| 1. |
|
|
| 2. |
- Becker, D., et al.
(författare)
-
Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum : the pH behaviour of Trichoderma reesei CeI7A and its E223S/A224H/L225V/T226A/D262G mutant
- 2001
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 356, s. 19-30
-
Tidskriftsartikel (refereegranskat)abstract
- The crystal structures of Family 7 glycohydrolases suggest that a histidine residue near the acid/base catalyst could account for the higher pH optimum of the Humicola insolens endoglucanase Cel7B, than the corresponding Trichoderma reesei enzymes. Modelling studies indicated that introduction of histidine at the homologous position in T. reesei Cel7A (Ala(224)) required additional changes to accommodate the bulkier histidine side chain. X-ray crystallography of the catalytic domain of the E223S/A224H/L225V/T226A/D262G mutant reveals that major differences from the wild-type are confined to the mutations themselves, The introduced histidine residue is in plane with its counterpart in H. insolens Cel7B, but is 1.0 Angstrom (= 0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 Angstrom contact between N-2 and O'(1). The pH variation of k(cat)/K-m for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wildtype and mutant, with pK(1) shifting from 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2) shifting from 5.99+/-0.02 to 6.78+/-0.02. With this poor substrate, the ionizations probably represent those of the free enzyme. The relative k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower k(cat)/K-m values for both lactosides and cellobiosides, and a marginally lower stability. However, k(cat) values for cellobiosides are higher for the mutant. This we attribute to reduced nonproductive binding in the +1 and +2 subsites; inhibition by cellobiose is certainly relieved in the mutant. The weaker binding of cellobiose is due to the loss of two water-mediated hydrogen bonds.
|
|
| 3. |
- Borriss, R., et al.
(författare)
-
Enzymatic synthesis of 4-methylumbelliferyl (1 -> 3)-beta-D-glucooligosaccharides - new substrates for beta-1,3-1,4-D-glucanase
- 2003
-
Ingår i: Carbohydrate Research. - : Elsevier BV. - 0008-6215 .- 1873-426X. ; 338:14, s. 1455-1467
-
Tidskriftsartikel (refereegranskat)abstract
- The transglycosylation reactions catalyzed by beta-1,3-D-glucanases (laminaranases) were used to synthesize a number of 4-methylumbelliferyl (MeUmb) (1 --> 3)-beta-D-gluco-oligosaccharides having the common structure [beta-D-Glcp-(1 --> 3)](n)-beta-D-Glcp-MeUmb, where n = 1-5. The beta-1,3-D-glucanases used were purified from the culture liquid of Oerskovia sp. and from a homogenate of the marine mollusc Spisula sachalinensis. Laminaran and curdlan were used as (1 --> 3)-beta-D-glucan donor substrates, while MeUmb-beta-D-glucoside (MeUmbGlcp) was employed as a transglycosylation acceptor. Modification of [beta-D-Glcp-(1 --> 3)](2)-beta-D-Glcp-MeUmb (MeUmbG(3)) gives 4,6-O-benzylidene-D-glucopyranosyl or 4,6-O-ethylidene-D-glucopyranosyl groups at the non-reducing end of artificial oligosaccharides. The structures of all oligosaccharides obtained were solved by H-1 and C-13 NMR spectroscopy and electrospray tandem mass spectrometry. The synthetic oligosaccharides were shown to be substrates for a beta-1,3-1,4-D-glucanase from Rhodothermus marinus, which releases MeUmb from beta-di- and beta-triglucosides and from acetal-protected beta-triglucosides. When acting upon substrates with d.p. > 3, the enzyme exhibits an endolytic activity, primarily cleaving off MeUrnbGlcP and MeUmbG(2).
|
|
| 4. |
- Carlén, Lars, et al.
