scholarly journals Intronless celB from the anaerobic fungus Neocallimastix patriciarum encodes a modular family A endoglucanase

1994 ◽  
Vol 297 (2) ◽  
pp. 359-364 ◽  
Author(s):  
L Zhou ◽  
G P Xue ◽  
C G Orpin ◽  
G W Black ◽  
H J Gilbert ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Catalytic Domain ◽  
Crystalline Cellulose ◽  
Anaerobic Fungus ◽  
Beta Glucan ◽  
Reading Frame ◽  
Neocallimastix Patriciarum ◽  
Rumen Fungus ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

The cDNA designated celB from the anaerobic rumen fungus Neocallimastix patriciarum contained a single open reading frame of 1422 bp coding for a protein (CelB) of M(r) 53,070. CelB expressed by Escherichia coli harbouring the full-length gene hydrolysed carboxymethylcellulose in the manner of an endoglucanase, but was most active against barley beta-glucan. It also released reducing sugar from xylan and lichenan, but was inactive against crystalline cellulose, laminarin, mannan, galactan and arabinan. The rate of hydrolysis of cellulo-oligosaccharides by CelB increased with increasing chain length from cellotriose to cellopentaose. The predicted structure of CelB contained features indicative of modular structure. The first 360 residues of CelB constituted a fully functional catalytic domain that was homologous with bacterial endoglucanases belonging to cellulase family A, including five which originate from three different species of anaerobic rumen bacteria. Downstream from this domain, and linked to it by a serine/threonine-rich hinge, was a non-catalytic domain containing short tandem repeats, homologous to the C-terminal repeats contained in xylanase A from the same anaerobic fungus. Unlike previous fungal cellulases, genomic celB was devoid of introns. This lack of introns and the homology of its encoded product with rumen bacterial endoglucanases suggest that acquisition of celB by the fungus may at some stage have involved horizontal gene transfer from a prokaryote to N. particiarum.

scholarly journals Xylanase B from Neocallimastix patriciarum contains a non-catalytic 455-residue linker sequence comprised of 57 repeats of an octapeptide

1994 ◽  
Vol 299 (2) ◽  
pp. 381-387 ◽  
Author(s):  
G W Black ◽  
G P Hazlewood ◽  
G P Xue ◽  
C G Orpin ◽  
H J Gilbert
Keyword(s):  
Dna Sequences ◽  
Tandem Repeats ◽  
Northern Blot Analysis ◽  
Catalytic Domain ◽  
Catalytic Properties ◽  
Single Gene ◽  
Reading Frame ◽  
Catalytic Domains ◽  
Neocallimastix Patriciarum ◽  
Derivatives Of

A Neocallimastix patriciarum cDNA library was screened for xylanase-expressing clones, which were distinct from the previously characterized N. patriciarum xynA cDNA encoding xylanase A. A single cDNA, designated xynB, which did not exhibit homology with xynA, was isolated. Northern-blot analysis of mRNA from Avicel-grown N. patriciarum showed that xynB hybridized to a 3.4 kb mRNA species. The nucleotide sequence of xynB revealed a single open reading frame of 2580 bp coding for a protein designated xylanase B (XYLB), of M(r) 88,066. The primary structure of XYLB was comprised of a 21-residue N-terminal signal peptide, followed by a 304-amino acid sequence that exhibited substantial homology with the catalytic domains of family F xylanases. The N-terminal domain was linked to a C-terminal 70-residue sequence by a putative linker region, comprising 12 tandem repeats of a sequence containing TLPG as the core sequence, followed by an octapeptide XSKTLPGG where X can be S, K or N, which was repeated in tandem 45 times. Truncated derivatives of xynB encoding the N-terminal 338 residues directed the synthesis of a functional xylanase, confirming that the region of XYLB, which exhibited homology with family F xylanases, constitutes the catalytic domain. To investigate the catalytic properties of XYLB, the catalytic domain was fused to the Escherichia coli maltose-binding protein, and the fusion protein purified by amylose affinity chromatography. The purified enzyme hydrolysed oat, rye and wheat arabinoxylan releasing primarily xylobiose, xylotriose and some xylose. The XYLB fusion did not cleave any cellulosic substrates. The data presented in this report suggest that the multiple xylanases of N. patriciarum arose, not through the duplication of a single gene, but by the transfer of distinct xylanase-encoding DNA sequences into the anaerobic fungus. The possible origin of the xynB gene is discussed.

