scholarly journals Two Bifunctional β-Xylosidase /α-L-Arabinofuranosidases From GH43 With Different Structures in the xylan Degradation Strain of Paenibacillus Physcomitrellae XB Displayed the Similar xylo-oligosaccharides Degradation Ability

2021 ◽  
Author(s):  
Xiao Jie Zhang ◽  
Su Wang ◽  
Zhi Ling Chen ◽  
Yan Hong Li
Keyword(s):  
Metal Ions ◽  
Physcomitrella Patens ◽  
Beech Wood ◽  
Xylanase Activity ◽  
Genomic Analysis ◽  
Exchange Chromatography ◽  
Glycoside Hydrolase Family ◽  
Xylan Degradation ◽  
Degradation Ability ◽  
Xylosidase Activity

Abstract BackgroundThe strain Paenibacillus physcomitrellae XB isolated from moss of Physcomitrella patens was found have the xylan degradation ability, but its degradation characteristics and the related mechanism has not been revealed.ResultsIn this study, Paenibacillus physcomitrellae XB exhibited different xylan degradation ability under the different substrates of corncob xylan (CCX), oat spet xylan (OSX), wheat flour arabinoxylan (AX) and beech wood xylan (BWX). Genomic analysis showed that ~ 38 genes were related to xylan degradation, and quantitative real time RT-PCR showed that two glycoside hydrolase family 43 genes (Pph_0602 and Pph_2344) were up-regulated on 1% CCX and xylose. Substrate-specific experiments with purified proteins Ppxyl43A (Pph_0602) and Ppxyl43B (Pph_2344) revealed that both of them exhibited β-xylosidase activity toward chromogenic substrate p-nitrophenyl–D-xylopyranoside and α-L-arabinofuranosidase activity toward p-nitrophenyl-α-L-arabinofuranoside, indicating at least bifunctionality. Combined their degradation features on the natural substrates of different xylans with the hydrolytic products separated by thin-layer chromatography and high-performance anion exchange chromatography profiles, it was found that both Ppxyl43A and Ppxyl43B were with the similar degradation ability on xylo-oligosaccharides (like CCX, OSX, xylohexaose and xylobiose). Both of them even could hydrolyze xylohexaose and xylobiose completely to xylose, but could not hydrolyze BWX and AX to produce xylooligosaccharides or xylose, suggesting they have no endo-xylanase activity and mainly hydrolyze xylo-oligosaccharides by β-xylosidase activity. Moreover, the kinetic parameters of β-xylosidase and α-L-arabinofuranosidase of both two proteins indicated their affinity with all the detected natural substrate (CCX) and chromogenic substrates were nearly similar. In addition, despite having no signal peptides, both of them might export outside the cell by the nonconventional pathways. However, Ppxy143B exhibited wider temperature and pH ranges, higher pH and thermostability, and was less influenced by metal ions than Ppxyl43A. Given its enzymatic characteristics and predicted structure, it is likely that the C-terminus domain (GH43_C2) of Ppxyl43B enhances the stability of the two enzymes and also restricts the substrates’ or metal ions’ access to the active sites.ConclusionsPpxyl43A and Ppxyl43B were β-xylosidase/α-L-arabinofuranosidase bifunctional enzymes with different structures from Paenibacillus physcomitrellae XB and exhibited similar xylo-oligosaccharides hydrolyse ability, which would be useful in the further lignocellulosic biomass conversion.

scholarly journals Optimization of Metal Ions in Sugarcane Bagasse Fermenting Medium for the Production of Streptokinase by Streptococcus equisimilis

2021 ◽  
Vol 9 (2) ◽  
pp. 24-30
Keyword(s):  
Metal Ions ◽  
Sugarcane Bagasse ◽  
Specific Activity ◽  
Gel Filtration ◽  
Value Added ◽  
Cost Effective ◽  
Fermentation Medium ◽  
Exchange Chromatography ◽  
Sds Page ◽  
The Cost

