Expression of Trichoderma reesei β-Xylanase in Escherichia coli and its Function in Degradation of Juncao Holocellulose

2011 ◽  
Vol 236-238 ◽  
pp. 1058-1062
Author(s):  
Li Hua Liu ◽  
Zhi Wei Lin ◽  
Ling Lin ◽  
Yan Ling Yang ◽  
Zhan Xi Lin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metal Ions ◽  
Molecular Mass ◽  
Trichoderma Reesei ◽  
Optimum Activity ◽  
Zymogram Analysis ◽  
Sds Page ◽  
Ph Range

In this study, the xyn2 gene, which encodes an endo-β-1,4-xylanase, was isolated with holocellulose extracted from Juncao Miscanthus floridulu as an inducer. The xyn2 gene expressed in Escherichia coli, with the estimated yield of 349 U·mL-1. Zymogram analysis showed that the purified Xyn2 had only one band on SDS-PAGE with an estimated molecular mass of 28 kDa. Enzymology analysis demonstrated that its optimum activity was at pH 6.0 and 60°C, with stability at pH range 4.0~7.0 and temperature up to 50°C. The metal ions Cu2+ and Mg2+ showed some inhibition effects, while Fe2+ and Fe3+ had small stimulating effects. Its values of Km and Vmax are 2.85 mM and 50.2 mM/min, respectively. Based on our results, we propose a novel way to convert Juncao biomass into energy and other useful products.

Isolation and purification of a Campylobacter upsaliensis autolysin

1999 ◽  
Vol 45 (1) ◽  
pp. 23-30
Author(s):  
Somchai Santiwatanakul ◽  
Noel R Krieg
Keyword(s):  
Escherichia Coli ◽  
Molecular Mass ◽  
Micrococcus Luteus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Isolation And Purification ◽  
Whole Cells ◽  
Sds Page ◽  
Autolytic Activity ◽  
Campylobacter Upsaliensis

Autolytic activity in the soluble and sediment fractions of sonicates of the spiral and the coccoid form of Campylobacter upsaliensis could not be demonstrated by native (nondenaturing) polyacrylamide gel electrophoresis (PAGE). Autolysins were detected, however, by using denaturing sodium dodecyl sulfate (SDS) - PAGE gels containing either purified Escherichia coli peptidoglycan or whole cells of Micrococcus luteus (Micrococcus lysodeikticus) as the turbid substrate, with subsequent renaturation by treatment with Triton X-100 buffer. In renaturing gels that contained Escherichia coli peptidoglycan, 14 putative autolytic bands ranging from 200 to 12 kDa were detected. In similar gels containing whole cells of M. luteus, only a single band appeared with a molecular mass of 34 kDa. This band corresponded to one of the bands present in the gels containing Escherichia coli peptidoglycan. This common autolysin was isolated by adsorbing it from Campylobacter upsaliensis soluble fractions onto M. luteus cells and then subjecting these cells to renaturing SDS-PAGE in gels containing Escherichia coli peptidoglycan. The 34-kDa autolysin differed from a single 51-kDa autolysin unique to the M. luteus cells, and when isolated from an SDS-PAGE gel, was pure when tested by isoelectric focusing. The N-terminal amino acid sequence analysis showed the first 15 amino acids of the 34-kDa autolysin to have 67% identity to a part of antigenic protein PEB4 of Campylobacter jejuni. The purified autolysin was used to immunize rabbits and the antibodies produced precipitated autolytic activity from cell lysates. The specificity of the antibodies was shown by Western blotting: only a single specific band occurred, with a molecular mass of 34 kDa, and thus it seems unlikely that the 34-kDa autolysin was derived from any of the other autolysins that were detected.Key words: autolysin, Campylobacter upsaliensis, zymogram, murein hydrolase.

PREPARATION AND PROPERTIES OF TRANSKETOLASE FROM PORK LIVER

1960 ◽  
Vol 38 (1) ◽  
pp. 115-124 ◽  
Author(s):  
F. J. Simpson
Keyword(s):  
Heavy Metal ◽  
Metal Ions ◽  
Heavy Metal Ions ◽  
Thiamine Pyrophosphate ◽  
Magnesium Ions ◽  
Optimum Activity ◽  
Pork Liver ◽  
High Concentrations ◽  
The Stability ◽  
Ph Range

Transketolase of pork liver has been purified 90-fold and separated from ribulose 5-phosphate 3-epimerase. The transketolase is most stable between pH 7.5 and 8.5 and below 40 °C. The pH range for optimum activity is between 7.6 and 8.1. Activation by magnesium ions or thiamine pyrophosphate could not be demonstrated, but thiamine pyrophosphate increased the stability of the enzyme. Sulphydryl agents, such as p-chloromercuriphenyl sulphonic acid and N-ethylmaleimide, and heavy metal ions, such as cupric, mercuric, and zinc, at relatively high concentrations inhibited the enzyme.