scholarly journals The cellodextrinase from Pseudomonas fluorescens subsp. cellulosa consists of multiple functional domains

1991 ◽  
Vol 279 (3) ◽  
pp. 793-799 ◽  
Author(s):  
L M A Ferreira ◽  
G P Hazlewood ◽  
P J Barker ◽  
H J Gilbert
Keyword(s):  
Pseudomonas Fluorescens ◽  
Catalytic Domain ◽  
Genomic Library ◽  
Terminal Region ◽  
Crystalline Cellulose ◽  
Nucleotide Sequencing ◽  
Reading Frame ◽  
Strong Homology ◽  
Cellulose Binding

A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA was constructed in pUC18 and Escherichia coli recombinants expressing 4-methylumbelliferyl beta-D-cellobioside-hydrolysing activity (MUCase) were isolated. Enzyme produced by MUCase-positive clones did not hydrolyse either cellobiose or cellotriose but converted cellotetraose into cellobiose and cleaved cellopentaose and cellohexaose, producing a mixture of cellobiose and cellotriose. There was no activity against CM-cellulose, insoluble cellulose or xylan. On this basis, the enzyme is identified as an endo-acting cellodextrinase and is designated cellodextrinase C (CELC). Nucleotide sequencing of the gene (celC) which directs the synthesis of CELC revealed an open reading frame of 2153 bp, encoding a protein of Mr 80,189. The deduced primary sequence of CELC was confirmed by the Mr of purified CELC (77,000) and by the experimentally determined N-terminus of the enzyme which was identical with residues 38-47 of the translated sequence. The N-terminal region of CELC showed strong homology with endoglucanase, xylanases and an arabinofuranosidase of Ps. fluorescens subsp. cellulosa; homologous sequences included highly conserved serine-rich regions. Full-length CELC bound tightly to crystalline cellulose. Truncated forms of celC from which the DNA sequence encoding the conserved domain had been deleted, directed the synthesis of a functional cellodextrinase that did not bind to crystalline cellulose. This is consistent with the N-terminal region of CELC comprising a non-catalytic cellulose-binding domain which is distinct from the catalytic domain. The role of the cellulose-binding region is discussed.

scholarly journals The non-catalytic C-terminal region of endoglucanase E from Clostridium thermocellum contains a cellulose-binding domain

1991 ◽  
Vol 273 (2) ◽  
pp. 289-293 ◽  
Author(s):  
A J Durrant ◽  
J Hall ◽  
G P Hazlewood ◽  
H J Gilbert
Keyword(s):  
Amino Acids ◽  
Clostridium Thermocellum ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Binding Domain ◽  
Full Length ◽  
Crystalline Cellulose ◽  
Cellulose Binding Domain ◽  
Beta Glucan ◽  
Cellulose Binding

Mature endoglucanase E (EGE) from Clostridium thermocellum consists of 780 amino acid residues and has an Mr of 84,016. The N-terminal 334 amino acids comprise a functional catalytic domain. Full-length EGE bound to crystalline cellulose (Avicel) but not to xylan. Bound enzyme could be eluted with distilled water. The capacity of truncated derivatives of the enzyme to bind cellulose was investigated. EGE lacking 109 C-terminal residues (EGEd) or a derivative in which residues 367-432 of the mature form of the enzyme had been deleted (EGEb), bound to Avicel, whereas EGEa and EGEc, which lack 416 and 246 C-terminal residues respectively, did not. The specific activity of EGEa, consisting of the N-terminal 364 amino acids, was 4-fold higher than that of the full-length enzyme. The truncated derivative also exhibited lower affinity for the substrate beta-glucan than the full-length enzyme. It is concluded that EGE contains a cellulose-binding domain, located between residues 432 and 671, that is distinct from the active site. The role of this substrate-binding domain is discussed.

