乳链菌肽高产菌株的选育及其基因定位

以乳酸乳酸球菌7962为原始菌株,用紫外线、LiCl、(60)~Co及8-MOP+NUV等多种理化诱变剂对其进行诱变处理,获得一株乳链菌肽高产突变株AL2。其效价稳定在2300～2500Iu/ml。经DNA杂交证实,编码乳链菌肽的前体基因位于染色体上,其遗传性状是稳定的。毒理试验表明AL2及其产物属于实际无毒类物质。     

THE EFFECT OF NISIN ON THE GROWTH OF CELLS AND SPORES OF CLOSTRIDIUM WELCHII IN GELATINE

SUMMARY: The action of various concentrations of nisin on the development of cells and spores from untreated and heated suspensions of Clostridium welchii added to gelatine has been investigated, using a tube colony count technique. The development of vegetative cells was prevented by the presence of 2 Reading Units (R.U.) of nisin/ml of the final culture medium, although the effective concentration may have been 6 R.U./ml. Approximately 40 R.U./ml prevented colony formation from spores. Gelatine containing nisin was dried. A considerable proportion of the activity of the antibiotic was still present after storage for 5 weeks.     

一个含有乳链菌肽抗性基因的乳酸乳球菌质粒pTS50的鉴定

在添加乳链菌肽、乳糖及溴甲酚紫的M1 7选择培养基上 ,从 1 97个新鲜牛奶样品中筛选到 3株乳链菌肽抗性菌株 ,PCR扩增证实它们都含有乳链菌肽抗性基因。菌种生理生化特性鉴定及特异性 1 6SrDNAPCR扩增产物的序列测定结果表明这 3株菌都属于乳酸乳球菌乳酸亚种。质粒转化实验发现乳酸乳球菌乳酸亚种TS 1 640中的乳链菌肽抗性基因位于一个约47kb的大质粒pTS50上。BamHI、EcoRI、HindⅢ、NcoI、PstⅠ酶切分析和Southern杂交 ,进一步将乳链菌肽抗性基因定位于pTS50的一个约 1 9kbEcoRI酶切片段中     

乳酸乳酸球菌AL2产生的乳链菌肽的提纯和性质

用NaCl饱和的乳酸乳酸球菌(Lactococcus lactis subsp. Lactis)AL2发酵液经正丙醇提取和CM-Sephadex C-25柱层析,得到聚丙烯酰胺凝胶电泳纯的乳链菌肽组分,比活力从24427IU/mg提高到39865IU/mg,活力回收为41.7％。Α—胰凝乳蛋白酶可使乳链菌肽丧失活性;在低pH条件下,乳链菌肽对热较稳定;对许多革兰氏阳性菌有强烈抑制作用,而对革兰氏阴性菌、酵母菌和霉菌没有作用。     

A SIMPLE PROCEDURE TO DETECT NISIN IN CHEESE

SUMMARY: A suitable amount of agar medium inoculated with a test organism sensitive to nisin ( Lactobacillus lactis or Streptococcus cremoris ) is poured on a cheese cylinder placed in a Petri dish. After a convenient incubation time the presence of a clear zone around the cheese cylinder will indicate the presence of nisin.
The method is applicable both to natural and processed cheese and can also be used for other antibiotics that may be present in cheese.     

固定化乳酸乳球菌连续生产Nisin的研究

以海藻酸钙为材料 ,固定乳酸乳球菌 (Lactococcuslactissubsp .lactis)SM5 2 6 ,研究不同条件对Nisin合成的影响。结果表明 ,利用 2 %海藻酸钠在 1 0mmol LCaCl2 条件下 ,得到的固定化细胞颗粒稳定性较好 ,可维持 90h无破裂 ;在发酵过程中SYS3培养基中的无机盐成分尤其磷酸盐对固定化颗粒有破坏作用 ;用mSYS3培养基代替SYS3 ,通过 72h三批次循环的半连续培养 ,Nisin活性为 85 0IU mL ,无明显的细胞渗漏现象。连续化生产 70h ,Nisin活性达 1 1 5 0IU mL ,相当于游离细胞的发酵水平。     

乳酸乳球菌AL2基因文库的构建及乳链菌肽生物合成基因簇的筛选

用本实验室获得的乳链菌肽高产菌株乳酸乳球菌AL2总DNA为供体,以λ EMBL3为载体,构建了该菌株的基因文库,共获得3900多个噬斑。通过Southern杂交、PCR扩增及DNA序列测定,证实从该文库中筛选到一个含有完整乳链菌肽生物合成基因簇的重组噬菌体λHJ\|3。     

THE KINETICS OF TRYPSIN DIGESTION : I. EXPERIMENTAL EVIDENCE CONCERNING THE EXISTENCE OF AN INTERMEDIATE COMPOUND.

