Extracellular amylolytic system of the yeast Lipomyces kononenkoae

Summary A strain of the yeast Lipomyces kononenkoae which converted starch into SCP with a high yield, produced three extracellular amylases which were purified from the culture fluid by Ficoll concentration, dialysis, isopropanol precipitation and DE-cellulose chromatography: an -amylase, a glucoamylase and a debranching transferase. The latter transferred -1,6-glucosyl units from panose to glucose forming maltose and appeared to have some debranching activity on amylopectin. The -amylase had the following properties: MW 38000 daltons; no effect of added calcium ions on activity; optimum temperature and pH for activity around 40°C and pH 5.5; H and S of heat inactivation 24360 cal mol^–1 and 29.2 cal deg^–1 mol^–1; range of pH stability pH 4–6.5; pI=7.1; final low molecular weight products of starch hydrolysis, maltose and glucose; K_m (40°C, pH 5.5) for starch 2.7 gl^–1, for maltotriose 109 gl^–1; uncompetitive inhibition by maltose with K_i (40°C, pH 5.5) 29.5 gl^–1. The glucoamylase had the following properties: MW 81500 daltons; optimum temperature and pH for activity around 50°C and pH 4.5: H and S of heat inactivation 20400 cal mol^–1 and 17.7 cal deg^–1 mol^–1; range of pH stability pH 4–6.5; pI=6.1; K_m (30°C, pH 4.5) for soluble starch 16.2 gl^–1, for maltose 0.36 gl^–1, for p-nitrophenyl--D-glucoside 0.35 gl^–1; competitive inhibition by glucose with K_i (30°C, pH 4.5) 4.7 gl^–1.     

Cell recycling studies for α-amylase production by Bacillus amyloliquefaciens

Production of -amylase by a strain of Bacillus amyloliquefaciens was investigated in a cell recycle bioreactor incorporating a membrane filtration module for cell separation. Experimental fermentation studies with the B. amyloliquefaciens strain WA-4 clearly showed that incorporating cell recycling increased -amylase yield and volumetric productivity as compared to conventional continuous fermentation. The effect of operating conditions on -amylase production was difficult to demonstrate experimentally due to the problems of keeping the permeate and bleed rates constant over an extended period of time. Computer simulations were therefore undertaken to support the experimental data, as well as to elucidate the dynamics of -amylase production in the cell recycle bioreactor as compared to conventional chemostat and batch fermentations. Taken together, the simulations and experiments clearly showed that low bleed rate (high recycling ratio) various a high level of -amylase activity. The simulated fermentations revealed that this was especially pronounced at high recycling ratios. Volumetric productivity was maximum at a dilution rate of around 0.4 h^–1 and a high recycling ratio. The latter had to exceed 0.75 before volumetric productivity was significantly greater than with conventional chemostat fermentation.List of Symbols a proportionality constant relating the specific growth rate to the logarithm of G (h) - a ₁ reaction order with respect to starch concentration - a ₂ reaction order with respect to glucose concentration - B bleed rate (h^–1) - C starch concentration (g/l) - C ₀ starch concentration in the feed (g/l) - D dilution rate (h^–1) - D _E volumetric productivity (KNU/(mlh)) - e intracellular -amylase concentration (g/g cell mass) - E extracellular -amylase concentration (KNU/ml) - F volumetric flow rate (l/h) - G average number of genome equivalents of DNA per cell - k _l intracellular equilibrium constant - k ₂ intracellular equilibrium constant - k _s Monod saturation constant (g/l) - k ₃ excretion rate constant (h^–1) - k _d first order decay constant (h^–1) - k _gl rate constant for glucose production - k _st rate constant for starch hydrolysis - k _t1 proportionality constant for -amylase production (gmRNA/g substrate) - k ₁ translation constant (g/(g mRNAh)) - KNU kilo Novo unit - m maintenance coefficient (g substrate/(g cell massh)) - n number of binding sites for the co-repressor on the cytoplasmic repressor - Q repression function K₁/K₂Q1.0 - R ratio of recycling - R _s rate of glucose production (g/lh) - r _c rate of starch hydrolysis (g/(lh)) - R _eX retention by the filter of the compounds X: starch or -amylase - r intracellular -amylase mRNA concentration (g/g cell mass) - r _C volumetric productivity of starch (g/lh) - r _E volumetric productivity of intracellular -amylase (KNU/(g cell massh)) - r _r volumetric productivity of intracellular mRNA (g/(g cell massh)) - r _e volumetric productivity of extracellular -amylase (KNU/(mlh)) - r _s volumetric productivity of glucose (g/(lh)) - r _X volumetric productivity of cell mass (g/(lh)) - S ₀ free reducing sugar concentration in the feed (g/l) - S extracellular concentration of reducing sugar (g/1) - t time (h) - V volume (l) - X cell mass concentration (g/l) - Y yield coefficient (g cell mass/g substrate) - Y _E/S yield coefficient (KNU -amylase/g substrate) - Y _E total amount of -amylase produced (KNU) - substrate uptake (g substrate/(g cell massh)) - specific growth rate of cell mass (h^–1) - _d specific death rate of cells (h^–1) - _m maximum specific growth rate of cell mass (h^–1) This study was supported by Bioprocess Engineering Programme of the Nordic Industrial Foundation and the Center for Process Biotechnology, the Technical University of Denmark.     

