Enhanced Production of β-Carotene by Recombinant Industrial Wine Yeast Using Grape Juice as Substrate

In this study, both recombinant Saccharomyces cerevisiae T73-63 and FY-09 derived from the industrial wine yeast T73-4 and laboratory yeast FY1679-01B, respectively, were constructed and compared for their β-carotene production in real grape juice. The results showed that highest β-carotene content (5.89 mg/g) was found in strain T73-63, which was 2.1 fold higher than that of strain FY-09. Although the cell growth was inhibited by the metabolic burden induced by the production of heterogeneous β-carotene, the pigment yield in T73-63 was still 1.7 fold higher than that of FY-09. Furthermore, high contents of ergosterol and fatty acid were also observed in T73-63. These results suggest that industrial wine yeast has highly active metabolic flux in mevalonate pathway, which leads to more carbon flux into carotenoid branch compared to that of laboratory yeast. The results of this study collectively suggest that in the application of recombinant strains to produce carotenoid using agro-industrial by-products as substrate, the suitable host strains should have active mevalonate pathway. For this purpose, the industrial wine yeast is a suitable candidate.     

Exploring the Influence of Carbon Nanoparticles on the Formation of β-Sheet-Rich Oligomers of IAPP22–28 Peptide by Molecular Dynamics Simulation

Recent advances in nanotechnologies have led to wide use of nanomaterials in biomedical field. However, nanoparticles are found to interfere with protein misfolding and aggregation associated with many human diseases. It is still a controversial issue whether nanoparticles inhibit or promote protein aggregation. In this study, we used molecular dynamics simulations to explore the effects of three kinds of carbon nanomaterials including graphene, carbon nanotube and C₆₀ on the aggregation behavior of islet amyloid polypeptide fragment 22–28 (IAPP_22–28). The diverse behaviors of IAPP_22–28 peptides on the surfaces of carbon nanomaterials were studied. The results suggest these nanomaterials can prevent β-sheet formation in differing degrees and further affect the aggregation of IAPP_22–28. The π–π stacking and hydrophobic interactions are different in the interactions between peptides and different nanoparticles. The subtle differences in the interaction are due to the difference in surface curvature and area. The results demonstrate the adsorption interaction has competitive advantages over the interactions between peptides. Therefore, the fibrillation of IAPP_22–28 may be inhibited at its early stage by graphene or SWCNT. Our study can not only enhance the understanding about potential effects of nanomaterials to amyloid formation, but also provide valuable information to develop potential β-sheet formation inhibitors against type II diabetes.     

Influence of physical and chemical factors during foraging and culture of the symbiont fungus in Atta sexdens rubropilosa （Hymenoptera： Formicidae）

The aim of this paper is to investigate the influence of physical and chemical factors on transport and use of substrate for Atta sexdens rubropilosa workers. Three types of rectangular fragments were used to study the physical influence factors： filter paper with paraffin, filter paper without paraffin and polyester film. To study the chemical factors, some fragments were impregnated with organic extract of orange albedo, others were soaked with soybean oil and for the remaining ones nothing was applied. The fonowing parameters were evaluated： （i） attractiveness of substrate for transport and number of loading workers per treatment; （ii） foraged material incorporation; （iii） rejection by numbers of fragments deposited in the garbage or beside the fungus garden. All the polyester film fragments carried out to the fungus garden were subsequently rejected. We verified that chemical factors of the substrate were more quickly detected by the workers, whereas physical factors were used as a criterion in the decision-making to reject or accept the substrate collected.     

Substituent Effect on the Absolute Stereochemistry of the Asymmetric Reduction of Fluorine-Containing β-Diketones by Bakers' Yeast

Fluorine-containing β-diketones (la-d) were reduced by free bakers' yeast (FBY) and immobilized baker yeast (IMBY) in water, to give optically-active fluorinated β-hydroxyketones (2a-d). It was found that the reaction is highly regioselective, and that the stereochemistry of the reduction is controlled by the R substituent.     

