A Novel Sesquiterpene,Tuberonone, from Solanum tuberosum L.

The effects of the ovarian hormones progesterone and estrone on the conversion of tryptophan to nicotinamide in rats were investigated. Female rats were fed for 35 days with a 20% casein diet, or with a 20% casein diet containing 0.1% progesterone, or 0.001% estrone, or 0.1% progestrone and 0.001% estrone. The conversion ratio of tryptophan to nicotinamide on the last day of the experiment was 2% in the groups fed with the 20% casein diet and the diet containing 0.1% progesterone, but around 1.2% in the group fed with 0.001% estrone, and 0.7% in the group fed with 0.1% progesterone and 0.001% estrone. These results demonstrated that administration of ovarian hormones significantly decreased the conversion of tryptophan to nicotinamide.     

Interaction and imbalance between indispensable amino acids in young piglets

Lowering protein level in diets for piglets urge to have knowledge on the piglet’s requirements for essential amino acids (AA) and their interactions. The present studies aimed to determine the interaction between the dietary level of valine (Val) and tryptophan (Trp) and the effect of AA imbalance at two levels of dietary Val on the growth performance of post-weaning piglets. In Experiment 1 (duration 4 weeks), the effects of supplementation of free l-Val (1.0 g/kg) and/or l-Trp (0.5 g/kg) in a low-CP diet (CP 17.7%), marginal in Trp and Val, was studied in a 2×2 factorial design and using an additional reference treatment (CP 19.5%). In Experiment 2 (duration 5 weeks), the influence of a stepwise increase in excess supply of isoleucine (Ile), histidine (His) and leucine (Leu), up to 10, 10% and 30% relative to their requirement values respectively, was evaluated at 60% or 70% standardized ileal digestible (SID) Val relative to SID lysine, using a 3×2 factorial design. In Experiment 1, over the whole experimental period, feed intake (FI) was affected by dietary Trp level (P<0.05) and feed conversion ratio (FCR) by both the level of Trp and Val in the diet (both P<0.05). Increasing Trp level increased FI and decreased FCR while increasing dietary Val level reduced FI and increased FCR. For BW gain (BWG), there was an interaction between dietary level of Trp and Val (P<0.05). Valine supplementation decreased BWG using a diet marginal in Trp, whereas it increased BWG when using a Trp sufficient diet. Piglets fed the low-CP diet with adequate levels of Val and Trp showed at least same performance compared to piglets fed the high CP reference diet. In Experiment 2, increasing dietary Val improved FI and BWG (P<0.001) and tended to improve FCR. Dietary AA excess for Ile, His and Leu reduced FI and BWG (P<0.05) and only affected FCR (P<0.01) in the 1^st week of the study. Dietary level of Val and AA excess did not show interactive effects, except for FCR over the final 2 weeks of the study (P<0.05). In conclusion, an interaction exists between dietary supply of Val and Trp on the zootechnical performance of post-weaning piglets and dietary AA excess for Ile, Leu and His, reduces growth performance of piglets in low-protein diets, independent of the dietary level of Val.     

Response of piglets to the valine content in diet in combination with the supply of other branched-chain amino acids

The branched-chain amino acids (BCAA) valine (Val) and isoleucine (Ile) are considered to be among the next-limiting amino acids for growth in piglets. In earlier studies, we estimated the standardized ileal digestible (SID) Val : Lys (lysine) requirement to be at least 70%, whereas the Ile : Lys requirement may be as low as 50%. Because the BCAA partially share a common route of catabolism, the supply of one BCAA may affect the availability of the other BCAA. Four experiments were conducted to determine the response of 6-week-old piglets to the Val supply in relation to the other BCAA. A deficient supply of Val or Ile typically results in a reduction in average daily feed intake (ADFI). Experiment 1 was designed to determine the effect of a limiting Val supply, independent of the effect on feed intake. In a dose-response study using restrictively fed piglets, nitrogen retention did not increase for an SID Val : Lys supply greater than 64%. In the remaining experiments, piglets were offered feed ad libitum using ADFI, average daily gain (ADG) and gain-to-feed ratio as response criteria. The interaction between the Val and leucine (Leu) was studied in Experiment 2 in a 2 × 2 factorial design (60% and 70% SID Val : Lys, and 111% and 165% SID Leu : Lys). Performance was considerably lower in piglets receiving 60% Val : Lys compared with those receiving 70% Val : Lys and was lowest in piglets receiving the diet with low Val and high Leu content. To further evaluate the interaction between Val and Leu, a dose-response study was carried out in which the response to Val supply was studied in combination with high Leu supply (165% Leu : Lys). Using a curvilinear-plateau model, the average SID Val : Lys requirement was 72%. However, low Val supply (60% SID Val : Lys) reduced performance by 13% to 38%, which was much greater than what we observed in earlier studies. Experiment 4 was carried out to test the hypothesis that the Val requirement is not affected by low Ile supply (50% SID Ile : Lys). Performance was not improved for Val : Lys supplies greater than 65%, which may indicate that Ile (and not Lys) was second-limiting in this study. In conclusion, the first response of piglets to deficient Val supply appears to be a reduction in ADFI, rather than a reduction in ADG or nitrogen retention. A large supply of Leu may not affect the Val requirement per se, but may aggravate the consequences of Val deficiency.     