1. The rate of hydrolysis of a casein solution by trypsin is not affected by the addition of gelatin. The trypsin, therefore, is not combined with the gelatin unless there is a separate enzyme for casein and for gelatin. 2. The presence of casein protects the gelatin-splitting power of trypsin from heat inactivation, and the presence of gelatin protects the casein-splitting power from heat inactivation. 3. It does not seem possible to account for both the above results by the assumption of an intermediate compound between enzyme and substrate, since, in order to account for the first result, a different enzyme must be assumed for each protein, while, to account for the second result, it must be assumed that the same enzyme attacks both.     

EFFECT OF THE FORMATION OF INERT PROTEIN ON THE KINETICS OF THE AUTOCATALYTIC FORMATION OF TRYPSIN FROM TRYPSINOGEN

A solution of crystalline trypsinogen in dilute buffer containing a trace of active trypsin when allowed to stand at pH 5.0–9.0 and 5°C. is gradually transformed partly into trypsin protein and partly into an inert protein which can no longer be changed into trypsin either by enterokinase or mold kinase. During the process of formation of trypsin and inert protein the ratio of the concentrations of the two products in any reaction mixture remains constant and is independent of the original concentration of trypsinogen protein. This ratio varies, however, with the pH of the solution, the proportion of trypsin formed being greater in the acid range of pH. The experimental curves for the rate of formation of trypsin, as well as for the rate of formation of inert protein are symmetrical S shaped curves closely resembling those of simple autocatalytic reactions. The kinetics of formation of trypsin and inert protein can be explained quantitatively on the theoretical assumptions that both reactions are of the simple unimolecular type, that in each case the reaction is catalyzed by trypsin, and that the rate of formation of each of the products is proportional to the concentration of trypsin as well as to the concentration of trypsinogen in solution.     

THE ANTIPROTEOLYTIC ACTIVITY OF SERUM : II. PHYSIOLOGICAL SIGNIFICANCE. THE INFLUENCE OF PURIFIED TRYPSIN INHIBITOR ON THE COAGULATION OF THE BLOOD

1. Serum antitrypsin and pancreatic trypsin inhibitor inhibited the coagulation of plasma in vitro. 2. This could be largely prevented by trypsin. 3. The anticoagulant action of the trypsin inhibitor was apparently due to its antiprothrombic action. It had no appreciable antithrombic action. 4. Examination of the blood of two hemophiliacs indicated that the prolonged coagulation time of their blood is not due to an excess of trypsin inhibitor. 5. Examination of the blood of heparinized dogs indicated that heparin does not appreciably contribute to the antiproteolytic activity of the serum.     

乳链菌肽的分离纯化和部分生物学性质

用乳酸链球菌ＳＭ５２６进行乳链菌肽的发酵生产,产量为４０～５０ｍｇ／ｌ．经中空纤维超滤器超滤,非极性大孔吸附树脂ＸＡＤ－２层析,ＣＭ－ＳｅｐｈａｄｅｘＣ－２５层析和ＳｅｐｈａｄｅｘＧ－５０层析纯化了该肽。ＳＤＳ－ＰＡＧＥ表明达均一,ＲＰ－ＨＰＬＣ表明其纯度不低于９５％。ＳＤＳ－ＰＡＧＥ测其Ｍｒ约为３６００,用ＩＥＦ测其等电点为９．５．酸性条件下稳定且抗热;对胰蛋白酶、胃蛋白酶和木瓜蛋白酶不敏感,但对α－胰凝乳酶和蛋白酶Ｋ敏感。乳链菌肽对多种革兰氏阳性菌有强烈的抑制作用;以枯草杆菌和金黄色葡萄球菌为指示菌,其作用方式是杀菌。     

Sensitivity of nisin-resistant Listeria monocytogenes to heat and the synergistic action of heat and nisin

Nisin, a bacteriocin produced by some strains of Lactococcus lactis, acts against foodborne pathogen Listeria monocytogenes. A single exposure of cells to nisin can generate nisin-resistant (Nisr) mutants, which may compromise the use of nisin in the food industry. The objective of this research was to compare the heat resistance of Nisr and wild type (WT) Listeria monocytogenes. The synergistic effect of heat-treatment (55 degrees C) and nisin (500 IU ml-1) on the Nisr cells and the WT L. monocytogenes Scott A was also studied. When the cells were grown in the absence of nisin, there was no significant (alpha = 0.05) difference in heat resistance between WT and Nisr cells of L. monocytogenes at 55, 60 and 65 degrees C. However, when the Nisr cells were grown in the presence of nisin, they were more sensitive to heat at 55 degrees C than the WT cells. The D-values at 55 degrees C were 2.88 and 2.77 min for Nisr ATCC 700301 and ATCC 700302, respectively, which was significantly (alpha = 0.05) lower than the D-value for WT, 3.72 min. When Nisr cells were subjected to a combined treatment of heat and nisin, there was approximately a four log reduction during the first 7 min of treatment.     

