发酵乳杆菌多铜氧化酶的异源表达及酶学性质

生物胺是一种存在于发酵食品中的含氮小分子有机化合物,过量摄入可能引起过敏或其他不良反应。利用酶法降解是减少发酵食品中生物胺含量从而保障食品安全的有效方法之一。文中成功克隆了来源于发酵乳杆菌的多铜氧化酶基因,在大肠杆菌中表达的酶活水平为484 U/L。通过镍柱亲和层析方法获得了此多铜氧化酶的纯酶。该多铜氧化酶的最适反应温度为50℃,最适反应pH为3.5,其K_m为1.3 mmol/L,V_(max)为7.67×10~(-2) mmol/(L·min)。对酶的应用特性研究表明,来源于发酵乳杆菌的多铜氧化酶对18%(W/F)NaCl有一定的耐受性,并可降解包括色胺、苯乙胺、腐胺、尸胺、组胺、酪胺和亚精胺在内的7种生物胺。其中它对组胺和酪胺的降解能力最高,分别为51.6%和40.9%。此外,该酶对酱油中的生物胺也有普遍降解作用,使用较低酶量(500 U/L)时,对酱油中总胺的降解率达到10.6%。多铜氧化酶具备降解发酵食品中生物胺的潜力,为进一步实现这类食品酶的实际应用奠定基础。     

Regulation of ornithine decarboxylase by ODC-antizyme in HTC cells.

Low concentrations of putrescine (10^?5M) blocked ornithine decarboxylase (ODC) in rat hepatoma (HTC) cells in culture, but the lower homologue of putrescine, 1, 3 diaminopropane, had no effect on ornithine decarboxylase at 10^?5M. Higher concentrations of both putrescine and 1, 3 diaminopropane induced approximately the same amount of soluble ODC antizyme type inhibitor. When concentrated dialyzed supernatants of cells grown in 10^?5M putrescine were treated with 250 mM NaCl and chromatographed on a superfine Sephadex G-75 column, both ODC and inhibitor were recovered. Spermidine, spermine and cadaverine also induced the inhibitor suggesting a low specificity of induction by amines.     

Apolar interaction of alpha-chymotrypsin with dimyristoyl phosphatidylcholine vesicles

The interaction of bovine pancreatic alpha-chymotrypsin with dimyristoyl phosphatidylcholine (PC) vesicles was measured turbimetrically. The protein interacted with the vesicles at NaCl concentrations of above 0.8 M. The turbidity reached a plateau on increase in the amount of either the protein or the vesicles in the presence of a fixed amount of the other component. The precipitates formed contained both PC and protein in ratios varying with the initial amount of each component. On mixing chymotrypsin and PC vesicles, time-dependent turbidity increase was high at below pH 2.5, but relatively small at neutral and alkaline pH values. Apolar interaction between the two components was confirmed by demonstrating an increase in fluorescence intensity of chymotrypsin in the presence of the PC in 1 M NaCl. The turbidity of a mixture of PC vesicles and bovine serum albumin (BSA) increased even in the absence of 1 M NaCl, whereas the turbidities of mixtures of the vesicles and lysozyme or alpha-lactalbumin did not change with time in the presence of 1 M NaCl at pH 8.0.     

Roots volatiles and fatty acids of coriander (<Emphasis Type="Italic">Coriandrum sativum</Emphasis> L.) grown in saline medium

An hydroponic culture was conducted to investigate the effect of saline stress on the essential oil and fatty acid composition of Tunisian coriander (Coriandrum sativum L.) roots. Ten days old coriander seedlings were treated during 3 weeks with different NaCl concentrations (0, 25, 50 and 75 mM). Roots volatile components and fatty acids were analyzed. The essential oil yield was 0.06% in the control, on the basis of dry matter weight, and did not changed at low concentration (25 mM), while it increased significantly with increasing NaCl concentrations to reach 0.12 and 0.21% at 50 and 75 mM NaCl, respectively. The major volatile component was (E)-2-dodecenal with 52% of total essential oil constituents, followed by decanal, dodecanal, (E)-2-tridecenal and (E)-2-dodecenal. Further, the amount of these compounds was affected differently by the NaCl level. Total fatty acid amount of coriander roots increased significantly only with 50 and 75 mM NaCl. Three major fatty acids: linoleic (43%), oleic (25.5%) and palmitic (21.6%) were identified. Linoleic acid amount remains unchanged at 25 mM, while it increased with raising NaCl concentrations. However, oleic acid amount decreased only at 25 mM and no effect was observed at 50 and 75 mM. Fatty acid percentages were differently affected by salt. The oleic/linoleic ratio was reduced with raising NaCl concentrations.     

