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Engineering the Non-catalytic Domain to Enhance Catalytic Activity and Thermal Stability of a Nκ2-Producing κ-Carrageenase

文献类型： 外文期刊

作者： Jiang, Chengcheng 1 ; Mao, Xiangzhao 2 ;

作者机构： 1.Chinese Acad Fishery Sci, Yellow Sea Fisheries Res Inst, Lab Marine Drugs & Byprod, Natl Lab Marine Sci & Technol,State Key Lab Maricu, Qingdao 266071, Peoples R China

2.Ocean Univ China, Coll Food Sci & Engn, Qingdao 266003, Peoples R China

关键词： kappa-carrageenase; product specificity; noncatalytic domains; substrate specificity

期刊名称：JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY （ 影响因子：6.2； 五年影响因子：6.4 ）

ISSN： 0021-8561

年卷期： 2024 年 72 卷 47 期

页码：

收录情况： SCI

摘要： kappa-Carrageenases play an important role in achieving the high-value utilization of carrageenan polysaccharides. They can be used in the preparation of even-numbered kappa-neocarrageenan oligosaccharides by degrading kappa-carrageenan (KC). We previously identified and characterized a kappa-carrageenase, CaKC16B, with high specificity for producing a single kappa-neocarrabiose. It can produce a single kappa-neocarrabiose from degrading KC. However, they also exhibited poor thermal stability and catalytic efficiency. To improve these properties, we introduced noncatalytic domains (nonCDs) from a heat-resistant kappa-carrageenase, MtKC16A, into the C-terminus of CaKC16B to construct CaKC16BUN and CaKC16BUNB. Compared to the original CaKC16B, both of them exhibited improved enzymatic properties, including optimal reaction temperature, thermal stability, substrate affinity, and catalytic efficiency. Remarkably, the k cat/K m values of 16BUN and 16BUNB increased by 127 and 290 times, respectively. Importantly, the addition of nonCDs did not alter the final products of degrading KC, retaining the high product specificity of CaKC16B. Interestingly, the addition of nonCDs changed CaKC16B's substrate specificity for hydrolyzing KC and beta kappa-carrageenan, with mutants exhibiting higher relative activity for beta kappa-carrageenan. We further observed through biolayer interferometry that the binding and dissociation process between MtUN nonCD and beta kappa-carrageenan is faster compared to that of KC. This may explain the change in the substrate specificity observed in the mutants of CaKC16B.

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