No abstract

No abstract

Abstract. Boontanom P, Chantarasiri A. 2020. Short Communication: Diversity of culturable epiphytic bacteria isolated from seagrass (Halodule uninervis) in Thailand and their preliminary antibacterial activity. Biodiversitas 21: 2907-2913. Epiphytic bacteria are symbiotic bacteria that live on the surface of seagrasses. This study presents the diversity of culturable epiphytic bacteria associated with the Kuicheai seagrass (H. uninervis) collected from Rayong Province in Eastern Thailand. Nine epiphytic isolates were identified into four phylogenetical genera based on their 16S rRNA nucleotide sequence analyses. They are considered firmicutes in the genera of Planomicrobium, Paenibacillus and Bacillus, and proteobacteria in the genus of Oceanimonas. Three species of epiphytic bacteria preliminarily exhibited antibacterial activity against the human pathogenic Staphylococcus aureus using the perpendicular streak method. The knowledge obtained from this study increases understanding the diversity of seagrass-associated bacteria in Thailand and suggests the utilization of these bacteria for further pharmaceutical applications.

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 β-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.

One impediment of large-scale biofuel production from lignocellulosic biomass is the insufficiency of cellulolytic microorganisms that can overcome extreme conditions during the industrial process. This study emphasized the isolation of a novel efficient cellulolytic bacterium. A new Bacillus methylotrophicus RYC01101, isolated from ruminant feces in Thailand, produced a hydrolysis capacity greater than that of known cellulolytic bacteria (Cellulomonas sp.). Cellulase activities were investigated on CMCase activity and FPase activity by 0.230±0.004 and 0.080±0.007 U/mL, respectively. B. methylotrophicus RYC01101 was co-cultured with Saccharomyces cerevisiae TISTR 5111 for bioethanol production. The productivity of the bioethanol was 1.38±0.40 g/L after 120 hours of fermentation. Moreover, B. methylotrophicus RYC01101 could be grown in the presence of 10% (w/v) NaCl, which could be applied in the pretreatment step of biofuel production. This study was the first report on cellulolytic activity and the halotolerant ability of B. methylotrophicus.

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.

This study proposed the isolation, screening and identification of cellulase-producing bacteria from Thai coastal wetland soil. Eighty seven bacterial strains which demonstrated cellulase-producing bacteria were investigated for cellulolytic properties. The bacterium identified as Bacillus cereus strain BR0302 exhibited the highest hydrolysis activity on carboxymethylcellulose agar plates. Cellulolytic performance for CMCase activity and FPase activity was 0.121 ± 0.006 and 0.057 ± 0.001 U/mL respectively. Cellulolytic characteristics showed that the B. cereus strain BR0302 could be used for the hydrolysis of cellulosic biomass for biofuel production.

No abstract