FORMATION OF HIGHER ALCOHOLS FROM 14C-LABELLED VALINE AND LEUCINE

The formation of isobutanol and 3-methylbutanol during alcoholic fermentation by brewer's yeast has been studied by adding uniformly ¹⁴C-labelled valine and leucine to the complex amino acid mixture which is used as nitrogen source. In the presence of ¹⁴C-leucine, only 3-methylbutanol becomes radioactive whereas, in the presence of ¹⁴C-valine, both isobutanol and 3-methylbutanol acquire label. The specific radioactivity of these compounds is, in all cases, inferior to that of the parent amino acids showing that a part of these higher alcohols is always formed by the synthetic functions of the yeast. The results suggest that the regulating effect of the valine and leucine level of the medium on the synthesis of these amino acids in yeast cells is rather limited. They furthermore indicate that intact assimilation of amino acids is not the only mode of their utilization, even in media with excessive levels of amino acids. An attempt is made to explain the dependence of the anabolic and catabolic modes of higher alcohol formation upon the nitrogenous nutrient level of the medium. The presence of radioactive isoleucine as impurity in the ¹⁴C-leucine used has allowed an approximative calculation of the formation of 2-methylbutanol from isoleucine which is very similar to the formation of isobutanol from valine.     

ABSORPTION OF AMINO ACIDS BY YEASTS FROM A SEMI-DEFINED MEDIUM SIMULATING WORT

The biosynthetic pathways involved when α-amino acids are absorbed by yeasts from a semi-defined medium simulating wort have been measured using ¹⁵N and ¹⁴C isotopes. When grown in this medium under brewery conditions, a complex transaminase equilibrium system operates within the cell, showing that the theory of intact assimilation of amino acids into yeast protein is invalid. A relatively high level of specificity was found in respect of the transfer of carbon skeletons of amino acids in the medium to carbon skeletons of yeast protein amino acids and the contributions in respect of each amino acid have been measured throughout growth. Simple sugars have been shown to contribute significantly to the carbon skeletons of most amino acids found in yeast and the extent of these syntheses has been quantitatively assessed.     

Simultaneous utilization of ammonia,free amino acids and peptides during fermentative growth of Saccharomyces cerevisiae

The efficiency of nitrogen use by yeast is one of the key determinants of the successful completion of alcoholic fermentations. In this work the growth of Saccharomyces cerevisiae S288c in a synthetic medium containing ammonia and free amino acids, supplemented with yeast hydrolysate, was studied. Experiments with ¹⁵NH₄Cl and ¹⁵N‐labelled yeast hydrolysate were carried out to gain insight into which of these three classes of assimilable nitrogen sources yeast cells prefer. Co‐consumption of all three sources was observed; approximately 40% of the total nitrogen in the yeast protein fraction originated from yeast hydrolysate, while free amino acids and ammonia contributed 40 and 20%, respectively. The results indicate that several amino acids are more readily obtained from peptides, most likely when the uptake of their free forms is competitively inhibited and/or repressed. During the second half of each fermentation, a decrease in the incorporation of yeast hydrolysate‐derived nitrogen was observed. These results highlight the nutritional role of peptides in various yeast fermentations. Copyright © 2016 The Institute of Brewing & Distilling     

Production of Candida utilis biomass as aquaculture feed

Fish offal-peat compost was hydrolysed and the resultant liquid extracts used as substrate in the cultivation of the yeast Candida utilis. The yeast was able to grow in this culture medium, attaining a growth yield of approximately 260 g biomass kg^?1 total carbohydrate consumed. The biomass produced had a good amino acid composition, with a protein content of 520 g kg^?1, and a relatively low lipid content. Dry yeast was used as a feed component in the diet of cultivated rainbow trout (Oncorhynchus mykiss) in which it substituted well for other, traditional sources of protein.     

Expanding the genetic code of Saccharomyces cerevisiae with methionine analogues

We replaced the single N‐terminal methionine in heterologously expressed human Cu/Zn superoxide dismutase with the non‐canonical methionine analogues homopropargylglycine and norleucine in the yeast Saccharomyces cerevisiae. Our non‐canonical amino acid incorporation protocol involves a two‐step procedure. In the first step, the methionine auxotrophic yeast cells are accumulated in synthetic medium containing methionine while the target protein production is shut off. After a short methionine depletion phase, the cells are transferred to inducing medium that contains the methionine analogue instead of methionine and target protein expression is switched on. The initially low level incorporation of ～12% could be elevated to 40% by increasing the non‐canonical amino acid concentration in the medium by 10‐fold. With this approach we were able to produce up to 5 mg substituted protein per litre of yeast culture. Copyright © 2008 John Wiley & Sons, Ltd.     