猕猴桃叶片耐热性指标研究

选择中华猕猴桃与美味猕猴桃共７个品种。对耐热性的形态与生理生化指标进行了探讨,结果表明：与耐热性较弱的品种相比,耐热性较强的品种有较大的叶片厚度、栅栏组织厚度,栅栏组织／海绵组织比例及较高的气孔密度,通过离体叶片温浴处理后细胞膜透性的变化拟合出的拐点温度高低可以鉴别品种耐热性的强弱,耐热性较强的品种在热胁迫下叶片游离脯氨酸累积率高于耐热性较弱的品种;品种耐热性与过氧化物酶活性之间无一致规律。     

THE EFFECT OF RADIO-ACTIVE RADIATIONS AND X-RAYS ON ENZYMES : I. THE EFFECT OF RADIATIONS FROM RADIUM EMANATION ON SOLUTIONS OF TRYPSIN.

A quantitative study has been made of the radiochemical decomposition of trypsin by the radiations from radium emanation. The following equation accounts quantitatively for the experimental results presented. See PDF for Equation It follows from this that the amount of trypsin decomposed by the radiations from radium emanation depends on the concentration of trypsin present and is proportional to the quantity of emanation expressed in millicuries and to the time of irradiation expressed in hours. It would seem that it is the active or undissociated trypsin that is affected. Evidence has been found which suggests that the beta radiations produce the decomposition observed for which the above statement holds. Qualitative evidence has been found which suggests that x-rays, gamma rays, and beta rays produce identical effects in dilute trypsin solutions.     

THE EFFECT OF CALCIUM AND OTHER IONS ON THE AUTOCATALYTIC FORMATION OF TRYPSIN FROM TRYPSINOGEN

Crystalline trypsinogen is completely transformed into trypsin by means of trypsin in the presence of calcium salts. The process follows the course of a pure autocatalytic unimolecular reaction. In the absence of calcium salts, the autocatalytic formation of trypsin from trypsinogen is complicated by the transformation of part of the trypsinogen into an inert protein which cannot be changed into trypsin by any known means. Salts increase or decrease the rate of both reactions so that the ultimate amount of trypsin formed varies with the nature and concentration of the salt used. With equivalent concentrations of salt the percentage of trypsinogen changed into trypsin is greatest in the presence of calcium ion followed in order by strontium; magnesium and sodium; rubidium, ammonium, lithium, and potassium; caesium and barium. With the anions the largest percentage of trypsinogen transformed into trypsin was found with the acetate, sulfate, oxalate, citrate, tartrate, fluoride, and chloride ions followed in order by bromide, nitrate, and iodide. The formation of inert protein is completely suppressed by concentrations of calcium ion greater than 0.02 M.     

THE ANTIPROTEOLYTIC ACTIVITY OF SERUM : III. PHYSIOLOGICAL SIGNIFICANCE. THE INFLUENCE OF TRYPSIN AND OF ANTIPROTEASE ON BACTERIAL GROWTH AND SULFONAMIDE ACTION

1. Heating diluted serum at 80° C. for 10 minutes made it a better medium for bacterial growth. This is believed to have been at least partly due to destruction of the serum antiprotease. 2. Growth was accelerated, and proceeded further, in the presence of trypsin. 3. Growth was somewhat retarded in the presence of pancreatic trypsin inhibitor. 4. The bacteriostatic action of sulfathiazole in serum was reduced by heating the serum at 80° C., and much more markedly (in any of the media studied) by adding trypsin. It was greater in serum and albumen than in peptone and meat infusion. 5. The significance of the experimental results was considered in relation to the possible influence of leucoprotease and of serum antiprotease on bacterial growth and sulfonamide action.     

The effect of nisin-sodium chloride interactions on the outgrowth of Bacillus licheniformis spores

The effect of salt (NaCl) on the efficacy of nisin in preventing outgrowth of Bacillus licheniformis spores was determined in Plate Count Agar (PCA). An equivalent liquid medium was used for heat activation. Nisin and salt were added to the heat-activation medium, the PCA, or both. The spores were extremely sensitive to nisin; outgrowth were completely inhibited in salt-free media when 10 iu/ml of nisin was present in both the heat-activation and the growth media or when 100 iu/ml nisin was present in either the heat-activation and the growth medium. In media supplemented with 1% salt, outgrowth occurred from 1% of spores exposed to 100 iu/ml nisin in either the heat-activation or the growth medium. A 3% salt supplement was necessary before detectable outgrowth occurred when both the heat-activation and the growth media contained 100 iu/ml nisin. Salt appears to antagonize the sporicidal action of nisin by interfering with nisin adsorption onto the spore.     

