Chondroitinase ABC I (csABC I) has attracted intensive attention because of its great potential in heparin refining and the enzymatic preparation of low‐molecular‐weight chondroitin sulfate (LMW‐CS). However, low thermal resistance (<30℃) restricts its applications. Herein, structure‐guided and sequence‐assisted combinatorial engineering approaches were applied to improve the thermal resistance of Proteus vulgaris csABC I. By integrating the deletion of the flexible fragment R166–L170 at the N‐terminal domain and the mutation of E694P at the C‐terminal domain, variant NΔ5/E694P exhibited 247‐fold improvement of its half‐life at 37℃ and a 2.3‐fold increase in the specific activity. Through batch fermentation in a 3‐L fermenter, the expression of variant NΔ5/E694P in an Escherichia coli host reached 1.7 g L⁻¹ with the activity of 1.0 × 10⁵ U L⁻¹. Finally, the enzymatic approach for the preparation of LMW‐CS was established. By modulating enzyme concentration and controlling depolymerization time, specifically distributed LMW‐CS (7000, 3400, and 1900 Da) with low polydispersity was produced, demonstrating the applicability of these processes for the industrial production of LMW‐CS in a more environmentally friendly way.

中文翻译：

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工程热稳定的软骨素酶以生产专门分布的低分子量硫酸软骨素

软骨素酶ABC I（csABC I）由于其在肝素精制和低分子量硫酸软骨素（LMW-CS）酶法制备中的巨大潜力而​​备受关注。但是，低热阻（<30℃）限制了其应用。本文采用结构指导和序列辅助的组合工程方法来提高寻常变形杆菌csABC I的耐热性。通过整合N端结构域柔性片段R166–L170的缺失和C处E694P的突变在其末端结构域，变体NΔ5/ E694P在37℃时的半衰期提高了247倍，比活性提高了2.3倍。通过在3L发酵罐中分批发酵，变体NΔ5/ E694P在大肠杆菌中的表达宿主达到1.7 g L ^-1，活性为1.0×10 ⁵ UL ^-1。最后，建立了用于制备LMW-CS的酶促方法。通过调节酶浓度并控制解聚时间，生产了具有低多分散性的特定分布的LMW-CS（7000、3400和1900 Da），从而证明了这些工艺在以更环保的方式工业化生产LMW-CS方面的适用性。