Effect of Inducers on the Production of 5‐Aminolevulinic Acid by Recombinant Escherichia coli

Abstract

Aminolevulinic acid (ALA) was produced by recombinant Escherichia coli BL21(DE3) (pET28‐A.R‐hemA) harboring the ALA synthase gene (hemA) from Agrobacterium radiobacter zju‐0121. The effects of inducers on the ALA synthase activity and ALA productivity were evaluated. The results indicated that a low isopropyl‐β‐D‐thiogalactoside (IPTG) concentration (0.05 mmol/L) was favorable for high expression of ALA synthase, which resulted in higher ALA productivity. For metabolic engineering applications, lactose was a better substitute of IPTG for active enzyme expression. When lactose concentration was 5 mmol/L, the specific ALA synthase activity and ALA productivity reached 16.7 nmol/(min · mg of protein) and 1.15 g/L, respectively, which were about 15% and 43% higher than those induced by IPTG.     

Influence of Spermine on DNA Conformation in a Molecular Dynamics Trajectory of d(CGCGAATTCGCG)2: Major Groove Binding by One Spermine Molecule Delays the A→B Transition

Abstract

The effect of spermine on the A-DNA to B-DNA transition in d(CGCGAATTCGCG)₂ has been investigated by five A-start molecular dynamics simulations, using the Cornell et al. potential. In the absence of spermine an A→B transition is initiated immediately and the DNA becomes equidistant from the A- and B-forms at 200ps. In three DNA-spermine simulations, when a spermine is located across the major groove of A-DNA in one of three different initial locations, the time taken to reach equidistance from the A- and B-forms is delayed until 800, 950 or 1000ps. In each case the A-form appears to be temporarily stabilized by spermine's electrostatic interactions with phosphates on both sides of the major groove. The onset of the A→B transition can be correlated with the spermine losing contact with phosphates on one side of the groove and with A-like → B-like sugar pucker transitions in the vicinity of the spermine bridge. However in the fifth trajectory, in which the spermine initially threads from the major groove via the backbone into the minor groove, the B→A transition occurs rapidly once again and the DNA is equidistant between the A- and B-forms within 300ps. This indicates that the mere presence of spermine is insufficient to delay the transition and that major groove binding stabilizes A-DNA.     

Influence of the ion-composition of the medium on alkaloid production by “hairy roots” ofDatura stramonium

The influence of culture age on biomass production and alkaloid yield of “hairy roots” obtained after infection ofDatura stramonium L. withAgrobacterium ATCC 15834 was investigated. Maximal hyoscyamine yield was obtained with roots harvested after six weeks. Fluctuations were found for tropine yield, the precursor of the ring moiety of hyoscyamine. These indicate a continuous conversion to hyoscyamine during the exponential growth phase. The effect of the ion-balance was investigated by preparing five different media that only differed in their ionic composition. The ionic interactions between macroelements, differently influenced biomass production and alkaloid yield. As a result, highest biomass yield was found with NO₃ ⁻ - and K⁺-dominance, whereas hyoscyamine yield was highest with the culture medium in which SO₄ ²⁻ and K⁺ were dominant. Shifting the intercationic balance to strongly towards Ca²⁺ caused an overall reduced metabolism, since as well biomass yield as hyoscyamine yield was lowest with the NO₃ ⁻Ca²⁺-medium. Also tropine yield was affected by the ion-balance, indicating that this culture parameter also influences alkaloid synthesis.[/p]     

Influence of culture conditions on the production of heterologous interleukin 1β by Kluyveromyces lactis

Summary Under the control of the repressible PHO5 promoter, the expression of gene encoding interleukin 1 (Il1) was derepressed when the medium was depleted of free inorganic phosphate (Pi). Maximum heterologous protein synthesis was obtained in the presence of 75 mg KH₂PO₄/1 (for 20 g glucose/l). The successful heterologous protein production greatly depends on nutritional culture conditions as Il1 production efficiency was increased by 83% through optimization of the growth medium. Comparison of different phosphate-limited cultivation strategies led to the development of a batch culture procedure with nutrient pulses to delay induced oxido-fermentative glucose metabolism and increase the Il1 production to 135 mg/l.     

