Gotowa bibliografia na temat „Pseudozyma antarctica”

Efficient production of optically pure (R)-3-substituted glutaric acid methyl monoesters, the multifunctional chiral building blocks used in the pharmaceutical industry, by manipulating the substrate pocket of Pseudozyma antarctica lipase B.

ABSTRACT Two amino acids, Leu149 and Val223, were identified as proteolytically sensitive when Pseudozyma antarctica lipase (PalB) was heterologously expressed in Escherichia coli. The functional expression was enhanced using the double mutant for cultivation. However, the recombinant protein production was still limited by PalB misfolding, which was resolved by DsbA coexpression.

Biodegradable plastics must be sufficiently stable to maintain functionality during use but need to be able to degrade rapidly after use. We previously reported that treatment with an enzyme named PaE, secreted by the basidiomycete yeast Pseudozyma antarctica can speed up this degradation. To facilitate the production of large quantities of PaE, here, we aimed to elucidate the optimal conditions of ethanol treatment for sterilization of the culture supernatant and for concentration and stabilization of PaE. The results showed that Pseudozyma antarctica completely lost its proliferating ability when incubated in ≥20% (v/v) ethanol. When the ethanol concentration was raised to 90% (v/v), PaE formed a precipitate; however, its activity was restored completely when the precipitate was dissolved in water. To reduce ethanol use, PaE was successfully concentrated and recovered by sequential ammonium sulfate precipitation and ethanol precipitation steps. Over 90% of the activity in the original culture supernatant was recovered and the specific activity was increased 3.4-fold. By preparing the enzyme solution at a final concentration of 20% (v/v) ethanol, about 60% of the initial activity was maintained at ambient temperature for over 6 months without growth of microbes. We conclude that ethanol treatment is effective for sterilization, concentration, and stabilization of PaE, and that concentrating PaE by sequential ammonium sulfate precipitation and ethanol precipitation substantially increases the PaE purity and decreases ethanol use.

The effect of mechanical wounding or foliar diseases caused by Sclerotinia homoeocarpa or Rhizoctonia solani on the epiphytic yeast communities on creeping bentgrass and tall fescue were determined by leaf washing and dilution plating. Total yeast communities on healthy bentgrass and tall fescue leaves ranged from 7.9 × 103 to 1.4 × 105 CFU·cm–2 and from 2.4 × 103 to 1.6 × 104 CFU·cm–2, respectively. Mechanically wounded leaves (1 of 2 trials) and leaves with disease lesions (11 of 12 trials) supported significantly larger communities of phylloplane yeasts. Total yeast communities on S. homoeocarpa infected or R. solani infected bentgrass leaves were 3.6–10.2 times and 6.2–6.4 times larger, respectively, than the communities on healthy leaves. In general, healthy and diseased bentgrass leaves supported larger yeast communities than healthy or diseased tall fescue leaves. We categorized the majority of yeasts as white-pigmented species, including Cryptococcus laurentii, Cryptococcus flavus, Pseudozyma antarctica, Pseudozyma aphidis, and Pseudozyma parantarctica. The percentage of pink yeasts in the total yeast community ranged from 2.6% to 9.9% on healthy leaves and increased to 32.0%–44.7% on S. homoeocarpa infected leaves. Pink-pigmented yeasts included Rhodotorula glutinis, Rhodotorula mucilaginosa, Sakaguchia dacryoidea, and Sporidiobolus pararoseus. Foliar disease significantly affected community size and composition of epiphytic yeasts on bentgrass and tall fescue.Key words: dollar spot, phylloplane, Rhizoctonia blight.