Fibrobacter succinogenes S85 possesses at least nine distinct glucanase genes

1993 ◽  
Vol 39 (9) ◽  
pp. 882-891 ◽  
Author(s):  
Laercio M. Malburg Jr. ◽  
Cecil W. Forsberg
Keyword(s):  
Escherichia Coli ◽  
Restriction Fragment ◽  
Carboxymethyl Cellulose ◽  
Southern Hybridization ◽  
Fibrobacter Succinogenes ◽  
Rumen Bacteria ◽  
Genomic Libraries ◽  
Zymogram Analysis ◽  
Sds Page ◽  
Specific Activities

The construction of genomic libraries of Fibrobacter succinogenes S85 in λ-Dash, λ-DashII, and pUC19, and the screening of recombinant clones for carboxymethylcellulose hydrolysis yielded 38 glucanase clones. These clones along with a collection of 10 glucanase clones in pUC8, were compared by restriction fragment and Southern hybridization analyses. Seven distinct glucanase clones (pCe14, pCe15, and pCe17 in pUC8, pCe16 in pUC19, and LCe18, LCel10, and LCel12 in λ-Dash) were identified, which were nonhomologous to previously studied cel3, lichenase, and cello-dextrinase genes from F. succinogenes S85. Specific activities of the encoded enzymes expressed in Escherichia coli were higher for carboxymethyl cellulose, barley β-glucan, and lichenan, and lower for acid-swollen cellulose, laminarin, and xylan. The enzymes were predominantly acidic, with pIs between 3.5 and 5.2, with the exception of the pCe16 enzyme, which was basic with a pI of about 8.4. As determined by zymogram analysis after SDS-PAGE, the LCe18 enzyme was 97 kDa, the pCel10 enzyme exhibited components of 70 and 45 kDa, and the pCel12 enzyme components were 69 and 65 kDa. These seven new glucanase clones, along with the cel3 and lichenase genes, indicate that F. succinogenes S85 possesses at least nine distinct endoglucanase genes.Key words: Fibrobacter succinogenes, rumen bacteria, cellulolytic, endoglucanases, glucanases, genes.

scholarly journals Purification and biochemical characterization of an extracellular lipase produced by a new strain of Rhizopus sp.

2006 ◽  
Vol 30 (3) ◽  
pp. 494-502 ◽  
Author(s):  
Maria Gabriela Bello Koblitz ◽  
Gláucia Maria Pastore
Keyword(s):  
Molecular Mass ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Extracellular Lipase ◽  
Optimum Activity ◽  
Ph Values ◽  
Sds Page ◽  
Rhizopus Sp

The present study had as a goal to purify and characterize the lipolytic fraction secreted by a strain of Rhizopus sp. Only 3 steps of purification were necessary to achieve SDS-PAGE homogeneity. One band with 37.5 KDa molecular mass and with 1446 U/mg specific activity was obtained. The purified fraction presented 2 lipase isoforms; both showed optimum activity at 50ºC, and were stable between 6.5 and 7.5 pH values and at temperatures below 50ºC and also kept their activity in hexane. The lipase was inactivated by Hg+2 and by n-bromosuccinimide and activated by Na+.

scholarly journals Spectroscopic studies on an oxygen-binding haemoglobin-like flavohaemoprotein from Escherichia coli

1992 ◽  
Vol 288 (2) ◽  
pp. 649-655 ◽  
Author(s):  
N Ioannidis ◽  
C E Cooper ◽  
R K Poole
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Spectroscopic Studies ◽  
Oxygen Binding ◽  
Histidine Residue ◽  
Nitrosyl Complex ◽  
Sds Page ◽  
Prosthetic Groups