Streptokinase is a fibrinolytic enzyme and a product of β-hemolytic Streptococci strains. This enzyme is used as a medication to break down clots in some cases of heart disease. Streptococcus equisimilis, a species of group C Streptococci, is widely used for the production of streptokinase by fermentation technology. In this study, the sugarcane bagasse fermentation medium was optimized for metal ions (KH2PO4, MgSO4.7H2O, CaCO3 and NaHCO3) at various levels to attain the maximal production of streptokinase. Sugarcane bagasse was used due to its profuse availability and as an ideal substrate for microbial processes for the manufacturing of value-added products. The results showed that maximal streptokinase production was found at 0.04% KH2PO4, 0.04% MgSO4.7H2O, 0.15% NaHCO3 and 0.04% CaCO3. Finally, the optimized medium resulted in 84.75 U/mg specific activity and 74.5% recovery. The purification process was carried out simultaneously using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. Finally, a purified sample of streptokinase was run on SDS-PAGE and resolute 47 kDa molecular weight. The use of β-hemolytic Streptococci to obtain streptokinase is not free from health risks and is related to anaphylaxis. This study provides a way forward for the cost-effective ways to obtain streptokinase for the treatment of thrombosis.

scholarly journals Phenylacetic and Phenylpropionic Acids Do Not Affect Xylan Degradation by Ruminococcus albus

2003 ◽  
Vol 69 (11) ◽  
pp. 6954-6958 ◽  
Author(s):  
Carine Reveneau ◽  
Sarah E. Adams ◽  
M. A. Cotta ◽  
M. Morrison
Keyword(s):  
Growth Medium ◽  
Cellulose Degradation ◽  
Xylanase Activity ◽  
Ruminococcus Albus ◽  
Carbohydrate Source ◽  
Ruminal Fluid ◽  
Xylan Degradation ◽  
Multiple Strains

ABSTRACT Since the addition of either ruminal fluid or a combination of phenylacetic and phenylpropionic acids (PAA/PPA) has previously been shown to dramatically improve cellulose degradation and growth of Ruminococcus albus, it was of interest to determine the effects of these additives on xylan-grown cultures. Although cell-bound xylanase activity increased when either PAA/PPA or ruminal fluid was added to the growth medium, total xylanase did not change, and neither of these supplements affected the growth or xylan-degrading capacity of R. albus 8. Similarly, neither PAA/PPA nor ruminal fluid affected xylan degradation by multiple strains of R. albus when xylan prepared from oat spelts was used as a carbohydrate source. These results show that the xylanolytic potential of R. albus is not conditional on the availability of PAA/PPA or other components of ruminal fluid.

scholarly journals A high-molecular-mass neutral endopeptidase-24.5 from human lung

1987 ◽  
Vol 241 (1) ◽  
pp. 129-135 ◽  
Author(s):  
R Zolfaghari ◽  
C R Baker ◽  
P C Canizaro ◽  
A Amirgholami ◽  
F J Bĕhal
Keyword(s):  
Metal Ions ◽  
Human Lung ◽  
Proteinase Inhibitors ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Neutral Endopeptidase ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Apparent Homogeneity

A high-Mr neutral endopeptidase-24.5 (NE) that cleaved bradykinin at the Phe5-Ser6 bond was purified to apparent homogeneity from human lung by (NH4)2SO4 fractionation, ion-exchange chromatography and gel filtration. The final enzyme preparation produced a single enzymically active protein band after electrophoresis on a 5% polyacrylamide gel. Human lung NE had an Mr of 650,000 under non-denaturing conditions, but after denaturation and electrophoresis on an SDS/polyacrylamide gel NE dissociated into several lower-Mr components (Mr 21,000-32,000) and into two minor components (Mr approx. 66,000). The enzyme activity was routinely assayed with the artificial substrate Z-Gly-Gly-Leu-Nan (where Z- and -Nan represent benzyloxycarbonyl- and p-nitroanilide respectively). NE activity was enhanced slightly by reducing agents, greatly diminished by thiol-group inhibitors and unchanged by serine-proteinase inhibitors. Human lung NE was inhibited by the univalent cations Na+ and K+. No metal ions were essential for activity, but the heavy-metal ions Cu2+, Hg2+ and Zn2+ were potent inhibitors. With the substrate Z-Gly-Gly-Leu-Nan a broad pH optimum from pH 7.0 to pH 7.6 was observed, and a Michaelis constant value of 1.0 mM was obtained. When Z-Gly-Gly-Leu-Nap (where -Nap represents 2-naphthylamide) was substituted for the above substrate, no NE-catalysed hydrolysis occurred, but Z-Leu-Leu-Glu-Nap was readily hydrolysed by NE. In addition, NE hydrolysed Z-Gly-Gly-Arg-Nap rapidly, but at pH 9.8 rather than in the neutral range. Although human lung NE was stimulated by SDS, the extent of stimulation was not appreciable as compared with the extent of SDS stimulation of NE from other sources.

