Serological differences between the multiple amine oxidases of yeasts and comparison of the specificities of the purified enzymes from Candida utilis and Pichia pastoris.

1. Antiserum to purified methylamine oxidase of Candida boidinii formed precipitin lines (with spurs) in double-diffusion tests with crude extracts of methylamine-grown cells of the following yeast species: Candida nagoyaensis, Candida nemodendra, Hansenula minuta, Hansenula polymorpha and Pichia pinus. No cross-reaction was observed with extracts of Candida lipolytica, Candida steatolytica, Candida tropicalis, Candida utilis, Pichia pastoris, Sporobolomyces albo-rubescens, Sporopachydermia cereana or Trigonopsis variabilis. Quantitative enzyme assays enabled the relative titre of antiserum against the various methylamine oxidases to be determined. 2. The amine oxidases from two non-cross-reacting species, C. utilis and P. pastoris, were purified to near homogeneity. 3. The methylamine oxidases, despite their serological non-similarity, showed very similar catalytic properties to methylamine oxidase from C. boidinii. Their heat-stability, pH optima, molecular weights, substrate specificities and sensitivity to inhibitors are reported. 4. The benzylamine oxidases of C. utilis and P. pastoris both oxidized putrescine, and the latter enzyme failed to show any cross-reaction with antibody to C. boidinii methylamine oxidase. Benzylamine oxidase from C. boidinii itself also did not cross-react with antibody to methylamine oxidase. The heat-stability, molecular weights, substrate specificities and sensitivity to inhibitors of the benzylamine/putrescine oxidases are reported. 5. The benzylamine/putrescine oxidase of C. utilis differed only slightly from that of C. boidinii. 6. Benzylamine/putrescine oxidase from P. pastoris differed from the Candida enzymes in heat-stability, subunit molecular weight and substrate specificity. In particular it catalysed the oxidation of the primary amino groups of spermine, spermidine, lysine, ornithine and 1,2-diaminoethane, which are not substrates for either of the Candida benzylamine oxidases that have been purified. 7. Spermine and spermidine were oxidized at both primary amino groups; in the case of spermidine this is a different specificity from that of plasma amine oxidase. 8. Under appropriate conditions, P. pastoris benzylamine/putrescine oxidase (which is very easy to purify) can be a useful analytical tool in measuring polyamines.     

A comparative study of glycerol and sorbitol as co-substrates in methanol-induced cultures of Pichia pastoris: temperature effect and scale-up simulation

Journal of Industrial Microbiology & Biotechnology - The production of recombinant proteins by Pichia pastoris under AOX1 promoter is usually performed using methanol together with either...     

Synthesis of allysine ethylene acetal using phenylalanine dehydrogenase from Thermoactinomyces intermedius

Allysine ethylene acetal [(S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (2)] was prepared from the corresponding keto acid by reductive amination using phenylalanine dehydrogenase (PDH) from Thermoactinomyces intermedius ATCC 33205. Glutamate, alanine, and leucine dehydrogenases, and PDH from Sporosarcina species (listed in order of increasing effectiveness) also gave the desired amino acid but were less effective. The reaction requires ammonia and NADH. NAD produced during the reaction was recyled to NADH by the oxidation of formate to CO(2) using formate dehydrogenase (FDH). PDH was produced by growth of T. intermedius ATCC 33205 or by growth of recombinant Escherichia coli or Pichia pastoris expressing the Thermoactinomyces enzyme. Using heat-dried T. intermedius as a source of PDH and heat-dried Candida boidinii SC13822 as a source of FDH,98%, but production of T. intermedius could not be scaled up. Using heat-dried recombinant E. coli as a source of PDH and heat-dried Candida boidinii 98%. In a third generation process, heat-dried methanol-grown P. pastoris expressing endogenous FDH and recombinant Thermoactinomyces98% ee.     

