caa a22 4500 605498474 CHVBK 20210128100546.0 cr unu---uuuuu 210128e20150801xx s 000 0 eng 10.1007/s00253-015-6421-8 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6421-8 Comprehensive characterization of sphingolipid ceramide N -deacylase for the synthesis and fatty acid remodeling of glycosphingolipids [Elektronische Daten] [Yun-Bin Han, Lie Wu, Jamie Rich, Feng-Tao Huang, Stephen Withers, Yan Feng, Guang-Yu Yang] Sphingolipid ceramide N-deacylase (SCDase) catalyzes reversible reactions in which the amide linkage in glycosphingolipids is hydrolyzed or synthesized. While SCDases show great value for the enzymatic synthesis of glycosphingolipids, they are relatively poorly characterized enzymes. In this work, the enzymatic properties of SCDase from Shewanella alga G8 (SA_SCD) were systematically characterized and compared with the commercially available SCDase from Pseudomonas sp. TK4 (PS_SCD). The optimal pH values for the hydrolytic and synthetic activity of SA_SCD were pH 6.0 and pH 7.5, respectively. Both activities were strongly inhibited by Zn2+ and Cu2+, while Fe2+, Co2+, Ni2+, Mn2+, Ca2+, and Mg2+ promoted the hydrolytic activity but inhibited the synthetic activity. SA_SCD showed very broad substrate specificity both in hydrolysis and synthesis. Importantly, SA_SCD has a broader specificity for acyl donor acceptance than does PS_SCD, especially for unsaturated fatty acids and fatty acids with very short or long acyl chains. Further kinetic analysis revealed that the k cat/K M value for the hydrolytic activity of SA_SCD was 8.9-fold higher than that of PS_SCD for GM1a, while the values for the synthetic activity were 38-fold higher for stearic acid and 23-fold higher for lyso-GM1a (d18:1) than those of PS_SCD, respectively. The broad fatty acid specificity and high catalytic efficiency, together with the ease of expression of SA_SCD in Escherichia coli, make it a better biocatalyst than is PS_SCD for the synthesis and structural remodeling of glycosphingolipids. Springer-Verlag Berlin Heidelberg, 2015 Glycosphingolipids nationallicence High-performance liquid chromatography nationallicence Sphingolipid ceramide N -deacylase nationallicence Substrate specificity nationallicence Han Yun-Bin State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 200240, Shanghai, China aut Wu Lie State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 200240, Shanghai, China aut Rich Jamie Department of Chemistry, University of British Columbia, V6T 1Z1, Vancouver, British Columbia, Canada aut Huang Feng-Tao State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 200240, Shanghai, China aut Withers Stephen Department of Chemistry, University of British Columbia, V6T 1Z1, Vancouver, British Columbia, Canada aut Feng Yan State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 200240, Shanghai, China aut Yang Guang-Yu State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 200240, Shanghai, China aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/16(2015-08-01), 6715-6726 0175-7598 99:16<6715 2015 99 253 https://doi.org/10.1007/s00253-015-6421-8 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6421-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Han Yun-Bin State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 200240, Shanghai, China aut NATIONALLICENCE 700 1- Wu Lie State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 200240, Shanghai, China aut NATIONALLICENCE 700 1- Rich Jamie Department of Chemistry, University of British Columbia, V6T 1Z1, Vancouver, British Columbia, Canada aut NATIONALLICENCE 700 1- Huang Feng-Tao State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 200240, Shanghai, China aut NATIONALLICENCE 700 1- Withers Stephen Department of Chemistry, University of British Columbia, V6T 1Z1, Vancouver, British Columbia, Canada aut NATIONALLICENCE 700 1- Feng Yan State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 200240, Shanghai, China aut NATIONALLICENCE 700 1- Yang Guang-Yu State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 200240, Shanghai, China aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/16(2015-08-01), 6715-6726 0175-7598 99:16<6715 2015 99 253