Thermostabilization of ovalbumin by an alkaline treatment: examination for the possible implications of an altered serpin loop structure

Ovalbumin, a member of the serpin superfamily, is transformed via an intermediate state into a non-cleaved, thermostabilized form (S-ovalbumin) during either the storage of unfertilized eggs or development of fertilized eggs; essentially the same thermostabilization also occurs upon in vitro incubation of isolated ovalubumin under alkaline conditions. To investigate the implications of a partial insertion of the alpha-helical serpin loop into beta-sheet A that has been proposed as a conformational mechanism for S-ovalbumin production, we examined the thermostabilization process of ovalbumin with different loop structures. When the thermostabilization processes were compared for the intact, P1-P1'-cleaved and P1-P1'/P8-P7-cleaved forms of egg white ovalbumin, both the rates for the conversion from the native to intermediate and from the intermediate to S-ovalbumin were almost indistinguishable among the three protein forms. Furthermore, the fully loop-inserted form of recombinant ovalbumin mutant R339T that had been thermostabilized by P1-P1' cleavage with Tm values from 72 to 88 degrees C was further thermostabilized by an alkaline treatment, yielding a final product (loop inserted S-ovalbumin) with a Tm value of 93 degrees C. No significant difference was found between native ovalbumin and S-ovalbumin in respect of the rate of proteolytic cleavage of the loop by elastase and subtilisin. These data strongly suggest that S-ovalbumin is produced by a mechanism other than that of the partial loop insertion model.     

Deglycosylation of ovalbumin prohibits formation of a heat-stable conformer

To study the influence of the carbohydrate-moiety of ovalbumin on the formation of the heat-stable conformer S-ovalbumin, ovalbumin is deglycosylated with PNGase-F under native conditions. Although the enzymatic deglycosylation procedure resulted in a complete loss of the ability to bind to Concavalin A column-material, only in about 50% the proteins lost their complete carbohydrate moiety, as demonstrated by mass spectrometry and size exclusion chromatography. Thermal stability and conformational changes were determined using circular dichroism and differential scanning calorimetry and demonstrated at ambient temperature no conformational changes due to the deglycosylation. Also the denaturation temperature of the processed proteins remained the same (77.4 +/- 0.4 degrees C). After heat treatment of the processed protein at 55 degrees C and pH 9.9 for 72 h, the condition that converts native ovalbumin into the heat-stable conformer (S-ovalbumin), only the material with the intact carbohydrate moiety forms this heat-stable conformer. The material that effectively lost its carbohydrate moiety appeared fully denatured and aggregated due to these processing conditions. These results indicate that the PNGase-F treatment of ovalbumin prohibits the formation and stabilization of the heat-stable conformer S-ovalbumin. Since S-ovalbumin in egg protein samples is known to affect functional properties, this work illustrates a potential route to control the quality of egg protein ingredients.     

Thermostabilization of porcine kidney D-amino acid oxidase by a single amino acid substitution

D-amino acid oxidase (DAO) is of considerable practical importance, such as bioconversion and enzymatic assay. In this study, we succeeded in obtaining a thermostable mutant DAO from porcine kidney by a single amino acid substitution. This mutant enzyme, F42C, was stable at 55 degrees C, while the wild-type enzyme was stable only up to 45 degrees C. The Km values of F42C for D-amino acids was about half of those of the wild-type enzyme. This mutant DAO with improved stability and affinity for its substrates is advantageous for the determination of D-amino acids.     

嗜硫色谱分离纯化碱性脂肪酶及其氨基酸序列测定

扩展青霉(Penicillium expansum)FS1884所产生的碱性脂肪酶经硫酸铵沉淀、Sephacryl S-200柱层析后,再经嗜硫色谱(Thiophilic Chromatography)柱层析,被纯化了173．8倍,最终比活为5694．9U／mg。纯化后的脂肪酶达到了SDS-PAGE电泳纯、PAGE电泳纯以及毛细管液相色谱(Capillary Liquid Chromatography)纯。该脂肪酶N-端氨基酸序列测定的结果是：A T A D A A A F P D L H R A A K L S S A,与来自青霉属其它真菌脂肪酶一级结构作了比较。     

