Expression and characterization of a raw-starch glucoamylase from Aspergillus fumigatus

Song, Weiyan; Tong, Yi; Li, Yang; Tao, Jin; Li, Jianghua; Zhou, Jingwen; Liu, Song

doi:10.1016/j.procbio.2021.10.024

Cited by 9 publications

(16 citation statements)

References 37 publications

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“…Similar increases in specific activity and substrate affinity mediated by glycosylation were also observed in the glycosylated glucoamylase. 41 To date, many studies have introduced positive mutations in the finger and palm regions to increase the thermostability of GH11 xylanases. 20,21 However, there were few reports regarding such mutations within the cord and thumb regions, 27 which were included in HFR I and II, respectively.…”

Section: ■ Discussionmentioning

confidence: 99%

Significantly Enhanced Thermostability of Aspergillus niger Xylanase by Modifying Its Highly Flexible Regions

Huang

Rao

et al. 2022

J. Agric. Food Chem.

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In this study, the thermostability of an acid-resistant GH11 xylanase (xynA) from Aspergillus niger AG11 was enhanced through systematic modification of its four highly flexible regions (HFRs) predicted using MD simulations. Among them, HFR I (residues 92−100) and HFR II (residues 121−130) were modified by iterative saturation mutagenesis (ISM), yielding mutants G92F/G97S/G100K and T121V/A124P/I126V/T129L/A130N, respectively. For HFR III, the N-(residues 1−37) and Ctermini (residues 179−188) were, respectively, substituted with the corresponding sequences from thermophilic EvXyn11 TS and Nesterenkonia xinjiangensis xylanase. N-Glycosylation was introduced into HFR IV (residues 50−70) through site-directed mutation (A55N/D57S/S61N) and the recombinant expression in A. niger AG11. Combining these positive mutations from each HFR yielded the variant xynAm1 with 137.6-and 1.3-fold increases in half-life at 50 °C and specific activity compared to the wild-type xynA, respectively. With the highest thermostability at 80 and 90 °C in reports, xynAm1 could be a robust candidate for industrial applications in functional foods, feed products, and bioethanol production.

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Section: ■ Discussionmentioning

confidence: 99%

Significantly Enhanced Thermostability of Aspergillus niger Xylanase by Modifying Its Highly Flexible Regions

Huang

Rao

et al. 2022

J. Agric. Food Chem.

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“…To induce the protein expression, 1% methanol was added to the culture medium every 24 h during the fermentation. All shake flask experiments were carried out in triplicate at 30 °C for 120 h …”

Section: Methodsmentioning

confidence: 99%

“…Raw starch glucoamylase (RSGA) can directly hydrolyze raw starch granules to glucose below the starch gelatinization temperature, reducing the energy consumption required for high-temperature cooking and simplifying the process flow in the traditional starch sugar process. − In recent years, researchers have screened and isolated various RSGAs from microbial populations such as Aspergillus niger, Aspergillus awamori, and Aspergillus flavus, which can act on different kinds of raw starch. Although these RSGAs can degrade raw starch granules, their optimal reaction temperature is mostly distributed between 65 and 75 °C, − , and the degradation efficiency of raw starch with a concentration of 15% is only 10%. This is not enough for its large-scale application .…”

Section: Introductionmentioning

confidence: 99%

“…niger were fused via a short peptide to obtain the fusion enzyme Amy-Glu with a 1.15-fold increase in the degradation efficiency of raw corn starch . N-glycosylation is a ubiquitous post-translational modification involving the covalent attachment of a carbohydrate unit at the asparagine residue within the sequon Asn-Xaa-Ser/Thr (where Xaa cannot be Pro) in eukaryotes. − Numerous studies have shown that N-glycosylation has an important effect on the activity of polysaccharide hydrolases. ,, The specific activity of xylanase from the GH10 family decreased by 20% after the elimination of glycan chains at three glycosylation sites . In cellobiose hydrolase, the presence of glycosylation site Asn137 increased the enzymatic activity by 40% .…”

Section: Introductionmentioning

confidence: 99%

“…In previous studies, we successfully expressed Aspergillus fumigatus RSGA (afRSGA) in Komagataella phaffii (formerly known as Pichia pastoris) and found that glycosylation had an important effect on its activity . In this study, 13 N-glycosylation sites were designed based on NetNGlyc 1.0, and three glycosylation mutants with the improved catalytic activity of raw starch were obtained.…”

Section: Introductionmentioning

confidence: 99%

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Improving the Catalytic Efficiency of Aspergillus fumigatus Glucoamylase toward Raw Starch by Engineering Its N-Glycosylation Sites and Saturation Mutation

Song

Tong

et al. 2022

J. Agric. Food Chem.

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Raw starch glucoamylase (RSGA) can degrade the raw starch below the starch gelatinization temperature. In this study, to improve the catalytic activity of raw corn starch, N-glycosylation was introduced into the RSGA from Aspergillus fumigatus through site-directed mutation and the recombinant expression in Komagataella phaffii. Among them, the mutants G101S (N99-L100-S101) and Q113T (N111-S112-T113) increased the specific activity of raw corn starch by 1.19- and 1.21-fold, respectively. The optimal temperature of Q113T decreased from 70 to 60 °C. Notably, the combined mutant G101S/Q113T increased the specific activity toward raw starch by 1.22-fold and reduced the optimal temperature from 70 to 60 °C. Moreover, the mutant Q113M with a 1.5-fold increase in the catalytic activity was obtained via saturation mutation at site 113. Thus, the N-glycosylation site engineering is an efficient method to improve the activity of RSGA toward raw starch.

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Solid-state fermentation of cassava (Manihot esculenta Crantz): a review

Egbune,

Ezedom,

Orororo

et al. 2023

World J Microbiol Biotechnol

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Expression and characterization of a raw-starch glucoamylase from Aspergillus fumigatus

Cited by 9 publications

References 37 publications

Significantly Enhanced Thermostability of Aspergillus niger Xylanase by Modifying Its Highly Flexible Regions

Significantly Enhanced Thermostability of Aspergillus niger Xylanase by Modifying Its Highly Flexible Regions

Improving the Catalytic Efficiency of Aspergillus fumigatus Glucoamylase toward Raw Starch by Engineering Its N-Glycosylation Sites and Saturation Mutation

Solid-state fermentation of cassava (Manihot esculenta Crantz): a review

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