Protein engineering of alcohol dehydrogenases: effects of amino acid changes at positions 93 and 48 of yeast ADH1.

By protein engineering we have investigated changes to two amino acid residues (Trp93 and Ser48) in the substrate pocket of yeast alcohol dehydrogenase 1. Upon changing Thr48 to serine we produced an enzyme which has markedly greater activity towards aliphatic alcohols with chain length up to 8, together with a general increase in catalytic activity (V/K). Changes at position 93 were less pronounced, with the Phe enzyme being more active than the parent towards the range of alcohols but with the alanine enzyme showing very little difference from the wild-type.

Protein engineering of alcohol dehydrogenase--1. Effects of two amino acid changes in the active site of yeast ADH-1.

One of the promises held out by protein engineering is the ability to alter predictably the properties of an enzyme to enable it to find new substrates or catalyse existing substrates more efficiently, such manipulations being of interest both enzymologically and, potentially, industrially. It has been postulated that in yeast alcohol dehydrogenase (YADH-1) certain amino acids such as Trp 93 and Thr 48 constrict the active site due to their bulky side chains and thus impede catalysis of molecules larger than ethanol. To study effects of enlarging the active site we have made two changes into YADH-1, replacing Trp 93 with Phe and Thr 48 with Ser.

Genomic clones of Aspergillus nidulans containing alcA, the structural gene for alcohol dehydrogenase and alcR, a regulatory gene for ethanol metabolism.

Our aim was to obtain from Aspergillus nidulans a genomic bank and then clone a region we expected from earlier genetic mapping to contain two closely linked genes, alcA, the structural gene for alcohol dehydrogenase (ADH) and alcR, a positive trans-acting regulatory gene for ethanol metabolism. The expression of alcA is repressed by carbon catabolites. A genomic restriction fragment characteristic of the alcA-alcR region was identified, cloned in pBR322, and used to select from a genomic bank in lambda EMBL3A three overlapping clones covering 24 kb of DNA.

Purification and preliminary characterization of alcohol dehydrogenase from Aspergillus nidulans.

Aspergillus alcohol dehydrogenase is produced in response to growth in the presence of a wide variety of inducers, of which the most effective are short-chain alcohols and ketones, e.g. butan-2-one and propan-2-ol.

The purification of urease from Aspergillus nidulans.

A purification procedure is described for Aspergillus urease, the most important step being affinity chromatography on hydroxyurea Sepharose. The enzyme exists as a single active species of Mr 240,000. The pure enzyme has an activity of 670 mumol urea hydrolysed/min, has a Km of 10(-3) M, an optimum pH of 8.