Effect of Adding the Limiting Amino Acids to an Amino Acid Diet Simulating Rice Protein on the Conversion of Tryptophan to Nicotinamide in Rats

The effect of amino acid composition on the conversion ratio of tryptophan to nicotinamide was investigated. The ratio in the group fed with an amino acid diet simulating rice protein was around 2.5%. This ratio was statistically decreased by the addition of the limiting amino acids, except for tryptophan, and increased by the addition of all the limiting amino acids, including tryptophan. The composition of amino acids proved to greatly affect the conversion ratio.     

Packing and hydrophobicity effects on protein folding and stability: effects of beta-branched amino acids, valine and isoleucine, on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers.

The aim of this study was to examine the differences between hydrophobicity and packing effects in specifying the three-dimensional structure and stability of proteins when mutating hydrophobes in the hydrophobic core. In DNA-binding proteins (leucine zippers), Leu residues are conserved at positions "d," and beta-branched amino acids, Ile and Val, often occur at positions "a" in the hydrophobic core. In order to discern what effect this selective distribution of hydrophobes has on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers, three Val or three Ile residues were simultaneously substituted for Leu at either positions "a" (9, 16, and 23) or "d" (12, 19, and 26) in both chains of a model coiled coil. The stability of the resulting coiled coils was monitored by CD in the presence of Gdn.HCl. The results of the mutations of Ile to Val at either positions "a" or "d" in the reduced or oxidized coiled coils showed a significant hydrophobic effect with the additional methylene group in Ile stabilizing the coiled coil (delta delta G values range from 0.45 to 0.88 kcal/mol/mutation). The results of mutations of Leu to Ile or Val at positions "a" in the reduced or oxidized coiled coils showed a significant packing effect in stabilizing the coiled coil (delta delta G values range from 0.59 to 1.03 kcal/mol/mutation). Our results also indicate the subtle control hydrophobic packing can have not only on protein stability but on the conformation adopted by the amphipathic alpha-helices. These structural findings correlate with the observation that in DNA-binding proteins, the conserved Leu residues at positions "d" are generally less tolerant of amino acid substitutions than the hydrophobic residues at positions "a."     

Interactive effect between dietary valine and isoleucine on growth performance,blood parameters and resistance against low salinity stress of Japanese flounder Paralichthys olivaceus

The interaction between dietary valine (Val) and isoleucine (Ile) on growth, blood parameters, and tolerance against low salinity stress of Japanese flounder Paralichthys olivaceus was studied in a 60-day feeding trial. Fish (initial body weight: 3.57 ± 0.05 g) were fed six experimental diets containing different proportions of Ile and Val, two levels of Ile 0% (LI) and 2% diet (HI), combine with three levels of Val 0% (LV), 0.83% (MV), and 2.27% diet (HV), respectively. The results revealed that significant interactive effect of Ile and Val was found on body weight gain and feed conversion ratio, also stimulative effect was found in high Ile groups. Meanwhile, interactive effect of these two amino acids was also observed on high-density lipoprotein (HDL), the maximum of Ile with maximum of Val group has the highest concentration of HDL, which was significantly higher than other groups (p < .05). Moreover, no interaction was found on whole body proximate composition, muscle Ile and Val concentration, and tolerance ability against low salinity stress (reflected by LT₅₀). But high level of Ile combined with high level of Val diet altered tolerance ability against low salinity stress of Japanese flounder. It would be indicated that dietary Ile and Val interacted in the diet of Japanese flounder, and the flounders fed the diet supplemented with 2% Ile and 2.27% Val was superior to the other groups on growth and blood parameters, and tolerance against low salinity stress.     