SILAC compatible strain of Pichia pastoris for expression of isotopically labeled protein standards and quantitative proteomics

The methylotrophic yeast Pichia pastoris is a powerful eukaryotic platform for the production of heterologous protein. Recent publication of the P. pastoris genome has facilitated strain development toward biopharmaceutical and environmental science applications and has advanced the organism as a model system for the study of peroxisome biogenesis and methanol metabolism. Here we report the development of a P. pastoris arg-/lys- auxotrophic strain compatible with SILAC (stable isotope labeling by amino acids in cell culture) proteomic studies, which is capable of generating large quantities of isotopically labeled protein for mass spectrometry-based biomarker measurements. We demonstrate the utility of this strain to produce high purity human serum albumin uniformly labeled with isotopically heavy arginine and lysine. In addition, we demonstrate the first quantitative proteomic analysis of methanol metabolism in P. pastoris, reporting new evidence for a malate-aspartate NADH shuttle mechanism in the organism. This strain will be a useful model organism for the study of metabolism and peroxisome generation.     

Effect of Chronic Ethanol Feeding on Oxysterols in Rat Liver

It was our hypothesis that, as a consequence of increased oxidative stress, cholesterol-derived hydroperoxides and oxysterols are increased in livers of rats exposed to ethanol. To test this we dosed Wistar rats (approximately 0.1 kg initial body weight) with ethanol chronically (rats fed a nutritionally complete liquid diet containing ethanol as 35% of total calories; sampled liver at approximately 6-7 weeks). We measured concentrations of 7 &#102 - and 7 &#103 -hydroperoxycholest-5-en-3 &#103 -ol (7 &#102 -OOH and 7 &#103 -OOH) as well as 7 &#102 - and 7 &#103 -hydroxycholesterol (7 &#102 -OH and 7 &#103 -OH), and 3 &#103 -hydroxycholest-5-en-7-one (also termed 7-ketocholesterol; 7-keto). In response to chronic alcohol feeding, there were significant elevations in the concentrations of 7 &#102 -OOH (+169%, P =0.005 ) and 7 &#103 -OOH (+199%, P =0.011 ). Increases in the concentrations of hepatic 7-keto (+74%, P =0.01 ) and decreases in cholesterol ( &#109 43%; P =0.03 ) also occurred. In contrast, the concentrations of both 7 &#102 -OH and 7 &#103 -OH were not significant (NS). However, when oxysterols in chronic ethanol-fed rats were expressed relative to cholesterol there were significant increases in 7-keto/cholesterol ( P =0.0006), 7 &#102 -OH/cholesterol ( P =0.0018) and 7 &#103 -OH/cholesterol ( P =0.0047). In conclusion, this is the first report of increased 7 &#102 -OOH, 7 &#103 -OOH, and 7-keto in liver of rats and their elevation in chronic experimental alcoholism represent evidence of increased oxidative stress.     

Production and purification of recombinant human granulocyte–macrophage colony stimulating factor (GM-CSF) from high cell density cultures of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Granulocyte–macrophage colony stimulating factor (GM-CSF) is a hematopoietic growth factor, which has been used as a therapeutic agent in clinical cases like neutropenia. In this study, we report the production of recombinant human GM-CSF in the methylotrophic yeast Pichia pastoris through secretory expression using the inducible AOX1 promoter. Recombinant P. pastoris GS115 cells were grown in fed batch cultures to obtain a biomass density of 55.6 gDCW L⁻¹ and a high volumetric activity of 131 mg L⁻¹ of GM-CSF. The protein migrated as a diffuse band on SDS-PAGE at the range of 28–35 kDa indicating differential glycosylation. The secreted protein was purified to 95% in two steps using cation exchange and size exclusion chromatography.     

A note on the yeast flora associated with fermentation of mango

Fifteen morphologically different groups of yeasts consisting of Metschnikowia pulcherrima, Trichosporon cutaneum, Kloeckera apiculata, Torulopsis Candida, Tor. glabrata. Tor. apicola, Candida tropicalis, Cand. krusei, Cand. sorbosa, Cand. diversa, Pichia terricola, Pic. membranaefaciens, Hyphopichia burtonii, Rhodotorula graminis and Aureobasidium pullulans were isolated from fresh, fermenting and fermented juice of two varieties of mango. This is the first report on the study of yeast flora of mango and also on the occurrence of Hyp. burtonii on fruits.     