Secretion of a variant of human single-chain urokinase-type plasminogen activator without an N-glycosylation site in the methylotrophic yeast,Pichia pastoris and characterization of the secreted product

Human single-chain urokinase-type plasminogen activator without an N-glycosylation site (scu-PA-Q³⁰²) was produced in the methylotrophic yeast, Pichia pastoris using the shortened prepeptide sequence of a fungal aspartic proteinase, Mucor pusillus rennin (MPR). The level of urokinase-type plasminogen activator (u-PA) immunoreactive material in YPM medium was 0·47 mg/l; however, most of the secreted product had been processed to smaller polypeptides. The N-terminal amino acid sequence of major species was identical to that of the low molecular weight two-chain u-PA. Some approaches to minimizing the proteolysis of scu-PA-Q³⁰² were attempted. Addition of Triton X-100, l-arginine and ammonium phosphate to the YPM medium minimized the proteolysis of scu-PA-Q³⁰² and increased the yield of immunoreactive material to approximately 5 mg/l. Use of proteinase A- or proteinase B-deficient strains of yeast did not reduce the degradation. Co-expression of scu-PA-Q³⁰² and urinary trypsin inhibitor resulted in partial reduction of the major species of proteolysis. Scu-PA-Q³⁰² was purified from the culture supernatant of the improved medium by two successive chromatographies on Phenyl-Sepharose and S-Sepharose. The purified protein had a molecular weight of 47 kDa. It did not contain detectable N-linked oligosaccharides, but contained O-linked oligosaccharides attached to the light chain. N-terminal amino acid sequencing of the purified preparation showed that the shortened prepeptide sequence of MPR was correctly processed by the Pichia yeast. Scu-PA-Q³⁰² closely resembles natural scu-PA with respect to its enzymatic activity against the chromogenic substrate S-2444 and its in vitro fibrinolytic properties.     

Cloning and characterization of the Hansenula polymorpha homologue of the Saccharomyces cerevisiae PMR1 gene

A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Hansenula polymorpha. The partial DNA fragment of the H. polymorpha homologue was initially obtained by a polymerase chain reaction and used to isolate the entire gene which encodes a protein of 918 amino acids. The putative gene product contains all ten of the conserved regions observed in P-type ATPases. The cloned gene product exhibits 60·3% amino acid identity to the S. cerevisiae PMR1 gene product and complemented the growth defect of a S. cerevisiae pmr1 null mutant in the EGTA-containing medium. The results demonstrate that the H. polymorpha gene encodes the functional homologue of the S. cerevisiae PMR1 gene product, a P-type Ca²⁺-ATPase. The DNA sequence of the H. polymorpha homologue has been submitted to GenBank with the Accession Number U92083. © 1998 John Wiley & Sons, Ltd.     

Regulating Flavor Compound Synthesis and Cofactor Recycling in Heterogeneous Enzymatic Reactions by Mixtures of Bacterial Cell-Free Extracts

Enzymatic cofactor recycling and generation of acetic acid, 3-methylbutanal, and 3-methylbutanol were evaluated using a mixture of cell-free extracts of Gluconobacter oxydans and Streptococcus lactis var. maltigenes. Ethanol was used as a substrate by cell-free extracts of G, oxydans to produce acetic acid and to generate reduced nicotinamide adenine dinucleotide from nicotinamide adenine dinucleotide. Leucine was converted by S. lactis var. maltigenes cell-free extracts to 3-methylbutanal and 3-methylbutanol and to regenerate nicotinamide adenine dinucleotides. Optimum pH to form 3-methylbutanal by the combined cell-free extracts was about 6.8 and that for 3-methylbutanol was 6.4. At 30°C, 3-methylbutanol reached a maximum of 73 ppm after 30 h but declined slightly at 48 h. Concentrations of 3-methylbutanol increased gradually over 48 h to a maximum of 56 ppm. Rates of production of these compounds at 12.8°C were about half that at 32°C. Concentrations of 3-methylbutanol were increased to about those of 3-methylbutanal by adjusting the concentrations of substrate, nicotinamide adenine dinucleotide, cell-free extract, and pH so as to produce large amounts of acetic acid and reduced nicotinamide adenine dinucleotide.     