Protein purification by bulk crystallization: the recovery of ovalbumin

Crystallization is used industrially for the recovery and purification of many inorganic and organic materials. However, very little is reported on the application of bulk crystallization for proteins. In this work, ovalbumin was selected as a model protein to investigate the feasibility of using bulk crystallization for the recovery and purification of proteins. A stirred 1-L seeded batch crystallizer was used to obtain the crystal growth kinetics of ovalbumin in ammonium sulfate solutions at 30 degrees C. The width of the metastable region, in which crystal growth can occur without any nucleation, is equivalent to a relative supersaturation of about 20. The bulk crystallizations were undertaken within this range (using initial relative supersaturations less than 10) and nucleation was not observed. The ovalbumin concentration in solution was measured by UV absorbance and checked by crystal content measurement. Crystal size distributions were measured both by using a Malvern Mastersizer and by counting crystals through a microscope. The crystal growth rate was found to have a second-order dependence upon the ovalbumin supersaturation. While there is no discernible effect of ammonium sulfate concentration at pH 4.90, there is a slight effect at higher pH values. Overall the effect of ammonium sulfate concentration is small compared to the effect of pH, for which there is a 10-fold increase in the growth rate constant, k(Gsigma) over the range pH 4.6-5.4. To demonstrate the degree of purification which can be achieved by bulk crystallization, ovalbumin was crystallized from a solution containing conalbumin (80,000 Da) and lysozyme (14, 600 Da). After one crystallization and a crystal wash, ovalbumin crystals were produced with a protein purity greater than 99%. No contamination by the other proteins was observed when using overloaded sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie blue stain and only trace amounts of lysozyme were observed using a silver stain. The presence of these other proteins in solution did not effect the crystal growth rate constant, k(Gsigma). The study demonstrates the feasibility of using bulk crystallization for the recovery and purification of ovalbumin. It should be readily applicable to other protein systems. (c) 1995 John Wiley & Sons, Inc.     

酸性和碱性酶稳定性机制及其识别

了解酸性和碱性酶稳定性机制并对其进行识别具有重要理论和实践意义。通过分析105条酸性酶和111条碱性酶序列的氨基酸组成, 结果表明: 酸性酶中Trp、Tyr、Thr和Ser的含量明显高于平均值, 而Glu、Lys、Met和Arg的含量则明显低于平均值; 碱性酶中Trp、Ala和Cys的含量略高于平均值, 而Lys、Arg和Glu的含量则略低于平均值; 酸性和碱性酶中Ala、Glu、Leu、Asn、Arg、Ser和Thr的含量存在较大差异。在此基础上, 发展了一种加权氨基酸组成的方法对两种酶进行识别, 其自一致性检验的识别精度可达86.1%, 5倍交叉验证的精度为83.3%。建立了一种基于序列识别酸性和碱性酶的新方法。     

利用荧光手性试剂（S）—TBMB甲酸分析氨基糖的D—和L—构型

D、L型氨基糖经荧光手性试剂 (S) ( ) 2 叔丁基 2 甲基 苯并 1 ,3 二氧杂环戊烷 4 甲酸 [(S) TBMB甲酸 ]标记、完全乙酰基化得到非对映的氨基糖完全乙酰基化N (S) TBMB羧酸衍生物。其1 HNMR图谱 ,特别是强的叔丁基及甲基信号峰被用于分析氨基糖的D、L构型。此外 ,还利用反相HPLC及同样的荧光标识方法创立了简便的高灵敏度氨基糖D、L构型分析方法。其全部分析操作时间在 2h内 ,检测极限为 0 .2 pmol     

Conversion of cysteinyl residues to unnatural amino acid analogs. Examination in a model system

Improved and efficient techniques have led to an explosive growth in the application of site-directed mutagenesis to the study of enzymes. However, the limited availability of only those 20 amino acids that are translated by the genetic code has prevented the systematic variation of an amino acid's properties in order to define more precisely its role in the catalytic mechanism of an enzyme. An approach is being examined that combines the high specificity of site-directed mutagenesis with the flexibility of chemical modification to overcome these limitations. A set of reagents has been synthesized and reacted with a cysteine model to produce a series of amino acid structural analogs at appreciable rates and in good overall yields. The selective incorporation of these analogs in place of important functional amino acids in a protein will allow a more detailed examination of the role of that amino acid.     

