Cloning And Expression Of An Acidophilic β-Mannanase Gene From Aspergillus Niger

β-mannanase （-1,4-D-mannan mannanohydrolase, EC3.2.1.78）, which randomly hydrolyzesthe β-1,4-D-mannosidic linkages within mannan, is a kind of hemicellulase. It can be produced byvarious microorganisms, and have wide applications in industrial processes, such as food, animalfeed, oil mining, biofuels and so on.The full-length cDNA encoding β-mannanase of Aspergillus niger LW-1was amplied by usinga pair of primers, Fman-F and Fman-R. The cDNA sequence was1417bp in length, containing a5′-untranslated region （41bp）, an open reading frame （ORF,1152bp） and a3′-untranslated region（224bp）. The sequence had been submitted to GenBank （accession no. JN123356）.A pair of primers, ManF and ManR, were designed to amply the gene （Anman5A） encoding themature peptide by using RT-PCR. The transformants of P. pastoris GS115/Anman5A weresuccessfully constructed and secreted β-mannanase with the highest activity of29.0U/mL afterinduction by0.5%menthanol for96h. The molecular weight of recombinant β-mannanase wasestimated to be52.0kDa by SDS-PAGE. PCR analysis of transformants revealled Anman5A wasintegrated into the P. pastoris GS115genome.The enzymatic properties of the recombinant β-mannanase were investigated. β-mannanasediplayed the highest activity at70℃and pH3.5. It was stable at a pH of2.5-7.5and below60℃.Its acvitity was not signficantly affected by an array of metal ions (Li⁺, Na⁺, Ba²⁺, Co²⁺, Cu²⁺, Mg²⁺,Zn²⁺and Fe³⁺) expect Fe²⁺and Mn²⁺, which inhibited β-mannannase, while EDTA and Ca²⁺couldactivate this enzyme. β-mannannase exhibited100%,79.1%and41.1%relative activity on locustbean gum, knjoc gum and guar gum, respectively, while its activity was negligible on birwoodxylan, carboxymethylcellulose and starch.β-mannannase gene （Anman5A） and xylanase gene （xynII） were firstly coexpressed in Pichiapastoris using the double-plasmid system. PCR analysis of transformants revealled that Anman5Aand xynII were integrated into the P. pastoris GS115genome. Genetic stability test was determinedthat the expression of β-mannanase gene and xylanase gene in P. pastoris GS115/Anman5A-xynⅡwas stable.One P. pastoris GS115/Anman5A-xynⅡ transformant, labeled as GSMX2, expressing thehighest activities of two enzymes in shake flasks was selected for optimizing expression conditions.The optimized expression conditions were as follows: initial pH of7.0, glycerol concentration of1.5%, methanol concentration of1.5%, induction for120h. With these conditions, the maximumactivities of β-mannanase and xylanase were up to37.1U/mL and193.6U/mL, respectively.