Alpha-thalassemia in Papua New Guinea

Summary A study of the distribution of -thalassemia in Papua New Guinea (PNG) was carried out by DNA analysis. A total of 664 DNA samples were screened for -thalassemia 2 and -thalassemia 1 caused respectively by either deletion of one or both of the duplicated -globin genes. -Thalassemia 2 was detected in high frequencies in coastal and lowland regions where malaria has been holo- to hyperendemic but in low frequencies in non-malarious highland regions. The highest frequency was observed in the north coast of PNG. The distribution of -thalassemia 2 seems to be in accordance with other conditions such as ovalocytosis and G6PD deficiency which are also prevalent in this population, suggesting that they may interact in protection against malaria. However, it appears to be negatively correlated with -thalassemia and -thalassemia 1, the latter being extremely rare in this population. Analysis of the types and subtypes of the single -globin gene deletion revealed a predominance of the –^4.2 type in general, except in some regions in the south where the –^3.7 type is prevalent. The –^3.7 I subtype is the common form of the –^3.7 deletion in the PNG mainland. The –^3.7 III subtype, previously reported to be unique in Melanesians and Polynesians, was detected in an offshore island of PNG. However, this subtype is very rare in Melanesians from the PNG mainland.     

Refinement of the protein backbone angle ψ in NMR structure calculations

Cross-correlated relaxation rates involving the C-H dipolar interaction and the carbonyl (C) chemical shift anisotropy (CSA) have been measured using two complementary 3D experiments. We show that the protein backbone angle can be directly refined against such cross-correlated relaxation rates (^{H C,C}) and the three-bond H/D isotope effect on the C chemical shifts (³C _(ND)). By simultaneously using both experimental parameters as restraints during NMR structure calculations, a unique value for the backbone angle is defined. We have applied the new refinement method to the -Spectrin SH3 domain (a -sheet protein) and to the Sgs1p HRDC domain (an -helical protein) and show that the quality of the NMR structures is substantially improved, judging from the atomic coordinate precision and the Ramachandran map. In addition, the -refined NMR structures of the SH3 domain deviate less from the 1.8 Å crystal structure, suggesting an improved accuracy. The proposed refinement method can be used to significantly improve the quality of NMR structures and will be applicable to larger proteins.     

Interrelationships Between Nitrogen Supply and Photosynthetic Parameters in Vicia faba L.