Roles of conserved basic amino acid residues and activation mechanism of the hyperthermophilic aspartate racemase at high temperature

X-ray crystallography has revealed two similar alpha/beta domains of the aspartate racemase from the hyperthermophilic archaeon, Pyrococcus horikoshii OT3. The active site is located in the cleft between the two domains where two cysteine residues face each other. This arrangement allows the substrate to enter the cleft and enables the two cysteine residues to act synergistically. However, the distance between their thiolates was estimated to be 9.6 angstroms, which is beyond the distance for cooperative action of them. We examined the molecular mechanism for the racemization reaction of this hyperthermophilic aspartate racemase by mutational analyses and molecular dynamics simulations. The mutational analyses revealed that Arg48 and Lys164 were essential for catalysis in addition to the putative catalytic cysteine residues. The molecular dynamics simulations revealed that the distance between the two active gamma-sulfur atoms of cysteine residues oscillate to periodically become shorter than the predicted cooperative distance at high temperature. In addition, the conformation of Tyr160, which is located at the entrance of the cleft and inhibits the entry of a substrate, changes periodically to open the entrance at 375 K. The opening of the gate is likely to be induced by the motion of the adjacent amino acid, Lys164. The entrance of an aspartate molecule was observed by molecular dynamics (MD) simulations driven by the force of the electrostatic interaction with Arg48, Lys164, and also Asp47. These results provide insights into the roles of amino acid residues at the catalytic site and also the activation mechanism of a hyperthermophilic aspartate racemase at high temperature.     

Dissociation of c‐Met phosphotyrosine sites in human cells in response to mouse hepatocyte growth factor but not human hepatocyte growth factor: the possible roles of different amino acids in different species

Hepatocyte growth factor (HGF) is essential for embryogenesis, tissue regeneration and tumour malignancy through the activation of its receptor, c‐Met. We previously demonstrated that HGF α‐chain hairpin–loop, K1 domain and β‐chain are required for c‐Met signalling. The sequential phosphorylation of tyrosine residues, from c‐Met kinase domain to multidocking regions, is required for HGF‐signalling transduction. Herein, we provide evidence that the disconcerted activation of c‐Met tyrosine regions fails to induce biological functions. When human cells were incubated with ‘mouse HGF’, kinase domain activation (i.e. phospho‐Tyr‐1230/34/35) became evident, but the multidocking site (i.e. Tyr‐1349) was not phosphorylated, resulting in unsuccessful induction of migration and mitogenesis. The binding ability of mouse HGF α‐chain, or of β‐chain, to human c‐Met was lower than that of human HGF, as evidenced by HGF–chimera assay. Notably, only four amino acid positions in HGF α‐chain hairpin–loop and K1 domain and six positions in β‐chain differed between human HGF and mouse HGF. The human‐specific amino acids (such as Gln‐95 in hairpin–loop, Arg‐134 in K1 domain and Cys‐561 in β‐chain) may be important for accurate c‐Met assembly and signalling transduction. Copyright © 2012 John Wiley & Sons, Ltd.     

Examination of Conditions Inhibiting the Formation of Acrylamide in the Model System of Fried Potato

Acrylamide (AAm) is produced in food through the reaction of asparagine and reducing sugar. We examined several methods of reducing the level of AAm using potato tubers. The fried model system that we employed consisted of thin slices that were first treated in water under different conditions before frying. A sufficient amount of water present in the fry material acts as an inhibitor against the formation of AAm and allows only a negligible amount of AAm to form. It was found that given the low content of water, the fry material temperature was sufficiently high to allow a relatively large level of AAm to form. Examination of water treatment prior to frying revealed that higher-temperature treatment water and longer treatment time resulted in the formation of lower levels of AAm. Moreover, removing some of the residual heat had an inhibiting effect on the formation of AAm.     

