Catalytic activity and thermostability of dehydrogenase conjugates with cortisol and progesterone.

The conjugates of glucose-6-phosphate dehydrogenase, lactate dehydrogenase, and malate dehydrogenase with progesterone and cortisol, containing 1-40 steroid molecules per enzyme molecule, were obtained by the reactions of N-succinimide esters of the 3-[O-(carboxymethyl)oximes)] of cortisol and progesterone with a protein in a water-DMFA (10%) medium. The catalytic activity and thermostability of dehydrogenases and their steroid conjugates were kinetically studied. The effects of the modification degree on the activity and thermostability of dehydrogenases by their hydrophobization were studied and discussed. Practical recommendations for using the dehydrogenase-steroid conjugates in enzyme immunoassay are given.     

一个耐热碱性磷酸酯酶突变型的研究

为了进一步揭示蛋白质的耐热机制,对含有耐热碱性磷酸酯酶（ＦＤ－ＴＡＰ）的表达质粒ｐＴＡＰ５０３Ｆ进行了随机诱变,用菌落原位显色法从约５０００个转化子中筛选到４个耐热性下降的突变型克隆,并对其中１克隆（ＴＡＰＭ３）进行了部分酶学性质、ＤＮＡ和氨基酸序列的研究。酶学性质研究表明,与野生型相比,该突变型酶的耐热性有较大幅度的下降,而热激活性无明显改变。ＤＮＡ序列分析表明在１２３９位ＴＡＰＭ３发生Ｇ→Ａ转     

Basis of thermostability in pig heart lactate dehydrogenase treated with O-methylisourea

Acetamidination of pig heart lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) with ethyl acetimidate resulted in an increase of thermostability, and covalent bridge formation between pairs of lysine residues is observed. Guanidination with O-methylisourea of the enzyme also increases the thermostability, but such a bridge seems not to be formed. Increased thermostability of guanidinated enzyme is considered to be due to the shift of the pK values of the lysine residues from 10.5 to 12.5 after guanidination. Modification experiments with carbodiimide reveals that the enzyme contains 4.6 pairs of neighboring lysine and carboxyl residues per subunit, and amide bonding between 3.2 pairs results in an increase of thermostability. Guanidination of 4.6 Lys/subunit of the enzyme yields an enzyme derivative with considerably increased thermostability. Salt bridge formation between the 4.6 pairs of neighboring carboxyl and guanidinated lysine residues per subunit might make a major contribution to the increased thermostability of the guanidinated enzyme.     

Fluorinated chloramphenicol acetyltransferase thermostability and activity profile: improved thermostability by a single-isoleucine mutant

A lysate-based thermostability and activity profile is described for chloramphenicol acetyltransferase (CAT) expressed in trifluoroleucine, T (CAT T). CAT and 13 single-isoleucine CAT mutants were expressed in medium supplemented with T and assayed for thermostability on cell lysates. Although fluorinated mutants, L82I T and L208I T, showed losses in thermostability, the L158I T fluorinated mutant demonstrated an enhanced thermostability relative to CAT T. Further characterization of L158I T suggested that T at position 158 contributed to a portion of the observed loss in thermostability upon global fluorination.     

Increased thermostability of microbial transglutaminase by combination of several hot spots evolved by random and saturation mutagenesis

The thermostability of microbial transglutaminase (MTG) of Streptomyces mobaraensis was further improved by saturation mutagenesis and DNA-shuffling. High-throughput screening was used to identify clones with increased thermostability at 55°C. Saturation mutagenesis was performed at seven "hot spots", previously evolved by random mutagenesis. Mutations at four positions (2, 23, 269, and 294) led to higher thermostability. The variants with single amino acid exchanges comprising the highest thermostabilities were combined by DNA-shuffling. A library of 1,500 clones was screened and variants showing the highest ratio of activities after incubation for 30 min at 55°C relative to a control at 37°C were selected. 116 mutants of this library showed an increased thermostability and 2 clones per deep well plate were sequenced (35 clones). 13 clones showed only the desired sites without additional point mutations and eight variants were purified and characterized. The most thermostable mutant (triple mutant S23V-Y24N-K294L) exhibited a 12-fold higher half-life at 60°C and a 10-fold higher half-life at 50°C compared to the unmodified recombinant wild-type enzyme. From the characterization of different triple mutants differing only in one amino acid residue, it can be concluded that position 294 is especially important for thermostabilization. The simultaneous exchange of amino acids at sites 23, 24, 269 and 289 resulted in a MTG-variant with nearly twofold higher specific activity and a temperature optimum of 55°C. A triple mutant with amino acid substitutions at sites 2, 289 and 294 exhibits a temperature optimum of 60°C, which is 10°C higher than that of the wild-type enzyme.     