Screening of local/exotic accessions of lentil (Lens culinaris Medic.) for salt tolerance at two growth stages

Screening of available local/exotic germplasm of a crop for salinity tolerance is of considerable value for the economic utilization of salt-affected soils of arid and semi-arid regions.The response of 133 lentil (Lens culinaris Medic.) accessions, to NaCl at the germination and seedling stage, was examined. A great amount of variation of NaCl tolerance in lentil was observed at both the growth stages but, in general, there was no consistent relationship between tolerance assessed at germination or at the seedling stage. In the NaCl treatment five accessions, ILL 5845, ILL 6451, ILL 6788, ILL 6793, and ILL 6796 produced significantly greater fresh and dry plant biomass in both absolute and relative terms than the others, but these accessions performed as well as other intermediate or low biomass producing accessions in total germination percentage and rate of germination. In view of the existence of the great amount of variability of tolerance to NaCl among lentil varieties improvement in NaCl tolerance in this species is possible.     

A simple procedure for solubilizing 3beta-hydroxysteroid dehydrogenase from microsomes of rat adrenals and testis interstitial cells

A simple procedure is described for solubilizing microsomal 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Microsomes from rat adrenals or from testicular interstitial cells were incubated for 1 or 2 h at 0 C in a buffer containing NaCl followed by overnight storage at -20 C. Maximum solubilization of 3beta-hydroxy-5beta-androstan-17-one-HSD (androstane-3beta-HSD) was obtained by incubating adrenal microsomes with 1 M NaCl and interstitial cell microsomes with 2 M NaCl. Incubation with NaCl for 1 or 2 h resulted in maximum solubilization; incubation with NaCl for 4, 8 or 24 h did not change the amount of enzyme solubilized. From adrenal microsomes incubated with 1 M NaCl, up to 80% (105.7 millimicron/mg microsomes) of the total androstane-3beta-HSD activity was recovered in the supernatant following centrifugation at 130,000 x g for 1 h. The maximum amount of androstane-3beta-HSD solubilized from interstitial cell microsomes was 56% (29.5 millimicron/mg microsomes) at 2 M NaCl. The "solubilized" androstane-3beta-HSD was retarded when chromatographed on a Sephadex G-200 column and it did not pellet out when centrifuged at 130,000 x g for 15 h. KCL appeared to be equally effective in solubilizing androstane-3beta-HSD from microsomes. Other steroid dehydrogenase activities such as pregnanolone-HSD and 3beta-hydroxy-5alpha-androstan-17-one-HSD were also found in the 130,000 x g supernatant.     

Growth, zearalenone production and some metabolic activities of Fusarium moniliforme under salt stress

With a increasing salinity, a decrease in the growth rate of Fusarium moniliforme was observed. The percentage of germinated conidia decreased with increasing salinity (% germination ranged from 80.3% at 0.0% NaCl to 0.0% at 15% NaCl). Concentration of 12.5% NaCl produced the highest chlamydospore-like structures. Concentration of 5% NaCl increased zearalenone production which decreased with increasing salt stress. Escherichia coli was more tolerant to the toxin than Bacillus subtilis. The total amount of lipids produced by F. moniliforme changed with increasing the concentration of NaCl in the growth medium. The genomic DNA of the control and treated samples showed a common band of more than 20 kilobases. Similar RAPD-PCR patterns were also produced.     

Sequence analysis of end-labeled DNA fragments by solvolysis in aqueous solutions of different amines.