We determined for Vicia faba L the influence of nitrogen uptake and accumulation on the values of photon saturated net photosynthetic rate (P _Nmax), quantum yield efficiency (), intercellular CO₂ concentration (C _i), and carboxylation efficiency (C _e). As leaf nitrogen content (N_L) increased, the converged onto a maximum asymptotic value of 0.0664±0.0049 mol(CO₂) mol(quantum)^–1. Also, as N_L increased the C _i value fell to an asymptotic minimum of 115.80±1.59 mol mol^–1, and C _e converged onto a maximum asymptotic value of 1.645±0.054 mol(CO₂) m^–2 s^–1 Pa^–1 and declined to zero at a N_L-intercept equal to 0.596±0.096 g(N) m^–2. fell to zero for an N_L-intercept of 0.660±0.052 g(N) m^–2. As N_L increased, the value of P _Nmax converged onto a maximum asymptotic value of 33.400±2.563 mol(CO₂) m^–2 s^–1. P _N fell to zero for an N_L-intercept of 0.710±0.035 g(N) m^–2. Under variable daily meteorological conditions the values for N_L, specific leaf area (_L), root mass fraction (R_f), P _Nmax, and remained constant for a given N supply. A monotonic decline in the steady-state value of R_f occurred with increasing N supply. _L increased with increasing N supply or with increasing N_L.     

Effects of osmotic stabilizers on the activities of mycolytic enzymes used in fungal protoplast liberation

Summary Assay methods for chitinase, -glucanase and -glucanase in the presence of the osmotic stabilizers used in fungal protoplast liberation were developed. Chitinase activity with an inorganic osmotic stabilizer system was in the order of NO ₃ ^– , Cl^–>SO ₄ ^2– >PO ₄ ^3– and Na⁺, K⁺>Ca²⁺, Mg²⁺. Monovalent anion salts with monovalent cations improved chitinase activity, whereas divalent and trivalent anion and cation salts caused appreciable inhibition; phosphate salts induced very serious inhibition. These phenomena suggest that a suitable electrical state is required for optimal chitinase activity. MgSO₄, KCl and NH₄Cl were equally efficient as stabilizers for protoplast liberation, although they had different effects on chitinase activity. -glucanase was inhibited more by sucrose than by mannitol and sorbitol; -glucanase was relatively stable to both organic and inorganic osmotic stabilizers. As chitin is the major component of the fungal cell wall, chitinase is thought to be more important for protoplast liberation than are -glucanase and -glucanase.

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Resumen Se han desarrollado técnicas para la determinación de: quitinasa, -glucanasa y -glucanasa en presencia de estabilizadores osmóticos usados en la liberación de protoplastos fúngicos. La actividad quitinasa con estabilizadores inorgánicos siguió el orden: NO ₃ ^– , Cl^–>SO ₄ ^2– >PO ₄ ^3– Na⁺, K⁺>Ca²⁺, Mg²⁺. Los aniones monovalentes junto con los cationes monovalentes mejoraron la actividad quitinasa, mientras que tanto aniones como cationes, divalentes y trivalentes causaron una inhibición apreciable. Las sales de fosfato indujeron inhibiciones muy severas. Estos fenómenos sugieren que un estado eléctrico adecuado es necesario para una actividad quitinasa óptima. MgSO₄, KCl y NH₄Cl fueron igualmente eficientes como estabilizadores para la liberación de protoplastos, aunque tuvieran distintos efectos en la actividad quitinasa. La -glucanasa se inhibió más por sucrosa que por azucares-alcoholes; la -glucanasa se mantuvo relativamente estable frente a estabilizadores osmóticos tanto orgánicos como inorgánicos. Al ser la quitina el componente mayoritario de la pared celular de los hongos, se cree que la quitinasa es más importante que la -glucoanasa y la -glucanasa en lo que concierne a la liberación de protoplastos.