Influence of nitrogen on the production of hypericins by St. John’s wort

The effect of nitrogen supply on the production of ‘hypericins’ (hypericin and pseudohypericin) in leaves of St. John’s wort (Hypericum perforatum L.) was examined with plants grown in sand culture and soil. In sand culture, 56-d growth of St. John’s wort plants with decreased nitrogen levels resulted in increased production of hypericins in leaves. A short-term low nitrogen stress in sand culture also resulted in increased production of leaf hypericins. While growth in a low nitrogen-containing soil resulted in elevated levels of hypericins, their production was decreased by supplementation of the soil with additional nitrogen. Increased production of hypericins in St. John’s wort leaves did not require the nitrogen supply to be decreased to levels that resulted in nitrogen deficiency symptoms. Moreover, alteration in the production of leaf hypericins occurring with changes in nitrogen supply did not alter the concentration ratio of pseudohypericin and hypericin. Increased production of leaf hypericins was not associated with any significant changes in the number of dark glands on the leaves and only a weak correlation was observed between leaf dark gland number and levels of leaf hypericins. These results are discussed in terms of the biochemistry of naphthodianthrone production by St. John’s wort plants and implications for growth environment effects during cultivated growth of this medicinal plant.     

Misfolding of glycoproteins is a prerequisite for peptide: N-glycanase mediated deglycosylation

Peptide:N-glycanase (PNGase) is a deglycosylating enzyme that catalyzes the hydrolysis of the beta-aspartylglycosylamine bond of aspargine-linked glycopeptides and glycoproteins. Earlier studies from our laboratory indicated that PNGase catalyzed de-N-glycosylation was limited to glycopeptide substrates, but recent reports have demonstrated that it also acts upon full-length misfolded glycoproteins. In this study, we utilized two glycoprotein substrates, yeast carboxypeptidase and chicken egg albumin (ovalbumin), to study the deglycosylation activity of yeast PNGase and its mutants. Our results provide further evidence that PNGase acts upon full-length glycoprotein substrates and clearly establish that PNGase acts only on misfolded or denatured glycoproteins.     

Site-directed mutagenesis study of yeast peptide:N-glycanase. Insight into the reaction mechanism of deglycosylation.

Yeast peptide:N-glycanase (Png1p; PNGase), a deglycosylation enzyme involved in the proteasome dependent degradation of proteins, has been reported to be a member of the transglutaminase superfamily based on sequence alignment. In this study we have investigated the structure-function relationship of Png1p by site-directed mutagenesis. Cys-191, His-218, and Asp-235 of Png1p are conserved in the sequence of factor XIIIa, where these amino acids constitute a catalytic triad. Point mutations of these residues in Png1p resulted in complete loss in activity, consistent with a role for each in catalyzing deglycosylation of glycoproteins. Other conserved amino acid residues, Trp-220, Trp-231, Arg-210, and Glu-222, were also vitally important for folding and structure stability of the enzyme as revealed by circular dichroism analysis. The potential effects of the mutations were predicted by mapping the conserved amino acids of Png1p within the known three-dimensional structure of factor XIIIa. Our data suggest that the lack in enzyme activity when any of the catalytic triad residues is mutated is either due to the absence of charge relay in the case of the triad or due to the disruption of the native fold of the enzyme. These findings strongly suggest a common evolutionary lineage for the PNGases and transglutaminases.     

A simple, sensitive in vitro assay for cytoplasmic deglycosylation by peptide: N-glycanase

A cytoplasmic peptide: N-glycanase (PNGase) has been implicated in the proteasomal degradation of aberrant glycoproteins synthesized in the endoplasmic reticulum. The reaction is believed to be important for subsequent proteolysis by the proteasome since bulky N-glycan chains on misfolded glycoproteins may impair their efficient entry into the interior of the cylinder-shaped 20S proteasome, where its active site resides. This cytoplasmic enzyme was first detected in 1993 by a simple, sensitive assay method using 14C-labeled glycopeptide as a substrate. The deglycosylation reaction by PNGase brings about two major changes on substrate the peptide; one is removal of the N-glycan chain and the other is the introduction of a negative charge into the core peptide by converting the glycosylated asparagine residue(s) into an aspartic acid residue(s). The assay method we developed monitors these major changes in the core peptide, and the respective changes were detected by distinct analytical methods: i.e., paper chromatography and paper electrophoresis. This chapter will describe the simple, sensitive in vitro assay method for PNGase.     

