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Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of L-tert-leucine.

التفاصيل البيبلوغرافية
العنوان: Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of L-tert-leucine.
المؤلفون: Liao L; Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, People's Republic of China., Zhang Y; Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, People's Republic of China.; College of Food and Biological Engineering, Jimei University, Xiamen, People's Republic of China., Wang Y; Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, People's Republic of China., Fu Y; Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, People's Republic of China., Zhang A; Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, People's Republic of China., Qiu R; Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, People's Republic of China., Yang S; Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, People's Republic of China., Fang B; Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, People's Republic of China. fbs@xmu.edu.cn.; The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, Fujian, People's Republic of China. fbs@xmu.edu.cn.
المصدر: Microbial cell factories [Microb Cell Fact] 2021 Jan 06; Vol. 20 (1), pp. 3. Date of Electronic Publication: 2021 Jan 06.
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: BioMed Central Country of Publication: England NLM ID: 101139812 Publication Model: Electronic Cited Medium: Internet ISSN: 1475-2859 (Electronic) Linking ISSN: 14752859 NLM ISO Abbreviation: Microb Cell Fact Subsets: MEDLINE
أسماء مطبوعة: Original Publication: London : BioMed Central, [2002-
مواضيع طبية MeSH: Bacillus cereus/*enzymology , Bacillus megaterium/*enzymology , Glucose 1-Dehydrogenase/*metabolism , Leucine/*biosynthesis , Leucine Dehydrogenase/*metabolism , Recombinant Fusion Proteins/*metabolism, Glucose 1-Dehydrogenase/chemistry ; Glucose 1-Dehydrogenase/genetics ; Leucine Dehydrogenase/chemistry ; Leucine Dehydrogenase/genetics ; Protein Conformation ; Recombinant Fusion Proteins/chemistry ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/isolation & purification
مستخلص: Background: Biosynthesis of L-tert-leucine (L-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of L-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully.
Results: In this work, a novel fusion enzyme (GDH-R3-LeuDH) for the efficient biosynthesis of L-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH-R3-LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of L-tle by GDH-R3-LeuDH was all enhanced by twofold. Finally, the space-time yield of L-tle catalyzing by GDH-R3-LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD + and 500 mM of a substrate including trimethylpyruvic acid and glucose).
Conclusions: It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize L-tle and reach the highest space-time yield up to now. These results demonstrated the great potential of the GDH-R3-LeuDH fusion enzyme for the efficient biosynthesis of L-tle.
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معلومات مُعتمدة: 21978245 National Natural Science Foundation of China; 22008084 National Natural Science Foundation of China; 21336009 Key Programme
فهرسة مساهمة: Keywords: Fusion enzyme; Glucose dehydrogenase; L-tert-leucine; Leucine dehydrogenase; Whole cells
المشرفين على المادة: 0 (Recombinant Fusion Proteins)
EC 1.1.1.47 (Glucose 1-Dehydrogenase)
EC 1.4.1.9 (Leucine Dehydrogenase)
GMW67QNF9C (Leucine)
تواريخ الأحداث: Date Created: 20210107 Date Completed: 20210906 Latest Revision: 20210906
رمز التحديث: 20221213
مُعرف محوري في PubMed: PMC7788806
DOI: 10.1186/s12934-020-01501-2
PMID: 33407464
قاعدة البيانات: MEDLINE
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