PubMed · 13058851
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M V TRACEY. 1953. Cellulases.. https://pubmed.ncbi.nlm.nih.gov/13058851/
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A method for the quantitative determination of oligogalacturonic acids (OGAs) by on-line high-performance liquid chromatographic (HPLC) separation and mass spectrometric detection via an electrospray interface (ESI-MS) without additional desalting steps was developed. Saturated OGAs up to a degree of polymerization (dp) of 3 were quantified by comparison with reference compounds. The calibration plots showed high linearity (R(2)>0.99), and the detection limits for dp 1, 2, and 3 were 11, 28, and 6 ng per injection, respectively. Non- and partially methyl-esterified OGAs with a dp of 3 and 4 were calculated semi-quantitatively as dp 3. The analytical system was used for the quantification of OGAs of digests obtained by incubation of polygalacturonic acid, pectin, and carrot pomace with commercial enzyme preparations. Furthermore, methyl-esterified OGAs up to a dp of 12 containing up to 4 methyl esters were detected in a pectin digest.
The conformational agenda harnessed by different glycosidases along the reaction pathway has been mapped by X-ray crystallography. The transition state(s) formed during the enzymic hydrolysis of glycosides features strong oxocarbenium-ion-like character involving delocalization across the C-1-O-5 bond. This demands planarity of C-5, O-5, C-1 and C-2 at or near the transition state. It is widely, but incorrectly, assumed that the transition state must be (4)H(3) (half-chair). The transition-state geometry is equally well supported, for pyranosides, by both the (4)H(3) and (3)H(4) half-chair and (2,5)B and B(2,5) boat conformations. A number of retaining beta-glycosidases acting on gluco -configured substrates have been trapped in Michaelis and covalent intermediate complexes in (1)S(3) (skew-boat) and (4)C(1) (chair) conformations, respectively, pointing to a (4)H(3)-conformed transition state. Such a (4)H(3) conformation is consistent with the tight binding of (4)E- (envelope) and (4)H(3)-conformed transition-state mimics to these enzymes and with the solution structures of compounds bearing an sp (2) hybridized anomeric centre. Recent work reveals a (1)S(5) Michaelis complex for beta-mannanases which, together with the (0)S(2) covalent intermediate, strongly implicates a B(2,5) transition state for beta-mannanases, again consistent with the solution structures of manno -configured compounds bearing an sp (2) anomeric centre. Other enzymes may use different strategies. Xylanases in family GH-11 reveal a covalent intermediate structure in a (2,5)B conformation which would also suggest a similarly shaped transition state, while (2)S(0)-conformed substrate mimics spanning the active centre of inverting cellulases from family GH-6 may also be indicative of a (2,5)B transition-state conformation. Work in other laboratories on both retaining and inverting alpha-mannosidases also suggests non-(4)H(3) transition states for these medically important enzymes. Three-dimensional structures of enzyme complexes should now be able to drive the design of transition-state mimics that are specific for given enzymes, as opposed to being generic or merely fortuitous.
The avi2 gene encoding Avi2, which is a major cellulase produced by Humicola insolens FERM BP-5977, was cloned and sequenced. Avi2 showed high homology with other family 6 cellulases. The expression vector pNCE4 containing the avi2 gene was constructed, and this strain was transformed using a protoplast method. As a result, the pNCE4 transformant secreted 8-fold more Avi2 than the recipient strain.