Cheng Chih-Yu, Tsai Chia-Huang, Liou Pei-Jyun, Wang Chi-Hang
Department of Marine Biotechnology, National Kaohsiung University of Science and Technology, Kaohsiung 81157, Taiwan.
Polymers (Basel). 2021 Jan 18;13(2):290. doi: 10.3390/polym13020290.
For pilot-scale production of chito-oligosaccharides, it must be cost-effective to prepare designable recombinant chitosanase. Herein, an efficient method for preparing recombinant chitosanase from by elimination of undesirable substances as a precipitate is proposed. After an optimized culture with IPTG (Isopropyl β-d-1-thiogalactopyranoside) induction, the harvested cells were resuspended, disrupted by sonication, divided by selective precipitation, and stored using the same solution conditions. Several factors involved in these procedures, including ion types, ionic concentration, pH, and bacterial cell density, were examined. The optimal conditions were inferred to be pH = 4.5, 300 mM sodium dihydrogen phosphate, and cell density below 10 cells/mL. Finally, recombinant chitosanase was purified to >70% homogeneity with an activity recovery and enzyme yield of 90% and 106 mg/L, respectively. When 10 L of 5% chitosan was hydrolyzed with 2500 units of chitosanase at ambient temperature for 72 h, hydrolyzed products having molar masses of 833 ± 222 g/mol with multiple degrees of polymerization (chito-dimer to tetramer) were obtained. This work provided an economical and eco-friendly preparation of recombinant chitosanase to scale up the hydrolysis of chitosan towards tailored oligosaccharides in the near future.
对于壳寡糖的中试规模生产，制备可设计的重组壳聚糖酶必须具有成本效益。在此，提出了一种通过将不需要的物质作为沉淀物去除来制备重组壳聚糖酶的有效方法。在用IPTG（异丙基β-D-1-硫代半乳糖苷）诱导进行优化培养后，收获的细胞重新悬浮，通过超声破碎，通过选择性沉淀进行分离，并在相同的溶液条件下储存。研究了这些过程中涉及的几个因素，包括离子类型、离子浓度、pH值和细菌细胞密度。推断最佳条件为pH = 4.5、300 mM磷酸二氢钠和细胞密度低于10个细胞/mL。最后，重组壳聚糖酶被纯化至>70%的纯度，活性回收率和酶产量分别为90%和106 mg/L。当在环境温度下用2500单位的壳聚糖酶水解10 L 5%的壳聚糖72小时时，获得了具有833±222 g/mol摩尔质量且具有多种聚合度（壳二聚体到四聚体）的水解产物。这项工作提供了一种经济且环保的重组壳聚糖酶制备方法，以便在不久的将来扩大壳聚糖向定制寡糖的水解规模。