Effect of mutations at Glu160 and Val198 on the thermostability of lactate oxidase.

We have obtained two types of thermostable mutant lactate oxidase - one that exhibited an E-to-G point mutation at position 160 (E160G) through error-prone PCR-based random mutagenesis, and another that exhibited an E-to-G mutation at position 160 and a V-to-I mutation at position 198 (E160G/V198I) through DNA shuffling-based random mutagenesis - both of which we have previously reported. Our molecular modeling of lactate oxidase suggests that the substitution of G for E at position 160 reduces the electrostatic repulsion between the negative charges of E160 and E130 in the (beta/alpha)8 barrel structure, but a thermal-inactivation experiment on the five kinds of single-mutant lactate oxidase at position 160 (E160A, E160Q, E160H, E160R, and E160K) showed that the side-chain volume of the amino acid at position 160 mainly contributes to the thermostability of lactate oxidase. We also produced V198I single-mutant lactate oxidase through site-directed mutagenesis, and analysed the thermostability of wild-type, V198I, E160G, and E160G/V198I lactate oxidase enzymes. The half-life of E160G/V198I lactate oxidase at 70 degrees C was about three times longer than that of E160G lactate oxidase, and was about 20 times longer than that of wild-type lactate oxidase. In contrast, the thermostability of the V198I lactate oxidase was almost identical to that of wild-type lactate oxidase. This indicates that the V198I mutation alone does not affect lactate oxidase thermostability, but does affect it when combined with the E160G mutation.     

Thermostability of manganese- and iron-superoxide dismutases from Escherichia coli is determined by the characteristic position of a glutamine residue.

The structurally homologous mononuclear iron and manganese superoxide dismutases (FeSOD and MnSOD, respectively) contain a highly conserved glutamine residue in the active site which projects toward the active-site metal centre and participates in an extensive hydrogen bonding network. The position of this residue is different for each SOD isoenzyme (Q69 in FeSOD and Q146 in MnSOD of Escherichia coli). Although site-directed mutant enzymes lacking this glutamine residue (FeSOD[Q69G] and MnSOD[Q146A]) demonstrated a higher degree of selectivity for their respective metal, they showed little or no activity compared with wild types. FeSOD double mutants (FeSOD[Q69G/A141Q]), which mimic the glutamine position in MnSOD, elicited 25% the activity of wild-type FeSOD while the activity of the corresponding MnSOD double mutant (MnSOD[G77Q/Q146A]) increased to 150% (relative to wild-type MnSOD). Both double mutants showed reduced selectivity toward their metal. Differences exhibited in the thermostability of SOD activity was most obvious in the mutants that contained two glutamine residues (FeSOD[A141Q] and MnSOD[G77Q]), where the MnSOD mutant was thermostable and the FeSOD mutant was thermolabile. Significantly, the MnSOD double mutant exhibited a thermal-inactivation profile similar to that of wild-type FeSOD while that of the FeSOD double mutant was similar to wild-type MnSOD. We conclude therefore that the position of this glutamine residue contributes to metal selectivity and is responsible for some of the different physicochemical properties of these SODs, and in particular their characteristic thermostability.     

Development of an Escherichia coli expression system and thermostability screening assay for libraries of mutant xylanase

A thermostability screening assay was developed using an Escherichia coli expression system to express Streptomyces lividans xylanase A (XlnA). The screening system was tested using mutants randomized at position 49 of the S. lividans XlnA gene, a position previously shown to confer thermostability with a I49P point mutation. The library was cloned into an E. coli expression vector and transformed into XL1-blue bacteria. The resulting clones were screened for increased thermostability with respect to wild-type XlnA. Using this assay, we isolated the I49P mutant previously shown to be thermostable, as well as novel I49A and I49C mutants. The I49A and I49C mutants were shown to have 2.8- to 8-fold increase in thermostability over that of wild-type XlnA. The results show that the screening assay can selectively enrich for clones with increased thermostability and is suitable for screening small- to medium-sized libraries of 5000–20,000 clones. Journal of Industrial Microbiology & Biotechnology (2000) 25, 310–314. Received 18 May 2000/ Accepted in revised form 19 September 2000     

