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Pseudomonas aeruginosa PAO1 has two possible catabolic pathways of spermidine and spermine; one includes the spuA and spuB products with unknown functions and the other involves spermidine dehydrogenase (SpdH; EC 1.5.99.6) encoded by an unknown gene. The properties of SpdH in P. aeruginosa PAO1 were characterized and the corresponding spdH gene in this strain identified. The deduced SpdH (620 residues, calculated M r of 68 861) had a signal sequence of 28 amino acids at the amino terminal and a potential transmembrane segment between residues 76 and 92, in accordance with membrane location of the enzyme. Purified SpdH oxidatively cleaved spermidine into 1,3-diaminopropane and 4-aminobutyraldehyde with a specific activity of 37 units (mg protein) "1 and a K m value of 36 mM. The enzyme also hydrolysed spermine into spermidine and 3-aminopropanaldehyde with a specific activity of 25 units (mg protein) "1 and a K m of 18 mM. Knockout of spdH had no apparent effect on the utilization of both polyamines, suggesting that this gene is minimally involved in polyamine catabolism. However, when spdH was fused to the polyamine-inducible promoter of spuA, it fully restored the ability of a spuA mutant to utilize spermidine. It is concluded that SpdH can perform a catabolic role in vivo, but P. aeruginosa PAO1 does not produce sufficient amounts of the enzyme to execute this function.

The engineering of enzymes with altered activity, specificity, and stability, using directed evolution techniques that mimic evolution on a laboratory timescale, is now well established. In vitro recombination techniques such as DNA shuffling, staggered extension process (StEP), random chimeragenesis on transient templates (RACHITT), iterative truncation for the creation of hybrid enzymes (ITCHY), recombined extension on truncated templates (RETT), and so on have been developed to mimic and accelerate nature's recombination strategy. This review discusses gradual advances in the techniques and strategies used for the directed evolution of biocatalytic enzymes aimed at improving the quality and potential of enzyme libraries, their advantages, and disadvantages.

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Two genes, cut1 and cut2, of Thermobifida fusca NRRL B-8184 with cutin-hydrolyzing activity were cloned and expressed in Escherichia coli BL21 (DE3) separately. Enhanced expression was achieved after screening of six different media, optimization of the culture conditions and medium components. Among the screened media, modified Terrific Broth was found to be the best for maximum production of recombinant cutinases in E. coli BL21 (DE3). Under optimal conditions, the production of recombinant Cut1 and Cut2 (cutinases) were found to be 318±0.73 and 316±0.90 U/ml, respectively. The production of recombinant cutinases was increased by 11-fold as compared with T. fusca NRRL B-8184 wild-type strain. Both the recombinant cutinases were purified to homogeneity. They were found to be thermostable, organic solvent, and surfactant tolerant. Both the cutinase were active in a broad range of temperature (40-80 °C) and pH (6.8-9) with optimum activity at pH 8.0 and 55 °C.

Glutaminase-free L-asparaginase is known to be an excellent anticancer agent. In the present study, statistically based experimental designs were applied to maximize the production of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428. Nine components of the medium were examined for their significance on the production of L-asparaginase using the Plackett-Burman experimental design. The medium components, viz., glucose, L-asparagine, KH2PO4, and MgSO(4).7H2O, were screened based on their high confidence levels (P<0.04). The optimum levels of glucose, L-asparagine, KH2PO4, and MgSO(4).7H2O were found to be 2.076, 5.202, 1.773, and 0.373 g L(-1), respectively, using the central composite experimental design. The maximum specific activity of L-asparaginase in the optimized medium was 27.88 U mg(-1) of protein, resulting in an overall 8.3-fold increase in the production compared to the unoptimized medium.

Subcritical water (SCW) treatment has gained enormous attention as an environmentally friendly technique for organic matter and an attractive reaction medium for a variety of applications. In the current work the process parameters were optimized by RSM model.