Ion-Exchange Chromatography of Metal Ions with Ethanolamine Eluents

1969 ◽  
Vol 4 (2) ◽  
pp. 111-117 ◽  
Author(s):  
Fred Hilgeman ◽  
Kazuko Shimomura ◽  
Harold F. Walton
Keyword(s):  
Ion Exchange ◽  
Metal Ions ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography

Structural elucidation of a dual-activity PAP phosphatase-1 fromEntamoeba histolyticacapable of hydrolysing both 3′-phosphoadenosine 5′-phosphate and inositol 1,4-bisphosphate

2014 ◽  
Vol 70 (7) ◽  
pp. 2019-2031 ◽  
Author(s):  
Khaja Faisal Tarique ◽  
Syed Arif Abdul Rehman ◽  
S. Gourinath
Keyword(s):  
Metal Ions ◽  
Metal Ion ◽  
Genomic Analysis ◽  
Inositol Polyphosphate ◽  
Inositol Monophosphatase ◽  
Native Form ◽  
Catalytic Loop ◽  
Important Regulator ◽  
Living Organisms ◽  
Toxic Byproduct

The enzyme 3′-phosphoadenosine 5′-phosphatase-1 (PAP phosphatase-1) is a member of the Li+-sensitive Mg2+-dependent phosphatase superfamily, or inositol monophosphatase (IMPase) superfamily, and is an important regulator of the sulfate-activation pathway in all living organisms. Inhibition of this enzyme leads to accumulation of the toxic byproduct 3′-phosphoadenosine 5′-phosphate (PAP), which could be lethal to the organism. Genomic analysis ofEntamoeba histolyticasuggests the presence of two isoforms of PAP phosphatase. The PAP phosphatase-1 isoform of this organism is shown to be active over wide ranges of pH and temperature. Interestingly, this enzyme is inhibited by submillimolar concentrations of Li+, while being insensitive to Na+. Interestingly, the enzyme showed activity towards both PAP and inositol 1,4-bisphosphate and behaved as an inositol polyphosphate 1-phosphatase. Crystal structures of this enzyme in its native form and in complex with adenosine 5′-monophosphate have been determined to 2.1 and 2.6 Å resolution, respectively. The PAP phosphatase-1 structure is divided into two domains, namely α+β and α/β, and the substrate and metal ions bind between them. This is a first structure of any PAP phosphatase to be determined from a human parasitic protozoan. This enzyme appears to function using a mechanism involving three-metal-ion assisted catalysis. Comparison with other structures indicates that the sensitivity to alkali-metal ions may depend on the orientation of a specific catalytic loop.

Identifying and characterizing the most significant β-glucosidase of the novel species Aspergillus saccharolyticus

2012 ◽  
Vol 58 (9) ◽  
pp. 1035-1046 ◽  
Author(s):  
Annette Sørensen ◽  
Birgitte K. Ahring ◽  
Mette Lübeck ◽  
Wimal Ubhayasekera ◽  
Kenneth S. Bruno ◽  
...  
Keyword(s):  
Hydrolytic Activity ◽  
Fungal Species ◽  
Culture Broth ◽  
Exchange Chromatography ◽  
Mass Spectrometry Analysis ◽  
Glycoside Hydrolase Family ◽  
Degenerate Primers ◽  
Spectrometry Analysis ◽  
Glucosidase Activity ◽  
Sds Page

The newly discovered fungal species Aspergillus saccharolyticus was found to produce a culture broth rich in β-glucosidase activity. In this present work, the main β-glucosidase of A. saccharolyticus responsible for the efficient hydrolytic activity was identified, isolated, and characterized. Ion exchange chromatography was used to fractionate the culture broth, yielding fractions with high β-glucosidase activity and only 1 visible band on an SDS–PAGE gel. Mass spectrometry analysis of this band gave peptide matches to β-glucosidases from aspergilli. Through a polymerase chain reaction approach using degenerate primers and genome walking, a 2919 bp sequence encoding the 860 amino acid BGL1 polypeptide was determined. BGL1 of A. saccharolyticus has 91% and 82% identity with BGL1 from Aspergillus aculeatus and BGL1 from Aspergillus niger , respectively, both belonging to Glycoside Hydrolase family 3. Homology modeling studies suggested β-glucosidase activity with preserved retaining mechanism and a wider catalytic pocket compared with other β-glucosidases. The bgl1 gene was heterologously expressed in Trichoderma reesei QM6a, purified, and characterized by enzyme kinetics studies. The enzyme can hydrolyze cellobiose, p-nitrophenyl-β-d-glucoside, and cellodextrins. The enzyme showed good thermostability, was stable at 50 °C, and at 60 °C it had a half-life of approximately 6 h.

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