Acidophilic Fungus Identification,Cloning And Expression Of Alpha-amylase Gene And Mannanase Gene

Aspergillus niger is an important industrial fermentation bacterium.Because of its rapid growth,short fermentation period,and the fact that it is not contaminated by bacteria and does not produce toxins during growth,it is known as one of GRAS strains.Strong enzyme-producing ability,and a wide variety of enzyme production,some extreme enzymes were produced from Aspergillus,which further laid a position in the fermentation industry.A special enzyme,acid-resistant?-amylase,can improve and modify the starch processing,reduce the cost of the production and help to solve their disposal and pollution problems etc.The special enzyme will have more value to development.Mannanase is a new type of industrial production enzyme,but due to its low activity of fermentation enzyme resulting in high cost,limiting the scope of application,therefore,the study of this enzyme has a certain value.In this experiment,a mold was isolated from an acidic leaching solution of a uranium deposit in Xinjiang.And it was named as MJ 5 in this experiment.The strain was identified based on its morphological features,rDNA ITS sequence analysis,and Biolog identification system.The Biolog identification plate provided the strain's consumption of the carbon source.The growth status and dominant carbon source of the strain could be analyzed.The alpha-amylase activity was measured in the crude enzyme solution of the strain,and based on the results of the whole genome sequencing,primers were designed to obtain sequence fragments of the acid-resistant alpha amylase and mannanase genes.Construction of Expression Vectors pPIC9K and Introduction into Yeast Receptor Cells for Expression.The results show:?1?Identification of mold MJ 5 as Aspergillus niger by combining three identification systems.And amino acids,lipids,and acids were use most in eight major carbon sources.The optimum carbon source for molds is p-hydroxyphenylacetic acid.The utilization of amino acids,lipids,and acids is higher than others.Then we do some research for the crude enzyme solution produced.The results showed that the strain produced acid amylase.The optimum pH and temperature of the enzyme-catalyzed reaction were 5.0 and 45°C.The reaction was incubated for 1 hour under the same temperature and different pH conditions.The results showed that the enzyme activity of MJ 5 amylase remained above 80%in the pH 3-7.When the incubation reaction was performed under the same pH and different temperature for 1 h,the enzyme activity of MJ 5 amylase remained more than 70%at 40°C,45°C,50°C and 55°C,which indicated that the MJ 5 amylase had better pH stability and thermal stability.?2?According to the Aspergillus niger genome,we can design suitable primers for acid-resistant?-amylase gene.The?-amylase gene-5 belonging to the glucoside hydrolase family13?GH 13?,and it was cloned by Polymerase Chain Reaction.Then this band was recycled puried connected to pPIC9K transformed into E.coli TransI-T1,screened positive colonies,from the results we can know the length of?-amy-5 was 1650bp,including the terminal condon,encoding 521 amino acids residues of the polypeptide chains,and its N-terminus had a signal peptide sequence encoding 28 amino acids residues.Then the pPIC9K-?-amy-5 plasmid was linearized and electroporated into yest competent cells of pichia pastoris host strains,but no recombinant enzyme activity was detected,the specific reason remains to be analyzed.?3?The mannanase gene man-5 which came from Aspergillus niger MJ 5 was belonging to the glucoside hydrolase family 26.The full-length of the gene is 1008bp,after removing the signal peptide sequence,the length of the DNA fragment and cDNA fragment was 954bp,encoding 317 amino acids residues of the polypeptide chains.From the bioinformatics analysis,it can be known that there are 4 glycosylation sites in this amino acid sequence,and the isoelectric point and molecular weight are predicted to be 5.71 and 37 kDa.According to the enzymatic properties,the optimal temperature and pH of this gene were 35°C and pH 5.0.In different buffers,after reacting for 1h at 37°C,80%of the enzyme activity remained between pH 5.0 and pH 7.0,and the pH tolerance range was not wide.When the reaction was carried out at 40°C for 1 h,there was almost no loss of enzyme activity,but as the temperature increased,the mannanase was almost inactivated.K⁺,?-mercaptoethanol,Na⁺,Pb²⁺,Mg²⁺,and EDTA can increase the activity of mannanase,Cr³⁺,Mn²⁺,Cu²⁺and Ca²⁺can inhibit the activity of the enzyme,?-mercaptoethanol has the strongest ability to increase enzyme activity.Increase of Man-5 activity by Fe³⁺is not affected by concentration.The other ions and reagents have almost no effect on the activity of the enzyme.Therefore,it can be seen that metal ions and chemical reagents affect MJ.5 mannanase production has little effect.The protein content of Man-5 was8.57 mg/mL,and the enzyme activity was 864 U/mL.Finally,the specific activity was 101U/mg.The Km and Vmax of Man-5 were 1.9 mg/mL and 1638?mol/min·mg.