Pichia pastoris

About: Pichia pastoris is a research topic. Over the lifetime, 7937 publications have been published within this topic receiving 162645 citations. The topic is also known as: Komagataella pastoris.

Characterization and heterologous gene expression of a novel esterase from Lactobacillus casei CL96.

Abstract: Microbial esterases and lipases are currently receiving considerable attention because of their potential applications in biotechnology for food processing, surfactant composition, detergents, paper, oil manufacture, diagnostics, and optically active drugs (27, 28). The enzymes that modify milk fat are lipases (triacylglycerol lipases; EC 3.1.1.3) and esterases (EC 3.1.1.1). Esterases are, by definition, enzymes that have the ability to hydrolyze ester substrates with short-chain fatty esters (≤C10), whereas lipases hydrolyze long-chain acylglycerols (≥C10) (54). For many years, scientists in the dairy field have tried to shorten the maturation period and to reduce the inherent costs, as well as to enhance flavor intensity, for various cheeses. So far, lipolytic and proteolytic enzymes have been used extensively in the dairy and food processing industries for hydrolysis of milk fat with the purpose of flavor enhancement or acceleration of ripening processes in cheeses and cheese-related products, but bitterness and rancidity have precluded their use on a large scale. A correct blend of alkanoic acid with carbon chains from C6 to C10 appears to impart a cheese-like flavor (41). The release of long-chain (C10 to C16) fatty acids by microbial lipase produces a soapy flavor, whereas short-chain fatty acids from animal esterases produce unclean flavors (50). The free fatty acids, which are liberated by the action of lipases or esterases on milk fat, give many dairy products their typical flavor characteristics. Upon further breakdown of fatty acids, reactions with other components of the maturing cheeses and fermented dairy products, which may contribute to the formation of various flavor components, are likely to occur (51). Esterases and lipases are widely present in various organisms from bacteria to higher eucaryotes. A common characteristic of these enzymes is that they contain a catalytic triad, composed of Ser, His, and Asp or Glu (15, 48). In addition, most of these enzymes have a structural motif, G-X-S-X-G, which contains the active-site serine residue (6). This motif is usually located between a β-strand and an α-helix, and it assumes an extremely sharp turn called a nucleophile elbow (42). Although many reports on the cloning and expression of microbial lipases and esterases have been published (31, 32), very little has been reported on lactic acid bacteria as a source of enzymes. Recently, Fenster et al. (19, 20) reported the nucleotide sequences of arylesterase (EstB) and intracellular esterase (EstC) from Lactobacillus casei LILA. Amino acid sequence analysis revealed that the mature proteins possess the GXSXG motif at the active site, and EstB selectivity for para-nitrophenyl (pNP) esters was greatest for C5 and C6 compounds, whereas EstC was most selective for C3 and C4 compounds. Other studies showed that the esterase in L. casei CL96 (10) possesses strong esterolytic activity for C6 and C8 compounds. These properties are somewhat different from those of L. casei LILA EstB and EstC. In this communication, we report the molecular properties and enzymatic characteristics of a novel esterase from L. casei CL96, and we also report the large-scale production of recombinant esterase in methylotrophic yeast (Pichia pastoris) and bacteria (Methylobacterium extorquens), since the Pichia expression system is well characterized (24) and M. extorquens (22, 23) has recently been shown to express high levels of recombinant protein in high-cell-density fed-batch culture.

Large-scale expression, purification and characterization of small fragments of thrombomodulin: the roles of the sixth domain and of methionine 388

Abstract: Fragments of human thrombomodulin (TM) have been expressed in large quantities in the Pichia pastoris yeast expression system and purified to homogeneity. Fermentation of P. pastoris resulted in yields of 170 mg/l TM. Purification to homogeneity resulted in an overall 10% yield, so that quantities of approximately 20 mg purified fragments can be readily obtained. Smaller fragments of TM, such as the individual fourth or fifth domains, were not active, nor were equimolar mixtures of the two domains. These results demonstrate that the fourth and fifth epidermal growth factor (EGF)-like domains together comprise the smallest active fragment of TM. The fragment containing the fourth and fifth EGF-like domains [TMEGF(4-5)] had 10% the specific activity of rabbit TM. Comparison of the M388L mutant TMEGF(4-5) fragment with the same mutant TMEGF(4-5-6) fragment showed that the fragment with the sixth domain had a 10-fold better Km value for thrombin than the fragment that did not contain the sixth domain; this factor completely accounts for the higher specific activity of the fragments containing the sixth domain. Comparison of the wild-type and M388L mutants showed that the M388L mutation resulted in a 2-fold increase in kcat for the activation of protein C by the thrombin-TM fragment complex, completely accounting for the 2-fold increase in specific activity of these mutant fragments.

Structural bases for the interaction and stabilization of the human amino acid transporter LAT2 with its ancillary protein 4F2hc

Abstract: Heteromeric amino acid transporters (HATs) are the unique example, known in all kingdoms of life, of solute transporters composed of two subunits linked by a conserved disulfide bridge. In metazoans, the heavy subunit is responsible for the trafficking of the heterodimer to the plasma membrane, and the light subunit is the transporter. HATs are involved in human pathologies such as amino acidurias, tumor growth and invasion, viral infection and cocaine addiction. However structural information about interactions between the heavy and light subunits of HATs is scarce. In this work, transmission electron microscopy and single-particle analysis of purified human 4F2hc/L-type amino acid transporter 2 (LAT2) heterodimers overexpressed in the yeast Pichia pastoris, together with docking analysis and crosslinking experiments, reveal that the extracellular domain of 4F2hc interacts with LAT2, almost completely covering the extracellular face of the transporter. 4F2hc increases the stability of the light subunit LAT2 in detergent-solubilized Pichia membranes, allowing functional reconstitution of the heterodimer into proteoliposomes. Moreover, the extracellular domain of 4F2hc suffices to stabilize solubilized LAT2. The interaction of 4F2hc with LAT2 gives insights into the structural bases for light subunit recognition and the stabilizing role of the ancillary protein in HATs.