α-L-阿拉伯呋喃糖苷酶在毕赤酵母中的表达及其对大麦麦芽过滤性能的影响

Expression of α-L-arabinofuranosidase in Pichia pastoris and its effect on filterability of barley malt

PCR技术从黑曲霉(Aspergillus niger)基因组扩增获得1个全长1 790 bp的α-L-阿拉伯呋喃糖苷酶基因,基因含有1个50 bp的内含子序列,其对应的蛋白质序列含有5个O糖基化位点和9个N糖基化位点,N端含有18个氨基酸的信号肽序列。将目的基因与表达载体pPICZαA连接并在毕赤酵母X-33诱导表达,获得重组α-L-阿拉伯呋喃糖苷酶。重组酶的相对分子质量为70 kDa,最适反应pH和温度分别为pH 5.5和50℃;金属离子Cu^(2+)、Zn^(2+)和Fe^(2+)对重组酶的酶活有抑制作用,Fe^(3+)对重组酶的酶活有促进作用;以4-Nitrophenylα-L-arabinofuranoside为底物测得酶的Km值和Vmax值分别为0.78 mmol/L和2.57μmol/(min·mg)。在大麦麦芽协定糖化的初始阶段添加31.2 mU/g重组酶,麦汁的过滤速度提高了12.8%。以大麦麦芽阿拉伯木聚糖为底物,α-L-阿拉伯呋喃糖苷酶与木聚糖酶有较好的协同作用。

The arabidofuranosidase gene was amplified from the Aspergillus niger genome by PCR. The gene length was 1 790 bp containing an intron of 50 bp. The corresponding protein sequence contained 9 N-glycosylation sites and 5 O-glycosylation sites,the N-terminus contained 18 amino acids of the signal peptide. Arabidofuranosidase gene was ligated with the expression vector pPICZαA and induced in Pichia pastor X-33. The recombinant enzyme was purified by Ni column affinity chromatography. The molecular size of the recombinant enzyme was 70 k Da,and the optimum reaction temperature and optimum pH of the recombinant enzyme were 50 ℃ and 5. 5 respectively.Cu^(2+),Zn^(2+) and Fe^(2+) had inhibitory effect on the activity of arabidofuranosidase,Fe^(3+)promoted the enzyme activity of arabidofuranosidase. Using 4-nitrophenyl α-L-arabinofuranoside as the substrate,the Kmand Vmaxvalues of the enzymes were 0. 78 mmol/L and 2. 57 μmol/(min·mg) respectively. Recombinant α-L-arabinofuranosidase was added at the initial stage of mashing with an amount of 31. 2 m U/g,wort filtration rate was increased by 12. 8%. α-L-arabinofuranosidase and xylanase had synergistic effect with barley malt arabinoxylans as substrate.