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A predominant β-CGTase G1 engineered to elucidate the relationship between protein structure and product specificity

Low reaction yields and the high cost of obtaining a single type of pure CD make γ-CD costly. Using rational design and with the aid of 3D modeling structures, recombinant CGTase from Bacillus sp. G1 was molecularly engineered with the aim of producing a higher percentage of γ-CD. A single mutation...

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Main Authors: Goh, Kian Mau, Mahadi, Nor Muhammad, Hassan, Osman, Raja Abdul Rahman, Raja Noor Zaliha, Md IIlias, Rosli
Format: Article
Language:English
Published: Elsevier 2009
Online Access:http://psasir.upm.edu.my/id/eprint/12782/1/A%20predominant%20%CE%B2.pdf
http://psasir.upm.edu.my/id/eprint/12782/
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spelling my.upm.eprints.127822016-09-28T01:43:52Z http://psasir.upm.edu.my/id/eprint/12782/ A predominant β-CGTase G1 engineered to elucidate the relationship between protein structure and product specificity Goh, Kian Mau Mahadi, Nor Muhammad Hassan, Osman Raja Abdul Rahman, Raja Noor Zaliha Md IIlias, Rosli Low reaction yields and the high cost of obtaining a single type of pure CD make γ-CD costly. Using rational design and with the aid of 3D modeling structures, recombinant CGTase from Bacillus sp. G1 was molecularly engineered with the aim of producing a higher percentage of γ-CD. A single mutation at subsite −3, denoted H43T, was found to increase γ-CD production from 10% to approximately 39% using tapioca starch. This novel increment was probably the result of reduced steric hindrance to the formation of γ-CD because of the shortened side chain together with the shortened loop at positions 86–89, at substrate-binding subsite −3. A mutation (Tyr188 → Trp) and a deletion at loop 139–144 showed little effect on product specificity; however, mutagenesis at these sites affected cyclization, coupling and hydrolysis activities as well as the kinetic properties of the mutant CGTase. Based on rational design, three further mutations of the mutant H43T (denoted H43T/Δ(139–144)/S134T/A137V/L138D/V139I, H43T/S85G and H43T/Y87F) were constructed and produced γ-CD with yields of 20%, 20% and 39%, respectively. The mutant H43T/Δ(139–144)/S134T/A137V/L138D/V139I had very low cyclization and coupling activities, however their hydrolysis activity was retained. Double mutation (H43T/S85G) caused the enzyme to exhibit higher starch hydrolysis activity, approximately 26 times higher than the native CGTase G1. Although the mutants H43T and H43T/Y87F could produce the same percentage (39%) of γ-CD, the latter was more efficient as the total amount of CD produced was higher based on the Vmax and kcat values. Elsevier 2009-10 Article PeerReviewed application/pdf en http://psasir.upm.edu.my/id/eprint/12782/1/A%20predominant%20%CE%B2.pdf Goh, Kian Mau and Mahadi, Nor Muhammad and Hassan, Osman and Raja Abdul Rahman, Raja Noor Zaliha and Md IIlias, Rosli (2009) A predominant β-CGTase G1 engineered to elucidate the relationship between protein structure and product specificity. Journal of Molecular Catalysis B: Enzymatic, 57 (1-4). pp. 270-277. ISSN 1381-1177; ESSN: 1873-3158 10.1016/j.molcatb.2008.09.016
institution Universiti Putra Malaysia
building UPM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Putra Malaysia
content_source UPM Institutional Repository
url_provider http://psasir.upm.edu.my/
language English
description Low reaction yields and the high cost of obtaining a single type of pure CD make γ-CD costly. Using rational design and with the aid of 3D modeling structures, recombinant CGTase from Bacillus sp. G1 was molecularly engineered with the aim of producing a higher percentage of γ-CD. A single mutation at subsite −3, denoted H43T, was found to increase γ-CD production from 10% to approximately 39% using tapioca starch. This novel increment was probably the result of reduced steric hindrance to the formation of γ-CD because of the shortened side chain together with the shortened loop at positions 86–89, at substrate-binding subsite −3. A mutation (Tyr188 → Trp) and a deletion at loop 139–144 showed little effect on product specificity; however, mutagenesis at these sites affected cyclization, coupling and hydrolysis activities as well as the kinetic properties of the mutant CGTase. Based on rational design, three further mutations of the mutant H43T (denoted H43T/Δ(139–144)/S134T/A137V/L138D/V139I, H43T/S85G and H43T/Y87F) were constructed and produced γ-CD with yields of 20%, 20% and 39%, respectively. The mutant H43T/Δ(139–144)/S134T/A137V/L138D/V139I had very low cyclization and coupling activities, however their hydrolysis activity was retained. Double mutation (H43T/S85G) caused the enzyme to exhibit higher starch hydrolysis activity, approximately 26 times higher than the native CGTase G1. Although the mutants H43T and H43T/Y87F could produce the same percentage (39%) of γ-CD, the latter was more efficient as the total amount of CD produced was higher based on the Vmax and kcat values.
format Article
author Goh, Kian Mau
Mahadi, Nor Muhammad
Hassan, Osman
Raja Abdul Rahman, Raja Noor Zaliha
Md IIlias, Rosli
spellingShingle Goh, Kian Mau
Mahadi, Nor Muhammad
Hassan, Osman
Raja Abdul Rahman, Raja Noor Zaliha
Md IIlias, Rosli
A predominant β-CGTase G1 engineered to elucidate the relationship between protein structure and product specificity
author_facet Goh, Kian Mau
Mahadi, Nor Muhammad
Hassan, Osman
Raja Abdul Rahman, Raja Noor Zaliha
Md IIlias, Rosli
author_sort Goh, Kian Mau
title A predominant β-CGTase G1 engineered to elucidate the relationship between protein structure and product specificity
title_short A predominant β-CGTase G1 engineered to elucidate the relationship between protein structure and product specificity
title_full A predominant β-CGTase G1 engineered to elucidate the relationship between protein structure and product specificity
title_fullStr A predominant β-CGTase G1 engineered to elucidate the relationship between protein structure and product specificity
title_full_unstemmed A predominant β-CGTase G1 engineered to elucidate the relationship between protein structure and product specificity
title_sort predominant β-cgtase g1 engineered to elucidate the relationship between protein structure and product specificity
publisher Elsevier
publishDate 2009
url http://psasir.upm.edu.my/id/eprint/12782/1/A%20predominant%20%CE%B2.pdf
http://psasir.upm.edu.my/id/eprint/12782/
_version_ 1643825133106757632
score 13.211869

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