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Engineering of the pH optimum of Bacillus cereus beta-amylase: conversion of the pH optimum from a bacterial type to a higher-plant type.

Hirata A, Adachi M, Utsumi S, Mikami B
Biochemistry 43 12523-31 (2004)
Related entries: 1vem, 1ven, 1veo

Cited: 13 times
EuropePMC logo PMID: 15449941

Abstract

The optimum pH of Bacillus cereus beta-amylase (BCB, pH 6.7) differs from that of soybean beta-amylase (SBA, pH 5.4) due to the substitution of a few amino acid residues near the catalytic base residue (Glu 380 in SBA and Glu 367 in BCB). To explore the mechanism for controlling the optimum pH of beta-amylase, five mutants of BCB (Y164E, Y164F, Y164H, Y164Q, and Y164Q/T47M/Y164E/T328N) were constructed and characterized with respect to enzymatic properties and X-ray structural crystal analysis. The optimum pH of the four single mutants shifted to 4.2-4.8, approximately 2 pH units and approximately 1 pH unit lower than those of BCB and SBA, respectively, and their k(cat) values decreased to 41-3% of that of the wild-type enzyme. The X-ray crystal analysis of the enzyme-maltose complexes showed that Glu 367 of the wild type is surrounded by two water molecules (W1 and W2) that are not found in SBA. W1 is hydrogen-bonded to both side chains of Glu 367 and Tyr 164. The mutation of Tyr 164 to Glu and Phe resulted in the disruption of the hydrogen bond between Tyr 164 Oeta and W1 and the introduction of two additional water molecules near position 164. In contrast, the triple mutant of BCB with a slightly decreased pH optimum at pH 6.0 has no water molecules (W1 and W2) around Glu 367. These results suggested that a water-mediated hydrogen bond network (Glu 367...W1...Tyr 164...Thr 328) is the primary requisite for the increased pH optimum of wild-type BCB. This strategy is completely different from that of SBA, in which a hydrogen bond network (Glu 380...Thr 340...Glu 178) reduces the optimum pH in a hydrophobic environment.

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  2. Directed evolution of an antitumor drug (arginine deiminase PpADI) for increased activity at physiological pH. Zhu L, Tee KL, Roccatano D, Sonmez B, Ni Y, Sun ZH, Schwaneberg U. Chembiochem 11 691-697 (2010)
  3. Extraction and purification of beta-amylase from stems of Abrus precatorius by three phase partitioning. Sagu ST, Nso EJ, Homann T, Kapseu C, Rawel HM. Food Chem 183 144-153 (2015)
  4. Engineering of the pH-dependence of thermolysin activity as examined by site-directed mutagenesis of Asn112 located at the active site of thermolysin. Kusano M, Yasukawa K, Hashida Y, Inouye K. J Biochem 139 1017-1023 (2006)
  5. Production of recombinant beta-amylase of Bacillus aryabhattai. Duan X, Shen Z, Zhang X, Wang Y, Huang Y. Prep Biochem Biotechnol 49 88-94 (2019)
  6. Modulating the pH Activity Profiles of Phenylalanine Ammonia Lyase from Anabaena variabilis by Modification of Center-Near Surface Residues. Zhang F, Huang N, Zhou L, Cui W, Liu Z, Zhu L, Liu Y, Zhou Z. Appl Biochem Biotechnol 183 699-711 (2017)
  7. Mechanism-based site-directed mutagenesis to shift the optimum pH of the phenylalanine ammonia-lyase from Rhodotorula glutinis JN-1. Zhu L, Zhou L, Cui W, Liu Z, Zhou Z. Biotechnol Rep (Amst) 3 21-26 (2014)
  8. Computational design of enhanced detoxification activity of a zearalenone lactonase from Clonostachys rosea in acidic medium. Lin M, Tan J, Xu Z, Huang J, Tian Y, Chen B, Wu Y, Tong Y, Zhu Y. RSC Adv 9 31284-31295 (2019)
  9. Two homologous fungal carbonyl reductases with different substrate specificities. Kristan K, Brunskole M, Stojan J, Rizner TL. Chem Biol Interact 178 295-302 (2009)
  10. Expression, biochemical and structural characterization of high-specific-activity β-amylase from Bacillus aryabhattai GEL-09 for application in starch hydrolysis. Duan X, Zhu Q, Zhang X, Shen Z, Huang Y. Microb Cell Fact 20 182 (2021)
  11. Biochemical Properties of β-Amylase from Red Algae and Improvement of Its Thermostability through Immobilization. Murakami M, Osanai T. ACS Omega 7 36195-36205 (2022)
  12. Combined Computer-Aided Predictors to Improve the Thermostability of Nattokinase. Li Y, Chen L, Tang X, Zhu W, Ma A, Shi C, Li J. Foods 12 3045 (2023)


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