scholarly journals beta-Glucosidase in cellulosome of the anaerobic fungus Piromyces sp. strain E2 is a family 3 glycoside hydrolase

2003 ◽  
Vol 370 (3) ◽  
pp. 963-970 ◽  
Author(s):  
Peter J.M. STEENBAKKERS ◽  
Harry R. HARHANGI ◽  
Mirjam W. BOSSCHER ◽  
Marlous M.C. van der HOOFT ◽  
Jan T. KELTJENS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Crystalline Cellulose ◽  
Anaerobic Fungi ◽  
Anaerobic Fungus ◽  
Glucosidase Activity ◽  
Biochemical Analyses ◽  
Beta Glucosidase

The cellulosomes of anaerobic fungi convert crystalline cellulose solely into glucose, in contrast with bacterial cellulosomes which produce cellobiose. Previously, a β-glucosidase was identified in the cellulosome of Piromyces sp. strain E2 by zymogram analysis, which represented approx. 25% of the extracellular β-glucosidase activity. To identify the component in the fungal cellulosome responsible for the β-glucosidase activity, immunoscreening with anti-cellulosome antibodies was used to isolate the corresponding gene. A 2737bp immunoclone was isolated from a cDNA library. The clone encoded an extracellular protein containing a eukaryotic family 3 glycoside hydrolase domain homologue and was therefore named cel3A. The C-terminal end of the encoded Cel3A protein consisted of an auxiliary domain and three fungal dockerins, typical for cellulosome components. The Cel3A catalytic domain was expressed in Escherichia coli BL21 and purified. Biochemical analyses of the recombinant protein showed that the Cel3A catalytic domain was specific for β-glucosidic bonds and functioned as an exoglucohydrolase on soluble substrates as well as cellulose. Comparison of the apparent Km and Ki values of heterologous Cel3A and the fungal cellulosome for p-nitrophenyl-β-d-glucopyranoside and d-glucono-1,5-Δ-lactone respectively indicated that cel3A encodes the β-glucosidase activity of the Piromyces sp. strain E2 cellulosome.

scholarly journals Crystal structure of a family 6 cellobiohydrolase from the basidiomycetePhanerochaete chrysosporium

2017 ◽  
Vol 73 (7) ◽  
pp. 398-403 ◽  
Author(s):  
Mikako Tachioka ◽  
Akihiko Nakamura ◽  
Takuya Ishida ◽  
Kiyohiko Igarashi ◽  
Masahiro Samejima
Keyword(s):  
Catalytic Domain ◽  
White Rot ◽  
White Rot Fungus ◽  
Crystalline Cellulose ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Linker Peptide ◽  
Ligand Free ◽  
Hydrolysis Of ◽  
Hydrolase Family

Cellobiohydrolases belonging to glycoside hydrolase family 6 (CBH II, Cel6A) play key roles in the hydrolysis of crystalline cellulose. CBH II from the white-rot fungusPhanerochaete chrysosporium(PcCel6A) consists of a catalytic domain (CD) and a carbohydrate-binding module connected by a linker peptide, like other known fungal cellobiohydrolases. In the present study, the CD ofPcCel6A was crystallized without ligands, andp-nitrophenyl β-D-cellotrioside (pNPG3) was soaked into the crystals. The determined structures of the ligand-free andpNPG3-soaked crystals revealed that binding of cellobiose at substrate subsites +1 and +2 induces a conformational change of the N-terminal and C-terminal loops, switching the tunnel-shaped active site from the open to the closed form.

