Cloning And Expression Of Arabinofuranosidase And Its Application In The Manufacture Of Wort

Arabinoxylan（AX）is the main non-starch polysaccharide in malt.The oxidation cross-linking of side chain groups in AX is one of the main factors leading to poor wort filtration.α-L-arabinofuranosidases（AnabfA）belongs to the glycosyl hydrolases family,that can hydrolyze from the non-reducing end of polymers like AX,arabinan,arabinogalactan and et al.Therefore,the present study cloned AnabfA gene from the genome of Aspergillus niger and expressed the recombinant AnabfA in Pichia pastoris X-33,for applying it in the manufacture of wort,which improves the wort filtration speed.The AnabfA gene is 1790 bp in length and contains a 50 bp intron.The corresponding enzyme contains an 18-amino acid N-terminal signal peptide sequence,9 N-glycosylation sites and 5 O-glycosylation sites.The DNA of AnabfA gene was amplified,modified and inserted into plasmid pPICZαA to construct expression vector pPICZαA-QAnabfA,which was then transformed into Pichia pastoris X-33 for recombinant expression of AnabfA.The recombinant AnabfA showed apparent molecular weight of 100 k Da,exhibited highest activity at pH 5.5,50℃,and great stability at pH 4.0-6.5.The recombinant AnabfA had 90 min half-life at 55°C.The K_m and V_maxax values of the enzyme were 0.78 mmol·L^-1 and2.57μmol·min^-1·mg^-1 respectively,using 4-Nitrophenylα-L-arabinofuranoside as the substrate.By supplementing recombinant AnabfA(31.2 mU·g^-1 malt)at the start of the European Brewery Convention（EBC）process,the filtration rate of wort was increased by 12.8%.We proposed that there were two mechanism for filterability improvement,one is that AnabfA could degrade the side chian of AX,which lead to reduction of the viscosity of AX aqueous solution by 10.9%,and decreace of arabinose and ferulic acid contents by 10%and 9.6%,respectively.And the other is the synergistic effect of AnabfA and xylanase on degradation of AX.However,partial degradation of AX side chains can increase the oxidative cross-linking between AX,and adversely affect the filtration rate of wort.The three main factors,temperature,pH and inducer concentration,which greatly influence production of recombinant AnabfA were determined by single factor experiment.The optimum fermentation parameters were determined by response surface analysis:temperature 28.6℃,inducer concentration 1.03%(v·v^-1),initial pH 6.01.With the optimum parameters,we got a fermentation broth with enzyme activity of 57.7±0.6 mU·mL^-1.