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  4. Parallel N- and C-Terminal Truncations Facilitate Purification and Analysis of a 155-kDa Cold-Adapted Type-I Pullulanase
 
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Parallel N- and C-Terminal Truncations Facilitate Purification and Analysis of a 155-kDa Cold-Adapted Type-I Pullulanase

Publikationstyp
Journal Article
Date Issued
2017-02-08
Sprache
English
Author(s)
Elleuche, Skander  
Krull, Annika  
Lorenz, Ute  
Antranikian, Garabed  
Institut
Technische Mikrobiologie V-7  
Biomechanik M-3  
TORE-URI
http://hdl.handle.net/11420/4235
Journal
Protein journal  
Volume
36
Issue
1
Start Page
56
End Page
63
Citation
Protein Journal 1 (36): 56-63 (2017)
Publisher DOI
10.1007/s10930-017-9703-4
Scopus ID
2-s2.0-85011933953
Publisher
Springer Science
The cold-adapted pullulanase Pul13A is an industrial useful amylolytic enzyme, but its low solubility is the major bottleneck to produce the protein in recombinant form. In a previous approach, a complex and time-consuming purification strategy including a step-wise dialysis procedure using decreasing concentrations of urea to renature the insoluble protein from inclusion bodies had been established. In this study, a truncation strategy was developed to facilitate the purification and handling of the type-I pullulanase. Pul13A has a size of 155-kDa with a multidomain architecture that is composed of the following predicted modules: CBM41/E-set/Amy-Pul/DUF3372/E-set/E-set/E-set, with CBM and E-set domains being putative carbohydrate-binding modules, Amy-Pul is the catalytic region and DUF is a domain of unknown function. Consecutive N- and C-terminal deletions of domains were applied to construct minimized enzyme variants retaining pullulanase activity and exhibiting improved renaturation efficiencies. A total of seven truncation constructs were generated and tested, which still led to the production of inclusion bodies. However, the parallel deletion of the exterior CBM41 and E-set domain enabled the direct refolding of active enzymes during one-step dialysis in urea-free buffer. Catalytic properties of truncation construct Pul13A-N1/C1 were not impaired indicating that this enzyme variant may be superior for industrial applications over the full-length pullulanase.
Subjects
cold-adaptation
purification efficiency
one-step renaturation
truncations
type-I pullulanase
DDC Class
500: Naturwissenschaften
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