Simple and rapid analytical method for detection of amino acids in blood using blood spot on filter paper, fast-GC/MS and isotope dilution technique

A simple and rapid method for quantitative analysis of amino acids, including valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met) and phenylalanine (Phe), in whole blood has been developed using GC/MS. In this method, whole blood was collected using a filter paper technique, and a 1/8 in. blood spot punch was used for sample preparation. Amino acids were extracted from the sample, and the extracts were purified using cation-exchange resins. The isotope dilution method using ²H₈-Val, ²H₃-Leu, ²H₃-Met and ²H₅-Phe as internal standards was applied. Following propyl chloroformate derivatization, the derivatives were analyzed using fast-GC/MS. The extraction recoveries using these techniques ranged from 69.8% to 87.9%, and analysis time for each sample was approximately 26 min. Calibration curves at concentrations from 0.0 to 1666.7 μmol/l for Val, Leu, Ile and Phe and from 0.0 to 333.3 μmol/l for Met showed good linearity with regression coefficients = 1. The method detection limits for Val, Leu, Ile, Met and Phe were 24.2, 16.7, 8.7, 1.5 and 12.9 μmol/l, respectively. This method was applied to blood spot samples obtained from patients with phenylketonuria (PKU), maple syrup urine disease (MSUD), hypermethionine and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), and the analysis results showed that the concentrations of amino acids that characterize these diseases were increased. These results indicate that this method provides a simple and rapid procedure for precise determination of amino acids in whole blood.     

A Simple Bioassay for Chemicals Active to the Photosynthetic or Respiratory Systems of Plants

After male rats of the Sprague Dawley strain, 5 weeks old, were fed a 20% casein diet with or without 0.5% nicotinamide for 13 days, 180 mg/kg body weight of alloxan was injected in- traperitoneally into the rats. The rats were kept for 18 days with the same diet. The level of blood glucose was increased 6-fold in the group on a 20% casein diet by the injection of alloxan, while there was only a 2-foid increase in the group on a nicotinamide-containing diet and the decreased body weight was also lower in the group on the nicotinamide diet than the group on the casein diet. The body weight was indirectly related to the concentration of blood glucose. A marked increase was observed in the activities of tryptophan oxygenase, aminocarboxymuconate-semialdehyde decarboxylase, and nicotinamide methyltransferase upon the injection of alloxan with both diets; on the other hand, the activities of kynureninase and NAD⁺ synthetase were decreased by the injection of alloxan. The activity of kynurenine aminotransferase increased in the group on the 20% casein diet by the injection of alloxan, while in the group on the nicotinamide-containing diet its activity was not increased by the injection. These changes in the above enzyme activities mean that the conversion ratio from tryptophan to niacin is lower in the alloxan diabetic rat than normal rat. It was found that the activities of tryptophan oxygenase, aminocarboxymuconate-semialdehyde decarboxylase, and nicotinamide methyltransferase were directly related to the concentration of blood glucose, and that the activities of kynureninase and NAD⁺ synthetase were inversely related. There was no difference in the activities of 3-hydroxyanthranilic acid oxygenase and nicotinamide mononucleotide adenylyltransferase upon the injection of alloxan with both diets.     

Structure-Guided Modification of Rhizomucor miehei Lipase for Production of Structured Lipids

To improve the performance of yeast surface-displayed Rhizomucor miehei lipase (RML) in the production of human milk fat substitute (HMFS), we mutated amino acids in the lipase substrate-binding pocket based on protein hydrophobicity, to improve esterification activity. Five mutants: Asn87Ile, Asn87Ile/Asp91Val, His108Leu/Lys109Ile, Asp256Ile/His257Leu, and His108Leu/Lys109Ile/Asp256Ile/His257Leu were obtained and their hydrolytic and esterification activities were assayed. Using Discovery Studio 3.1 to build models and calculate the binding energy between lipase and substrates, compared to wild-type, the mutant Asp256Ile/His257Leu was found to have significantly lower energy when oleic acid (3.97 KJ/mol decrease) and tripalmitin (7.55 KJ/mol decrease) were substrates. This result was in accordance with the esterification activity of Asp256Ile/His257Leu (2.37-fold of wild-type). The four mutants were also evaluated for the production of HMFS in organic solvent and in a solvent-free system. Asp256Ile/His257Leu had an oleic acid incorporation of 28.27% for catalyzing tripalmitin and oleic acid, and 53.18% for the reaction of palm oil with oleic acid. The efficiency of Asp256Ile/His257Leu was 1.82-fold and 1.65-fold that of the wild-type enzyme for the two reactions. The oleic acid incorporation of Asp256Ile/His257Leu was similar to commercial Lipozyme RM IM for palm oil acidolysis with oleic acid. Yeast surface-displayed RML mutant Asp256Ile/His257Leu is a potential, economically feasible catalyst for the production of structured lipids.     

