Cyanide hydratase from Aspergillus niger K10: Overproduction in Escherichia coli, purification, characterization and use in continuous cyanide degradation

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• 作者：Anna Riná ; gelová ; ^a ; ^b ; ^{Rinagelova.A@seznam.cz" class="auth_mail} ; Ond艡ej Kaplan^a ; ¹ ; Alicja B. Veselá ; ^a ; ^c ; Martin Chmá ; tal^a ; Alena K艡enková ; ^a ; ^c ; Ond艡ej Plí ; hal^a ; ² ; Fabrizia Pasquarelli^a ; ^d ; Maria Cantarella^d ; Ludmila Martí ; nková ; ^a ; ^{martinko@biomed.cas.cz" class="auth_mail}
• 关键词：aa ; amino acid ; CHT ; cyanide hydratase ; CSMR ; continuous stirred membrane reactor ; CynDpum ; cyanide dihydratase from Bacillus pumilus ; CynDstu ; cyanide dihydratase from Pseudomonas stutzeri ; NitAn1 ; cyanide hydratase/nitrilase from Aspergillus niger K10 ; NitAn1-C14 ; NitAn1-C18 ; NitAn1-C22 ; NitAn1-C26 ; NitAn1-C30 and NitAn1-C34 ; variants of NitAn1 missing 14 ; 18 ; 22 ; 26 ; 30 and 34 C-terminal aa residues ; respectively ; Nit(p) ; nitrilase from Pseudomonas fluorescens EBC 191 ; Nit(r) ; nitrilase from
• 刊名：Process Biochemistry
• 出版年：March, 2014
• 年：2014
• 卷：49
• 期：3
• 页码：445-450
• 全文大小：603 K

文摘

A cyanide hydratase from Aspergillus niger K10 was expressed in Escherichia coli and purified. Apart from HCN, it transformed some nitriles, preferentially 2-cyanopyridine and fumaronitrile. V_max and K_m for HCN were ca. 6.8 mmol min^鈭? mg^鈭? protein and 109 mM, respectively. V_max for fumaronitrile and 2-cyanopyridine was two to three orders of magnitude lower than for HCN (ca. 18.8 and 10.3 渭mol min^鈭? mg^鈭?, respectively) but K_m was also lower (ca. 14.7 and 3.7 mM, respectively). Both cyanide hydratase and nitrilase activities were abolished in truncated enzyme variants missing 18-34 C-terminal aa residues. The enzyme exhibited the highest activity at 45 掳C and pH 8-9; it was unstable at over 35 掳C and at below pH 5.5. The operational stability of the whole-cell catalyst was examined in continuous stirred membrane reactors with 70-mL working volume. The catalyst exhibited a half-life of 5.6 h at 28 掳C. A reactor loaded with an excess of the catalyst was used to degrade 25 mM KCN. A conversion rate of over 80% was maintained for 3 days.