基因组整合与游离质粒联合应用介导阿魏酸酯酶A在马克斯克鲁维酵母中高效分泌表达

High-efficient Secretory Expression of Feruloyl Esterase A in Kluyveromyces marixanus via the Combination of Genomic Integration and Episomal Plasmid

阿魏酸是植物中广泛存在的酚类化学物质,具有抗氧化活性.阿魏酸酯酶能够水解植物细胞壁半纤维素和阿魏酸之间的酯键,释放阿魏酸,在食品、医药、饲料加工等领域具有广泛的应用价值.马克斯克鲁维酵母(Kluyveromyces marxianus)是一种生长迅速、蛋白分泌能力强的新型食品安全级酵母,具有高效分泌表达菊粉酶的能力,本研究利用基因组菊粉酶位点INU1整合与游离质粒联合应用的方式,在马克斯克鲁维酵母中实现了黑曲霉(Aspergillus niger)阿魏酸酯酶AnFaeA的高效表达,发酵液上清中的酶活达到164.4U/mL.利用马克斯克鲁维酵母重组表达的阿魏酸酯酶能够高效降解玉米芯释放阿魏酸,释放量可达1.81mg/g,并且在β-木聚糖酶的协助下可进一步提高阿魏酸的释放量,达到2.8mg/g.

Ferulic acid is a natural antioxidant hydroxycinnamic acid that found in cereals such as brown rice,whole wheat and oats.Feruloyl esterases can release ferulic acid from the plant cell walls and have received an increasing interest in industrial applications such as in the food,medicine,feed processing and other fields.As a generally recognized as safe(GRAS)yeast,Kluyveromyces marxianus is of fast growth and high capability of secreting proteins,and can be used as novel host for expressing heterologous proteins.In this study,we combined the methods of genomic inulinase site integration and episomal plasmid in K.marxianus to achieve the high-efficient expression of the feruloyl esterase AnfaeA fromAspergillus niger.The strain FIM-1/inu∶∶AnfaeA/pUKDN132-AnfaeA,integrated with a copy of the AnfaeAat the site of inulase gene INU1 and harboured a recombinant plasmid pUKDN132-AnfaeA,could highly produce the feruloyl esterase AnFaeA with 164.4 U/mL during the high cell-density fermentation.The capability of the recombinant AnFaeA for releasing ferulic acid from the corn cob was further investigated,and results showed that 1.81 mg/g ferulic acid was obtained by the AnFaeA after 24 h.The amount of ferulic acid release increased to 2.8 mg/g when the corn cob was degraded by the AnFaeA supplemented with theβ-xylanase.