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Résumé Des méthodes ont été mises au point pour mesurer les activités -et -glucanase en présence des stabilisateurs osmotiques utilisés pour la production de protoplastes. En présence d'un système minéral de stabilisation osmotique, l'activité chitinase est dans l'ordre NO ₃ ^– , Cl^–>SO ₄ ^2– >PO ₄ ^3– et Na⁺, K⁺>Ca²⁺, Mg²⁺. Les anions monovalents ainsi que les cations monovalents améliorent l'activité chitinase, tandis que les anions et les cations di- et tri-valents l'inhibent de façon appréciable; les phosphates sont fortement inhibiteurs. Ces phénomènes suggèrent que l'activité chitinase optimale exige un état électrique approprié. MgSO₄, KCl et NH₄Cl stabilisent de façon identique la formation des protoplastes, bien que leurs effets respectifs sur l'activité chitinase soient différents. L' -chitinase est inhibée par le saccharose davantage que par les sucres-alcools; la -glucanase est relativement stable en présence des stabilisateurs organiques ou minéraux. Comme la chitine est le constituant majeur des parois cellulaires fungiques, on considère que la chitinase est plus importante que la -et l' -glucanase pour la formation des protoplastes.

     

Skeletal muscle pH dynamics during arousal from hibernation measured by31P NMR spectroscopy

Summary The time-course of changes in skeletal muscle pH during arousal from hibernation in the Columbian ground squirrel was studied using³¹P NMR spectroscopy. In hibernation (T _re 7–9°C), shoulder/neck muscle pH was 7.45±0.03 and _Im was 0.60. In euthermia (T _re 37°C), muscle pH was 7.24±0.05 and _Im was 0.75. Thus the overall pH-temperature coefficient was-0.009 pH units/°C, indicating acidification of the muscle in hibernation. During the transition from hibernation to euthermia, however, the muscle shows a nonlinear pattern of pH change. In early arousal (T _sh<20–25°C,T _re<15°C) muscle pH does not change and muscle _Im increases to 0.72. In later arousal (T _sh>20–25°C,T _re>15°C) muscle pH decreases gradually toward the euthermic value and muscle _Im increases only slightly from 0.72 to 0.75. These results support the hypothesis that intracellular acidification of the muscle, present during hibernation, is reversed in early arousal. This may facilitate an increase in muscle metabolism and the contribution of maximal shivering thermogenesis to rewarming of the animal.Abbreviations _Im dissociation ratio of protein imidazole buffergroups - NST non-shivering thermogenesis - BAT brown adipose tissue - dp H/dT temperature coefficient of pH - pH _i intracellular pH - ³¹ P NMR ³¹Phosphorus nuclear magnetic resonance - P _i chemical shift of inorganic phosphate relative to PCr - PCr phosphocreatine - T _b body temperature - T _re rectal temperature - T _sh subcutaneous shoulder temperature - T _a ambient temperature     

Tolerance regions with segmented fermentation processes

Many microbial fermentation processes exhibit different phases (e.g. adaption phase, main growth phase, main production phase). The process variables e.g. the biomass vary randomly about their mean. The experimentalist is interested to know the break points of the different phases, and a tolerance region, i.e. a range of possible values of the process variable that can be considered as normal. This paper deals with statistical methods for determining break points and tolerance regions.List of Symbols a _i intercept in phasei - b _i specific growth rate in phasei - e _t deviation of a measurement in timet - tEX expectation of variableX - r number of phases of fermentation - T _i break point of phaseit - t _ij time of measurementj in phasei - t _n–2.1–/2 quantile oft distribution - Y(t) logarithm of measurement at timet Greek Letters 1 – cover probability of tolerance region - 1 – part covered by the tolerance region - ² variance ofe _t - (·) standard normal distribution - quantile of chisquare distribution     

A new mouse T-cell receptorα chain variable region family

Sequence analysis of the rearranged T-cell receptor a chain gene segments from an influenza reactive T-cell clone T2.5-5 and a hemin chloride reactive T-cell hybrid SJL-HE-1.1 have revealed a previously undescribedV gene family. We have designated this familyV 15. Southern hybridization analysis has indicated that this family most probably contains only two members, and that these are conserved in each of six mouse strains representing three previously describedV haplotypes:V ^a ,V ^b , andV ^c .     