The Effects of Amines on the Esterase Activities of Thrombin and Thrombokinase and on Several Clotting Tests

1974 ◽  
Vol 31 (02) ◽  
pp. 309-318
Author(s):  
Phyllis S Roberts ◽  
Raphael M Ottenbrite ◽  
Patricia B Fleming ◽  
James Wigand
Keyword(s):  
Choline Chloride ◽  
Polymerization Process ◽  
Ammonium Bromide ◽  
Bovine Thrombin ◽  
One Stage ◽  
Human Proteins ◽  
Rate Of Hydrolysis ◽  
The One ◽  
Hydrolysis Of Esters ◽  
Hydrolysis Of

Summary1. Choline chloride, 0.1 M (in 0.25 M Tris. HCl buffer, pH 7.4 or 8.0, 37°), doubles the rate of hydrolysis of TAME by bovine thrombokinase but has no effect on the hydrolysis of this ester by either human or bovine thrombin. Only when 1.0 M or more choline chloride is present is the hydrolysis of BAME by thrombokinase or thrombin weakly inhibited. Evidence is presented that shows that these effects are due to the quaternary amine group.2. Tetramethyl ammonium bromide or chloride has about the same effects on the hydrolysis of esters by these enzymes as does choline chloride but tetra-ethyl, -n.propyl and -n.butyl ammonium bromides (0.1 M) are stronger accelerators of the thrombokinase-TAME reaction and they also accelerate, but to a lesser degree, the thrombin-TAME reaction. In addition, they inhibit the hydrolysis of BAME by both enzymes. Their effects on these reactions, however, do not follow any regular order. The tetraethyl compound is the strongest accelerator of the thrombokinase-TAME reaction but the tetra-ethyl and -butyl compounds are the strongest accelerators of the thrombin-TAME reaction. The ethyl and propyl compounds are the best (although weak) inhibitors of the thrombokinase-BAME and the propyl compound of the thrombin-BAME reactions.3. Tetra-methyl, -ethyl, -n.propyl and -n.butyl ammonium bromides (0.01 M) inhibit the clotting of fibrinogen by thrombin (bovine and human proteins) at pH 7.4, imidazole or pH 6.1, phosphate buffers and they also inhibit, but to a lesser degree, a modified one-stage prothrombin test. In all cases the inhibition increases regularly as the size of the alkyl group increases from methyl to butyl. Only the ethyl com pound (0.025 M but not 0.01 M), however, significantly inhibits the polymerization of bovine fibrin monomers. It was concluded that inhibition of the fibrinogen-thrombin and the one-stage tests by the quaternary amines is not due to any effect of the com pounds on the polymerization process but probably due to inhibition of thrombin’s action on fibrinogen by the quaternary amines.

The effect of polymeric and model imidazolium halides on the rate of hydrolysis of 4-acetoxy-3-nitrobenzoic acid

1985 ◽  
Vol 50 (4) ◽  
pp. 845-853 ◽  
Author(s):  
Miloslav Šorm ◽  
Miloslav Procházka ◽  
Jaroslav Kálal
Keyword(s):  
Catalyst Concentration ◽  
Low Molecular Weight ◽  
Imidazolium Chloride ◽  
First Order ◽  
Nitrobenzoic Acid ◽  
Rate Of Hydrolysis ◽  
Acid Catalyzed ◽  
Hydrolysis Of ◽  
Ethanol In Water ◽  
Direct Attack

