title: Purification and characterization of a novel glutamate dehydrogenase from Geotrichum candidum with higher alcohol and amino acid activity.
abstract: Crude enzyme from Geotrichum candidum S12 exhibited high activity towards hexanol at pH 4.0, distinguishing it from currently known enzymes. To identify the dominant enzyme contributing to this activity, the crude enzyme extract was separated into different fractions by ammonium sulfate precipitation, MonoQ anion-exchange chromatography, and Sephacryl S-200 gel filtration chromatography. Afraction with high activity towards hexanol at pH 4.0 was obtained, exhibiting 38-fold improved purity and a specific activity of 3802.7 U/mg. After electrophoretic analysis, the fraction showed a molecular weight of 223 kDa by Native-PAGE and 51.4 kDa by SDS-PAGE. The purified fraction was identified as a glutamate dehydrogenase (GDH) by peptide mass fingerprinting data. This fraction showed high activity towards glutamate, α-ketoglutarate, hexanol, and isoamyl alcohol with a Km value of 41.74, 4.01, 20.37, and 19.37 mM, respectively, but with no activity towards methanol, ethanol, 1-propanol, and isobutanol. As a comparison, the GDH from yeast had no activity towards hexanol and other alcohols. Kinetic analysis revealed that glutamate and hexanol served as competitive inhibitors to each other for the purified GDH. The GDH showed the highest activity towards hexanol at pH 4.0 and 30 °C, and was the most stable at pH 2.2-7.0 and ≤40 °C. The presence of ADP, Fe2+, K+, and Zn2+increased the enzymatic activity towards hexanol and EDTA, Pb2+, Mn2+, ATP, and DTT decreased the activity. These novel characteristics expand the reported properties of GDH and suggest the newly characterized GDH has unique potential for practical application.
authors.affiliation: Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi'an, 710072, Shaanxi, China.|School of Food Science, Xinyang Agriculture and Forestry University, New 24 street of yangshan new district, Xinyang, 464000, Henan, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhu
authors.affiliation: Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi'an, 710072, Shaanxi, China.
authors.forename: Kuan
authors.initials: K
authors.lastname: Lu
authors.affiliation: Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi'an, 710072, Shaanxi, China.
authors.forename: Xiaoguang
authors.initials: X
authors.lastname: Xu
authors.affiliation: College of Enology, Northwest A&F University, 23 Xinong Road, Yangling, 712100, Shaanxi, China.
authors.forename: Xinglong
authors.initials: X
authors.lastname: Wang
authors.affiliation: Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi'an, 710072, Shaanxi, China. sjlshi2004@nwpu.edu.cn.
authors.forename: Junling
authors.initials: J
authors.lastname: Shi
keywords: Characteristics; Geotrichum candidum; Glutamate dehydrogenase; Hexanol; Identification