Interaction between Duodenase and α1-Proteinase Inhibitor

The interaction between duodenase, a newly recognized serine proteinase belonging to the small group of Janusfaced proteinases, and ₁-proteinase inhibitor (₁-PI) from human serum was investigated. The stoichiometry of the inhibition was 1.2 mol/mol. The presence of a stable enzyme–inhibitor complex was shown by SDS-PAGE. The mechanism of interaction between duodenase and ₁-PI was shown to be of the suicide type. The equilibrium and inhibition constants are 13 ± 3 nM and (1.9 ± 0.3)·10⁵ M^–1·sec^–1, respectively. Based on the association rate constant of the enzyme–inhibitor complex and localization of duodenase and ₁-PI in identical compartments, ₁-PI is suggested to be a duodenase inhibitor in vivo.     

A new α2HS-glycoprotein typing by isoelectric focusing

Summary Isoelectric focusing (pH 4.0–5.0) of serum ₂HS-glycoprotein on polyacrylamide gels has been found to be a useful tool in population genetics and forensic science. Using this method, we isolated three common types, ₂HS 1-1, ₂HS 2-1 and ₂HS 2-2, and showed that ₂HS types are determined by two autosomal codominant alleles, ₂ HS ¹ and ₂ HS ². The method is simple, fast and easy to perform. Results of typing for the two alleles, ₂ HS ¹ and ₂ HS ², are described for a Japanese population sample (n=1003).     

GTP-binding proteins regulate high conductance anion channels in rat bile duct epithelial cells

Summary Epithelial cells from the intrahepatic bile duct contribute to bile formation, but little is known of the cellular mechanisms responsible. In these studies, we have characterized the endogenous GTP-binding proteins (G proteins) present in these cells and evaluated their role in regulation of high conductance anion channels. G proteins were identified in purified plasma membranes of isolated bile duct epithelial cells using specific antisera on Western blots, and ion channel activity was measured in excised inside-out membrane patches using patch-clamp recording techniques. In patches without spontaneous channel activity, addition of cholera toxin to the cytoplasmic surface had no effect (n=10). Addition of pertussis toxin caused an activation of channels in 13/34 (38%) attempts, as detected by an increase in channel open probability. Activated channels were anion selective (gluconate/Cl^– permeability ratio of 0.17±0.04) and had a unitary conductance of 380 pS. Channel open probability was also increased by the nonhydrolyzable GDP analogue guanosine 5-0-(2-thiodiphosphate) in 8/14 (57%) attempts. In contrast, channel open probability was rapidly and reversibly decreased by the nonhydrolyzable analogue of GTP 5guanylylimidodiphosphate in 7/9 (78%) attempts. Western blotting with specific antisera revealed that both G_i –2 and G_i –3 were present in significant amounts, whereas G_i –1 and G_o were not detected. These studies indicate that in bile duct epithelial cells, high conductance anion channels are inhibited, in a membrane-delimited manner, by PTX-sensitive G proteins.We gratefully acknowledge the assistance of Marwan Farouk, M.D. in the preparation of bile duct epithelial cells, Lucy Seger in the identification of the G proteins, C.F. Starmer in channel analysis, and P.J. Casey for the gift of bacteria expressing the different G-protein -subunits. This work was supported in part by grants from the National Institutes of Health DK43278 (to J.G.F.), DK42486 (to T.W.G.), and DK07568 (to J.M.M.); American Gastroenterological Association/G.D. Searle Research Scholar Award (to J.G.F.) and an American Gastroenterological Association Advanced Research Training Award (J.M.M.).     