Structure‐based replacement of methionine residues at the catalytic domains with serine significantly improves the oxidative stability of alkaline amylase from alkaliphilic Alkalimonas amylolytica

The alkaline amylase requires high resistance towards chemical oxidation for use in the detergent and textile industries. This work aims to improve the oxidative stability of alkaline amylase from alkaliphilic Alkalimonas amylolytica by site‐directed mutagenesis based on the enzyme structure model. Five mutants were created by individually replacing methionine at positions 145, 214, 229, 247, and 317 in the amino acid sequence of alkaline amylase with oxidative‐resistant serine. The pH stability of the mutant enzymes was almost the same as that of the wild‐type (WT) enzyme (pH 7.0–11.0). The stable temperature range of the mutant enzymes M145S and M247S decreased from <50°C of the WT to <40°C, while the thermal stability of the other three mutant enzymes (M214S, M229S, and M317S) was almost the same as that of the WT enzyme. The catalytic efficiency (k_cat/K_m) of all the mutant enzymes decreased when compared to WT enzyme. The mutant enzymes showed increased activity in the presence of surfactants Tween‐60 and sodium dodecyl sulfate. When incubated with 500 mM H₂O₂ at 35°C for 5 h, the WT enzyme retained only 13.3% of its original activity, while the mutant enzymes M145S, M214S, M229S, M247S, and M317S retained 55.6, 70.2, 54.2, 62.5, and 46.4% of the original activities, respectively. The results indicated that the substitution of methionine residues at the catalytic domains with oxidative‐resistant serine can significantly improve the oxidative stability of alkaline amylase. This work provides an effective strategy to improve the oxidative stability of amylase, and the high oxidation resistance of the mutant enzymes shows their potential applications in the detergent and textile industries. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Kinetic resolution of esters of amino acids in t-butanol containing 5% water catalyzed by a stable industrial alkaline protease

We developed a procedure for the resolution of esters of amino acids in 95% t-butanol, followed by saponification of the unreacted esters to afford both enantiomers with high yield and optical purity. The hydrolysis, catalyzed by alkaline protease, was conducted in a mixture of t-butanol (95%) and water (5%) at 25°C, with a pH controlled at pH 8.5 by the addition of NaOH (2 M). The hydrolyzed L -amino acid, which was insoluble under these conditions, precipitated during the course of hydrolysis. After separation of the precipitate, the pH of the filtrate was adjusted to 11.5 to saponify the unreacted ester. The D -antipode precipitated at pH 6.2–6.5. Both optically pure antipodes were obtained with high enantiomeric excesses and yields by simple filtration. © 1994 Wiley-Liss, Inc.     

Two-dimensional near-IR correlation spectroscopy study of molten globule-like state of ovalbumin in acidic pH region: simultaneous changes in hydration and secondary structure