The Escherichia coli haemoglobin-like flavohaemoprotein (Hmp) has been purified to near homogeneity using two chromatographic steps. The prosthetic groups are identified as FAD and protohaem IX. SDS/PAGE has indicated a molecular mass of 44 kDa for the monomeric protein consistent with the amino-acid sequence deduced from the hmp+ gene. The protein, as isolated, is in the Fe(III) state, exhibiting absorbance maxima at 403.5, 540 (shoulder) and 627 nm. The ferrous and carbonmonoxyferrous states resemble those of haemoglobin, showing maxima at 431.5 and 558 nm, and 421, 542 and 566 nm respectively. Upon aerobic addition of NAD(P)H, the ferric state is reduced to the oxygenated Fe(II) state, characterized by maxima at 413, 544 and 580 nm. This oxy form is not stable and slowly decays to the ferric state. Addition of dithionite and nitrite to the ferric protein results in the formation of a nitrosyl complex, whose e.p.r. characteristics indicate that the b-type haem is attached to the protein through a nitrogenous ligand, probably originating from a histidine residue.

scholarly journals Partial Purification of a Catalase from an Improved Nigerian Sorghum Grain Variety

2020 ◽  
pp. 30-37
Author(s):  
C. I. Nnamchi ◽  
B. C. Nwanguma ◽  
O. C. Amadi
Keyword(s):  
Crude Protein ◽  
Specific Activity ◽  
Partial Purification ◽  
Optimum Activity ◽  
Preliminary Purification ◽  
Sds Page ◽  
Cellular Detoxification ◽  
Activity Temperature ◽  
Ph Range ◽  
Sorghum Grain

Catalases are key components of cellular detoxification pathways that prevent the formation of highly reactive hydroxyl radicals through catalyzing the decomposition of hydrogen peroxide into water and molecular oxygen. Their presence in brewery grains prevent the inactivation of important brewery enzymes and also stop lipid peroxidation. To determine their occurrence and establish some of its properties in sorghum, which has become as an important brewery grain similar to barley, crude catalase was obtained from a sorghum grain variety. Preliminary purification of catalase from the sorghum grain variety used, NRL-3, showed that the enzyme was purified 3.2-fold from the crude protein to give a 49% yield of the partially purified enzyme, with a final specific activity of 32 Umg-1 proteins. There was also a positive indication of sorghum catalase presence on SDS PAGE with positive bands occurring between the range of 48-62 kDa. Therefore, the molecular weight of sorghum catalase most likely falls within the two bands. The enzyme showed a narrow pH range with optimum activity occurring at pH 7. Similarly, its optimum activity temperature occurred at 40°C.  This work is the first reported attempt at purifying catalase from sorghum.

Cloning of a xylanase gene from the ruminal fungus Neocallimastix patriciarum 27 and its expression in Escherichia coli

1993 ◽  
Vol 39 (1) ◽  
pp. 134-139 ◽  
Author(s):  
J. M. Tamblyn Lee ◽  
Y. Hu ◽  
H. Zhu ◽  
K. J. Cheng ◽  
P. J. Krell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Xylanase Activity ◽  
Hydrolytic Activity ◽  
Crystalline Cellulose ◽  
Xylanase Gene ◽  
Amorphous Cellulose ◽  
Zymogram Analysis ◽  
Sds Page ◽  
Neocallimastix Patriciarum ◽  
Temperature Optima

An endo-β-1,4-xylanase gene was cloned from Neocallimastix patriciarum 27 in the bacteriophage vector λgtWESλB and was subcloned into the plasmid vectors pUC18 and pUC19 in which xylanase activity was expressed in both orientations. The xylanase was located in the periplasmic space of the host, Escherichia coli HB101. The pH and temperature optima for periplasmic xylanase activity were 6.2 and 40 °C, respectively, and the Km for oat spelt xylan hydrolysis was 0.89 mg∙mL−1. It also exhibited hydrolytic activity on carboxymethyl cellulose that was equivalent to 28% of the activity exhibited by the enzyme on xylan. It bound to crystalline cellulose, but lacked hydrolytic activity on amorphous cellulose. SDS-PAGE followed by zymogram analysis showed active bands of 68, 58, and 51 kDa. Isoelectric focusing in gels combined with zymogram analysis showed one band of xylanase activity with a pI of 3.6.Key words: Neocallimastix patriciarum, xylanase, gene.

