Klebsiella and Enterobacter Isolated from Mangrove Wetland Soils in Thailand and Their Application in Biological Decolorization of Textile Reactive Dyes.

Wastewater released from textile and dye-based industries is one of the major concerns for human and aquatic beings. Biological decolorization using ligninolytic bacteria has been considered as an effective and alternative approach for the treatment of dyeing wastewater. This study aimed to assess the isolation, characterization and application of soil bacteria isolated from mangrove wetlands in Thailand. Four active bacteria were genetically identified and designated as Klebsiella pneumoniae strain RY10302, Enterobacter sp. strain RY10402, Enterobacter sp. strain RY11902 and Enterobacter sp. strain RY11903. They were observed for ligninolytic activity and decolorization of nine reactive dyes under experimental conditions. All bacteria exhibited strong decolorization efficiency within 72 h of incubation at 0.01% (w/v) of reactive dyes. The decolorization percentage varied from 20% (C.I. Reactive Red 195 decolorized by K. pneumoniae strain RY10302) to 92% (C.I. Reactive Blue 194 decolorized by Enterobacter sp. strain RY11902) in the case of bacterial monoculture, whereas the decolorization percentage for a mixed culture of four bacteria varied from 58% (C.I. Reactive Blue 19) to 94% (C.I. Reactive Black 1). These findings confer the possibility of using these bacteria for the biological decolorization of dyeing wastewater.

Effects of halophilic peptide fusion on solubility, stability, and catalytic performance of D-phenylglycine aminotransferase.

D-Phenylglycine aminotransferase (D-PhgAT) from Pseudomonas stutzeri ST-201 is useful for enzymatic synthesis of enantiomerically pure D-phenylglycine. However, its low protein solubility prevents its application at high substrate concentration. With an aim to increase the protein solubility, the N-terminus of D-PhgAT was genetically fused with short peptides (A1 α- helix, A2 α-helix, and ALAL, which is a hybrid of A1 and A2) from a ferredoxin enzyme of a halophilic archaeon, Halobacterium salinarum. The fused enzymes A1-D-PhgAT, A2-D-PhgAT, and ALAL-D-PhgAT displayed a reduced pI and increased in solubility by 6.1-, 5.3-, and 8.1- fold in TEMP (pH 7.6) storage, respectively, and 5-, 4.5-, and 5.9-fold in CAPSO (pH 9.5) reaction buffers, respectively, compared with the wild-type enzyme (WT-D-PhgAT). In addition, all the fused D-PhgAT displayed higher enzymatic reaction rates than the WT-DPhgAT at all concentrations of L-glutamate monosodium salt used. The highest rate, 23.82 ± 1.47 mM/h, was that obtained from having ALAL-D-PhgAT reacted with 1,500 mM of the substrate. Moreover, the halophilic fusion significantly increased the tolerance of D-PhgAT in the presence of NaCl and KCl, being slightly in favor of KCl, where under the same condition at 3.5 M NaCl or KCl all halophilic-fused variants showed higher activity than WT-D-PhgAT.

Effective improvement of D-phenylglycine aminotransferase solubility by protein crystal contact engineering.

Structure-guided genetic engineering of D-phenylglycine aminotransferase (D-PhgAT) aimed at increasing protein solubility was attempted. In silico analyses predicted the Asn439 and Gln444 as highly solvent-exposed ß-turn residues involved with protein crystal contact (CC) potential candidates for solubility-improving mutations. They were replaced with Asp and Glu creating the N439D and Q444E single mutants, and N439D/Q444E double mutant with 2.5-, 3.3- and 5.9-fold increases in solubility, respectively. The protein CC prevention effect rather than the net charge effect accounted for the dramatically improved solubility since the N439D, Q444E and N439D/Q444E mutations altered the isoelectric point of D-PhgAT by only 0.1, 0.1 and 0.3 units, respectively. Examination of the D-PhgAT structural model revealed that the N439D mutation weakened the CC attraction force and the Q444E mutation created electrostatic repulsion at the CC point. Analysis of circular dichroism spectra, melting temperature, and D-PhgAT-specific activity showed that the mutations posed no unfavorable effect on the conformational stability and catalytic performance of the enzyme. The protein solubility-improving strategy employed on D-PhgAT in this study was successful with minimal protein structure modification required. It should be applicable with a high chance of success for other proteins, especially those with 3-D structural models available.