Fusion of a family 20 carbohydrate‐binding module (CBM20) with cyclodextrin glycosyltransferase of <i>Geobacillus</i> sp. CHB1 improves catalytic efficiency

Jia, Xiaoling; Guo, Yonghua; Lin, Xi Zhang; You, Minsheng; Chao, Lin; Chen, Longjun; Chen, Jichen

doi:10.1002/jobm.201600628

Cited by 9 publications

(3 citation statements)

References 30 publications

(37 reference statements)

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“…A CBM not only contributes to substrate binding but also stabilizes the molecular structure at high temperatures [ 27 ]. A CBM can also affect the stability and activity of the enzyme [ 15 ], and Jia et al were able to obtain a fusion enzyme with greatly increased activity and thermostability by linking a CBM to the C-terminus of cyclodextrin glycosyltransferase [ 28 ]. Similarly, the CBM1 module was deleted from a GH5 β-mannanase from T. leycettanus JCM12802, resulting in a recombinant enzyme with reduced thermal tolerance at 80 °C [ 29 ].…”

Section: Resultsmentioning

confidence: 99%

Engineering a carbohydrate-binding module to increase the expression level of glucoamylase in Pichia pastoris

et al. 2022

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Background Glucoamylase is an important industrial enzyme for the saccharification of starch during sugar production, but the production cost of glucoamylase is a major limiting factor for the growth of the starch-based sugar market. Therefore, seeking strategies for high-level expression of glucoamylase in heterologous hosts are considered as the main way to reduce the enzyme cost. Results ReGa15A from Rasamsonia emersonii and TlGa15B-GA2 from Talaromyces leycettanus have similar properties. However, the secretion level of ReGa15A was significantly higher than TlGa15B-GA2 in Pichia pastoris. To explore the underlying mechanisms affecting the differential expression levels of glucoamylase in P. pastoris, the amino acid sequences and three-dimensional structures of them were compared and analyzed. First, the CBM region was identified by fragment replacement as the key region affecting the expression levels of ReGa15A and TlGa15B-GA2. Then, through the substitution and site-directed mutation of the motifs in the CBM region, three mutants with significantly increased expression levels were obtained. The eight-point mutant TlGA-M4 (S589D/Q599A/G600Y/V603Q/T607I/V608L/N609D/R613Q), the three-point mutant TlGA-M6 (Q599A/G600Y/V603Q) and the five-point mutant TlGA-M7 (S589D/T607I/V608L/N609D/R613Q) have the same specific activity with the wild-type, and the enzyme activity and secretion level have increased by 4–5 times, respectively. At the same time, the expression levels were 5.8-, 2.0- and 2.4-fold higher than that of wild type, respectively. Meanwhile, the expression of genes related to the unfolded protein responses (UPR) in the endoplasmic reticulum (ER) did not differ significantly between the mutants and wild type. In addition, the most highly expressed mutant, TlGA-M7 exhibits rapidly and effectively hydrolyze raw corn starch. Conclusions Our results constitute the first demonstration of improved expression and secretion of a glucoamylase in P. pastoris by introducing mutations within the non-catalytic CBM. This provides a novel and effective strategy for improving the expression of recombinant proteins in heterologous host expression systems.

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Section: Resultsmentioning

confidence: 99%

Engineering a carbohydrate-binding module to increase the expression level of glucoamylase in Pichia pastoris

et al. 2022

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“…SBDs are found in several amylolytic enzymes (Christiansen et al , 2009) and the activity of these enzymes may be further engineered by adding additional starch-binding CBMs, or CBMs with high affinity for starch (Valk et al , 2016). Similarly, glycosyltransferases may gain additional activity when fused with a CBM20 (Jia et al , 2017). Such an approach may provide useful enzymatic modifications of starches on an industrial scale.…”

Section: Discussionmentioning

confidence: 99%

Expression of starch-binding factor CBM20 in barley plastids controls the number of starch granules and the level of CO2 fixation

Zhong

Sagnelli

Topbjerg

et al. 2019

Journal of Experimental Botany

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“…Among them, CBM20 is the best studied and first discovered SBD, identified at the C-terminal domain of Aspergillus niger glucoamylase [6,7]. The CBM20 domains bring special interest as they have been reported to play a role not only in binding but also in the disruption of the surface of starch granules, making the substrate Molecules 2023, 28, 5033 2 of 18 more accessible to be cleaved by starch degrading enzymes and perform degradation at a higher rate [8,9]. This is reported for A. niger glucoamylase [10,11], Cryptococcus sp α-amylase [12], and AA13 polysaccharide monooxygenases of Neurospora crassa [13].…”

Section: Introductionmentioning

confidence: 99%

Novel Design of an α-Amylase with an N-Terminal CBM20 in Aspergillus niger Improves Binding and Processing of a Broad Range of Starches

et al. 2023

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In the starch processing industry including the food and pharmaceutical industries, α-amylase is an important enzyme that hydrolyses the α-1,4 glycosidic bonds in starch, producing shorter maltooligosaccharides. In plants, starch molecules are organised in granules that are very compact and rigid. The level of starch granule rigidity affects resistance towards enzymatic hydrolysis, resulting in inefficient starch degradation by industrially available α-amylases. In an approach to enhance starch hydrolysis, the domain architecture of a Glycoside Hydrolase (GH) family 13 α-amylase from Aspergillus niger was engineered. In all fungal GH13 α-amylases that carry a carbohydrate binding domain (CBM), these modules are of the CBM20 family and are located at the C-terminus of the α-amylase domain. To explore the role of the domain order, a new GH13 gene encoding an N-terminal CBM20 domain was designed and found to be fully functional. The starch binding capacity and enzymatic activity of N-terminal CBM20 α-amylase was found to be superior to that of native GH13 without CBM20. Based on the kinetic parameters, the engineered N-terminal CBM20 variant displayed surpassing activity rates compared to the C-terminal CBM20 version for the degradation on a wide range of starches, including the more resistant raw potato starch for which it exhibits a two-fold higher Vmax underscoring the potential of domain engineering for these carbohydrate active enzymes.

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Fusion of a family 20 carbohydrate‐binding module (CBM20) with cyclodextrin glycosyltransferase of Geobacillus sp. CHB1 improves catalytic efficiency

Cited by 9 publications

References 30 publications

Engineering a carbohydrate-binding module to increase the expression level of glucoamylase in Pichia pastoris

Engineering a carbohydrate-binding module to increase the expression level of glucoamylase in Pichia pastoris

Expression of starch-binding factor CBM20 in barley plastids controls the number of starch granules and the level of CO2 fixation

Novel Design of an α-Amylase with an N-Terminal CBM20 in Aspergillus niger Improves Binding and Processing of a Broad Range of Starches

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