title: High level production of β-galactosidase exhibiting excellent milk-lactose degradation ability from Aspergillus oryzae by codon and fermentation optimization.
abstract: A β-galactosidase gene from Aspergillus oryzae was engineered utilizing codon usage optimization to be constitutively and highly expressed in the Pichia pastoris SMD1168H strain in a high-cell-density fermentation. After fermentation for 96 h in a 50-L fermentor using glucose and glycerol as combined carbon sources, the recombinant enzyme in the culture supernatant had an activity of 4,239.07 U mL(-1) with o-nitrophenyl-β-D-galactopyranoside as the substrate, and produced a total of extracellular protein content of 7.267 g L(-1) in which the target protein (6.24 g L(-1)) occupied approximately 86 %. The recombinant β-galactosidase exhibited an excellent lactose hydrolysis ability. With 1,000 U of the enzyme in 100 mL milk, 92.44 % lactose was degraded within 24 h at 60 °C, and the enzyme could also accomplish the hydrolysis at low temperatures of 37, 25, and 10 °C. Thus, this engineered strain had significantly higher fermentation level of A. oryzae lactase than that before optimization and the β-galactosidase may have a good application potential in whey and milk industries.
authors.affiliation: Shandong Academy of Pharmaceutical Science, No. 989, Xinluo Dajie, Ji'nan, 250101, Shandong Province, People's Republic of China, qqzhao1023@hotmail.com.
authors.forename: Qianqian
authors.initials: Q
authors.lastname: Zhao
authors.forename: Fei
authors.initials: F
authors.lastname: Liu
authors.forename: Zhongwen
authors.initials: Z
authors.lastname: Hou
authors.forename: Chao
authors.initials: C
authors.lastname: Yuan
authors.forename: Xiqiang
authors.initials: X
authors.lastname: Zhu
mesh_terms: D000818:Animals; D001236:Aspergillus oryzae / Q000737:chemistry / Q000201:enzymology*; D001483:Base Sequence; D003062:Codon; D005285:Fermentation; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D005818:Genetic Engineering; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D007785:Lactose / Q000737:chemistry*; D008892:Milk / Q000737:chemistry*; D008969:Molecular Sequence Data; D009598:Nitrophenylgalactosides / Q000737:chemistry; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D013379:Substrate Specificity; D013696:Temperature; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
chemical_list: D003062:Codon; D005656:Fungal Proteins; D011994:Recombinant Proteins; C055012:2-nitrophenylgalactoside; D009598:Nitrophenylgalactosides; D001616:beta-Galactosidase; D005947:Glucose; D007785:Lactose; D005990:Glycerol