Cleavage of 3'-end-labeled DNA in hot aqueous solutions of different amines is comparatively examined for overall rate of DNA scission as well as for potential differences in the preference of the various amines for cleavage at the different bases. Under comparable conditions (0.5 M amine, 0.3 M NaCl, 90 degrees C), piperidine, diethylamine, morpholine, and ethylenediamine produce the same set of labeled fragments, at approximately equal overall cleavage rates. The same set of fragments is also obtained with diisopropylamine, triethylamine, and 1,4-diazabicyclo[2.2.2]octane, but at markedly lower overall cleavage rates. Solvolysis in aqueous piperidine or aqueous diethylamine leads to DNA scission predominantly at A sites, followed by G and C sites, and least frequently at T sites. In contrast, morpholine, ethylenediamine, diisopropylamine, triethylamine, and diazabicyclo[2.2.2]octane cleave the DNA predominantly at G sites. Therefore, use of one of the latter amines allows clear distinction of G bands and C bands, which could not be distinguished by the criterion of band intensity in the original one-lane sequencing method based on cleavage in hot aqueous piperidine (B. Ambrose and R. Pless (1985) Biochemistry 24, 6194-6200). The effect of varying the salt concentration on the cleavage distribution obtained with various amines is also examined, and a rationale is given for the influence of salt concentration and amine basicity on the relative rate of cleavage at G sites.     

Acid unfolding and self-association of recombinant Escherichia coli derived human interferon gamma

The secondary and tertiary structure of recombinant human interferon gamma, determined by far- and near-UV circular dichroism, showed a transition from the native state to an unfolded state below pH 4.5. The acid unfolding was extensively studied at pH 3.5 as a function of NaCl concentration. Addition of 0.05-0.2 M NaCl to a pH 3.5 sample increased the amount of beta-sheet structure with no change in the amount of alpha-helix and also induced reversible self-association of interferon gamma to form large aggregates from the monomer. When samples at pH 3.5 were dialyzed against 0.1 M ammonium acetate (pH 6.9) to refold interferon gamma, the samples that contained NaCl in acid formed aggregates upon dialysis while those without NaCl formed a dimer apparently identical with the starting protein (i.e., before acid treatment). Thus, the self-association of interferon gamma in acid is closely correlated with its aggregation behavior in 0.1 M ammonium acetate after removal of acid.     

氯化钠处理对原壳小球藻生长及油脂积累的影响

采用Basal培养基,通过光学显微镜、电子显微镜、激光共聚焦显微镜以及尼罗红染色定量等方法研究了不同浓度氯化钠(0、150、300、600 mmol/L)对小球藻属原壳小球藻的生长状态、脂滴分布、总脂含量的影响。结果表明,添加不同浓度的氯化钠对原壳小球藻的生长有明显的影响,随着氯化钠浓度的增加,小球藻的生长速度受到明显的抑制,600 mmol/L氯化钠处理时生长几乎完全被抑制。在显微镜下观察,可见氯化钠浓度的增加会导致小球藻聚集成团,这种现象在150 mmol/L和300 mmol/L氯化钠培养下比较明显;通过电子显微镜下观察,可以发现培养初期,随着氯化钠浓度的增加,小球藻细胞壁增厚,脂滴增多。通过尼罗红染色对脂含量进行定量,处理初期脂滴的合成量在600 mmol/L时最高,但到后期,随着藻生物量的增加,150 mmol/L和300 mmol/L处理下脂合成量逐渐升高,而对照小球藻脂合成量基本不变。稳定期后,从生物量(干重)和脂总量来看,300 mmol/L氯化钠培养处理的小球藻虽然生物量只有对照的73.55%,但是总脂含量却是对照的2.22倍,可见一定浓度的氯化钠处理一定时间可显著提高原壳小球藻的油脂含量。     

Relationships of Temperature and Sodium Chloride Concentration to the Survival of Vibrio parahaemolyticus in Broth and Fish Homogenate

The interaction of temperature and NaCl concentration in affecting the survival of three strains of Vibrio parahaemolyticus was studied in Trypticase soy broth and fish homogenate. Cells of V. parahaemolyticus suspended in Trypticase soy broth without NaCl were quite unstable and readily killed. The presence of NaCl appeared to be protective to the cells at 48 +/- 1 C, with the optimal concentration strain-dependent for the 3 to 12% range tested. Temperatures of 5 +/- 1, -5 +/- 1, and -18 +/- 1 C reduced the number of viable organisms per milliliter regardless of the NaCl concentration. In the presence of NaCl, viable cells, in numbers ranging up to 580 per ml, were still detected at the end of 30 days of storage. Similar results were obtained for cells suspended in fish homogenate, except that fish homogenate itself was protective as compared with Trypticase soy broth. This protection was significantly lower than that provided by NaCl in any amount tested.     