Orientation and helical conformation of a tissue-specific hunter-killer peptide in micelles

Hunter-killer peptides are chimeric synthetic peptides that selectively target specific cell types for an apoptotic death. These peptides, which are models for potential therapeutics, contain a homing sequence for receptor-mediated interactions and a pro-apoptotic sequence. Homing domains have been designed to target angiogenic tumor cells, prostate cells, arthritic tissue and, most recently, adipose tissue. After a receptor-mediated internalization, the apoptotic sequence, which contains D-enantiomer amino acids, initiates apoptosis through mitochondrial membrane disruption. We have begun structure and functional studies on a peptide (HKP1) that specifically targets angiogenic tumor cells for apoptosis. As a model for mitochondrial membrane disruption, we have examined peptide-induced leakage of a calcein fluorophore from large unilamellar vesicles. These experiments demonstrate more potent leakage activity by HKP1 than the peptide lacking the homing domain. Circular dichroism and 2D homonuclear NMR experiments demonstrate that this tumor-specific HKP adopts a left-handed amphipathic helix in association with dodecylphosphorylcholine micelles in a parallel orientation to the lipid-water interface with the homing domain remaining exposed to solvent. The amphipathic helix of the apoptotic domain orients with nonpolar leucine and alanine residues inserting most deeply into the lipid environment.     

Synthesis and CD studies of an 88-residue peptide containing the main receptor binding site of HTLV-I SU-glycoprotein

Essential HTLV-I biological functions, like host-cell receptor recognition, depend on the structural motives on the surface glycoprotein gp46. We defined a peptide of 88 amino acids [Arg¹⁴⁷-Leu²³⁴] corresponding to the central part of the protein sequence, where major neutralizing epitopes are localized. After evaluating the feasibility of its chemical synthesis, the chosen sequence was realized using the stepwise solid-phase methodology. Multiple chromatographic purification steps were required to obtain a sample suitable for structural analysis. Correct folding was supported by strong binding of monooclonal antibodies, recognizing known exposed immunodominant regions. Circular dichroism studies confirmed a non-random conformation of at least 70–80% of the synthetic peptide. Investigation of the 3D-structure of the synthetic peptide will provide useful information for future vaccine and drug-design strategies. © 1997 European Peptide Society and John Wiley & Sons, Ltd. J. Pep. Sci.3: 347–353 No. of Figures: 5. No. of Tables: 0. No. of References: 23     

Circular dichroism study of ribonuclease A mutants containing the minimal structural requirements for dimerization and swapping

Four residues Pro19, Leu28, Cys31 and Cys32 proved to be the minimal structural requirements in determining the dimeric structure and the N-terminal segment swapping of bovine seminal ribonuclease, BS-RNase. We analyzed the content of secondary and tertiary structures in RNase A, P-RNase A, PL-RNase A, MCAM-PLCC-RNase A and MCAM-BS-RNase, performing near and far-UV CD spectra. It results that the five proteins have very similar native conformations. Thermal denaturation at pH 5.0 of the proteins, studied by means of CD measurements, proved reversible and well represented by the two-state ND transition model. Thermodynamic data are discussed in the light of the structural information available for RNase A and BS-RNase.     

Effect of protein conformation on rate of deamidation: ribonuclease A

The effect of the folded conformation of a protein on the rate of deamidation of a specific asparaginyl residue has been determined. Native and unfolded ribonuclease A (RNase A) could be compared under identical conditions, because stable unfolded protein was generated by breaking irreversibly the protein disulfide bonds. Deamidation of the labile Asn-67 residue of RNase A was followed electrophoretically and chromatographically. At 80 degrees C, similar rates of deamidation were observed for the disulfide-bonded form, which is thermally unfolded, and the reduced form. At 37 degrees C and pH 8, however, the rate of deamidation of native RNase A was negligible, and was more than 30-fold slower than that of reduced, unfolded RNase A. This demonstrates that the Asn-67 residue is located in a local conformation in the native protein that greatly inhibits deamidation. This conformation is the beta-turn of residues 66-68.     

