method to degrade cellulose is more feasible and efficient in the use of cellulose.Neurospora crassa is a kind of model microorganism that can produce cellulase,which can degrade cellulose efficiently.In our study,10 mutants were selected from the mutant library of Neurospora crassa which gene suspected to be relevant with cellulase.Further screening test proved that protein encoded by NCU07340 gene has cellulase?CBH,exoglucanase?activity.In order to investigate the details of cellulase,we designed primers to clone NCU07340 gene according to its gene sequence reported from Neurospora Crassa Database,and then connected it to a vector--qa-2.5myc.6His dH.The newly constructed gene vector qa-2.5myc.6HisdH-NCU07340 is 8705bp in length,it has a qa promoter sequence,an Amp sequence and His3 sequence.The newly constructed vector was transformed into a histidine-deficient strain301-6 of Neurospora crassa.Western blotting was used to detect the protein clones,and Corn stalks as the carbon source to induce the production of the extracellular and intracellular enzyme activity.The results of cellulase activity detection and SDS-PAGE showed that the amount of protein expressed by over-expressed strain was significant increased,the enzyme activity was also increased.with CBH activity reached 38.1U/mL,increased by 11%compared to 87-3.When changing the carbon source to rice straw and rice husk the activity of EC and BG cellulase respectively reached 29.06U/mL and 25.14U/mLMeanwhile,the influence factor was also optimized on cellulase extracted by ammonium sulfate precipitation,and the temperature,pH,and metal ions were investigated.The results showed that the optimum temperature of the CBH was50?,and the thermal stability was relatively stable between 40?-50?.The optimum pH values were 5.0,it was relatively stable between 4-6.Metal ions Na⁺,Mg²⁺,Mn²⁺can promote the CBH activity.Except of CBH activity,the other 2 cellulase activity were also measured.The results showed BG??-1,4-glucosidase?and EG?endo-glucanase?activity respectively reached 38.3U/mL,14.35U/mL,increased by 2.2U/mL and 1.1U/mL compared to87-3.The ptimum temperature of the BG and EG was 50?,and the thermal stability was relatively stable between 40?-50?.Metal ions Zn²⁺can promote the EG activity and Na⁺?Mg²⁺can promote the BG activity.