Molecules of nucleic acids coding a dextran-saccharase catalysing the synthesis of dextran having alpha-1,2 osidic branching

摘要

New isolated polypeptides (I), with glycosyl transferase activity suitable for producing dextrans having alpha (1-2) branches from sucrose, alpha -fluoroglucose, p-nitrophenyl-alpha -D-glucopyranoside, alpha -D-glucopyranoside-alpha -D-sorbopyranoside or alpha -D-galactopyranosyl-sucrose, and at least one glucan bonding domain (GBD) and a catalytic domain (CD) downstream of the GBD, are new. Independent claims are also included for: (1) an isolated nucleic acid encoding (I), having at least one nucleotide sequence encoding a CD and with at least 50% (preferably at least 80%) homology with a sequence of 2568 bases given in the specification (S3), located at 3' relative to a sequence encoding a GBD; (2) an expression vector containing a nucleic acid as in (1) (preferably under the control of sequences allowing expression in prokaryotic or eukaryotic cells); (3) host cells transformed by a nucleic acid as in (1) or a vector as in (2); (4) methods for the production of (I) (specifically dextran saccharases); (5) glycosyl transferases forming at least 30% alpha (1-2) bonds obtained as in (4); (6) the production of a dextran saccharase (I'), suitable for forming oligosides or dextrans having more than 30% alpha (1-2) bonds, involving modification of a fully defined sequence of 8506 bases given in the specification (S4) by addition, deletion and mutation, such that when the reading frame is not modified, the following aminoacids are retained: W in positions 425 and 2122 (encoded by TGG in positions 1273 and 6364), E in positions 430, 565, 2127 and 2248 (encoded by GAA in positions 1288, 1693, 6379 and 6742), D in positions 487, 489, 527, 638, 2170, 2172, 2210 and 2322 (encoded by GAT in positions 1459, 1465, 1579, 1912, 6508 and 6628), H in positions 637 and 2321 (encoded by CAT in position 1909 and CAC in position 6961) and Q in positions 1019 and 2694 (encoded by CAA in position 3055 and CAG in position 8080); and (7) the production of a glycosyl transferase (I''), suitable for forming oligosides or dextrans having more than 30% alpha (1-2) bonds, by random mutation of (S4), forming a library of variants, expressing these modified sequences in a suitable host cell, screening the hosts for expression of (I'') and isolating the improved genes. ACTIVITY : Cytostatic; dermatological; antiseborrheic. No biological data is given. MECHANISM OF ACTION : None given.