Individual tyrosine side-chain contributions to circular dichroism of ribonuclease

Experimental and theoretical studies using site-directed mutants of ribonuclease A (RNase A) offer more extensive information on the tyrosine side-chain contributions to the circular dichroism (CD) of the enzyme. Bovine pancreatic RNase A has three exposed tyrosine residues (Tyr73, Tyr76, and Tyr115) and three buried tyrosine residues (Tyr25, Tyr92 and Tyr97). The difference CD spectra between the wild type and the mutants at pH 7.0 (Deltaepsilon(277,wt) - Deltaepsilon(277,mut)) show bands with more negative DeltaDeltaepsilon(277) values for Y73F and Y115F than those for Y25F and Y92F and bands with positive DeltaDeltaepsilon(277) values for Y76F and Y97F. The theoretical calculations are in good semiquantitative agreement for all the mutants. The pH difference spectrum (pH 11.3-7.0) for the wild type shows a negative band at 295 nm and an enhanced positive band at 245 nm. The three mutants at buried tyrosine sites and one mutant at an exposed tyrosine site (Y76F) exhibit pH-difference spectra that are similar to that of the wild type. In contrast, two mutants at exposed tyrosine sites (Y73F and Y115F) exhibit diminished 295-nm negative bands and, instead of positive bands at 245 nm, negative bands are observed. Our results indicate that Tyr73 and Tyr115, two of the exposed tyrosine residues, are the largest contributors to the 277- and 245-nm CD bands of RNaseA, but the buried tyrosine residues and the one remaining exposed residue also contribute to these bands. Disulfide contributions to the 277- and 240-nm bands and the peptide contribution to the 240-nm band are confirmed theoretically.     

Separation and analysis of the glycoform populations of ribonuclease B using capillary electrophoresis

The development of methods to separate, analyse and monitor changes in glycoform populations is essential if a more detailed understanding of the structure, function and processing of glycoproteins is to emerge. In this study, intact ribonuclease B was resolved by borate capillary electrophoresis into five populations according to the particular oligomnnose structure associated with each glycoform. The relative proportions of these populations are correlated with the percentages obtained indirectly by analysis of the hydrazine released oligosaccharides using Bio-Gel P-4 gel filtration, matrix assisted laser desorption mass spectrometry and high performance anion exchange chromatography. Alterations in the composition of the glycoform populations during digestion of ribonuclease B withA. saitoi (1–2)mannosidase were monitored by capillary electrophoresis (CE). Digestion of the free oligosaccharides under the same conditions, monitored by anion exchange chromatography, revealed a difference in rate, allowing some insight into the role of the protein during oligosaccharide processing. In conjunction with other methods, this novel application of CE may prove a useful addition to the techniques available for the study of glycoform populations.     

Large-scale purification and characterization of recombinant Pseudomonas ceramidase: regulation by calcium

Ceramidases (CDases) hydrolyze ceramide to sphingosine (SPH) and fatty acid. Pseudomonas CDase (pCDase) is a homolog of mammalian neutral ceramidases and may play roles in disease pathogenesis. In this study, pCDase was cloned and expressed in Escherichia coli (E. coli). The expressed recombinant pCDase was solubilized by optimizing several factors, including culture medium, the concentration of isopropyl-beta-thiogalactopyranoside (IPTG), temperature, and time of induction, which were identified to be critical for the optimal production of recombinant pCDase. The recombinant pCDase was purified using nickel-nitrilotriacetic acid affinity, phenyl-Sepharose, and Q-Sepharose column chromatography, which gave an overall yield of 0.45 mg/l purified protein of starting culture. The activity of the recombinant pCDase followed classical Michaelis-Menten kinetics, with optimum activity in the neutral pH range. Both the hydrolytic and the reverse activities of CDase were stimulated by calcium with an affinity constant (K(a)) of 1.5 microM. Kinetics studies showed that calcium caused a decrease of K(m) and an increase in V(max) of pCDase. Calcium and D-erythro-sphingosine caused significant changes in the near ultraviolet circular dichroism (CD) spectra and the changes were inhibited in the presence of EGTA. These results identify important interactions between calcium and pCDase, which may play an essential role in the interaction of pCDase and its substrate.