Effects of fatty liver induced by niacin-free diet with orotic acid on the metabolism of tryptophan to niacin in rats

The effects of dietary orotic acid on the metabolism of tryptophan to niacin in weaning rats was investigated. The rats were fed with a niacin-free, 20% casein diet containing 0% (control diet) or 1% orotic acid diet (test diet) for 29 d. Retardation of growth, development of fatty liver, and enlargement of liver were observed in the test group in comparison with the control group. The concentrations of NAD and NADP in liver significantly decreased, while these in blood did not decrease compared to the control group. The formation of the upper metabolites of tryptophan to niacin such as anthranilic acid, kynurenic acid, and 3-hydroxyanthranilic acid were not affected, but the quinolinic acid and beyond, such as nicotinamide, N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and N1-methyl-4-pyridone-3-carboxamide, were significantly reduced by the administration of orotic acid. Therefore, the conversion ratio of tryptophan to niacin significantly decreased in the test group in comparison with the control group.     

The response of weaned piglets to dietary valine and leucine

Valine (Val) is considered to be the fifth-limiting amino acid in a maize–soyabean meal diet for pigs. Excess leucine (Leu) levels often occur in commercial diets, which may attenuate the effect of Val deficiency because of an increased oxidation of Val. The objective of the present experiment was to determine the effect of increasing concentrations of Leu on the response of young piglets to dietary Val. In all, 75 Large White×Landrace entire male pigs, 44 days of age and with a mean starting weight of 13.5 kg, were used. Three of these were sacrificed at the start to determine their mean initial chemical composition. A summit feed first limiting in Val was serially diluted with a non-protein diluent to produce a series of five digestible Val concentrations of 11.9, 10.1, 8.3, 6.6 and 4.8 g/kg, with a sixth treatment being added to test that the feeds were limiting in Val. Three identical Val series, each with six levels of Val, were supplemented with increasing amounts of Leu (23, 45 and 67 g/kg), thus 18 treatments in total. All pigs were killed at the end of the trial after 18 days for analysis of water, protein, lipid and ash in the carcass. The levels of Val and Leu and their interaction significantly influenced all the measurements taken in the trial. Daily gain in liveweight, water and protein, and feed conversion efficiency all increased with dietary Val content, whereas feed intake decreased as both Val and Leu contents increased. The deleterious effect of increased Leu on feed intake and growth was more marked at lower levels of Val. Supplementing the feed with the lowest Val content with additional Val largely overcame the effect of excess Leu. The efficiency of utilisation of Val for protein growth was unaffected by the level of Leu in the feed, the primary response to excess Leu being a reduction in feed intake. An intake of around 9 g Val/day yielded maximal protein growth during the period from 44 to 62 days of age in pigs of the genotype used in this trial.     

Conversion of Biotin Diaminocarboxylic Acid into Biotin and Bisnorbiotin by Resting Cell System of Microorganisms

The effects of dietary fat and protein levels on the conversion of Trp-Nam were investigated. In rats fed with 20% casein diets, the Trp-Nam conversion ratio [(urinary excretion of Nam + MNA + 2-Py + 4-Py in μmol/day)/(daily Trp intake during urine collection in μmol/day) × 100] was about 4.3% for the groups fed with the 20% corn oil and 20% soybean oil diets, 2.8% for the group fed with the 20% lard diet, and 2.1 % for the group fed with the no fat diet. In rats fed with 40% casein diets, a similar phenomenon was observed, but the ratios were 2.0%, 2.4%, 1.6%, and 0.8% for the groups fed with the 20% corn oil, 20% soybean oil, 20% lard, and no fat diets, respectively. From these results, it was found that an increase in fat intake elevated the conversion ratio regardless of the dietary protein level, while an increase in protein intake reduced it.     