The course of hydrolysis of an ester, 4-acetoxy-3-nitrobenzoic acid catalyzed with poly(1-methyl-3-allylimidazolium bromide) (IIa), poly[l-methyl-3-(2-propinyl)imidazolium chloride] (IIb) and poly[l-methyl-3-(2-methacryloyloxyethyl)imidazolium bromide] (IIc) in a 28.5% aqueous ethanol was investigated as a function of pH and compared with low-molecular weight models, viz., l-methyl-3-alkylimidazolium bromides (the alkyl group being methyl, propyl, and hexyl, resp). Polymers IIb, IIc possessed a higher activity at pH above 9, while the models were more active at a lower pH with a maximum at pH 7.67. The catalytic activity at the higher pH is attributed to an attack by the OH- group, while at the lower pH it is assigned to a direct attack of water on the substrate. The rate of hydrolysis of 4-acetoxy-3-nitrobenzoic acid is proportional to the catalyst concentration [IIc] and proceeds as a first-order reaction. The hydrolysis depends on the composition of the solvent and was highest at 28.5% (vol.) of ethanol in water. The hydrolysis of a neutral ester, 4-nitrophenyl acetate, was not accelerated by IIc.

Kinetics of hydrolysis of peroxodisulphate ions in acidic medium to peroxomonosulphuric acid

1981 ◽  
Vol 46 (5) ◽  
pp. 1229-1236 ◽  
Author(s):  
Jan Balej ◽  
Milada Thumová
Keyword(s):  
Ionic Strength ◽  
Rate Constants ◽  
Acidic Medium ◽  
Measured Data ◽  
Molar Ratios ◽  
Starting Solution ◽  
Kinetics Of Hydrolysis ◽  
Rate Of Hydrolysis ◽  
Kinetics Of ◽  
Hydrolysis Of

The rate of hydrolysis of S2O82- ions in acidic medium to peroxomonosulphuric acid was measured at 20 and 30 °C. The composition of the starting solution corresponded to the anolyte flowing out from an electrolyser for production of this acid or its ammonium salt at various degrees of conversion and starting molar ratios of sulphuric acid to ammonium sulphate. The measured data served to calculate the rate constants at both temperatures on the basis of the earlier proposed mechanism of the hydrolysis, and their dependence on the ionic strength was studied.

Polarographic study of hydrolysis of [8-lysine]vasopressin and its derivatives with blood serum of pregnant women

1980 ◽  
Vol 45 (4) ◽  
pp. 1099-1108 ◽  
Author(s):  
Mikuláš Chavko ◽  
Michal Bartík ◽  
Evžen Kasafírek
Keyword(s):  
Blood Serum ◽  
Pregnant Women ◽  
Degree Of Hydrolysis ◽  
Polarographic Study ◽  
Ph Optimum ◽  
Lysine Vasopressin ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Vasopressin Analogues

A polarographic study of the hydrolysis of [8-lysine]vasopressin and some hormonogens of the vasopressin series with the blood serum of women in the last week of pregnancy was studied. The dependence of hydrolysis on pH (pH optimum: 7.4-7.50, substrate concentration (Km 1.2 . 10-5M), pH stability and thermal stability were determined. The rate of hydrolysis of individual vasopressin analogues decreases in the order: [8-lysine]vasopressin > Nα-glycyl-prolyl[8-lysine]-vasopressin > Nα-leucyl-[8-lysine]vasopressin > Nα-alanyl-[8-lysine]vasopressin > Nα-phenyl alanyl-[8-lysine]vasopressin > Nα-diglycyl-[8-lysine]vasopressin > Nα-prolyl-[8-lysine]vasopressin > Nα-triglycyl-[8-lysine]vasopressin > Nα-sarcosyl-glycyl-[8-lysine]vasopressin. The degree of hydrolysis gradually increases to a multiple with the length of the pregnancy in consequence of the presence of oxytocine. However, vasopressin is also hydrolysed to a small extent with the enzymes from the blood sera of non-pregnant women. Under similar analytical conditions oxytocin was not hydrolysed with the sera of non-pregnant women and therefore oxytocin is a more suitable substrate than vasopressin for polarographic determination of serum oxytocinase.

Sign in / Sign up

Export Citation Format

Share Document