Intranuclear distribution of the human myeloid cell nuclear differentiation antigen in HL-60 cells

Based on solubility properties, the human myeloid cell nuclear differentiation antigen exists as at least two distinct populations. Most is easily extracted from isolated nuclei in 0.35 M NaCl, while 20 percent resists such treatment. Compared to undigested nuclei, both the amount of myeloid cell nuclear differentiation antigen (MNDA) released from nuclei after DNase I treatment and the amount resisting further extraction in 0.35 M NaCl increased after DNA was digested with DNase I. Under these conditions, there was a concomitant decrease in the amount of MNDA that was extractable with 0.35 M NaCl. Mixing nuclear protein extracts that contain MNDA with nuclei from cells that do not express this protein demonstrated that the MNDA redistributes from the freely soluble form to the nuclear residual fraction as a consequence of DNase I digestion. These data are consistent with a model in which the amount of MNDA that is tightly bound to salt-washed nuclei is held constant in the presence of an excess of unassociated MNDA in the nucleus, and that the level of MNDA binding to this nuclear fraction increases in proportion to the extent of DNA damage resulting from DNase I digestion.     

Effect of NaCl on fatty acids, phenolics and antioxidant activity of Nigella sativa organs

The objective of this study was to investigate the effect of salinity on growth, fatty acid composition, phenol content and antioxidant activity of Nigella sativa organs. Plants were grown hydroponically under NaCl stress (0, 20 40 and 60 mM). The results indicated that salinity affected N. sativa growth. The fatty acid composition of the leaves and the roots was investigated for the first time and major fatty acids were linolenic acid (58.1%) in the leaves and linoleic (43.9%) and palmitic (33.3%) acids and in the roots. Total fatty acid (TFA) content of the leaves decreased at 60 mM NaCl while root TFA increased at 20 and 40 mM NaCl. Moreover, the fatty acid composition was affected by NaCl; in leaves, the double bond index (DBI) decreased accompanied by a decrease of the level of linolenic acid which reached 14% at 60 mM NaCl. However, root DBI degree increased at 40 at 60 mM NaCl provoked mainly by the increase of the amount of linoleic acid by 15 and 8%, respectively, and the decrease of the amount of palmitic acid by 20 and 14%, respectively. Salt stress increased total polyphenol and individual phenolic acid contents in shoots. Moreover, the antiradical activity of the shoots (DPPH) increased at 60 mM NaCl. However, in roots, the total polyphenol content and the antiradical activity decreased sharply with increasing NaCl doses. Data reported here revealed the variation of fatty acids and phenolic compound contents in different organs of N. sativa, and the possible role of theses changes in the plant salt response were discussed.     

Growth and soluble amino acid composition inPenicillium corylophilum andHalobacterium halobium grown under salt stress

Penicillium corylophilum can grow in a medium containing NaCl up to 3 mol/L, whereasHalobacterium halobium can grow at 5 mol/L NaCl. At 1 and 3 mol/L NaCl the number of spores decreased with constrictions in the conidiophore and damage in phialides and metulae while there were no morphological changes in bacterial cells grown in the presence of NaCl. Addition of some amino acids or sugar alcohol to the growth medium enhanced the growth of both organisms in the presence of NaCl. Total protein in the fungal cells grown at 1 mol/L NaCl increased by 50% more than in the control cells but it decreased in the bacterial cells with increasing NaCl concentration in the growth medium. Total saccharides and lipids increased in both organisms with increasing NaCl in the growth medium. The endogenous amino acid pool increased in fungal and bacterial cells in the presence of NaCl. Proline was the major amino acid in the fungal cells at 1 mol/L NaCl, representing 41.3% of the total identified amino acids at this concentration, being 8.4 times higher than in the control cells. Glutamate and aspartate showed the highest amount in bacterial cells at 3 mol/L NaCl. Isoleucine showed the highest increase at 3 mol/L NaCl, being 54 times higher than in the control cells.     

