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Hardiman E.,Research Frontiers | Gibbs M.,Research Frontiers | Gibbs M.,Applimex Systems Pty Ltd | Reeves R.,Research Frontiers | And 5 more authors.
Applied Biochemistry and Biotechnology | Year: 2010

Characteristics that would make enzymes more desirable for industrial applications can be improved using directed evolution. We developed a directed evolution technique called randomdriftmutagenesis (RNDM).Mutant populations are screened and all functionalmutants are collected and put forward into the next round ofmutagenesis and screening. The goal of this technique is to evolve enzymes by rapidly accumulating mutations and exploring a greater sequence space by providing minimal selection pressure and high-throughput screening. The target enzyme was a β-glucosidase isolated from the thermophilic bacterium, Caldicellulosiruptor saccharolyticus that cleaves cellobiose resulting from endoglucanase hydrolysis of cellulose. Our screening method was fluorescence-activated cell sorting (FACS), an attractive method for assaying mutant enzyme libraries because individual cells can be screened, sorted into distinct populations and collected very rapidly. However, FACS screening poses several challenges, in particular, maintaining the link between genotype and phenotype because most enzyme substrates do not remain associated with the cells. We employed a technique where whole cells were encapsulated in cell-like structures along with the enzyme substrate. We used RNDM, in combination with whole cell encapsulation, to create and screen mutant β-glucosidase libraries. A mutant was isolated that, compared to the wild type, had higher specific and catalytic efficiencies (k cat/K M) with p-nitrophenol-glucopyranoside and -galactopyranoside, an increased catalytic turnover rate (k cat) with cellobiose, an improvement in catalytic efficiency with lactose and reduced inhibition (K i) with galactose and lactose. This mutant had three amino acid substitutions and one was located near the active site. © Humana Press 2009. Source


Gibbs M.D.,Applimex Systems Pty Ltd | Gibbs M.D.,Macquarie University | Reeves R.A.,Applimex Systems Pty Ltd | Choudhary P.R.,Macquarie University | And 3 more authors.
New Biotechnology | Year: 2010

We reported previously that the activities of several glycosyl hydrolase family 11 xylanases claimed to be active under alkaline conditions, were found to have optima in the pH 5-6 range when assayed under optimal conditions. One enzyme, BadX, had enhanced activity at pHs greater than 7 compared to other family 11 xylanases. Gene shuffling between badX and Dictyoglomus thermophilum xynB6 was performed in an attempt to elucidate regions conferring alkaline activity to BadX, and potentially, to increase the alkaline activity of the highly thermophilic XynB6. Segment substitution using degenerate oligonucleotide gene shuffling (DOGS) experiments with combinations of input parental gene fragments from xynB6 and badX was not able to improve the activity of XynB6 at alkaline pH. With one exception, the replacement of a single segment of BadX with the equivalent segment from XynB6 reduced the alkaline activity BadX. The results indicate that it might not be possible to alter significantly the alkaline pH characteristics of family 11 xylanases by one or a few mutations and that family 11 xylanases showing enhanced activity at alkaline pH's require multiple sequence adaptations across the protein. © 2010 Elsevier B.V. Source


Gibbs M.D.,Applimex Systems Pty Ltd | Gibbs M.D.,Macquarie University | Reeves R.A.,Applimex Systems Pty Ltd | Hardiman E.M.,Macquarie University | And 5 more authors.
New Biotechnology | Year: 2010

Xylanases have several industrial uses, particularly in baking, modification of animal feed and in pulp bleaching in the paper industry. Process conditions in kraft pulp bleaching generally favour an enzyme that is active at high pH values. The activities of several glycosyl hydrolase family 11 xylanases reported to be active under alkaline conditions were determined under optimal conditions and found to have optima in the pH 5-6 range. Only one enzyme tested, BadX, was shown to have an alkaline pH optimum. Significant activity at pH values higher than 8 appears often to be the result of excess enzyme added to the reaction mixtures so that substrate is limiting. The different nature of laboratory and industrial substrates needs to be taken into consideration in designing assay conditions. In some cases, significant differences were observed in pH profiles generated using a small-molecule substrate when compared to those generated using xylan. We conclude that small-molecule substrates are not a suitable proxy for determining the pH profiles of family 11 xylanases. © 2010 Elsevier B.V. Source

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