Studies on Wheat Steep Liquor Produced During Starch Production

Chemical analysis of wheat steeping liquor, produced from soaking of wheat grains revealed the presence of 1% total soluble solids with 7,15% proteins. A method was devised to obtain large amounts of precipitates with protein content up to 9,84%. Glutamic acid and lysine constitute the largest proportions of the amino acids. Nine essential amino acids were found. The raw wheat steeping liquor and the supernatant resulting after separation of the protein were used as a growth medium for the production of fodder yeast Candida tropicalis.     

Microencapsulation of Cell-Free Extracts to Demonstrate the Feasibility of Heterogeneous Enzyme Systems and Cofactor Recycling for Development of Flavor in Cheese

A mixture of a cell-free extract from Gluconobacter oxydans that produced acetic acid from ethanol and a cell-free extract from Streptococcus lactis var. maltigenes, which converted leucine to 3-methylbutanal and 3-methylbutanol, were coencapsulated with substrate and nicotinamide adenine dinucleotide in a milk fat coat. The capsules were incubated as 12.8, 22, and 32°C to measure production of 3-methylbutanal, 3-methylbutanol, and acetic acid and recycling of nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide. Maximum amounts of 3-methylbutanal and 3-methylbutanol were produced after 48 h at 22°C, demonstrating the feasibility of encapsulating a combined CFE to recycle cofactors and produce an array of products. Capsules containing the combined cell-free extract were added to skim milk and milk containing 1.1% fat to produce cheeses with reduced fat. About 16% of the capsules were lost in whey during manufacturing of cheese from skim milk with added capsules but less for cheese made with 1.1% fat plus capsules. During ripening at 7.2 and 12.8°C for 1, 3, and 6 mo, cheeses containing whole capsules with a complete cell-free extract mixture contained more 3-methylbutanal and 3-methylbutanol and exhibited a stronger malty taste than cheeses made with broken capsules or an incomplete cell-free extract mixture.     

AMINO ACID BALANCE IN YEAST FERMENTATION WITH A SYNTHETIC AMINO ACID MIXTURE AS NITROGEN SOURCE

Two brewery yeasts, one bottom- and one top-fermenting strain, were allowed to ferment an 8% glucose solution containing as nitrogen source an amino acid mixture simulating that obtained when yeast was autolysed. The amounts given were approximately twice as high as the expected requirements. After completion of fermentation the total amounts of each amino acid in the whole system, i. e., in medium and yeast, were determined. The results show that the yeast had not taken up amino acids according to its own composition. The amino acids previously found to be rapidly absorbed from brewery wort were present in the whole system in considerably smaller amounts than in the original medium, indicating that these acids had been utilized as a nitrogen souce or for other purposes. The acids which are taken up slowly from brewery wort were present in larger amounts than in the original medium, indicating that they had been synthesized despite the excess in the medium. The two strains showed relatively similar behaviour.     

MACERATION ACTIVITY OF AN ENDOPOLYGALACTURONASE FROM CANDIDA MACEDONIENSIS

The yeast Candida macedoniensis produces constitutively an extracellular pectinolytic enzyme with a high maceration activity; the culture filtrate is free from foreign enzyme activity. The enzyme formation is optimal under strictly anaerobic conditions with N₂ gassing. The culture medium for optimal enzyme recovery is a nutrient solution containing 1% yeast extract, 2% peptone and 10% sucrose, at pH 3, 28°C. Characterization of the enzyme showed it to be an endopolygalacturonase. The pH and temperature optima of the enzyme differ for pectic acid cleavage and maceration activity, these being 4.5 and 50—53°C and 2.5 and 40°C, respectively. The enzyme activities could not be separated from one another by protein chemical methods (analytical and preparative isoelectric focussing). Dialysis of the enzyme-containing culture filtrate did not decrease the enzyme activity. The endopolygalacturonase from Candida macedoniensis leads to the release of plant cells from the tissue, without destroying the cells by lysing the cell walls. After two hours incubation of the substrate (carrot slices), the tissue mass consisted of cell clumps of up to 15 cells.     

ENZYMIC PROTEIN HYDROLYSATES FROM MALT SPROUTS

Recent literature on the use of malt sprouts is reviewed. Studies on the hydrolysis of malt sprouts by alkaline protease were carried out to determine the optimal conditions required. Under these conditions an overall 40% solubilisation of the malt sprouts was achieved which incorporated a 65% solubilisation of the total protein content. Amino nitrogen content of the malt sprouts reached 0.8% on hydrolysis. The extract from the hydrolysed malt sprouts contained approximately 40% hydrolysed protein, 40% dextrins and salts. Evaporation of this extract produces a viscous syrup and spray drying produces a hydroscopic powder containing 1.8–2.0% amino nitrogen. The protein part of the hydrolysate extract is balanced with respect to its essential amino acid content and has a low bitter taste. Data from experiments carried out on the effect of the extract on yeast fermentation indicate a significant stimulation and possible applications in the food industry.     