Beta-branched residues adjacent to GG4 motifs promote the efficient association of glycophorin A transmembrane helices

Protein transmenembrane (TM) segments participating in helix-helix packing commonly contain small residue patterns (termed GG4 or "small-xxx-small" motifs) at i and i + 4 positions. Within many TM segments - such as the glycophorin A (GpA) sequence L75IxxGVxxGVxxT87- the G17y-xxx-Gly83 motif often occurs in combination with large, usually beta3-branched aliphatic residues at adjacent positions, typified here by Val30 and Val84 residues. To explore the importance of local P-branched character on GpA dimerization, we made systematic replacements to all 16 combinations of single or double Ile, Leu, and AIa residues at GpA TM Val/Val positions 80 and 84. Using the TOXCAT system to assay self-oligomerization in the Escherichia coli inner membrane--we observed that (i) combinations of Val and lie residues maintained, or improved dimerization levels; (ii) single Ala or Leu mutant combinations with Val or Ile maintained near-wild type dimerization affinities; and (iii) in the absence of beta-branching, i.e., Leu/Leu, Ala/Ala and Ala/Leu combinations, GpA dimerization was significantly diminished. An apparent capacity of lle-containing mutants to increase GpA dimerization versus WT likely arises from improved van der Waals packing (vs. Val) within the locus of helix contact, consistent with correlations we noted in lipid accessibility measurements. Examination of several synthetic peptides with sequences corresponding to selected GpA mutants (VV VI, IV II, and LL) confirmed their dimerization on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The overall results reinforce the importance of a beta-branch-containing "ridge" residue to complement a "small-xxx-small groove" in promotion of TM-TM interactions.     

Studies on limiting essential amino acids in grieves as source of dietary protein for rainbow trout (oncorhynchus mykiss)

Diets were computed to contain equal concentrations of digestible crude protein either of wheat gluten (diet 1) or of grieves (diets 2–8). Per kg dry diet, 41 g crystalline amino acids were supplemented. All diets contained at least 1.2 g Lys per MJ digestible energy (DE). In diet 2, ratios of Met + Cys, Trp, Leu, Ile and Phe to Lys were about equal to those in diet 1. In each of diets 3–7, one of the respective amino acids, in diet 8 all five were replaced by Glu in the supplemented mixture of amino acids.

Each diet was fed to triplciate groups of 20 trout during a trial lasting 66 days. Trout fed the diet containing wheat gluten consumed more dry matter and showed higher growth rates as well as higher protein contents in their gained body mass than trout fed diets based on grieves. Supplementing Met plus Trp significantly improved dry matter intake, growth rate and protein content of gain, though not to the level of trout fed the wheat gluten diet, whereas Leu, Ile and Phe showed no such effect. When grieves were not supplemented with both Met and Trp, gain in body mass contained significantly more lipids. DE required per kg gain by trout fed wheat gluten, grieves + Met + Trp or grieves without supplementation of Met and Trp was 20.1, 21.2 and 29.9 MJ, respectively.     

Amino acid supplementation of methanol-grown dried microbial cells in diets for young chicks

Growth rates and efficiency of food conversion of young chicks fed on diets marginally limiting in total nitrogen and containing 150 g/kg diet of flash-dried microbial cells (MC) with and without amino acid supplements, were measured in three experiments. Performance of these animals was compared with that of groups fed on a methioninefortified soya bean meal (SBM) control diet. In all experiments, chicks fed on the SBM control diet grew faster and were more efficient than chicks fed on the basal unsupplemented MC diet. In Experiment 1, arginine supplementation markedly enhanced weight gain and efficiency of food utilisation of chicks offered the basal MC diet; methionine had no effect. The second experiment demonstrated that a supplement containing methionine, arginine and tryptophan was more effective in augmenting the nutritional value of MC than either methionine with arginine or tryptophan with arginine. In the final experiment, weight gain and food intake of chicks fed on MC with supplements of arginine, methionine and tryptophan were increased markedly by additions of lysine and glutamic acid but not by addition of lysine alone. In all experiments, performance of animals in MC supplemented groups was lower than that of animals fed on the SBM control diet.     

Growth-promoting activity of pyrazinoic acid,a putative active compound of antituberculosis drug pyrazinamide,in niacin-deficient rats through the inhibition of ACMSD activity

We have recently reported that the antituberculosis drug, pyrazinamide (PZA), caused a significant increase in the conversion ratio of tryptophan to niacin in rats. In the present work, we investigated whether or not pyrazinoic acid (POA), a putative metabolite of PZA, increased the conversion ratio of tryptophan to niacin. Weaning rats were fed with a niacin-free and tryptophan-limited diet (negative control diet), or with the negative control diet supplemented with 0.003% nicotinic acid (positive control diet) or 1% POA (test diet) for 27 days. The growth rate was almost same between the groups fed on the positive control diet and the test diet. Dietary POA significantly increased the conversion ratio of tryptophan to niacin. Although POA did not directly inhibit the activity of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD), the rate-limiting enzyme in the tryptophan-niacin pathway, liver ACMSD activity was only not detected in the test diet group. These results suggest that a derivative of POA metabolized by rats inhibited the ACMSD activity.     

