CBM21 starch-binding domain: A new purification tag for recombinant protein engineering

Li, Chia-Jung; Lin, I-Ping; Chou, Wei-Yuan; Hsieh, Chen-An; Liu, Shi-Hwei; Huang, Rong-Yuan; Sheu, Chia‐Chin; Chang, Margaret Dah‐Tsyr

doi:10.1016/j.pep.2009.01.008

Cited by 20 publications

(8 citation statements)

References 26 publications

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“…The DNA fragment of An SBD was subsequently ligated into the pET15b expression vector at the Nde I and Xho I sites to generate pET15b- An SBD. Expression and purification of An SBD was carried out as for Ro SBD [59].…”

Section: Methodsmentioning

confidence: 99%

Two Unique Ligand-Binding Clamps of Rhizopus oryzae Starch Binding Domain for Helical Structure Disruption of Amylose

Jiang

Chou

et al. 2012

PLoS ONE

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The N-terminal starch binding domain of Rhizopus oryzae glucoamylase (RoSBD) has a high binding affinity for raw starch. RoSBD has two ligand-binding sites, each containing a ligand-binding clamp: a polyN clamp residing near binding site I is unique in that it is expressed in only three members of carbohydrate binding module family 21 (CBM21) members, and a Y32/F58 clamp located at binding site II is conserved in several CBMs. Here we characterized different roles of these sites in the binding of insoluble and soluble starches using an amylose-iodine complex assay, atomic force microscopy, isothermal titration calorimetry, site-directed mutagenesis, and structural bioinformatics. RoSBD induced the release of iodine from the amylose helical cavity and disrupted the helical structure of amylose type III, thereby significantly diminishing the thickness and length of the amylose type III fibrils. A point mutation in the critical ligand-binding residues of sites I and II, however, reduced both the binding affinity and amylose helix disruption. This is the first molecular model for structure disruption of the amylose helix by a non-hydrolytic CBM21 member. RoSBD apparently twists the helical amylose strands apart to expose more ligand surface for further SBD binding. Repeating the process triggers the relaxation and unwinding of amylose helices to generate thinner and shorter amylose fibrils, which are more susceptible to hydrolysis by glucoamylase. This model aids in understanding the natural roles of CBMs in protein-glycan interactions and contributes to potential molecular engineering of CBMs.

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Section: Methodsmentioning

confidence: 99%

Two Unique Ligand-Binding Clamps of Rhizopus oryzae Starch Binding Domain for Helical Structure Disruption of Amylose

Jiang

Chou

et al. 2012

PLoS ONE

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“…One of the most direct industrial applications of CBMs is their use as purification tags to catch unrelated proteins. CBMs from different families, such as those that are able to recognize cellulose and starch, have been used.…”

Section: Applications Of Cbms In Biotechnologymentioning

confidence: 99%

Advances in molecular engineering of carbohydrate-binding modules

et al. 2017

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Carbohydrate-binding modules (CBMs) are non-catalytic domains that are generally appended to carbohydrate-active enzymes. CBMs have a broadly conserved structure that allows recognition of a notable variety of carbohydrates, in both their soluble and insoluble forms, as well as in their alpha and beta conformations and with different types of bonds or substitutions. This versatility suggests a high functional plasticity that is not yet clearly understood, in spite of the important number of studies relating protein structure and function. Several studies have explored the flexibility of these systems by changing or improving their specificity toward substrates of interest. In this review, we examine the molecular strategies used to identify CBMs with novel or improved characteristics. The impact of the spatial arrangement of the functional amino acids of CBMs is discussed in terms of unexpected new functions that are not related to the original biological roles of the enzymes. Proteins 2017; 85:1602-1617. © 2017 Wiley Periodicals, Inc.

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“…Immobilized metal affinity chromatography and other affinity chromatography, such as using starch and β-cyclodextrin as ligands might simplify the purification procedure as well as using binding to starch granules [57,58]. On the other hand even RSDA that contains SBD are not always fully recovered from raw starch granule when raw starch was used as affinity support [59]. Recently, by using mixed mode Nuvia™ cPrime™ resin with the idea to exploit the hydrophobic patches on RSDA surface, we were able to simultaneously concentrate, remove pigment and to purify RSDA with yields of 96% directly from the fermentation broth in a single step [56].…”

Section: Recent Advances In Rsda Upstream and Downstream Processingmentioning

confidence: 99%

Raw starch degrading α-amylases: an unsolved riddle

Božić

Lončar

Slavić

et al. 2017

Amylase

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Starch is an important food ingredient and a substrate for the production of many industrial products. Biological and industrial processes involve hydrolysis of raw starch, such as digestion by humans and animals, starch metabolism in plants, and industrial starch conversion for obtaining glucose, fructose and maltose syrup or bioethanol. Raw starch degrading α-amylases (RSDA) can directly degrade raw starch below the gelatinization temperature of starch. Knowledge of the structures and properties of starch and RSDA has increased significantly in recent years. Understanding the relationships between structural peculiarities and properties of RSDA is a prerequisite for efficient application in different aspects of human benefit from health to the industry. This review summarizes recent advances on RSDA research with emphasizes on representatives of glycoside hydrolase family GH13. Definite understanding of raw starch digesting ability is yet to come with accumulating structural and functional studies of RSDA.

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CBM21 starch-binding domain: A new purification tag for recombinant protein engineering

Cited by 20 publications

References 26 publications

Two Unique Ligand-Binding Clamps of Rhizopus oryzae Starch Binding Domain for Helical Structure Disruption of Amylose

Two Unique Ligand-Binding Clamps of Rhizopus oryzae Starch Binding Domain for Helical Structure Disruption of Amylose

Advances in molecular engineering of carbohydrate-binding modules

Raw starch degrading α-amylases: an unsolved riddle

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