Menghiu Gheorghita, Ostafe Vasile, Prodanovic Radivoje, Fischer Rainer, Ostafe Raluca
Institute for Biology VII, Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany; Advanced Environmental Research Laboratories, Department of Biology - Chemistry, West University of Timisoara, Oituz 4, 300086, Timisoara, Romania.
Advanced Environmental Research Laboratories, Department of Biology - Chemistry, West University of Timisoara, Oituz 4, 300086, Timisoara, Romania.
Protein Expr Purif. 2019 Feb;154:25-32. doi: 10.1016/j.pep.2018.09.007. Epub 2018 Sep 17.
Chitin is an abundant biopolymer found mainly in the exoskeleton of crustaceans and insects. The degradation of chitin using chitinases is one way to address the accumulation of chitin waste streams in the environment, and research has therefore focused on the identification, improvement and expression of suitable enzymes. Here we describe the production, purification and characterization of Bacillus licheniformis chitinase A in the Pichia pastoris expression system. Optimal enzyme activity occurred at pH 4.0-5.0 and within the temperature range 50-60 °C. With colloidal chitin as the substrate, the K (2.307 mM) and V (0.024 mM min) of the enzyme were determined using a 3,5-dinitrosalicylic acid assay. The degradation products of colloidal chitin and hexa-N-acetylchitohexaose were compared by thin-layer chromatography. The activity of the glycosylated enzyme produced in P. pastoris was compared with the in vitro deglycosylated and aglycosylated version produced in Escherichia coli. We showed that the glycosylated chitinase was more active than the deglycosylated and aglycosylated variants.
几丁质是一种丰富的生物聚合物，主要存在于甲壳类动物和昆虫的外骨骼中。使用几丁质酶降解几丁质是解决环境中几丁质废物流积累问题的一种方法，因此研究集中在合适酶的鉴定、改进和表达上。在此，我们描述了地衣芽孢杆菌几丁质酶A在毕赤酵母表达系统中的生产、纯化和特性。最佳酶活性出现在pH 4.0 - 5.0以及50 - 60°C的温度范围内。以胶体几丁质为底物，使用3,5 - 二硝基水杨酸测定法测定该酶的K（2.307 mM）和V（0.024 mM min）。通过薄层色谱法比较了胶体几丁质和六 - N - 乙酰壳六糖的降解产物。将毕赤酵母中产生的糖基化酶的活性与大肠杆菌中产生的体外去糖基化和无糖基化版本进行了比较。我们表明糖基化几丁质酶比去糖基化和无糖基化变体更具活性。
