A new procedure for enzymatic semisynthesis of human insulin by hydrolysis of single‐chain des‐(b‐30)‐lnsulin precursor with lysyl endopeptidase

Morihara, Kazuyuki; Ueno, Yoshihiko

doi:10.1002/bit.260370712

Cited by 5 publications

(3 citation statements)

References 11 publications

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“…Stable serine proteases are used industrially in diverse areas ranging from medical applications to food processing and laundry. For example, Achromobacter lyticus protease I has been used to convert pig insulin to human insulin by specific removal and replacement of amino acid 30 in the insulin Bchain (Morihara & Ueno, 1991) or as a step during the total synthesis of human insulin (Tofteng et al, 2008). Biochemical applications such as primary-structure analysis, peptide mapping, in-gel digestion, cleavage of fusion proteins and protein synthesis require specific proteases that are resilient to denaturation even in the presence of significant concentrations of denaturants and that operate under a broad range of conditions (Kuhlman et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Atomic resolution structure of a lysine-specific endoproteinase fromLysobacter enzymogenessuggests a hydroxyl group bound to the oxyanion hole

Asztalos

Müller

Hölke

et al. 2014

Acta Cryst D Biol Crystallogr

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Lysobacter enzymogenes lysyl endoproteinase (LysC) is a trypsin-type serine protease with a high pH optimum that hydrolyses all Lys-Xaa peptide bonds. The high specificity of LysC renders it useful for biotechnological purposes. The K30R variant of a related lysyl endoproteinase from Achromobacter lyticus has favourable enzymatic properties that might be transferrable to LysC. To visualize structural differences in the substrate-binding sites, the crystal structures of wild-type and the K30R variant of LysC were determined. The mutation is located at a distance of 12 Å from the catalytic triad and subtly changes the surface properties of the substrate-binding site. The high pH optimum of LysC can be attributed to electrostatic effects of an aromatic Tyr/His stack on the catalytic aspartate and is a general feature of this enzyme subfamily. LysC crystals in complex with the covalent inhibitor N(α)-p-tosyl-lysyl chloromethylketone yielded data to 1.1 and 0.9 Å resolution, resulting in unprecedented precision of the active and substrate-binding sites for this enzyme subfamily. Error estimates on bond lengths and difference electron density indicate that instead of the expected oxyanion a hydroxyl group binds to the partially solvent-exposed oxyanion hole. Protonation of the alkoxide catalytic intermediate might be a recurring feature during serine protease catalysis.

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Section: Introductionmentioning

confidence: 99%

Atomic resolution structure of a lysine-specific endoproteinase fromLysobacter enzymogenessuggests a hydroxyl group bound to the oxyanion hole

Asztalos

Müller

Hölke

et al. 2014

Acta Cryst D Biol Crystallogr

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“…( Morihara et al 1980;Morihara and Ueno 1991). Also, the use of carboxypeptidase A for the same procedure was described (Andresen et al 1983).…”

Section: Insulin and Insulin Analoguesmentioning

confidence: 99%

Biocatalyzed synthesis of antidiabetic drugs: A review

Alcántara

2017

Biocatalysis and Biotransformation

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“…A further investigation was undertaken to determine whether DAI can be formed by hydrolysis of SCI with trypsin or Ach (Morihara and Ueno, 1991). As shown in Fig.…”

Section: Use Of Single-chain Insulin Precursor For Conversionmentioning

confidence: 99%

Enzymatic semisynthesis of human insulin: An update

Morihara

1990

J of Molecular Recognition

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Peptide bond formation can be enzymatically catalysed by the reverse reaction of proteases. Application is seen in the industrial production of human insulin. Human insulin derivative can be enzymatically prepared using either porcine insulin or the single chain B(1-29)-A(1-21) insulin precursor as the starting material. This is accomplished by either (1) digesting the starting material at Lys29 with Achromobacter lyticus protease I (Ach) and then coupling with Thr-X (X = blocking residue) (two-step reaction) or (2) subjecting Ala-B30 of porcine insulin or Gly-A1 of the single chain insulin precursor to transpeptidation with Thr-X (one-step reaction). Trypsin and Ach can be used for either type of reaction and, in the immobilized form, for the two-step reaction. Since the single chain insulin precursor can be produced by gene technology (yeast), use of immobilized trypsin or Ach and the two-step reaction using the single chain insulin precursor as the starting material ensures the continuous production of human insulin making it a feasible method for industrial manufacture.

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A new procedure for enzymatic semisynthesis of human insulin by hydrolysis of single‐chain des‐(b‐30)‐lnsulin precursor with lysyl endopeptidase

Cited by 5 publications

References 11 publications

Atomic resolution structure of a lysine-specific endoproteinase fromLysobacter enzymogenessuggests a hydroxyl group bound to the oxyanion hole

Atomic resolution structure of a lysine-specific endoproteinase fromLysobacter enzymogenessuggests a hydroxyl group bound to the oxyanion hole

Biocatalyzed synthesis of antidiabetic drugs: A review

Enzymatic semisynthesis of human insulin: An update

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