Gene cloning, expression enhancement in Escherichia coli and biochemical characterization of a highly thermostable amylomaltase from Pyrobaculum calidifontis

• Published in: International journal of biological macromolecules 2020-12, Vol.165 (Pt A), p.645-653
• Main Authors: Mehboob, Sumaira, Ahmad, Nasir, Munir, Sajida, Ali, Ramzan, Younas, Hooria, Rashid, Naeem
• Format: Article
• Language: English
• Subjects: 4-α-glucanotransferases; Archaeal Proteins - biosynthesis; Archaeal Proteins - chemistry; Archaeal Proteins - genetics; Archaeal Proteins - isolation & purification; Cloning, Molecular; Enzyme Stability; Escherichia coli - enzymology; Escherichia coli - genetics; Family GH77; Gene Expression; Glycogen Debranching Enzyme System - biosynthesis; Glycogen Debranching Enzyme System - chemistry; Glycogen Debranching Enzyme System - genetics; Glycogen Debranching Enzyme System - isolation & purification; Hot Temperature; Pyrobacullum calidifontis; Pyrobaculum - enzymology; Pyrobaculum - genetics; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2020.09.071

Pcal_0768 gene encoding an amylomaltase, a 4-α-glucanatransferase belonging to family 77 of glycosyl hydrolases, from Pyrobaculum calidifontis was cloned and expressed in Escherichia coli. The recombinant protein was produced in E. coli in soluble and active form. However, the expression level was not very high. Analysis of the mRNA of initial seven codons at the 5′-end of the gene revealed the presence of a hair pin like secondary structure. This secondary structure was removed by site directed mutagenesis, without altering the amino acids, which resulted in enhanced expression of the cloned gene. Recombinant Pcal_0768 exhibited optimal amylomaltase activity at 80 °C and pH 6.9. Under these conditions, the specific activity was 690 U/ mg. Recombinant Pcal_0768 was highly thermostable with a half-life of 6 h at 100 °C. It exhibited the highest kcat value among the characterized glucanotransferases. No metal ions were required for activity or stability of the enzyme. Recombinant Pcal_0768 was successfully employed in the synthesis of modified starch for producing thermoreversible gel. To the best of our knowledge, till now this is the most thermostable enzyme among the characterized amylomaltases. High thermostability and starch modification potential make it a novel and distinct amylomaltase.