The molten globule-like states of ovalbumin (OVA) in acid aqueous solutions are investigated by generalized two-dimensional (2D) Fourier transform near-IR (FT-NIR) correlation spectroscopy. This new method allows us to explore the changes in hydration and the secondary structure simultaneously. FT-NIR spectra are measured for OVA aqueous solutions with concentrations of 1, 2, 3, 4, and 5 wt % over a pH range of 2.4-5.4. Concentration-perturbed 2D correlation spectra are calculated for the spectra in the 4850-4200 and 7500-5350 cm(-1) regions at different pH values. The 2D NIR synchronous spectrum in the 4850-4200 cm(-1) region shows a significant change upon going from pH 5.4 to 3.6. An autopeak at 4265 cm(-1) that is due to a combination of a symmetric CH(2) stretching mode and a CH(2) bending mode of side chains seen at pH 5.0 disappears completely in the synchronous spectrum at pH 3.6. This suggests that some amino acid residues of OVA are subjected to microenvironmental changes with decreasing pH. More remarkable changes are observed in the synchronous spectra at pHs below 2.8. A band near 4600 cm(-1) arising from a combination of amide B and amide II modes (amide B/II) shifts downward with considerable broadening between pH 3.0 and 2.4, suggesting that the strength of the hydrogen bonds of amide groups of OVA changes significantly. The synchronous and asynchronous spectra in the 4850-4200 cm(-1) region show that the intensities of the bands attributable to amide groups and side chains of OVA and that of the band near 4800 cm(-1) arising from water change in phase with the increase in the concentration above pH 2.8, but they vary out of phase below pH 2.8. The 2D synchronous map in the 7500-5350 cm(-1) region also shows marked changes upon going from pH 2.8 to 2.6. A broad autopeak at around 6950 cm(-1) assigned to free water and bound water with weak hydrogen bonds becomes very weak in the synchronous spectrum at pH 2.6, while broad autopeaks around 6450 cm(-1) suddenly appear that are due to bound water with several hydrogen bonds and the first overtone of an NH stretching mode of the amide groups of OVA. Therefore, it is very likely that protein hydration and the hydrogen bonds of amide groups change simultaneously in a narrow pH region of 2.8-2.6. It is probably that below pH 2.6 the protein assumes a molten globule-like state in which the whole molecule is very flexible, and side chains (but not the backbone chain) fluctuate significantly.     

Thermostabilization by replacement of specific residues with lysine in a Bacillus alkaline cellulase: building a structural model and implications of newly formed double intrahelical salt bridges

An alkaline, mesophilic endo-1,4-beta-glucanase from alkaliphilic Bacillus sp. strain KSM-64 was significantly thermostabilized by replacement of both Asn179 and Asp194 with lysine by site-directed mutagenesis. Structural remodeling of the mutant enzyme newly generated by the double mutation suggested that Glu175-->Lys179 and Glu190-->Lys194 were the most plausible ion pairs, both of which involved side chains at the i and i + 4 positions on the alpha(4)-helix from Glu175 to Ser195. By molecular dynamics simulations, the N(zeta) hydrogens of Lys179 and Lys194 were found to coordinate with the carbonyl O(varepsilon1) and O(varepsilon2) of Glu175 and the carbonyl O(varepsilon1) of Glu190, respectively, with distances of around 2 A for all. These results confirm that the formation of these double intrahelical ion pairs (salt bridges) is responsible for the thermostabilization by the double mutation.     

Improvement of antibody affinity by introduction of basic amino acid residues into the framework region

Antibodies are widely used not only as therapeutic agents but also as research tools and diagnostic agents, and extensive efforts have been made to generate antibodies that have higher affinity. It was recently reported that introduction of charged residues into the framework region of an antibody improved its affinity; however, the underlying molecular mechanism has not been elucidated. In this study, we used kinetic and thermodynamic analyses of the antibody–antigen interaction to investigate the molecular mechanism by which an antibody with introduced charged residues recognizes its antigen with higher affinity. The introduction of basic amino acid residues resulted in improvement of the affinity whereas the introduction of acidic residues weakened the interaction. For two mutant antigen-binding fragments (Fabs) with improved affinity (named K5- and R5-mutants), the balance between the association rate constant k_on and the dissociation rate constant k_off was distinct despite each mutant having the same number of charged residues. Moreover, thermodynamic analysis of the interactions in the transition state revealed a difference between the K5- and R5-mutants in terms of enthalpic energy change following formation of the encounter complex with the antigen. These results suggest that the affinity of the K5- and R5-mutants is improved by distinct mechanisms. Although the mutations destabilize the Fab and necessitate further studies, our strategy is expected to become a versatile and simple means to improve the affinity of antibodies to their antigens.     

An improved synthesis of (2S, 4S)‐ and (2S, 4R)‐2‐amino‐4‐methyldecanoic acids: assignment of the stereochemistry of culicinins

An improved synthesis of (2S, 4S)‐ and (2S, 4R)‐2‐amino‐4‐methyldecanoic acids was accomplished using a glutamate derivative as starting material and Evans' asymmetric alkylation as the decisive step. The NMR data of the two diastereomers were measured and compared with those of the natural product. As a result, the stereochemistry of this novel amino acid unit in culicinins was assigned as (2S, 4R). Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Haptenization of ovalbumin with the skin sensitizer methyl octanesulfonate: characterization of the methylated OVA323-339 T-cell epitope at His331.

