RESUMO
Trehalose-6-phosphate phosphatase (TPP) catalyzes the final step in the biosynthesis of the anti-stress sugar trehalose. An 82 kDa TPP enzyme was isolated from Candida utilis with 61% yield and 43-fold purification. The protein sequence, determined by N-terminal sequencing and MALDI-TOF analysis, showed significant homology with known TPP sequences from related organisms. The full length gene sequence of TPP of C. utilis was identified using rapid amplification of cDNA ends-PCR reaction (RACE-PCR). The gene was cloned and expressed in Escherichia coli BL21. Recombinant TPP enzyme was isolated using affinity chromatography. CD spectroscopy and steady-state fluorescence revealed that the structural and conformational aspects were identical in both native and recombinant forms. The biochemical properties of the two forms were also similar. Km was determined to be ~0.8 mM. Optimum temperature and pH were found to be 30 °C and 8.5, respectively. Activity was dependent on the presence of divalent cations and inhibited by metal chelators. Methylation-mediated regulation of TPP enzyme and its effect on the overall survival of the organism under stress were investigated. The results indicated that enhancement of TPP activity by methylation at the Cysteine residues increased resistance of Candida cells against thermal stress. This work involves extensive investigations toward understanding the physico-chemical properties of the first TPP enzyme from any yeast strain. The mechanism by which methylation regulates its activity has also been studied. A correlation between regulation of trehalose synthesis and survivability of the organism under thermal stress was established.
Assuntos
Candida/enzimologia , Proteínas Fúngicas/metabolismo , Resposta ao Choque Térmico , Monoéster Fosfórico Hidrolases/metabolismo , Trealose/biossíntese , Sequência de Aminoácidos , Candida/genética , Quelantes/farmacologia , Cromatografia de Afinidade , Dicroísmo Circular , Clonagem Molecular , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Concentração de Íons de Hidrogênio , Cinética , Metilação , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/isolamento & purificação , Conformação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , TemperaturaRESUMO
A soil bacterium, designated strain AKS31, was isolated on the plastic polyurethane (PUR) and based on the molecular and biochemical analysis was tentatively assigned to the genus Pseudomonas. Preliminary studies suggested that strain AKS31 had the capability of biodegrading polyurethane (PUR) and low-density polyethylene (LDPE). This observation was confirmed by the analysis of the biodegradation products. The hydrolyzed products of PUR analyzed sequentially by High-Performance Liquid Chromatography (HPLC) and Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) showed the presence of diethylene glycol suggesting the presence of an esterase. A gene that could be involved in producing an esterase-like activity (PURase gene) was identified after the amplification and sequencing of a PCR product. Fourier Transformed Infrared (FTIR) spectrophotometric analysis of AKS31-treated LDPE film revealed the incorporation of hydroxyl groups suggesting the involvement of a hydroxylase in the degradation of LDPE. It is established that plastics form microplastics and microbeads in soils which negatively impact the health of living organisms and there have been concentrated research efforts to remediate this problem. Microcosm studies revealed that when strain AKS31 was bioaugmented with soil both the polymers were degraded during which time the heterotrophic plate counts, soil respiration and soil organic carbon content increased but this was not the case with the control nonbioaugmented microcosm. The results demonstrate that the strain AKS31 may have the potential in biodegradation of PUR and LPDE present as plastic microbeads and thereby improving soil health. Further studies in this direction are warranted. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02592-9.