Kang Chao, Yu Xiao-Wei, Xu Yan
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China,
J Ind Microbiol Biotechnol. 2015 Feb;42(2):263-72. doi: 10.1007/s10295-014-1571-8. Epub 2014 Dec 30.
A novel prolyl endopeptidase gene from Aspergillus oryzae was cloned and expressed in Pichia pastoris. Amino acid sequence analysis of the prolyl endopeptidase from Aspergillus oryzae (AO-PEP) showed that this enzyme belongs to a class serine peptide S28 family. Expression, purification and characterization of AO-PEP were analyzed. The optimum pH and temperature were pH 5.0 and 40 °C, respectively. The enzyme was activated and stabilized by metal ion Ca(2+) and inhibited by Zn(2+), Mn(2+), Al(3+), and Cu(2+). The K m and k cat values of the purified enzyme for different substrates were evaluated. The results implied that the recombinant AO-PEP possessed higher affinity for the larger substrate. A fed-batch strategy was developed for the high-cell-density fermentation and the enzyme activity reached 1,130 U/l after cultivation in 7 l fermentor. After addition of AO-PEP during the fermentation phase of beer brewing, demonstrated the potential application of AO-PEP in the non-biological stability of beer, which favor further industrial development of this new enzyme in beer stabilization, due to its reducing operational costs, as well as no beer losses unlike regeneration process and beer lost with regenerated polyvinylpolypyrrolidone system.
从米曲霉中克隆出一个新的脯氨酰内肽酶基因，并在毕赤酵母中进行表达。对米曲霉脯氨酰内肽酶（AO-PEP）的氨基酸序列分析表明，该酶属于丝氨酸肽S28家族。对AO-PEP进行了表达、纯化及特性分析。其最适pH和温度分别为pH 5.0和40℃。该酶被金属离子Ca(2+)激活并稳定，被Zn(2+)、Mn(2+)、Al(3+)和Cu(2+)抑制。评估了纯化后的酶对不同底物的K m和k cat值。结果表明，重组AO-PEP对较大的底物具有更高的亲和力。开发了一种分批补料策略用于高细胞密度发酵，在7升发酵罐中培养后酶活达到1130 U/l。在啤酒酿造的发酵阶段添加AO-PEP后，证明了AO-PEP在啤酒非生物稳定性方面的潜在应用，这有利于这种新酶在啤酒稳定化方面的进一步工业开发，因为它降低了运营成本，且与再生过程不同，不会损失啤酒，也不像再生聚乙烯聚吡咯烷酮系统那样会损失啤酒。