Our interest is focused on the induction of allergic contact dermatitis (ACD) by the strong skin sensitizer, methyl octanesulfonate, which is a potent methyl transfer agent, especially to histidine and methionine residues. We are particularly interested to study the effect of methylation on the presentation and recognition of the ovalbumin (OVA) T-cell epitope, OVA323-339, by the T-cell receptor (TCR). Here we report the synthesis of the modified monomer N-alpha-Fmoc-N-tau-methyl-L-histidine and its incorporation by solid phase synthesis into the three possible methylated analogues of OVA323-339, that were needed as references for the subsequent studies. Native OVA was haptenized by methyl octanesulfonate. Using classical protein chemistry techniques (trypsin digestion, gel permeation, HPLC, MS and Edman sequencing) we were able to show that OVA323-339 was selectively methylated at His331. Circular dichroism (CD) studies showed that the methylation has no influence on the secondary structure of the peptide.     

Enantioselectivity of bunitrolol 4‐hydroxylation is reversed by the change of an amino acid residue from valine to methionine at position 374 of cytochrome P450‐2D6

The enantioselectivity of 4‐hydroxylation of bunitrolol (BTL), a β‐adrenoceptor blocking drug, was studied in microsomes from human liver, human hepatoma (Hep G2) cells expressing CYP2D6, and lymphoblastoid cells expressing CYP2D6. Kinetics in human liver microsomes showed that the Vmax value for (+)‐BTL was 2.1‐fold that of (−)‐BTL, and that the Km value for (+)‐BTL was lower than that for the (−)‐antipode, resulting in the intrinsic clearance (Vmax/Km) of (+)‐BTL being 2.1‐fold over its (−)‐antipode. CYP2D6 (CYP2D6‐met) expressed in Hep G2 cells had a methionine residue at position 373 of the amino acid sequence and a rat‐type N‐terminal peptide (MELLNGTGLWSM) instead of the human‐type (MGLEALVPLAVIV), and showed enantioselectivity of [(+)‐BTL < (−)‐BTL] for the rate of BTL 4‐hydroxylation. In contrast, enantioselectivity [(+)‐BTL > (−)‐BTL] for Hep G2‐CYP2D6 (CYP2D6‐val) with a human‐type N‐terminal peptide that had a valine residue at 374, which corresponds to the methionine of the CYP2D6‐met variant, was the same as that for human liver microsomes. We further confirmed that CYP2D6‐met and CYP2D6‐val expressed in human lymphoblastoid cells, both of which have methionine and valine, respectively, at position 374 and a human‐type N‐terminal peptide, exhibited the same enantioselectivities as those obtained from CYP2D6‐met and CYP2D6‐val expressed in the Hep G2 cell system. These results indicate that the amino acid at 374 of CYP2D6 is one of the key factors influencing the enantioselectivity of BTL 4‐hydroxylation. Chirality 11:1–9, 1999. © 1999 Wiley‐Liss, Inc.     

Application of alkaline treatment for sludge decrement and humic acid recovery

A new method was introduced to reduce waste activated sludge and extract humic acid for liquid fertilizer. Sludge was disintegrated with NaOH (0.4 g/g dry solid, 8 h) and then centrifuged to obtain the supernatant. The residual sludge was then dewatered, while the supernatant was used to extract humic acid with an ultrafiltration membrane. The results showed that the alkaline treatment dissolved more than half of the sludge organic matter, which was composed of 24% humic acid by mass. After the supernatant was concentrated 20 times using a membrane with a molecular weight cutoff of 1000, the retentate contained 94.5% of the dissolved organics and could be used to produce humic acid fertilizer. Additionally, only 26% of the NaOH was consumed and the residual NaOH in the permeate flux could be reused. Due to the removal of water and organics, the dewatered sludge could be reduced by 60% when compared to samples that did not receive the alkaline treatment.