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

Abstract

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.

Streptomyces small laccase expressed in Aspergillus niger as a new addition for the lignocellulose bioconversion toolbox

“…A.niger strain MGG029 pyrE − , derived from A. niger MGG029 (26) was used as the transformation host for heterologous expression of SLAC genes, while Escherichia coli DH5α was employed for plasmid propagation (27). E. coli DH5α was grown on Luria-Bertani medium supplemented with ampicillin at 100 µg/mL.…”

“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in 10% precast polyacrylamide gels (Bio-Rad, Mini-PROTEAN® TGX™, #4561033) as described in (27). The A. niger transformants culture medium sample was mixed with SDS-PAGE loading buffer containing 50 mM Tris-HCL pH 6.8, 25% glycerol, 0.05% bromophenol blue, 1% SDS, in a 3:1 ratio, without boiling and then loaded into the precast polyacrylamide gel.…”

“…The electrophoresis was run at 120 V for 1 hour at room temperature. Afterwards, the SDS-PAGE gel staining process was conducted as addressed in (27) using Sypro Ruby staining solution (Bio-Rad, Cat# 1703125). The Gel imaging was performed with the BioRad GelDoc™ EZ Imager.…”

HPLC-DAD-MS Characterization, Antioxidant Activity, α-amylase Inhibition, Molecular Docking, and ADMET of Flavonoids from Fenugreek Seeds

“…The process involves the hydrolysis of carbohydrates by the salivary α-amylase enzyme to disaccharides and oligosaccharides, which are further hydrolyzed by the α-glycosidase enzyme to monosaccharides like glucose, which are absorbed into the blood through the small intestine [25]. The pancreatic α-amylase then hydrolyzes the remaining oligosaccharides to glucose and maltose [26][27][28]. So, α-amylase is a key enzyme for starch digestion [29].…”

Immobilization of Aspergillus fumigatus α-Amylase via Adsorption onto Bentonite/Chitosan for Stability Enhancement

“…Fungi from the genus Aspergillus are the most important sources for the commercial production of α-amylase. Some well-known examples are Aspergillus terreus, A. ochraceus, A. tamarii MTCC5152, A. flavus AUMC10636, A. niger, A. oryzae, and A. fumigatus [8][9][10][11][12][13][14]. Of the various aforementioned sources of α-amylase, A. fumigatus is of particular importance since this fungus is known to have a high ability to endure heat, grow rapidly in a fundamentally nutrient-rich habitat, and require no special nutrition [15].…”