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Functional characterisation of cellobiohydrolase I (Cbh1) from Trichoderma virens UKM1 expressed in Aspergillus niger

Wahab AFFA, Abdul Karim NA, Ling JG, Hasan NS, Yong HY, Bharudin I, et al.

Protein Expr Purif, 2019 02;154:52-61.
PMID: 30261309 DOI: 10.1016/j.pep.2018.09.014

Cellobiohydrolases catalyze the processive hydrolysis of cellulose into cellobiose. Here, a Trichoderma virens cDNA predicted to encode for cellobiohydrolase (cbhI) was cloned and expressed heterologously in Aspergillus niger. The cbhI gene has an open reading frame of 1518 bp, encoding for a putative protein of 505 amino acid residues with a calculated molecular mass of approximately 54 kDa. The predicted CbhI amino acid sequence has a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region and showed high sequence homology with glycoside hydrolase family 7 proteins. The partially purified enzyme has an optimum pH of 4.0 with stability ranging from pH 3.0 to 6.0 and an optimum temperature of 60 °C. The partially purified CbhI has a specific activity of 4.195 Umg-1 and a low Km value of 1.88 mM when p-nitrophenyl-β-D-cellobioside (pNPC) is used as the substrate. The catalytic efficiency (kcat/Km) was 5.68 × 10-4 mM-1s-1, which is comparable to the CbhI enzymes from Trichoderma viridae and Phanaerochaete chrysosporium. CbhI also showed activity towards complex substrates such as Avicel (0.011 Umg-1), which could be useful in complex biomass degradation. Interestingly, CbhI also exhibited a relatively high inhibition constant (Ki) for cellobiose with a value of 8.65 mM, making this enzyme more resistant to end-product inhibition compared to other fungal cellobiohydrolases.

Matched MeSH terms: Aspergillus niger/genetics
Understanding thermostability factors of Aspergillus niger PhyA phytase: a molecular dynamics study

Noorbatcha IA, Sultan AM, Salleh HM, Amid A

Protein J, 2013 Apr;32(4):309-16.
PMID: 23636517 DOI: 10.1007/s10930-013-9489-y

Molecular dynamics simulation was used to study the dynamic differences between native Aspergillus niger PhyA phytase and a mutant with 20 % greater thermostability. Atomic root mean square deviation, radius of gyration, and number of hydrogen bonds and salt bridges are examined to determine thermostability factors. The results suggest that, among secondary structure elements, loops have the most impact on the thermal stability of A. niger phytase. In addition, the location rather than the number of hydrogen bonds is found to have an important contribution to thermostability. The results also show that salt bridges may have stabilizing or destabilizing effect on the enzyme and influence its thermostability accordingly.

Matched MeSH terms: Aspergillus niger/genetics
Elucidation of Pyranonigrin Biosynthetic Pathway Reveals a Mode of Tetramic Acid, Fused γ-Pyrone, and exo-Methylene Formation

Yamamoto T, Tsunematsu Y, Noguchi H, Hotta K, Watanabe K

Org. Lett., 2015 Oct 16;17(20):4992-5.
PMID: 26414728 DOI: 10.1021/acs.orglett.5b02435

Successful activation of the pyranonigrin biosynthetic gene cluster and gene knockout in Aspergillus niger plus in vivo and in vitro assays led to isolation of six new products, including a spiro cyclobutane-containing dimeric compound, which served as the basis for the proposed comprehensive pyranonigrin biosynthetic pathway. Two redox enzymes are key to forming the characteristic fused γ-pyrone core, and a protease homologue performs the exo-methylene formation.

Matched MeSH terms: Aspergillus niger/genetics*
Fulltext Bioprocess optimization for pectinase production using Aspergillus niger in a submerged cultivation system

El Enshasy HA, Elsayed EA, Suhaimi N, Malek RA, Esawy M

BMC Biotechnol, 2018 11 09;18(1):71.
PMID: 30413198 DOI: 10.1186/s12896-018-0481-7

BACKGROUND: Pectinase enzymes present a high priced category of microbial enzymes with many potential applications in various food and oil industries and an estimated market share of $ 41.4 billion by 2020.
RESULTS: The production medium was first optimized using a statistical optimization approach to increase pectinase production. A maximal enzyme concentration of 76.35 U/mL (a 2.8-fold increase compared with the initial medium) was produced in a medium composed of (g/L): pectin, 32.22; (NH4)2SO4, 4.33; K2HPO4, 1.36; MgSO4.5H2O, 0.05; KCl, 0.05; and FeSO4.5H2O, 0.10. The cultivations were then carried out in a 16-L stirred tank bioreactor in both batch and fed-batch modes to improve enzyme production, which is an important step for bioprocess industrialization. Controlling the pH at 5.5 during cultivation yielded a pectinase production of 109.63 U/mL, which was about 10% higher than the uncontrolled pH culture. Furthermore, fed-batch cultivation using sucrose as a feeding substrate with a rate of 2 g/L/h increased the enzyme production up to 450 U/mL after 126 h.
CONCLUSIONS: Statistical medium optimization improved volumetric pectinase productivity by about 2.8 folds. Scaling-up the production process in 16-L semi-industrial stirred tank bioreactor under controlled pH further enhanced pectinase production by about 4-folds. Finally, bioreactor fed-batch cultivation using constant carbon source feeding increased maximal volumetric enzyme production by about 16.5-folds from the initial starting conditions.