Adrenergic regulation of chloride secretion across the opercular epithelium: The role of cyclic AMP

Summary Activation of the -adrenergic receptors of the opercular epithelium ofFundulus heteroclitus stimulates Cl^– secretion, while activation of the -adrenergic receptors inhibits Cl^– secretion (Degnan and Zadunaisky, 1979). The possible involvement of adenosine 3, 5-monophosphate (cAMP) in these adrenergic responses was investigated. Isolated opercular epithelia incubated in Ringer, containing 10 mM theophylline, had cAMP levels ranging between 5.3 and 19.3 pmoles·mg protein^–1 (mean=9.5±1.0 pmoles·mg protein^–1). Activation of the -receptors by 10^–5 M isoproterenol increased the mean cAMP level 430% (P<0.001). Blockage of the -receptors with propranolol greatly reduced the increase in cAMP in response to isoproterenol. Activation of the -receptors by 10^–5 M arterenol stimulated the mean cAMP level 270% (P<0.01). However, when the -receptors were blocked with propranolol, arterenol had no effect on the cAMP level. The possible involvement of Ca⁺⁺ in these adrenergic responses was investigated. Neither the stimulatory effect of isoproterenol, nor the inhibitory effect of arterenol on the Cl^– secretion were diminished in the absence of extracellular Ca⁺⁺. The Ca⁺⁺ ionophore, A23187, and the calmodulin inhibitor, trifluoperazine, had no effects on the Cl^– secretion. The Ca⁺⁺-channel blocker, D600, had a significant inhibitory effect (P<0.005). Guanosine 3,5-monophosphate (cGMP) had no effect on the Cl^– secretion.The results indicate that -adrenergic stimulation of Cl^– secretion across the opercular epithelium is accompanied by an elevation in tissue cAMP levels. -adrenergic inhibition of Cl^– secretion does not involve changes in the tissue cAMP. Neither of these responses appear to require Ca⁺⁺.     

A solid-phase synthesis of three aza-, iminoaza- and reduced aza-peptides from the same precursor

Using the Boc-strategy, a step-by-step synthesis on the PAM solid supportof three aza-, iminoaza- and reduced aza-peptide homologues is described.From the same hydrazinocarbonyl peptide-PAM precursor, the coupling ofeither a Boc-amino acid or a Boc-amino aldehyde gives rise to an aza-peptideor an iminoaza-peptide containing theC-CO-NH-N-CO-NH-C orC-CH=N-N-CO-NH-C surrogate of the peptide motif, respectively. In situreduction of the latter by NaBH₃CN leads to a reducedaza-peptide containing theC-CH₂-NH-N-CO-NH-C moiety. The key step synthesis of thehydrazinocarbonyl peptide-PAM precursor is carried out by coupling on thegrowing peptide chain the N-Boc-aza-amino acid chloride obtained by theaction of triphosgene on the corresponding N-Boc-hydrazine. Thesemodifications have been introduced in position 1-2 of the YLGYLEQLLRbenzodiazepine-like decapeptide     

Angular dependence of 1J(Ni,Calphai) and 2J(Ni,Calpha(i-1)) coupling constants measured in J-modulated HSQCs

A new method to measure ¹J(N_i,C _i) and ²J(N_i,C _{(i – 1)}) coupling constants in proteins based on a J-modulated sensitivity enhanced HSQC was introduced. Coupling constants were measured in the denatured and in the native state of ubiquitin and found to depend on the conformation of the protein backbone. Using a combined data set of experimental coupling constants from ubiquitin and staphylococcal nuclease (Delaglio et al., 1991), the angular dependence of the coupling constants on the backbone angles and was investigated. It was found that the size of ²J(N_i,C _{(i – 1)}) correlates strongly with the backbone conformation, while only a weak conformational dependence on the size of ¹J(N_i,C _i) coupling constants was observed. Coupling constants in the denatured state of ubiquitin were uniform along the sequence of the protein and not dependent on a given residue type. Furthermore it was shown that the observed coupling constants were in good agreement with predicted coupling constants using a simple model for the random coil.     