THE INFLUENCE OF PROTEINS ON THE REACTIVATION OF YEAST INVERTASE

1. Acid-inactivated yeast invertase could not be regenerated in the presence of the proteolytic enzymes trypsin, pepsin, and chymotrypsin. 2. Certain foreign proteins of non-enzymatic nature partially inhibited the reactivation of acid-inactivated invertase. 3. Certain proteins as gelatin, lacto-globulin, and carbohydrate-free horse crystalbumin did not prevent the reactivation of invertase at all. 4. Highly purified reactivated invertase was shown to exhibit an effect typical of original native invertase; that is, acceleration of its activity in presence of foreign protein at pH 3.0. 5. Native invertase was not digested by trypsin and chymotrypsin. 6. The addition of trypsin and chymotrypsin to reactivating invertase did not affect the invertase which had already reverted to the active form, but prevented further reactivation of inactive invertase.     

Modifications of membrane phospholipid composition in nisin-resistant Listeria monocytogenes Scott A.

A nisin-resistant (NISr) variant of Listeria monocytogenes Scott A was isolated by stepwise exposure to increasing concentrations of nisin in brain heart infusion (BHI) broth. The NISr strain was about 12 times more resistant to nisin than was the wild-type (WT) strain. Accordingly, higher nisin concentrations were required to dissipate both components of the proton motive force in the NISr strain than in the WT strain. Comparison of the membrane fatty acyl composition of the sensitive strain with that of its NISr derivative revealed no significant differences. From phospholipid head group composition analysis and phospholipid biosynthesis measurements during growth in the absence and presence of nisin, it could be inferred that the NISr strain produces relatively more phosphatidylglycerol (PG) and less diphosphatidylglycerol (DPG) than the parent strain does. Monolayer studies with pure lipid extracts from both strains showed that nisin interacted more efficiently with lipids derived from the WT strain than with those derived from the NISr strain, reflecting qualitative differences in nisin sensitivity. Involvement of the cell wall in acquisition of nisin resistance was excluded, since the WT and NISr strains showed a comparable sensitivity to lysozyme. Recently, it has been demonstrated that nisin penetrates more deeply into lipid monolayers of DPG than those of other lipids including PG, phosphatidylcholine, phosphatidylethanolamine, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol (R.A. Demel, T. Peelen, R.J. Siezen, B. de Kruijff, and O.P. Kuipers, Eur. J.Biochem. 235:267-274, 1996). Collectively, the mechanism of nisin resistance in this L. monocytogenes NISr strain is attributed to a reduction in the DPG content of the cytoplasmic membrane.     

THE INACTIVATION OF TRYPSIN : II. THE EQUILIBRIUM BETWEEN TRYPSIN AND THE INHIBITING SUBSTANCE FORMED BY ITS ACTION ON PROTEINS.

1. A study has been made of the equilibrium existing between trypsin and the substances formed in the digestion of proteins which inhibit its action. 2. This substance could not be obtained by the hydrolysis of the proteins by acid or alkali. It is dialyzable. 3. The equilibrium between this substance (inhibitor) and trypsin is found to agree with the equation, trypsin + inhibitor ⇌ trypsin-inhibitor The equilibrium is reached instantaneously and is independent of the substrate concentration. If it be further assumed that the rate of hydrolysis is proportional to the concentration of the free trypsin and that the equilibrium conforms to the law of mass action, it is possible to calculate the experimental results by the application of the law of mass action. 4. The equilibrium has been studied by varying (a) the concentration of the inhibiting substance, (b) the concentration of trypsin, (c) the concentration of gelatin, and (d) the concentration of trypsin and inhibitor (the relative concentration of the two remaining the same). In all cases the results agree quantitatively with those predicted by the law of mass action. 5. It was found that the percentage retarding effect of the inhibiting substance on the rate of hydrolysis is independent of the hydrogen ion concentration between pH 6.3 and 10.0. 6. The fact that the experimental results agree with the mechanism outlined under 3, is contrary to the assumption that any appreciable amount of trypsin is combined with the gelatin at any one time; i.e., the velocity of the hydrolysis must depend on the time required for such a compound to form rather than for it to decompose. 7. The experiments may be considered as experimental proof of the validity of Arrhenius'' explanation of Schütz''s rule as applied to trypsin digestion. 8. Inactivated trypsin does not enter into the equilibrium.