Matched MeSH terms: Aspergillus niger/genetics
Fulltext Optimizing culture conditions for production of intra and extracellular inulinase and invertase from Aspergillus niger ATCC 20611 by response surface methodology (RSM)

Dinarvand M, Rezaee M, Foroughi M

Braz J Microbiol, 2017 Jul-Sep;48(3):427-441.
PMID: 28359854 DOI: 10.1016/j.bjm.2016.10.026

The aim of this study was obtain a model that maximizes growth and production of inulinase and invertase by Aspergillus niger ATCC 20611, employing response surface methodology (RSM). The RSM with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. Results showed that the experimental data could be appropriately fitted into a second-order polynomial model with a coefficient of determination (R2) more than 0.90 for all responses. This model adequately explained the data variation and represented the actual relationships between the parameters and responses. The pH and temperature value of the cultivation medium were the most significant variables and the effects of inoculum size and agitation speed were slightly lower. The intra-extracellular inulinase, invertase production and biomass content increased 10-32 fold in the optimized medium condition (pH 6.5, temperature 30°C, 6% (v/v), inoculum size and 150rpm agitation speed) by RSM compared with medium optimized through the one-factor-at-a-time method. The process development and intensification for simultaneous production of intra-extracellular inulinase (exo and endo inulinase) and invertase from A. niger could be used for industrial applications.

Matched MeSH terms: Aspergillus niger/genetics
Comprehensive studies on optimization of cellulase and xylanase production by a local indigenous fungus strain via solid state fermentation using oil palm frond as substrate

Tai WY, Tan JS, Lim V, Lee CK

Biotechnol Prog, 2019 05;35(3):e2781.
PMID: 30701709 DOI: 10.1002/btpr.2781

The high cost of cellulases remains the most significant barrier to the economical production of bio-ethanol from lignocellulosic biomass. The goal of this study was to optimize cellulases and xylanase production by a local indigenous fungus strain (Aspergillus niger DWA8) using agricultural waste (oil palm frond [OPF]) as substrate. The enzyme production profile before optimization indicated that the highest carboxymethyl cellulose (CMCase), filter paper (FPase), and xylanase activities of 1.06 U/g, 2.55 U/g, and 2.93 U/g were obtained on day 5, day 4, and day 5 of fermentation, respectively. Response surface methodology was used to study the effects of several key process parameters in order to optimize cellulase production. Of the five physical and two chemical factors tested, only moisture content of 75% (w/w) and substrate amount of 2.5 g had statistically significant effect on enzymes production. Under optimized conditions of 2.5 g of substrate, 75% (w/w) moisture content, initial medium of pH 4.5, 1 × 106 spores/mL of inoculum, and incubation at ambient temperature (±30°C) without additional carbon and nitrogen, the highest CMCase, FPase, and xylanase activities obtained were 2.38 U/g, 2.47 U/g, and 5.23 U/g, respectively. Thus, the optimization process increased CMCase and xylanase production by 124.5 and 78.5%, respectively. Moreover, A. niger DWA8 produced reasonably good cellulase and xylanase titers using OPF as the substrate when compared with previous researcher finding. The enzymes produced by this process could be further use to hydrolyze biomass to generate reducing sugars, which are the feedstock for bioethanol production.

Matched MeSH terms: Aspergillus niger/genetics
Fungal solid state culture of palm kernel cake

Iluyemi FB, Hanafi MM, Radziah O, Kamarudin MS

Bioresour Technol, 2006 Feb;97(3):477-82.
PMID: 16216731

Palm kernel cake (PKC), an agro-industrial by-product used extensively in the animal feed industry, has limited use in fish feeds due to its high fiber and low protein contents. In this study, PKC was processed under solid state culture conditions with five fungal strains and the effect of this fungal culturing on the amino acid, fatty acid, cellulose and hemicellulose fractions was evaluated. Fungal strains used were Sclerotium rolfsii, Trichoderma harzianum, Trichoderma longiobrachiatum, Trichoderma koninggi and Aspergillus niger. Fungal growth was carried out at 50% moisture level and 1% inoculum level for 7 days. A significant increase in protein content from 18.76% to 32.79% was obtained by growing T. longibrachiatum on PKC. Cellulose level decreased significantly from 28.31% to 12.11% for PKC cultured with T. longibrachiatum, and hemicellulose from 37.03% to 19.01% for PKC cultured with A. niger. Fungal culturing of PKC brought about an increase in the level of unsaturated- and a decrease in the level of the saturated-fatty acids.

Matched MeSH terms: Aspergillus niger/genetics