Difference in rehydration process due to salt concentration of drinking water in rats

Albino rats were thermally dehydrated (approximately 8% of body wt), divided into five groups, and given tap water or 0.2, 0.45, 0.9, or 2.0% NaCl solution ad libitum for 16 h. Rats given 0.9 or 0.45% NaCl solution regained fluid loss completely in 3-3.5 h, whereas those given 0.2% solution became fully rehydrated at 10 h. The rats in the tap water and 2.0% NaCl groups were only 78 and 59% rehydrated, respectively, within 16 h. Na balance was positive in the 0.9% NaCl group by about five times the amount of the cations lost during the dehydration period. A positive balance of Na was also observed in the 0.45 (approximately 250%) and 2.0% NaCl groups (300%), whereas the 0.2% NaCl group regained lost water and Na simultaneously at 10 h. With tap water, additional loss of cations was observed. These findings show that for the replacement of water due to thermal dehydration there is a range of NaCl concentration with which the rats can rehydrate with the mutual cooperation of thirst, salt appetite, and kidney function.     

Isolation of tissue collagenase from homogenates of embryonic chick bones

An enzyme capable of digesting undenatured collagen in solution and in the solid state as reconstituted collagen fibrils at neutral pH was extracted from demineralized embryonic chick bone homogenates in 1.0M NaCl at neutral pH. The enzyme could be dissociated from the small amount of collagen which was also solubilized in 1.0M NaCl by the serial use of Diaflo XM-300 and PM-10 membranes, which procedures also concentrated the enzyme. The enzymatic activity was inhibited by EDTA, cysteine and horse serum, and was enhanced by the addition of heparin.     

NaCl-aided Hoechst 33258 staining method for DNA quantification and its application

We investigated the effect of salt on the fluorescence staining procedure for quantification of the amount of DNA in cell nuclei in situ. For this, NaCl was added at various concentrations to the Hoechst 33258 fluorochrome (Hoe) medium for staining DNA. The fluorescence intensity of free DNA-Hoe solution was not changed by the addition of NaCl, but that of the nuclei-Hoe complex in situ increased 4-fold on increasing the NaCl concentration up to 1 M. SDS polyacrylamide gel electrophoresis showed that histones H1, H2A, and H2B dissociated from cell nuclei in the presence of 1 M NaCl, resulting in increasing accessibility of DNA to the fluorochrome. The applicability of the NaCl-aided fluorescence staining method was evaluated by measuring the ploidy classes of various cells. The amount of DNA in spermatozoa is half that in 2 n hepatocytes, but by the conventional Hoe staining procedure the fluorescence intensity of spermatozoa is higher than that of 2 n hepatocytes, due to differences in accessibility of the dye to DNA. In contrast, by the NaCl-aided procedure, the fluorescence intensity of 2 n hepatocytes was twice that of spermatozoa. The effectiveness of the NaCl-aided Hoe staining method was checked using cultivated human gingival cells and hepatocytes of LEC rats with hereditary hepatitis. In all cases, reasonable proportionality between the fluorescence intensity and the amount of DNA was observed.     

NaCl胁迫对唐古特白刺愈伤组织的生理效应

以唐古特白刺(Nitraria tangutorum Bobr.)愈伤组织为材料,研究低(75 mmol/L)、中(150 mmol/L)、高(300 mmol/L)浓度NaCl处理下其膜脂过氧化、抗氧化酶活性及渗透性调节物含量的变化,试图从细胞水平揭示唐古特白刺适应盐环境的生理机制。结果显示:(1)唐古特白刺愈伤组织中MDA含量在低浓度NaCl处理下与对照无显著性差异,而在中高浓度下显著升高。(2)白刺愈伤组织超氧化物歧化酶(SOD)活性在各浓度NaCl胁迫下均比对照显著升高,且此效应无浓度依赖性;其过氧化氢酶(CAT)活性随胁迫浓度的增加呈先升高后降低的变化趋势,在中低浓度下比对照显著增加,高浓度下显著降低为对照的64%;其过氧化物酶(POD)活性在低浓度下无显著性变化,在中高浓度显著下降为对照的55%和29%。(3)随NaCl胁迫浓度的增加,白刺愈伤组织中脯氨酸、可溶性糖和可溶性蛋白含量均呈先升高后降低的变化趋势,且除高浓度下可溶性蛋白含量约降为对照的87%外,其余NaCl胁迫处理的3种渗透调节物含量均高于对照。研究发现,白刺愈伤组织在低浓度的盐胁迫下具有较强的抗氧化酶活性和渗透性调节作用,因而表现出较强的抗氧化作用,但高浓度NaCl胁迫对白刺愈伤组织造成了显著的氧化损害。