Lipid Peroxidation Inhibition Reduces NF-κB Activation and Attenuates Cerulein-induced Pancreatitis

Increased lipid peroxidation, enhanced nuclear factor kappa-B (NF- &#115 B) activation and augmented tumor necrosis factor- &#102 (TNF- &#102 ) production have been implicated in cerulein-induced pancreatitis. We investigated whether lipid peroxidation inhibition might reduce NF- &#115 B activation and the inflammatory response in cerulein-induced pancreatitis. Male Sprague-Dawley rats of 230-250 g body weight received administration of cerulein (80 &#119 g/kg s.c. for each of four injections at hourly intervals). A control group received four s.c. injections of 0.9% saline at hourly intervals. Animals were randomized to receive either raxofelast, an inhibitor of lipid peroxidation (20 mg/kg i.p. administered with the first cerulein injection) or its vehicle (1 ml/kg of a 10% DMSO/NaCl solution). All these rats were sacrificed 2 h after the last injection of either cerulein or its vehicle. Raxofelast administration (20 mg/kg i.p. with the first cerulein) significantly reduced malondialdehyde (MDA) levels, an index of lipid peroxidation (CER+DMSO=3.075 &#45 0.54 &#119 mol/g; CER+raxofelast= 0.693 &#45 0.18 &#119 mol/g; p <0.001 ), decreased myeloperoxidase (MPO) activity ( CER+DMSO=22.2 &#45 3.54 mU/g; CER+raxofelast=9.07 &#45 2.05 mU/g; p <0.01 ), increased glutathione levels (GSH) (CER+DMSO= 5.21 &#45 1.79 &#119 mol/g; CER+raxofelast=15.71 &#45 2.14 &#119 mol/g; p <0.001 ), and reduced acinar cell damage evaluated by means of histology and serum levels of both amylase ( CER+DMSO=4063 &#45 707.9 U/l; CER+raxofelast=1198 &#45 214.4 U/l; p <0.001 ), and lipase (CER+DMSO=1654 &#45 330 U/l; CER+raxofelast= 386 &#45 118.2 U/l; p <0.001 ), Furthermore, raxofelast reduced pancreatic NF- &#115 B activation and the TNF- &#102 mRNA levels and tissue content of mature protein in the pancreas. Indeed, lipid peroxidation inhibition might be considered a potential therapeutic approach to prevent the severe damage in acute pancreatitis.     

Production of large quantities of isotopically labeled protein in Pichia pastoris by fermentation

Heterologous expression in Pichia pastoris has many of the advantages of eukaryotic expression, proper folding and disulfide bond formation, glycosylation, and secretion. Contrary to other eukaryotic systems, protein production from P.pastoris occurs in simple minimal defined media making this system attractive for production of labeled proteins for NMR analysis. P.pastoris is therefore the expression system of choice for NMR of proteins that cannot be refolded from inclusion bodies or that require post-translational modifications for proper folding or function. The yield of expressed proteins from P.pastoris depends critically on growth conditions, and attainment of high cell densities by fermentation has been shown to improve protein yields by 10–100-fold. Unfortunately, the cost of the isotopically enriched fermentation media components, particularly 15NH4OH, is prohibitively high. We report fermentation methods that allow for both 15N- labeling from (15NH4)2SO4 and 13C-labeling from 13C-glucose or 13C-glycerol of proteins produced in Pichia pastoris. Expression of an 83 amino acid fragment of thrombomodulin with two N-linked glycosylation sites shows that fermentation is more cost effective than shake flask growth for isotopic enrichment.     

Sul folobus sol fataticus P2嗜热嗜酸α-淀粉酶在大肠杆菌和毕赤酵母中的表达

通过PCR方法从Sulfolobus solfataticus P2中扩增到2．6kb的α-淀粉酶基因（SS01172）,将其分别克隆到表达载体pET32a（＋）和pPICZaA,并在E．coliRosetta和Pichia pastoris GS115中进行表达。结果表明α-淀粉酶基因在Rosetta中得到了高效表达,酶活为143．466U／mL;而在GS115中表达量稍低,发酵液酶活力为98．102U／mL。     

A note on the presence of novel DNA species in the spoilage yeasts Zygosaecharomyces bailii and Pichia membranaefaciens

P ainting , K.A. & K irsop , B arbara , 1984. A note on the presence of novel DNA species in the spoilage yeasts Zygosaecharomyces bailii and Pichia membranaefaciens. Journal of Applied Bacteriology 56 , 331–336.
Two novel covalently closed circular DNA species of 5.4 and 6.0 kilobases were detected in strains of Zygosaecharomyces bailii with a rapid small scale isolation procedure. The 5.4 kb species was found in four strains and both species were found in three strains. A novel, covalently-closed circular DNA species of 6.9 kb was detected in four of 12 strains of Pichia membranaefaciens . Plasmid DNA (2 μm) (that is CCC DNA of approximately 6 kb in Saccharomyces cerevisiae) was detected in 38 of 40 strains of Sacch. cerevisiae confirming reports of the widespread distribution of this plasmid.     