(författare)
-
CHICSi - a compact ultra-high vacuum compatible detector system for nuclear reaction experiments at storage rings. III. readout system
- 2004
-
Ingår i: Nuclear Instruments & Methods in Physics Research. Section A: Accelerators, Spectrometers, Detectors, and Associated Equipment. - : Elsevier BV. - 0167-5087 .- 0168-9002. ; 516:2-3, s. 327-347
-
Tidskriftsartikel (refereegranskat)abstract
- (CHICSi) Celsius Heavy Ion Collaboration Si detector system is a high granularity, modular detector telescope array for operation around the cluster-jet target/circulating beam intersection of the CELSIUS storage ring at the The. Svedberg Laboratory in Uppsala, Sweden. It is able to provide identity and momentum vector of up to 100 charged particles and fragments from proton-nucleus and nucleus-nucleus collisions at intermediate energies, 50-1000A MeV. All detector telescopes as well as the major part of electronic readout system are placed inside the target chamber in ultra-high vacuum (UHV, 10(-9)-10(-7) Pa). This requires Very Large Scale Integrated (VLSI) microchip for the spectroscopic signal processing and the generation and transport of digital control signals. Eighteen telescopes, read out with chip-on-board technique by ceramics Mother Boards (MB) and corresponding 18 microchips are mounted on a 450 x 45 mm(2) Grand Mother Board (GMB), processed on FR4 glass-fibre material. Each of these 28 GMB units contains a daisy-chain organisation of the VLSI chips and associated protection circuits. Analogue-to-digital conversion of the spectroscopic signals is performed on a board outside the chamber which is connected on one side to a power distribution board, directly attached to a UHV mounting flange, and on the other side to the VME-based data acquisition system (CHICSiDAQ). This in its turn is connected via a fibre-optic link to the general TSL acquisition system (SVEDAQ), and in this way data from auxiliary detector systems, read out in CAMAC mode, can be stored in coincidence with CHICSi data.
|
|
| 5. |
- Eneyskaya, E. V., et al.
(författare)
-
Enzymatic synthesis of beta-xylanase substrates : transglycosylation reactions of the beta-xylosidase from Aspergillus sp
- 2003
-
Ingår i: Carbohydrate Research. - 0008-6215 .- 1873-426X. ; 338:4, s. 313-325
-
Tidskriftsartikel (refereegranskat)abstract
- A beta-D-xylosidase with molecular mass of 250 +/- 5 kDa consisting of two identical subunits was purified to homogeneity from a cultural filtrate of Aspergillus sp. The enzyme manifested high transglycosylation activity in transxylosylation with p-nitrophenyl P-D-xylopyranoside (PNP-X) as substrate, resulting in regio- and stereoselective synthesis of p-nitrophenyl (PNP) beta-(1 --> 4)-D-xylooligosaccharides with dp 2-7. All transfer products were isolated from the reaction mixtures by HPLC and their structures established by electrospray mass spectrometry and H-1 and C-13 NMR spectroscopy. The glycosides synthesised, beta-Xyl-1 --> (4-beta-Xyl-1 -->)(n)4-beta-Xyl-OC6H4NO2-p (n = 1 - 5), were tested as chromogenic substrates for family 10 beta-xylanase from Aspergillus orizae (XynA) and family 11 beta-xylanase I from Trichoderma reesei (XynT) by reversed-phase HPLC and UV-spectroscopy techniques. The action pattern of XynA against the foregoing PNP beta-(1 --> 4)-D-xylooligosaccharides differed from that of XynT in that the latter released PNP mainly from short PNP xylosides (dp 2 - 3) while the former liberated PNP from the entire set of substrates synthesised.
|
|
| 6. |
- Golubev, Pavel, et al.
(författare)
-
CHICSi - a compact ultra-high vacuum compatible detector system for nuclear reaction experiments at storage rings. II. Detectors
- 2003
-
Ingår i: Nuclear Instruments & Methods in Physics Research. Section A: Accelerators, Spectrometers, Detectors, and Associated Equipment. - 0167-5087. ; 500:1-3, s. 96-115
-
Tidskriftsartikel (refereegranskat)abstract
- We describe the detectors for identification of charged particles and fragments in CHICSi, a large solid angle multitelescope system mounted inside an ultra-high vacuum (UHV), cluster-jet target chamber. CHICSi performs nuclear reaction experiments at storage rings. The telescopes consist of a first very thin, 10-14 mum Si detector, a second 300 mum (or possibly 500 pm) ion implanted Si detector supplemented by a 6 mm GSO(Ce) scintillator read out by a photodiode (PD) or by a third 300 mum Si detector. The telescopes provide full charge separation up to Z = 17 and mass resolution up to A = 9 in the energy range 0.7-60A MeV. The thin p-i-n diode detector, etched out from a 280 mum Si wafer, and the GSO/PD detector, both exclusively developed for CHICSi, provide an energy resolution less than or equal to 8%, while the standard 300 mum detectors have less than or equal to 2% energy resolution. Radiation stability of the Si detectors is confirmed up to an integrated flux of 10(10) alpha particles. The GSO detector has 70% light collection efficiency with the optical coupling to the PD a simple open, 0.2 mm, gap. A new method, developed to perform absolute energy calibration for the GSO/PD detector is presented. (C) 2003 Elsevier Science B.V. All rights reserved.
|
|
| 7. |
- Hart, D. O., et al.