A 37·5 kb region of yeast chromosome X includes the SME1, MEF2, GSH1 and CSD3 genes,a TCP-1-related gene,an open reading frame similar to the DAL80 gene,and a tRNAArg

The complete DNA sequence of cosmid clone p59 comprising 37,549 bp derived from chromsome X was determined from an ordered set of subclones. The sequence contains 14 open reading frames (ORFs) containing at least 100 consecutive sense codons. Four of the ORFs represent already known and sequenced yeast genes: B645 is identical to the SME1 gene encoding a protein kinase, required for induction of meiosis in yeast, D819 represents the MEF2 gene probably encoding a second mitochondrial elongation factor-like protein, D678 is identical to the yeast GSH1 gene encoding γ-glutamylcysteine synthetase and B746 is identical to the CSD3 gene, which plays an as yet unidentified role in chitin biosynthesis and/or its regulation. The deduced amino acid sequence of A550 is 63% identical to the Ccη subunit of a murine TCP-1-containing chaperonin and more than 35% identical to thermophilic factor 55 from Sulfolobus shibatae, as well as to a number of proteins belonging to the chaperonin TCP-1 family. Open reading frame F551 exhibits homology to two regions of the DAL80 gene located on yeast chromosome XI encoding a pleiotropic negative regulatory protein. In addition, extensive homology was detected in three regions including parts of ORFs A560, B746/CSD3 and the incomplete ORF C852 to three consecutive ORFs of unknown function in the middle of the right arm of chromosome XI. Finally, the sequence contained a tRNA^Arg3 (AGC) gene. The nucleotide sequence data reported in this paper have been deposited in the EMBL and GenBank databases under the accession number X85021.     

SOME CONSIDERATIONS OF THE FLOCCULATION CHARACTERISTICS OF ALE AND LAGER YEAST STRAINS*

While some ale yeast strains are able to flocculate when cultured in a defined medium of glucose, ammonium salts, vitamins and ions, others require the presence of a nitrogen-containing inducer in the growth medium. On the other hand, all flocculent lager strains examined to date are able to flocculate after being cultured in a defined medium and do not appear to require the addition of inducer material to the growth medium. The inducer material present in wort has been identified as peptide. By the use of ion exchange chromatography the peptide fraction that induces flocculation has been found to contain a high level of acidic amino acid residues with a very similar structure to that reported for the α-factor involved in sexual agglutination of haploid α and a cells of Sacch. cerevisiae. Studies on the adsorption of Ca⁺⁺ ion by the cell wall failed to reveal any significant differences in total uptake between flocculent and non-flocculent cultures. It would appear that Ca⁺⁺ ions are bound less tightly by non-flocculent cells than by flocculent cells. The contribution of calcium to flocculation is not the absolute amount of this ion adsorbed by the yeast cell wall but rather the stereo-specific manner by which it is bound, i.e., its position relative to the three-dimensional structure of the yeast cell wall.     

BIOSYNTHETIC FORMATION OF HIGHER ALCOHOLS BY YEAST. DEPENDENCE ON THE NITROGENOUS NUTRIENT LEVEL OF THE MEDIUM

In previous investigations it has been shown that in batch fermentations the yield of higher alcohols formed biosynthetically (in the sense that yeast synthesizes the carbon skeletons needed) is mainly negatively correlated with the intial level of nitrogenous nutrients in the medium. The validity of this relationship has been established with different nitrogen sources and sugars, and at different sugar concentrations. Its causes have been studied in simple systems, using single, rapidly-absorbed nitrogen sources, usually ammonium salts. Under such conditions it is the amount, rather than the concentration, of nitrogen source that is of importance. Yields of higher alcohols per unit weight sugar consumed are practically identical at varying nitrogen levels as long as nitrogen is available in the medium, but they increase sharply after nitrogen exhaustion, by a factor of about 2–3. However, if allowance is made for the production of valine, leucine, and isoleucine during the period when nitrogen is present, there is no rise in the total production of carbon skeletons along these pathways after the nitrogen is exhausted. On the contrary, the yields of valine-isobutanol and isoleucine-2-methylbutanol skeletons per unit weight of sugar are appreciably reduced in the absence of nitrogen, whereas the yields of leucine-3-methylbutanol skeletons are nearly unchanged.     

