PubMed · 3174446
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R F van Gorcom, C A van den Hondel. 1988-09-26. Expression analysis vectors for Aspergillus niger.. https://doi.org/10.1093/nar%2F16.18.9052
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Aspergillus niger is an important industrial fungus widely used for citric acid production and a range of biotechnological applications. In this study, a protoplast-based DNA isolation protocol was optimized for a gamma-irradiated A. niger AN-L103_M1 mutant strain, followed by whole-genome sequencing and functional genome analysis. Protoplast yield was strongly influenced by enzyme concentration and the molarity of the osmotic stabilizer. The highest yield was achieved at an enzyme concentration of 50 mg/mL (2.487 ± 0.04 × 10⁸ cells/mL) and 0.8 M KCl (2.550 ± 0.06 × 10⁸ cells/mL), with both factors showing significant effects (p < 0.0001) in GraphPad Prism 11.0.0. Whole-genome sequencing performed using an Illumina NovaSeq 6000 platform yielded a 37.06 Mb draft genome assembled into 537 contigs, with an N50 of 363,084 bp and a GC content of 48.2%. BUSCO 14 analysis showed high completeness (97.95% complete BUSCOs). Functional annotation and KEGG pathway mapping identified genes involved in glycolysis, the tricarboxylic acid cycle, and citrate biosynthesis, while biosynthetic gene cluster analysis revealed diverse potential for secondary metabolite production. These findings provide an optimized workflow for protoplast-based DNA isolation and genome-scale functional analysis in A. niger, proposing a basis for future comparative genomics, transformation studies, and experimentally validated metabolic engineering.
Beta-glucosidase was purified from a crude cellulase preparation from Aspergillus niger by affinity chromatography on a methacrylamide-N-methylene-bis-methacrylamide copolymer bearing cellobiamine. The purified enzyme was a dimer with an isoelectric point of 4.0. The molecular mass of the enzyme was estimated to be 240 kDa by gel-permeation chromatography. The enzyme hydrolyzed specifically beta-glucosidic bonds and catalyzed transglucosylation of the beta-glucosyl group of cellobiose to yield 4-O-beta-gentiobiosylglucose in the presence of organic solvents or under neutral conditions.
The presteady-state and steady-state kinetics of the binding and hydrolysis of substrates, maltose and isomaltose, and the transition-state analogue, gluconolactone, by glucoamylase from Aspergillus niger were investigated using initial-rate, stopped-flow and steady-state methods. The change in the intrinsic fluorescence of the enzyme was monitored. Distinct mechanistic differences were observed in the interaction of the enzyme with maltose compared to isomaltose. Hydrolysis of maltose requires a three-step mechanism, whereas that of isomaltose involves at least one additional step. The rates of an observed conformational change, which is the second discernible step of the reactions, clearly show a tighter binding of maltose compared to isomaltose, probably because the reverse rate constants differ. Compared to the non-enzymic hydrolysis the transition-state stabilization energy of glucoamylase is approximately -66 kJ/mol with maltose and only -14 kJ/mol with isomaltose. Kinetic analysis of the binding of the inhibitor, gluconolactone, implies that independent interactions of two molecules occur. One of these, apparently, is a simple, fast association reaction in which gluconolactone is weakly bound. The other resembles binding of maltose, involving a fast association followed by a conformational change. Based on the results obtained, we propose new reaction mechanisms for Aspergillus glucoamylase.