Cloning, sequence analysis, and characterization of the 'lysyl oxidase' from Pichia pastoris

Lysyl oxidase from Pichia pastoris has been successfully overexpressed. EPR and resonance Raman experiments have shown that copper and TPQ are present, respectively. Lysyl oxidase from P. pastoris has a similar substrate specificity to the mammalian enzyme (both have been shown to oxidize peptidyl lysine residues) and is 30% identical to the human kidney diamine oxidase (the highest of any non-mammalian source). This enzyme also has a relatively broad substrate specificity compared to other amine oxidases. Molecular modeling data suggest that the substrate channel in lysyl oxidase from P. pastoris permits greater active site access than observed in structurally-characterized amine oxidases. This larger channel may account for the diversity of substrates that are turned over by this enzyme.     

Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris: functional characterisation of a novel promoter

Applied Microbiology and Biotechnology - As Pichia pastoris (syn. Komagataella sp.) yeast can secrete pure recombinant proteins at high rates, it is a desirable production system. The function of a...     

Biosynthetic production of type II fish antifreeze protein: fermentation by Pichia pastoris

Sea raven type II antifreeze protein (SRAFP) is one of three different fish antifreeze proteins isolated to date. These proteins are known to bind to the surface of ice and inhibit its growth. To solve the three-dimensional structure of SRAFP, study its ice-binding mechanism, and as a basis for engineering these molecules, an efficient system for its biosynthetic production was developed. Several different expression systems have been tested including baculovirus, Escherichia coli and yeast. The latter, using the methylotrophic organism Pichia pastoris as the host, was the most productive. In shake-flask cultures the levels of SRAFP secreted from Pichia were up to 5 mg/l. The recombinant protein has an identical activity to SRAFP from sea raven serum. In order to increase yields further, four different strategies were tested in 10-l fermentation vessels, including: (1) optimization of pH and dissolved oxygen, (2) mixed feeding of methanol and glycerol with Mut^s clones, (3) supplementation of amino acid building blocks, and (4) methanol feeding with Mut⁺ clones. The mixed-feeding/Mut^s strategy proved to be the most efficient with SRAFP yields reaching 30 mg/l. Received: 19 November 1996 / Received revision: 29 January 1997 / Accepted: 7 March 1997     

A simple structured model for recombinant IDShr protein production in Pichia pastoris

A simple structured model is proposed for the methanol production phase of the iduronate 2-sulphate sulfatase recombinant enzyme (IDShr) in Pichia patoris Mut(+). The model is mainly focused in oxidative stress phenomenon due to methanol consumption and based on extracellular experimental information and the basic knowledge of methanol metabolism in Pichia pastoris yeast (P. pastoris). The model's prediction shows a reasonable accuracy as compared with the experimental data. Likewise, it was proved that this model is able to simulate the production of other recombinant protein in P. pastoris.     

球孢白僵菌丝氨酸蛋白酶基因CDEP-1在毕赤酵母中的表达

我们从球孢白僵菌中克隆了丝氨酸蛋白酶Pr1类基因CDEP-1。为明确CDEP-1的功能、评价其在害虫生物防治中的潜力,需要大量制备具有生物活性的CDEP-1编码蛋白。由于大肠杆菌系统表达真核基因存在产物复性困难的问题,本文利用毕赤酵母系统来表达CDEP-1。结果表明,CDEP-1可在毕赤酵母中高效的分泌表达,而且产物活性高,甲醇诱导48h后上清液中的酶活即可达到38,266U/L。诱导表达的上清液经浓缩后进行凝胶过滤层析,得到了CDEP-1的初纯品,蛋白质含量为50mg/L。将纯化的蛋白酶CDEP-1免疫家兔,制备了CDEP-1的抗血清。Westernblotting分析表明,制备的抗血清可特异性地检测CDEP-1。     