Efficient and precise measurement of H(alpha)-C(alpha), C(alpha)-C', C(alpha)-C(beta) and H(N)-N residual dipolar couplings from 2D H(N)-N correlation spectra

A suite of experiments are presented for the measurement of H–C, C–C, C–C and H^N–N couplings from uniformly ¹⁵N, ¹³C labeled proteins. Couplings are obtained from a series of intensity modulated two-dimensional H^N–N spectra equivalent to the common ¹H–¹⁵N–HSQC spectra, alleviating many overlap and assignment issues associated with other techniques. To illustrate the efficiency of this method, H–C, C–C, and H^N–N isotropic scalar couplings were determined for ubiquitin from data collected in less than 4.5 h, C–C data collection required 10 h. The resulting couplings were measured with an average error of ±0.06, ±0.05, ±0.04 and ±0.10 Hz, respectively. This study also shows H–C and C–C couplings, valuable because they provide orientation of bond vectors outside the peptide plane, can be measured in a uniform and precise way. Superior accuracy and precision to existing 3D measurements for C–C couplings and increased precision compared to IPAP measurements for H^N–N couplings are demonstrated. Minor modifications allow for acquisition of modulated H^N–C 2D spectra, which can yield additional well resolved peaks and significantly increase the number of measured RDCs for proteins with crowded ¹H–¹⁵N resonances.     

Comparison of the carbohydrate moieties of recombinant soluble Fcε receptor (sFcε RII/sCD23) expressed inSaccharomyces cerevisiae and Chinese hamster ovary cells. Different O-glycosylation sites are used by yeast and mammalian cells

Recombinant human soluble low affinity receptor for the Fc portion of IgE (sFcRII/sCD23) was produced inSaccharomyces cerevisiae or Chinese hamster ovary cells and subjected to carbohydrate analysis. Applied methods included analytical SDS-PAGE, reversed phase HPLC, methylation analysis and sequential degradation with exoglycosidases. The results revealed that sFcRII derived from Chinese hamster ovary cells is glycosylated exclusively at Ser-147, containing mainly the trisaccharide Sia(2–3)Gal(1–3)GalNAc, whereas the yeast derived glycoprotein was glycosylated at Ser-167 and contained only -mannosyl residues. It is shown here for the first time that different amino acids of a given protein can be O-glycosylated when expressed in yeast or Chinese hamster ovary cells.     

Phylogeny and evolutionary rates of G protein α subunit genes

Summary Rooted phylogenetic trees for a total of 34 genes encoding the stimulatory (s), inhibitory (i), transducin (t), G_x (x), G_z (z), G₁₁ (11), G₁₂ (12), G₁₃ (13), G₁₆ (16), G_q (q), and other (o) G protein a subunits have been constructed. The analysis shows that the G₁₂ (12 and 13), G_q (11, 16, and q), and G_s (s genes) groups form one cluster, and the G_x (x and z genes), G_i (i genes), G_t (t1 and t2), and G_o (o genes) groups form another cluster. During mammalian evolution, the rates of synonymous substitutions for these genes were estimated to be between 1.77 × 10^–9/site/year and 5.63 × 10^–9/site/year, whereas those of nonsynonymous substitutions were between 0.008 × 10^–9/site/year and 0.067 × 10^–9/site/year. These evolutionary rates are similar to those for histone genes, suggesting equally important biological functions of the G protein a subunits. Offprint requests to: S. Yokoyama     

Biotechnological production of unnatural L-amino acids from D,L-5-monosubstituted hydantions. II. L-α- and L-β-naphthylalanine

Summary Resting cells ofArthrobacter sp. (DSM 3745) with the ability to form L-tryptophan from D,L-5-(3-indolylmethy)hydantoin were used for the bioconversion of D,L-5-- and D,L-5--naphthylmethylhydantoin (D,L-5-- and D,L-5--NMH) to the corresponding L-amino acids. Under the optimal reaction conditions of pH 9.7 and 40°C specific productivities of 0.2 (-naphtylalanine) and 0.6 (-naphtylalanine) mM amino acid x g cell dry mass^–1 x h^–1 were obtained in a 0.1 M Na₂CO₃/NaHCO₃-buffer in a strirred bioreactor.