PREPARATION AND PROPERTIES OF TRANSKETOLASE FROM PORK LIVER

1960 ◽  
Vol 38 (2) ◽  
pp. 115-124 ◽  
Author(s):  
F. J. Simpson
Keyword(s):  
Heavy Metal ◽  
Metal Ions ◽  
Heavy Metal Ions ◽  
Thiamine Pyrophosphate ◽  
Magnesium Ions ◽  
Optimum Activity ◽  
Pork Liver ◽  
High Concentrations ◽  
The Stability ◽  
Ph Range

Transketolase of pork liver has been purified 90-fold and separated from ribulose 5-phosphate 3-epimerase. The transketolase is most stable between pH 7.5 and 8.5 and below 40 °C. The pH range for optimum activity is between 7.6 and 8.1. Activation by magnesium ions or thiamine pyrophosphate could not be demonstrated, but thiamine pyrophosphate increased the stability of the enzyme. Sulphydryl agents, such as p-chloromercuriphenyl sulphonic acid and N-ethylmaleimide, and heavy metal ions, such as cupric, mercuric, and zinc, at relatively high concentrations inhibited the enzyme.

scholarly journals Optimization Of Expression Conditions Of The Acetylesterase CE16 From Hypocrea Jecorina Encoded By A Synthetic Gene And Expressed In Escherichia coli Cells

2015 ◽  
Vol 14 (2) ◽  
pp. 201-211
Author(s):  
Daniela Jamrichová ◽  
Andrej Godány ◽  
Ľubica Urbániková
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Molecular Mass ◽  
Trichoderma Reesei ◽  
Active Form ◽  
Synthetic Gene ◽  
Hypocrea Jecorina ◽  
Secretion Signal ◽  
The Difference ◽  
C 30

Abstract Acetylesterase CE16 was identified as a part of the enzymatic cocktail secreted by fungus Hypocrea jecorina (anamorph: Trichoderma reesei) during its growth on cellulose. Later it was classified as the first member of a newly organized carbohydrate esterase family CE16. Further studies showed that acetylesterase is crucial for complete deacetylation of naturally acetylated xylans enabling their saccharification by xylanases. To study the relationship between structure and function of acetylesterase, highly purified recombinant enzyme produced by Trichoderma reesei Rut C-30 was prepared. The enzyme was composed of 348 amino acid residues from which the 1 - 19 formed a secretion signal peptide. Determined molecular mass of purified recombinant acetylesterase (Aes1) was 45 kDa which was more than molecular mass calculated from amino acid sequence. As it has been proved later, the difference was caused by the enzyme glycosylation. Glycosylation of proteins increases their stability, but it can also be a source of heterogeneity, which might be a problem during crystallization. To make the future X-ray study of the enzyme easier, recombinant non-glycosylated enzyme needed to be prepared. For these purposes, a synthetic gene optimized for protein expression in Escherichia coli was designed and synthetized. The first nonglycosylated acetylesterase obtained by the expression of its synthetic gene in E. coli cells was mostly insoluble or aggregated. Conditions of cell cultivation, induction of gene expression and cells disruption were necessary to optimize. Presently, after optimization of all mentioned steps, the non-glycosylated recombinant CE16 acetylesterase was prepared in the soluble and active form, ready for further downstream procedures, involving protein purification and crystallization.

Purification and characterization of cycloisomaltotetraose-forming glucanotransferases from Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006

2021 ◽  
Vol 85 (3) ◽  
pp. 600-610
Author(s):  
Akihiro Fujita ◽  
Akira Kawashima ◽  
Yuuki Mitsukawa ◽  
Noriaki Kitagawa ◽  
Hikaru Watanabe ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Culture Supernatant ◽  
Coupling Reaction ◽  
The Other ◽  
Purification And Characterization ◽  
Other Hand ◽  
Sds Page ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Glucanotransferases that can synthesize cyclo-{→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→} (CI4) from dextran were purified to homogeneity from the culture supernatant of Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006. The molecular mass of both enzymes was estimated to be 86 kDa by SDS-PAGE. The glucanotransferase, named CI4-forming enzyme, from Agreia sp. exhibited the highest activity at pH 6.0 and 40 °C. The enzyme was stable on the pH range of 4.6-9.9 and up to 40 °C. On the other hand, the enzyme from M. trichothecenolyticum exhibited the highest activity at pH 5.7 and 40 °C. The enzyme was stable on the pH range of 5.0-6.9 and up to 35 °C. Both enzymes catalyzed 4 reactions, namely, intramolecular α-1,6-transglycosylation (cyclization), intermolecular α-1,6-transglycosylation, hydrolysis of CI4, and coupling reaction. Furthermore, the CI4-forming enzyme produced CI4 from α-1,6-linked glucan synthesized from starch by 6-α-glucosyltransferase. These findings will enable the production of CI4 from starch.

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