Extractability and functionality of protein from yeast cells grown on cassava hydrolysate

An extraction of protein from two strains of yeast, Candida tropicalis and C. utilis, grown by batch method on cassava hydrolysate was carried out by a combination of mechanical and mild chemical treatment yielding about 71% and 68% protein for C. tropicalis and C. utilis, respectively. The process resulted in about 75–80% reduction in the nucleic acid level of the freeze dried whole yeast cells of both organisms. The protein concentrates showed a good amino acid profile comparable to egg protein with methionine and tryptophan as the limiting amino acids. The functional characteristics of the two yeast proteins were studied and compared with soy protein concentrate. They showed functional properties possessing very good wettability, emulsion capacity and whippability but poor emulsion stability.     

Microfiltration of whey proteins adsorbed on yeast cells

Soluble whey proteins (WPs), adsorbed on yeast cells, were recovered by a crossflow microfiltration (MF) technique using a cellulose nitrate membrane with a pore size of 0.45 μm. The crossflow velocity was 1.5 m s^?1 with a transmembrane pressure of 200 kPa at 25 °C. A series of protein rejections occured at various pH values ranging from 2 to 8. WPs adsorbed more on to yeast cells at low pH (pH < 4) than at high pH values, probably because they were positively charged at low pH. It was also shown that permeate flux increased and Modified Membrane Fouling Index values decreased at low pH levels. When the yeast concentration was 50 g L^?1, the flux decreased five times compared with that in the absence of yeast. Protein recovery increased with increasing yeast concentrations. The highest protein recovery was found to be 85% at a yeast concentration of 50 g L^?1 at a steady state flux rate of 10^?6 m s^?1 at 25 °C. When diluted solutions of whey were used, the same rejection of protein, adsorbed on yeast cells, was achieved at ten times lower amounts of yeast cells. This technique not only provides for the recovery of protein but also may give rise to the direct use of yeast cells, which are rich in protein, in the baking industry. WPs absorbed by yeast cells can be used to produce nutritionally rich products in areas where yeasts have been already used.     

MODIFICATION OF THE LOW-MOLECULAR WEIGHT BASIC ALBUMIN FRACTION FROM RAPESEED (BRASSICA NAPUS L.) BY ACETYLATION PART 1. CHEMICAL AND PHYSICOCHEMICAL ASPECTS1

The chemical and physicochemical changes of the low-molecular weight basic albumin fraction from rapeseed, as a function of degree of acetylation, were studied using amino and ester groups analyses, PAGE electrophoresis, isoelectric focusing, viscometry, circular dichroism and fluorescence spectroscopy. The surface hydrophobicity was evaluated by means of the ANS fluorescence probe technique. The protein was readily acetylated at the amino groups by addition of acetic anhydride. Acetylation of amino acid hydroxyl groups was significantly slower and proceeded in the presence of an excess of the reagent after the amino groups had already been blocked. Acetylation resulted in protein species with isoelectric points at pH 7.6, 6.6, 5.95 and 5.4. The intrinsic viscosity of the native protein fraction dropped from 0.159 dlg^?1 to 0.038 dlg^?1 at a moderate degree of modification. The secondary structure of the protein, characterized by a content of 40–45% helix conformation, was not significantly influenced by acetylation. Modification did not result in wavelength shifts of the peaks in the near ultraviolet CD and fluorescence spectra. However, the negative ellipticities in the 250–270 nm region of the CD spectrum increased markedly with increasing degree of acetylation. The surface hydrophobicity increased linearly with the amount of acetyl groups introduced into the protein.     

重组降血压肽发酵培养基及发酵条件的优化实验研究

以纯化的融合蛋白包涵体表达量为考核指标,对重组降血压肽发酵培养基的组成和发酵条件进行了优化实验研究。以富含疏水性氨基酸的麦胚水解物为基础培养基,通过正交实验,优化得到大肠杆菌BL21-MF3A3制备重组降血压肽的麦胚培养基配方为:酵母粉0.5%、麦胚酶解物C2.0%、葡萄糖0.5%和NaCl0.5%。对发酵过程中的诱导条件进行正交优化实验,得到最佳的诱导条件为诱导温度32℃、诱导起始OD6001.4和诱导时间10h,在此条件下包涵体表达量可达到535mg·L-1。