Journal of Microbiology

Purification and Characterization of an Alkaline Protease produced by a Xanthomonas sp. YL-37: Lee, Chang Ho , Kim, Hee Sik , Kwon, Gi Soek , Oh, Hee Mock , Kang, Sang Mo , Kwon, Tae Jong , Yoon, Byung Dae; J. Microbiol. 1995;33(2):115-119.

Abstract: The alkaline protease of Xanthomonas sp. YL-37 has been purified, and the properties of the enzyme investigated. The alkaline protease of Xanthomonas sp. YL-37 was purified form crude enzyme by ammonium sulfate fractionation, CM-cellulose ion exchange chromatography, and Sephadex G-100 gel filtration. Through the series of chromatographies, the enzyme was purified to homogenecity with specific activity of 4.23 fold higher than that of the crude broth. The molecular weight of the purified protease has been estimated to be 62 KDa on SDS-polyacrylamide gel electrophoresis. The optimal pH and temperature for alkaline protease activity were 11.0 and 50℃, respectively. The enzyme was stable between pH 5.0 and 10.0 and up to 50℃. Enzyme activity was lost up to 50% on heating at 70℃ for 30 minutes. The activity of alkaline protease was inhibited by Cu^2+, Zn^2+, Hg^2+, PMSF, and activated by Mn^2+ and Ca^2+. The K_m value for casein as a substrate was 4.0 mg/ml.

Controlled Expression and Secretion of Aspergillus oryzae Alkaline Protease in Aspergillus nidulans: Eun Ah Kim , Jeong Goo Lee , Mi Kyung Whang , Hee Moon Park , Jeong Yoon Kim , Suhn Kee Chae , Pil Jae Maeng; J. Microbiol. 2001;39(2):95-101.

Abstract: In an effort to develop an efficient expression and secretion system for heterologous proteins in Aspergillus nidulans, the PCR-amplified coding sequence for alkaline protease (AlpA) of A. oryzae was cloned into a fungal expression vector downstream of A. nidulans alcA (alcohol dehydrogenase) promoter to yield pRAAlp. Transformation of A. nidulans with pRAAlp gave stable transformants harboring various copy numbers (3 to 10) of integrated alpA gene, from among which 6 representatives were selected. On a medium containing 0.8% ammonium sulfate that represses the expression of the hosts own protease, the alcA promoter-controlled AlpA expression was strongly induced by threonine but repressed by glucose. The level of AlpA secretion was highest (approximately 666 mU/ml) in transformant ALP6 containing the largest copy number integrated alpA. However, the level of AlpA secretion was not necessarily proportional to the copy numbers of the integrated alpA genes. The N-terminal sequence of the secreted mature AlpA was determined to be Gly-Leu-Thr-Thr-Gln-Lys-Ser and its molecular mass to be approximately 34 kDa, indicating that AlpA is properly processed by the removal of 121 N-terminal amino acids.

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