Biochemical characterisation and kinetic properties of a purified lipase from Aspergillus niger in bulk phase and monomolecular films

2002 ◽  
Vol 30 (7) ◽  
pp. 902-909 ◽  
Author(s):  
Esta van Heerden ◽  
Derek Litthauer ◽  
Robert Verger
Keyword(s):  
Aspergillus Niger ◽  
Bulk Phase ◽  
Kinetic Properties ◽  
Biochemical Characterisation ◽  
Monomolecular Films

scholarly journals Biochemical characterisation of a glucoamylase from Aspergillus niger produced by solid-state fermentation

2011 ◽  
Vol 54 (3) ◽  
pp. 559-568 ◽  
Author(s):  
Christiane Trevisan Slivinski ◽  
Alex Vinicius Lopes Machado ◽  
Jorge Iulek ◽  
Ricardo Antônio Ayub ◽  
Mareci Mendes de Almeida
Keyword(s):  
Solid State ◽  
Aspergillus Niger ◽  
Solid State Fermentation ◽  
Biochemical Characterisation

scholarly journals Teladorsagia circumcincta 1,6-Bisphosphate Aldolase: Molecular and Biochemical Characterisation, Structure Analysis and Recognition by Immune Hosts

Parasitologia ◽  
2021 ◽  
Vol 1 (1) ◽  
pp. 1-11
Author(s):  
Saleh Umair ◽  
Charlotte Bouchet ◽  
Nikola Palevich ◽  
Heather Simpson
Keyword(s):  
Binding Sites ◽  
Kinetic Properties ◽  
Dictyocaulus Viviparus ◽  
Teladorsagia Circumcincta ◽  
Multiple Alignments ◽  
Sds Page ◽  
Biochemical Characterisation ◽  
Optimum Ph ◽  
Fructose 1,6 Bisphosphate ◽  
Substrate Binding Sites

A 1095 bp full length cDNA encoding Teladorsagia circumcincta aldolase (TciALDO-1) was cloned and expressed in Escherichia coli. Recombinant TciALDO-1 was purified, and its kinetic properties determined. The predicted protein consisted of 365 amino acids, and was present as a single band of about 44 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TciALDO-1 with homologues from other helminths showed the greatest similarity (93%) to the aldolases of Haemonchus contortus and Dictyocaulus viviparus, 82–86% similarity to the other nematode sequences, and 68–71% similarity to cestode and trematode enzymes. Substrate binding sites and conserved regions were identified, and were completely conserved in other homologues. At 30 °C, the optimum pH for TciALDO-1 activity was pH 7.5, the Vmax was 432 ± 23 nmol × min−1 × mg−1 protein, and the apparent Km for the substrate fructose 1,6-bisphosphate was 0.24 ± 0.01 µM (mean ± SEM, n = 3). Recombinant TciALDO-1 was recognized by antibodies in both serum and saliva from field-immune sheep in ELISA, however, that was not the case with nematode-naïve sheep. Teladorsagia circumcincta fructose 1,6-bisphosphate aldolase appears to have potential as a vaccine candidate to control this common sheep parasite.

Carboxyl group modification significantly altered the kinetic properties of purified carboxymethylcellulase from Aspergillus niger

2000 ◽  
Vol 27 (7) ◽  
pp. 467-474 ◽  
Author(s):  
Khawar Sohail Siddiqui ◽  
Abdul Aala Najmus Saqib ◽  
Mohammad Hamid Rashid ◽  
Mohammad Ibrahim Rajoka
Keyword(s):  
Aspergillus Niger ◽  
Kinetic Properties ◽  
Carboxyl Group ◽  
Group Modification

scholarly journals The rapid alteration by tri-iodo-l-thyronine in vivo of both the ADP/O ratio and the apparent H+/O ratio in hypothyroid-rat liver mitochondria

1987 ◽  
Vol 241 (3) ◽  
pp. 657-661 ◽  
Author(s):  
A Crespo-Armas ◽  
J Mowbray
Keyword(s):  
Liver Mitochondria ◽  
Proton Exchange ◽  
Bulk Phase ◽  
Rat Liver Mitochondria ◽  
Kinetic Properties ◽  
Protonmotive Force ◽  
Proton Pumps ◽  
Pulse Technique ◽  
Physiological Dose

Mitochondria from the livers of thyroidectomized rats have a lowered ADP/O ratio, which can be restored to normal within 15 min after intravenous injection of a near-physiological dose of tri-iodothyronine. Thyroidectomy lowered the measured delta pH, which appears to be compensated by a rise (not statistically significant) in the membrane potential, so that the protonmotive force is unaltered. A simple simulation technique is described for use in estimating H+/O ratios by the oxygen-pulse technique, which circumvents the problem that this ratio can be seriously underestimated because of re-uptake of protons from the bulk phase by the mitochondria before their expulsion is complete. By this procedure the H+/O ratio of hypothyroid mitochondria is shown to be lowered by the same factor as the ADP/O ratio, and both these ratios are very rapidly restored in parallel by hormone administration. Although these findings could be consistent with a proposal that tri-iodothyronine rapidly modulates by some mechanism the efficiency of the respiratory-chain-linked proton pumps, the kinetic properties of the proton exchange suggest that the bulk-phase protons measured may not reflect faithfully those that drive the ATP synthetase.