Multifunctional enzymes have alternative functions or activities, known as “moonlighting” or “promiscuous”, which are often hidden behind a native enzyme activity and therefore only visible under special environmental conditions. In this thesis, the active-site of Pseudozyma (formerly Candida) antarctica lipase B was explored for a promiscuous conjugate addition activity. Pseudozyma antarctica lipase B is a lipase industrially used for hydrolysis or transacylation reactions. This enzyme contains a catalytic triad, Ser105-His224-Asp187, where a nucleophilic attack from Ser105 on carboxylic acid/ester substrates cause the formation of an acyl enzyme. For conjugate addition activity in Pseudozyma antarctica lipase B, replacement of Ser105 was assumed necessary to prevent competing hemiacetal formation. However, experiments revealed conjugate addition activity in both wild-type enzyme and the Ser105Ala variant. Enzyme-catalyzed conjugate additions were performed by adding sec-amine, thiols or 1,3-dicarbonyl compounds to various α,β-unsaturated carbonyl compounds in both water or organic solvent. The reactions followed Michaelis-Menten kinetics and the native ping pong bi bi reaction mechanism of Pseudozyma antarctica lipase B for hydrolysis/transacylation was rerouted to a novel ordered bi uni reaction mechanism for conjugate addition (Paper I, II, III). The lipase hydrolysis activity was suppressed more than 1000 times by the replacement of the nucleophilic Ser105 to Ala (Paper III).

Protein kinases, the third most populous protein family, are a major class of enzymes that regulate a wide range of cellular processes by phosphorylating multiple cellular proteins. There are many conserved sequence motifs within the catalytic domain that are essential for the regulation of the proteins, and the catalytic core is commonly shared across all typical protein kinases. In this thesis, we present identification and comprehensive analyses of Ser/Thr or Tyr (STY) protein kinases encoded in many pathogenicity causing organisms. The role of kinases in causing pathogenicity has been elucidated in many of the previous studies. With the emergence of diverse pathogenic species in the last few decades, the protein kinases of the pathogens and the respective hosts might have evolved to adapt to the hostile environment and tolerate stress response. Hence, it is essential to even perform a comparative study of kinases within pathogens against those encoded in their host species. Protein kinases can be identified using sequence-based approaches. However, some of the kinase subfamilies have highly diversified, and one requires sensitive homology detection approaches for their identification. Therefore, we developed a fresh protocol named "Master Blaster" for the detection of distantly related proteins. The performance of Master Blaster is evaluated by comparing against widely-used profile based and hidden Markov model-based methods. An improvement in fold coverage using Master Blaster is reported. We also used artificially designed sequences for detecting distantly related proteins using Master Blaster. Use of the designed sequences was found to be useful in connecting protein families that are highly diverged at sequence level. We applied the developed protocol to identify protein kinases encoded in Candida albicans and performed an extensive analysis of these kinases using sequence-based methods. A comparative study of kinases within C. albicans, pathogenic and non-pathogenic non-albicans Candida species, Baker's yeast and Human was performed. This study has resulted in identifying organism-specific kinases and signature motifs in kinases of pathogenicity causing Candida species. Domain architectures of kinases are also found to be organism-specific. Some of the protein kinases within industrial relevant Candida species such as Pseudozyma antarctica are found to be similar to those encoded in human. Kinases within each subfamily can recognize and phosphorylate multiple substrates and the substrates that are recruited could be specific to a particular kinase subfamily based on their functional role. Phosphorylation of specific substrates is possible due to the conserved substrate binding residues within in each kinase subfamily. Most of the kinase classification schemes depends on the conservation within the catalytic domain region and does not take into account the conservation within the substrate binding regions. This could lead to misclassification of some of the kinases. Hence, we proposed a new approach to classify kinases based on conservation of their substrate binding residues. Using the proposed scheme, we could identify signature regions within some of the kinase subfamilies and these regions can be used for the re-classification of protein kinases. In this thesis work, we also identified kinases encoded in viral genomes using rigorous sequence-based methods and reported the sequence similarities and differences between kinases within various viral genomes. Kinases are detected in viruses having double stranded DNA genome and in some of the subfamilies of retroviruses. In none of the host infecting viruses, kinases could be detected. The putative protein kinases from giant viruses are found to be similar to the retroviral oncogenic kinases. The substrate binding regions within some of the viral kinases are identified using the new classification scheme. This work can be extended by applying developed protocols for studying protein kinases encoded in different organisms.