Blood-brain barrier transfer of L-Trp and alpha-MTrp in Li-treated rats.

Blood-brain barrier (BBB) transport for L-Trp and alpha-methyl-L-tryptophan was evaluated in Li-treated rats. Five different brain areas as well as left to right differences were examined. No left to right difference in the PS product was observed. Lithium treatment had a significant effect on the plasma concentration of Val, Leu and Ile but no effect on plasma total or free Trp. The ratio of plasma Trp to the sum of Leu, Val, Ile, Phe, Met and Tyr is increased in the Li-treated rats but not significantly. However, the ratio of Trp/(Val+Leu+Ile) is significantly increased in the Li-treated rats. The Km apparent (Kmapp) for the BBB Trp transport is significantly decreased (affinity of the carrier for Trp is increased) in the Li-treated rats. A decrease in the Kmapp is one of the possible factors responsible for an increase in the brain Trp concentration and subsequent increase in the brain serotonin synthesis in Li-treated rats.     

A diurnal component to the variation in sieve tube amino acid content in wheat

We have used high-sensitivity capillary electrophoresis coupled to a laser-induced fluorescence detection method to quantify 16 amino acids in wheat (Triticum aestivum) sieve tube (ST) samples as small as 2 nL collected by severing the stylets of feeding aphids. The sensitivity of the method was sufficient to determine a quantitative amino acid profile of individual STs without the need to bulk samples to produce larger volumes for analysis. This allowed the observation of the full range of variation that exists in individual STs. Some of the total concentrations of amino acids recorded are higher than those reported previously. The results obtained show variation in the concentrations of phenylalanine (Phe), histidine/valine (His/Val), leucine/isoleucine (Leu/Ile), arginine, asparagine, glutamine, tyrosine (Tyr), and lysine (Lys) across the ST samples. These could not be explained by plant-to-plant variation. Statistical analyses revealed five analytes (Tyr, Lys, Phe, His/Val, and Leu/Ile) that showed striking covariation in their concentrations across ST samples. A regression analysis revealed a significant relationship between the concentrations of Tyr, Lys, Phe, Leu/Ile, His/Val, asparagine, arginine, and proline and the time of collection of ST samples, with these amino acids increasing in concentration during the afternoon. This increase was confirmed to occur in individual STs by analyzing samples obtained from stylet bundles exuding for many hours. Finally, an apparent relationship between the exudation rate of ST sap and its total amino acid concentration was observed: samples containing higher total amino acid concentrations were observed to exude from the severed stylet bundles more slowly.     

Construction of a screening system for selecting lysozyme mutants unable to form a stable structure from random mutants.

To collect folding information, we screened and analyzed the recombinant hen lysozyme mutants which were not secreted from yeast. As model mutants, Leu8Arg, Ala10Gly, and Met12Arg were prepared by site-directed mutagenesis and analyzed as to whether they were secreted from yeast or not. Consequently, Ala10Gly was found to be secreted from yeast, but Leu8Arg and Met12Arg were not. Next, these mutants were expressed in Escherichia coli and refolded in vitro. As a result, Ala10Gly folded as the wild-type did. Leu8Arg efficiently refolded in renaturation buffer containing glycerol. Met12Arg did not refold even in the presence of glycerol. These results show that the Ala10Gly mutation does not affect folding or stability, that Leu8Arg is too unstable to be secreted from yeast, and that Met12Arg may be very unstable or the mutation affects the folding pathway. We screened the mutants that were not secreted by yeast from a randomly mutated lysozyme library, and obtained Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln. These two mutants were expressed in E. coli and then refolded in the presence of urea or glycerol. These mutants were refolded only in the presence of glycerol. Each single mutant of Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln was independently prepared and folded in vitro. The results showed that Leu25Arg and Leu56Gln were the dominant mutations, respectively, which cause destabilization. These results show that the mutant lysozymes which were not secreted from yeast may be unstable or have a defect in the folding pathway. Thus, we established a screening system for selecting mutants which are unable to form a stable structure from random mutants.