残基突变对冷休克蛋白热稳定性影响的计算分析研究(英文)

残基突变是提高蛋白质热稳定性最直接有效的方式。在本文中,我们选取一对冷休克蛋白质作为研究对象,其中一个来自嗜温的Bacillus subtilis(Bs-CspB),另一个来自嗜热的Bacillus caldolyticus(Bc-Csp),这两个蛋白质在序列和结构上具有高度的相似性,但两者的耐热能力却相差很大。我们利用全原子模型计算残基突变前后蛋白质的自由能和氨基酸之间相互作用能的变化,分析残基突变对冷休克蛋白热稳定性的影响。通过对比两个蛋白质对应位置上残基的能量,我们成功鉴别出对Bc-Csp的高热稳定性有突出贡献的残基。我们计算了这些残基突变前后,该残基的静电相互作用和范德华相互作用的变化,以分析该残基对Bc-Csp高热稳定性的主要贡献。同时,我们分析了离子键对蛋白质热稳定性的贡献。我们的计算结果和实验结果吻合得很好,关键在于利用该方法可以详细地说明残基突变影响蛋白质热稳定性的根本原因。本文为研究残基突变对蛋白质热稳定性的影响提供了一种计算思路和方法,并有助于设计具有高耐热能力的蛋白质。     

Correlation of enzyme thermostability and its surface hydrophobicity (using a modified alpha-chymotrypsin as an example)

Basing on the hypothesis that contact of hydrophobic surface clusters of proteins with water is thermodynamically disadvantageous, it is suggested to carry out the hydrophilization of protein surface by covalent modification in order to increase its thermostability. Hydrophilic fragments were introduced into the surface of alpha-chymotrypsin using acylation by anhydrides of aromatic carboxylic acids and reductive alkylation by aliphatic aldehydes. As a result of the hydrophilization the stability of the enzyme against irreversible thermoinactivation increased thousand-fold. The correlation is observed between the degree of hydrophilization of the protein surface and the increase in thermostability of modified alpha-chymotrypsin. The level of thermostability achieved by covalent modification of alpha-chymotrypsin is practically equal to thermostability of proteinases from extreme thermophiles, the most stable proteolytic enzymes currently known.     

Improved thermostability and the optimum temperature of Rhizopus arrhizus lipase by directed evolution

To expand the functionality of lipase from Rhizopus arrhizus (RAL) we have used error-prone PCR and DNA shuffling methods to create RAL mutants with improved thermostability and the optimum temperature. One desirable mutant with three amino acids substitution was obtained. The mutated lipase was purified and characterized. The optimum temperature of the mutant lipase was higher by 10 °C than that of the wild-type RAL (WT-RAL). In addition, the thermostability characteristic of the mutant was also improved as the result of directed evolution. The half-life (T_1/2) at 50 °C of the mutant exceeded those of WT-RAL by 12-fold. To confirm which substitution contributed to enhance thermostability and the optimum temperature for lipase activity, three chimeric lipases: chimeric lipase 1(CL-1; A9T), chimeric lipase 2 (CL-2; E190V) and chimeric lipase 3 (CL-3; M225I) from the WT-RAL gene were constructed. Each of the chimeric enzymes was purified and characterized. Amino acid substitution at position 190 was determined to be critical for lipase thermostability and the optimum temperature, while the residue at position 9 and 225 had only marginal effect. The mutational effect is interpreted according to a simulated three-dimensional structure for the mutant lipase.     

Introduction of a stabilizing 10 residue beta-hairpin in Bacillus subtilis neutral protease.

A 10 residue beta-hairpin, which is characteristic of thermostable Bacillus neutral proteases, was engineered into the thermolabile neutral protease of Bacillus subtilis. The recipient enzyme remained fully active after introduction of the loop. However, the mutant protein exhibited autocatalytic nicking and a 0.4 degree C decrease in thermostability. Two additional point mutations designed to improve the interactions between the enzyme surface and the introduced beta-hairpin resulted in reduced nicking and increased thermostability. After the introduction of both additional mutations in the loop-containing mutant, nicking was largely prevented and an increase in thermostability of 1.1 degrees C was achieved.     