Use of HDEL-tagged Trichoderma reesei mannosyl oligosaccharide 1,2-alpha-D-mannosidase for N-glycan engineering in Pichia pastoris

Therapeutic glycoprotein production in the widely used expression host Pichia pastoris is hampered by the differences in the protein-linked carbohydrate biosynthesis between this yeast and the target organisms such as man. A significant step towards the generation of human-compatible N-glycans in this organism is the conversion of the yeast-type high-mannose glycans to mammalian-type high-mannose and/or complex glycans. In this perspective, we have co-expressed an endoplasmic reticulum-targeted Trichoderma reesei 1,2-alpha-D-mannosidase with two glycoproteins: influenza virus haemagglutinin and Trypanosoma cruzi trans-sialidase. Analysis of the N-glycans of the two purified proteins showed a >85% decrease in the number of alpha-1,2-linked mannose residues. Moreover, the human-type high-mannose oligosaccharide Man(5)GlcNAc(2) was the major N-glycan of the glyco-engineered trans-sialidase, indicating that N-glycan engineering can be effectively accomplished in P. pastoris.     

Quantification of glycosidase activities in selected yeasts and lactic acid bacteria

Using a model system, the activities of α-L-arabinofuranosidase, β-glucosidase, and α-L-rhamonopyranosidase were determined in 32 strains of yeasts belonging to the genera Aureobasidium, Candida, Cryptococcus, Hanseniaspora, Hansenula, Kloeckera, Metschnikowia, Pichia, Saccharomyces, Torulaspora and Brettanomyces (10 strains); and seven strains of the bacterium Leuconostoc oenos. Only one Saccharomyces strain exhibited β-glucosidase activity, but several non-Saccharomyces yeast species showed activity of this enzyme. Aureobasidium pullulans hydrolyzed α-L-arabinofuranoside, β-glucoside, and α-L-rhamnopyranoside. Eight Brettanomyces strains had β-glucosidase activity. Location of enzyme activity was determined for those species with enzymatic activity. The majority of β-glucosidase activity was located in the whole cell fraction, with smaller amounts found in permeabilized cells and released into the growth medium. Aureobasidium pullulans hydrolyzed glycosides found in grapes. Received 02 February 1999/ Accepted in revised form 26 June 1999     

Yeast diversity and killer activity dispersed in fecal pellets from marsupials and rodents in a Brazilian tropical habitat mosaic

Yeasts with counts of above 10⁶ g⁻¹ wet weight and high diversity were found in the fecal pellets of rodents and marsupials from a mosaic of forest fragments, grasslands, cultivated fields and pasture in Rio de Janeiro. The most frequently isolated yeasts were Debaryomyces hamsenii, Pichia membranaefaciens and Issatchenkia orientalis (and its anamorph Candida krusei ), probably reflecting a high fruit content in the diet of these animals. The opportunistic pathogens Candida albicans, Candida glabrata and Candida tropicalis were isolated at lower frequency. Some Pichia anomala and P. membranaefaciens cultures had killer activity affecting many of the other isolates. These animals can be involved in dispersal of yeasts.     

Pseudomonas rhodesiae PF1: A New and Efficient Biocatalyst for Production of Isonovalal from α-pinene Oxide

A new bacterial strain, identified as Pseudomonas rhodesiae PF1 and deposited under the accession number CIP 107491, is presented. It is very active for the production of the acyclic compound Z-2-methyl-5-isopropyl-hexa-2,5-dien-1-al (isonovalal) from &#102 -pinene oxide. Enzyme synthesis is induced by culturing cells on &#102 -pinene; growing bacteria also have the ability to synthesize the epoxide derivative of &#102 -pinene. Isonovalal production was performed without aeration and with concentrated resting cells previously frozen at &#109 20°C, and subsequently thawed in a water-organic solvent, two-phase system. The organic layer was hexadecane, the volume ratio being 1:1. The best results achieved allowed recovery of c.a. 60 g/l organic solvent of isonovalal in 2.5 h operation, which is the most efficient process to date in the area of terpene biotransformations.