(författare)
-
Identification of Asp-130 as the catalytic nucleophile in the main alpha-galactosidase from Phanerochaete chrysosporium, a family 27 glycosyl hydrolase
- 2000
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 39:32, s. 9826-9836
-
Tidskriftsartikel (refereegranskat)abstract
- Characterization of the complete gene sequence encoding the alpha-galactosidase from Phanerochaete chrysosporium confirms that this enzyme is a member of glycosyl hydrolase family 27 [Henrissat, B., and Bairoch, A. (1996) Biochem. J. 316, 695-696]. This family, together with the family 36 alpha-galactosidases, forms glycosyl hydrolase dan GH-D, a superfamily of alpha-galactosidases, alpha-N-acetylgalactosaminidases, and isomaltodextranases which are likely to share a common catalytic mechanism and structural topology. Identification of the active site catalytic nucleophile was achieved by labeling with the mechanism-based inactivator 2',4',6'-trinitrophenyl 2-deoxy-2,2-difluoro-alpha-D- lyxo-hexopyranoside; this inactivator was synthesized by anomeric deprotection of the known 1,3,4,6-tetra-O-acetyl-2-deoxy-2,2-difluoro-D-lyxo-hexopyranoside [McCarter, J. D., Adam, M. J., Braun, C., Namchuk, M., Tull, D., and Withers, S. G. (1993) Carbohydr. Res. 249, 77-90], picrylation with picryl fluoride and 2,6-di-tert-butylpyridine, and O-deacetylation with methanolic HCl. Enzyme inactivation is a result of the formation of a stable 2-deoxy-2,2-difluoro-beta-D-lyxo-hexopyranosyl-enzyme intermediate. Following peptic digestion, comparative liquid chromatographic/mass spectrometric analysis of inactivated and control enzyme samples served to identify the covalently modified peptide. After purification of the labeled peptide, benzylamine was shown to successfully replace the 2-deoxy-2,2-difluoro-D-lyxo-hexopyranosyl peptidyl ester by aminolysis. The labeled amino acid was identified as Asp-130 of the mature protein by further tandem mass spectrometric analysis of the native and derivatized peptides in combination with Edman degradation analysis. Asp-130 is found within the sequence YLKYDNC, which is highly conserved in all known family 27 glycosyl hydrolases.
|
|
| 8. |
- Polívka, Tomáš, et al.
(författare)
-
Direct observation of the S1 level of the carotenoid spheroidene using near-infrared femtosecond spectroscopy
- 2001
-
Ingår i: Ultrafast Phenomena XII. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 9783642625121 - 9783642565465 ; 66, s. 668-670
-
Bokkapitel (refereegranskat)abstract
- In this work, we have determined the energy of the S1 state of the carotenoid spheroidene. The energy of this state is 13,400 ± 90 cm-1 at both 293 K and 186 K, showing that there is no temperature-induced shift of the S1 level. A discrepancy of about 800 cm-1 between the S1 energy determined here and that obtained from previous fluorescence and resonance Raman measurements is explained in terms of the different conformational species co-existing in the S1 excited state.
|
|
| 9. |
- Polívka, Tornáš, et al.
(författare)
-
Near-infrared time-resolved study of the S-1 state dynamics of the carotenoid spheroidene
- 2001
-
Ingår i: The Journal of Physical Chemistry Part B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 105:5, s. 1072-1080
-
Tidskriftsartikel (refereegranskat)abstract
- Using a novel experimental approach based on near-infrared femtosecond absorption spectroscopy, we have determined the energy of the S-1 state of the carotenoid spheroidene. The energy of this state is 13 400 +/- 90 cm(-1) at both 293 and 186 K, showing that there is no temperature-induced shift of the S-1 level. A discrepancy of about 800 cm(-1) between the S-1 energy determined here and that obtained from previous fluorescence and resonance Raman measurements is explained in terms of the different conformational species coexisting in the S-1 excited state. Measurements of kinetics in the near-infrared region revealed that at least three relaxation processes take place after excitation of spheroidene into its S-2 state. Ultrafast S-2 --> S-1 internal conversion occurs within the first 300 fs, followed by vibrational cooling in the SI state, which occurs on a time scale of similar to 700 fs. The S-1 lifetime is 8 ps at 293 K, in good agreement with previous measurements of the S-1 --> S-N transition. A somewhat longer S-1 lifetime of 9.5 ps is observed at 186 K.
|
|
| 10. |
|
|