Catalysis and thermostability of mitochondrial F1-ATPase in toluene-phospholipid-low-water systems

Soluble mitochondrial F1-ATPase from bovine heart can be transferred to systems composed of a nonpolar solvent (toluene), phospholipid, and water at concentrations between 0.02 and 0.05% (volume of water per volume toluene). In these systems, F1 becomes resistant to cold denaturation and acquires a remarkable thermostability; i.e., its half-life at 70 degrees C is more than 24 h. Thermostability is due to the low content of water, since increases of water concentration bring about a progressive decrease in thermostability. At 0.04% water, the enzyme fails to catalyze a single splitting of ATP per enzyme. Gradual increases in water concentration up to 2.5% result in a progressive increase of hydrolytic activity. However, even at 2.5% water, the activity is orders of magnitude lower than in totally aqueous media. At various concentrations of water (0.1-2.5% v/v) and Mg-ATP, it was found that water affects the Vmax, but not the Km. The results show that, at levels of water below 0.04% (v/v), the enzyme is in a state that does not carry out catalysis and possesses high thermostability. As the water content is increased, the enzyme acquires the progressive flexibility that is required for catalysis and for undergoing rapid thermal denaturation.     

Third degree obstetric anal sphincter tears: risk factors and outcome of primary repair.

OBJECTIVES--To determine (i) risk factors in the development of third degree obstetric tears and (ii) the success of primary sphincter repair. DESIGN--(i) Retrospective analysis of obstetric variables in 50 women who had sustained a third degree tear, compared with the remaining 8553 vaginal deliveries during the same period. (ii) Women who had sustained a third degree tear and had primary sphincter repair and control subjects were interviewed and investigated with anal endosonography, anal manometry, and pudendal nerve terminal motor latency measurements. SETTING--Antenatal clinic in teaching hospital in inner London. SUBJECTS--(i) All women (n = 8603) who delivered vaginally over a 31 month period. (ii) 34 women who sustained a third degree tear and 88 matched controls. MAIN OUTCOME MEASURES--Obstetric risk factors, defecatory symptoms, sonographic sphincter defects, and pudendal nerve damage. RESULTS--(i) Factors significantly associated with development of a third degree tear were: forceps delivery (50% v 7% in controls; P = 0.00001), primiparous delivery (85% v 43%; P = 0.00001), birth weight > 4 kg (P = 0.00002), and occipito-posterior position at delivery (P = 0.003). No third degree tear occurred during 351 vacuum extractions. Eleven of 25 (44%) women who were delivered without instruments and had a third degree tear did so despite a posterolateral episiotomy. (ii) Anal incontinence or faecal urgency was present in 16 women with tears and 11 controls (47% v 13%; P = 0.00001). Sonographic sphincter defects were identified in 29 with tears and 29 controls (85% v 33%; P = 0.00001). Every symptomatic patient had persistent combined internal and external sphincter defects, and these were associated with significantly lower anal pressures. Pudendal nerve terminal motor latency measurements were not significantly different. CONCLUSIONS--Vacuum extraction is associated with fewer third degree tears than forceps delivery. An episiotomy does not always prevent a third degree tear. Primary repair is inadequate in most women who sustain third degree tears, most having residual sphincter defects and about half experiencing anal incontinence, which is caused by persistent mechanical sphincter disruption rather than pudendal nerve damage. Attention should be directed towards preventive obstetric practice and surgical techniques of repair.     

Estimating total genic diversity in the house mouse

In a survey of variation in both electrophoretic charge and thermostability at 14 structural loci in 40 strains of Mus musculus, 27 electromorphs (polypeptides differing in electrophoretic charge) and 20 thermomorphs (polypeptides differing in thermostability) were found. Electrophoresis detected 11 new variants within thermomorphs, and the heat denaturation technique detected four new variants within electromorphs. From these data, and making the assumption that both techniques are independent of each other, it is estimated that the actual total number of alleles at the 14 loci is 53, or an average of 3.79 per locus (1.96 per electromorph), and that electrophoresis apparently detects one-third of the variants, thus describing about 50% of the alleles at structural gene loci in the house mouse.     

Mutations in the alpha-amylase gene of Bacillus amyloliquefaciens, leading to a decrease in the temperature of protein inactivation

Alpha-amylase genes of Bacillus amyloliquefaciens, coding proteins with reduced thermostability, had been obtained as a result of hydroxylamine mutagenesis. Temperature, pH and starch concentration dependences of two mutant alpha-amylases were investigated. The synthesis of the alpha-amylases by several B. subtilis strains with different levels of extracellular proteases was also studied. The mutation containing fragments were localized and the structures of the mutations were determined. It was found that the decrease of thermostability of mutant No 141 was due to Asp to Asn change at the position No 194 of the mature protein, and for mutant No 191--due to Glu to Lys change at the position No 185.     