scholarly journals Cellobiohydrolase B of Aspergillus niger over-expressed in Pichia pastoris stimulates hydrolysis of oil palm empty fruit bunches

2017 ◽  
Author(s):  
James Sy-Keen Woon ◽  
Mukram M Mackeen ◽  
Rosli M Illias ◽  
Nor M Mahadi ◽  
William J Broughton ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Pichia Pastoris ◽  
Oil Palm ◽  
Biomass Conversion ◽  
Methylotrophic Yeast ◽  
Cellulase Production ◽  
Crystalline Cellulose ◽  
Empty Fruit Bunches ◽  
Biochemical Characterisation ◽  
Hydrolysis Of

Background. Aspergillus niger along with many other lignocellulolytic fungi, has been widely used as a commercial workhorse for cellulase production. A fungal cellulase system generally includes three major classes of enzymes i.e. β-glucosidases, endoglucanases and cellobiohydrolases. Cellobiohydrolases (CBH) are vital to the degradation of crystalline cellulose present in lignocellulosic biomass. However, A. niger naturally secretes low levels of CBH. Hence, recombinant production of A. niger CBH is desirable to increase CBH production yield and also to allow biochemical characterisation of the recombinant CBH from A. niger. Methods. In this study, the gene encoding a cellobiohydrolase B (cbhB) from A. niger ATCC 10574 was cloned and expressed in the methylotrophic yeast Pichia pastoris X-33. The recombinant CbhB was purified and characterised to study its biochemical and kinetic characteristics. To evaluate the potential of CbhB in assisting biomass conversion, CbhB was supplemented into a commercial cellulase preparation (Cellic® CTec2) and was used to hydrolyse oil palm empty fruit bunch (OPEFB), one of the most abundant lignocellulosic waste from the palm oil industry. To attain maximum saccharification, enzyme loadings were optimised by response surface methodology and the optimum point was validated experimentally. Hydrolysed OPEFB samples were analysed using attenuated total reflectance FTIR spectroscopy (ATR-FTIR) to screen for any compositional changes upon enzymatic treatment. Results. Recombinant CbhB was over-expressed as a hyperglycosylated protein attached to N-glycans. CbhB was enzymatically active towards soluble substrates such as 4-methylumbelliferyl-β-D-cellobioside (MUC), p-nitrophenyl-cellobioside (pNPC) and p-nitrophenyl-cellobiotrioside (pNPG3) but was not active towards crystalline substrates like Avicel® and Sigmacell cellulose. Characterisation of purified CbhB using MUC as the model substrate revealed that optimum catalysis occurred at 50 oC and pH 4 but the enzyme was stable between pH 3 to 10 and 30 to 80 oC. Although CbhB on its own was unable to digest crystalline substrates, supplementation of CbhB (0.37%) with Cellic® CTec2 (30%) increased saccharification of OPEFB by 27%. Compositional analyses of the treated OPEFB samples revealed that CbhB complementation reduced peak intensities of both crystalline cellulose Iα and Iβ in the treated OPEFB samples. Discussion. Since CbhB alone was inactive against crystalline cellulose, these data suggested that it might work synergistically with other components of Cellic® CTec2. CbhB supplements were desirable as they further increased hydrolysis of OPEFB when the performance of Cellic® CTec2 was theoretically capped at an enzyme loading of 34% in this study. Hence, A. niger CbhB was identified as a potential supplementary enzyme for the enzymatic hydrolysis of OPEFB.

scholarly journals Teladorsagia circumcincta 1,6 bisphosphate aldolase: molecular and biochemical characterisation, structure analysis and recognition by immune hosts

2020 ◽  
Author(s):  
S. Umair ◽  
C.L.G. Bouchet ◽  
N. Palevich ◽  
J.S. Knight ◽  
H.V. Simpson
Keyword(s):  
Recombinant Protein ◽  
Kinetic Properties ◽  
Dictyocaulus Viviparus ◽  
Teladorsagia Circumcincta ◽  
Multiple Alignments ◽  
Sds Page ◽  
Biochemical Characterisation ◽  
Optimum Ph ◽  
Fructose 1,6 Bisphosphate ◽  
Substrate Binding Sites

ABSTRACTA 1095 bp full length cDNA encoding Teladorsagia circumcincta aldolase (TciALDO) was cloned, expressed in Escherichia coli, the recombinant protein purified and its kinetic properties determined. A phylogenetic tree was constructed using helminth aldolase sequences. The predicted protein consisted of 365 amino acids and was present as a single band of about 44 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TciALDO with homologues from other helminths showed that the greatest similarity (93%) to the aldolases of Haemonchus contortus and Dictyocaulus viviparus, 82-86% similarity to the other nematode sequences and 68-71% similarity to cestode and trematode enzymes. Substrate binding sites and conserved regions were identified and were completely conserved in other homologues. At 25 °C, the optimum pH for TciALDO activity was pH 7.5, the Vmax was 432 ± 23 nmoles.min−1.mg−1 protein and the apparent Km for the substrate fructose 1,6-bisphosphate was 0.24 ± 0.01 μM (mean ± SEM, n = 3). Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TciALDO in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to native aldolase indicates similar antigenicity of the two proteins.

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