Characterization And Directed Evolution Of N-actylglucosamine Deacetylase From Cronobacter Sakazakii

D-glucosamine（GlcN） is the only alkaline monosaccharide in nature. In human body, GlcN mainly exists in articular cartilage and connective tissues, which major function is to provide protection and lubrication for human. Because of its multiple biological activity, GlcN has been widely used in many fields, including food industry, health products and biological pharmaceutical field. The purpose of this study is to find a highly active N-actylglucosamine-6-phosphate deacetylase gene and produce D-glucosamine by engineering bacteria with recombinant plasmid, which can improve the conversion rate and decrease the cost.An N-acetylglucosamine-6-phosphate deacetylase gene（CsnagA: 1149bp） was cloned from Cronobacter sakazakii, which encodes CsNAGPase of 382 amino acids with an estimated molecular weight of 41.3kDa. CsNAGPase belongs to the metal-dependent hydrolases superfamily and shows the highest identity（87%） with EcNAGPase which was from E. coli K-12 and was used to produce GlcN in industry. CsNAGPase showed optimum activity at 50℃ and pH 8.0 respectively, same as EcNAGPase from E. coli K-12. CsNAGPase and EcNAGPase retained almost 90% of its original activity at 40℃, 50℃, 60℃ for 1h. Meanwhile, CsNAGPase and EcNAGPase were quite stable in pH 5.0-9.0. About the metal ions, Co²⁺ showed dramatically activation to the CsNAGPase（1mM, 164.7%; 5mM, 174.0%）. The kcat/Km value of CsNAGPase without Co²⁺ was 72.12% higher than EcNAGPase. Furthermore, the maximum conversion rates of CsNAGPase reached 78.85% while EcNAGPase reached 58.45%. The maximum conversion rate of CsNAGPase was 34.90% higher than EcNAGPase. When adding Co²⁺ into reaction system, Cs NAGPase conversion rates reached 93.56%, 18.66% higher than CsNAGPase without Co²⁺.CsNAGPase with improved acid resistance was obtained by directed evolution with high-throughput screening method. Variant 1-B2 was acquired from the mutant library which contained more than 9000 clones. The mutant 1-B2 remained 30% relative activity after incubating with pH 4.0 buffer at 30℃ for 2h while for the WT only 15% relative activity was left. After treated with 1% acetic acid for 2h, there was still more than 90% activity left in 1-B2 while residual activity of the WT was about 75%. As is often the case, GlcN was unstable in under alkaline conditions and acid resistance of mutant 1-B2 had practical value in improving the stability of GlcN.