Effects of changing the interaction between subdomains on the thermostability of Bacillus neutral proteases.

Variants of the thermolabile neutral protease (Npr) of B. subtilis (Npr-sub) and the thermostable neutral protease of B. stearothermophilus (Npr-ste) were produced by means of site-directed mutagenesis and the effects of the mutations on thermostability were determined. Mutations were designed to alter the interaction between the middle and C-terminal subdomain of these enzymes. In all Nprs a cluster of hydrophobic contacts centered around residue 315 contributes to this interaction. In thermostable Nprs (like Npr-ste) a 10 residue beta-hairpin, covering the domain interface, makes an additional contribution. The hydrophobic residue at position 315 was replaced by smaller amino acids. In addition, the beta-hairpin was deleted from Npr-ste and inserted into Npr-sub. The changes in thermostability observed after these mutations confirmed the importance of the hydrophobic cluster and of the beta-hairpin for the structural integrity of Nprs. Combined mutants showed that the effects of individual mutations affecting the interaction between the subdomains were not additive. The effects on thermostability decreased as the strength of the subdomain interaction increased. The results show that once the subdomain interface is sufficiently stabilized, additional stabilizing mutations at the same interface do not further increase thermostability. The results are interpreted on the basis of a model for the thermal inactivation of neutral proteases, in which it is assumed that inactivation results from the occurrence of local unfolding processes that render these enzymes susceptible to autolysis.     

Number, activity and thermostability of the electrophoretic forms of acid phosphatase in Amoeba proteus, cultured at different temperatures

In free-living amoebae (Amoeba proteus, strain B), cultured at 10 and 25 degrees C, we compared the number, activity, and thermostability of separate electromorphs of Triton-soluble acid phosphatase (AcP) revealed by disc-electrophoresis in polyacrylamide gel using 2-naphthyl phosphate (pH 4.0) as a substrate. No differences in the number of AcP electromorphs and their mobility were observed at both these temperatures. The total activity of AcP electromorphas per unit of cellular protein and their total thermostability were lower in amoebae acclimated to 10 degrees C than to 25 degrees C. The above decrease may be a consequence of a simultaneous decrease in the activity and thermostability of two tartrate-sensitive electromorphs, both being of lysosomal nature. The total activity and thermostability of tartrate-resistant AcP electromorphs did not differ in amoebae acclimated to the two above temperatures. In amoebae cultured at 10 degrees C the fall of activity and thermostability of lysosomal AcP correlates with the decrease in their primary cell thermoresistance and phagocytic activity. The obtained results confirm the earlier conclusion (Vysotskaya et al., 1994) that lysosomes may be involved in acclimation of electrothermal animals to changing environmental temperatures.     

编码序列的（G＋C）%与蛋白质的耐热性相关性分析

运用计算机统计方法,对以木糖异构酶为主的几个蛋白质家族的核酸和氨基酸序列进行分析,发现密码子各位上的（Ｇ＋Ｃ）％与编码序列的（Ｇ＋Ｃ）％成线性正相关,大多数氨基酸的含量与编码序列的（Ｇ＋Ｃ）％也存在相关性,按其相关性,将氨基酸分为正相关,负相关和不相关３类,对木糖异构酶氨基酸序列和酶的耐热性的统计发现,那些在统计学上显著的,可能提高蛋白质耐热性的氨基酸替换,往往伴随关编码序列中ＧＣ含量的上升,这提     

Third degree laceration at delivery; etiological considerations,and a technique for repair

In a series of 14,080 cases in which either median or mediolateral episiotomy was used to facilitate delivery, third degree extension occurred in 75 cases (0.5 per cent). In related data extension of laceration was observed to occur in an inordinately high proportion of cases in association with use of forceps, greater than normal anterior pelvic depth, delivery of a large baby, primiparity, abnormal position and presentation, use of median incision (although extension also occurred in some cases in which mediolateral episiotomy was done), and hyperflexion and extreme abduction of the thighs. The method of immediate postpartum repair employed was associated with a minimal amount of postpartum discomfort, and late complications were almost nil.