nisin Z部分操纵子基因超表达对nisin Z合成的影响

目的:构建表达载体pMG76e-nisABTCI,合成重组菌,分析nisin合成基因簇中的nisABTCI超表达对nisin生物合成的影响。方法:将nisABTCI基因克隆与表达载体pMG76e酶切后连接转化,并通过Bio-Rad Gene Pulsor电穿孔法将重组质粒载体转入Lactococcus Lactis N401中,得到重组菌株N401C。对比工程菌和原始产生菌的nisin Z产量。以16S rRNA为内参基因,通过半定量RT-PCR方法,比较分析原始菌株N401和3株重组菌株中nisin合成基因簇中的11个基因的表达情况。结果:成功构建重组菌株,重组菌株的nisin Z产量提高了70%左右。半定量RT-PCR结果表明不同菌株的nisin合成基因的表达表现出了不同的特征。与菌株N401相比,3株重组菌株的大部分基因上调了。结论:nisABTCI超表达明显地提高nisin产量约70%。上调的基因可能对nisin合成效率的提高具有关键作用。     

乳链菌肽(nisin)抗性机制的研究进展

乳链菌肽(nisin)是某些乳酸乳球菌产生的一种羊毛硫细菌素。其对包括食品腐败菌和致病菌在内的许多革兰氏阳性菌具有强烈的抑制作用,是目前世界上唯一被允许用作食品添加剂的细菌素。nisin的广泛使用虽未引发大范围的抗性,但在自然界或实验室的选择压力下,某些非nisin产生菌也获得了抵御nisin攻击的抗性机制。nisin抗性机制通常涉及两种方式,即非特异性的生理适应机制和特异性蛋白酶介导的主动防御机制。本文综述了近年来nisin抗性机制的研究进展。     

乳酸菌食品级nisin控制的基因表达系统NICE

乳酸菌安全应用于人们的生产和生活已有上千年的历史,是一种食品级的微生物。在过去二十年里,其生理及遗传学特性已被彻底研究。由于其遗传可行且操作简单,乳酸菌除了其传统应用外已被广泛用于表达异源基因,在食品、农业及医药工程领域具有重要的应用前景。人们已开发了一系列乳酸菌食品级基因表达系统。本文主要介绍了乳酸菌,重点是其模式菌Lactococcus lactis最常见的食品级诱导表达系统--nisin控制的基因表达系统NIC E及其食品级诱导物nisin、食品级的宿主及表达载体系统,以及NICE系统在表达异源基因方面的应用。     

The lantibiotic nisin, a special case or not?

Nisin is a 34-residue-long peptide belonging to the group A lantibiotics with antimicrobial activity against Gram-positive bacteria. The presence of dehydrated residues and lanthionine rings (thioether bonds) in nisin, imposing structural restrains on the peptide, make it an interesting case for studying the mode of action. In addition, the relatively high activity (nM range) of nisin against Gram-positive bacteria indicates that nisin may be a special case in the large family of pore-forming peptides antibiotics. In this review, we attempted to dissect the mode of action of nisin concentrating on studies that used model membranes or biological membranes. The picture that emerges suggests that in model membrane systems, composed of only phospholipids, nisin behaves similar to the antimicrobial peptide magainin, albeit with an activity that is much lower as compared to its activity towards biological membranes. This difference can be contributed to a missing factor which nisin needs for its high activity. Novel results have identified the factor as Lipid II, a precursor in the bacterial cell wall synthesis. The special high affinity interaction of nisin with Lipid II resulting in high activity and the active role of Lipid II in the pore-formation process make nisin a special case.     

nisp基因在nisin生物合成终末成熟过程中的控制作用

本研究应用分子克隆和基因缺失突变技术首创了一个乳酸链球菌ｎｉｓｉｎ生物合成基因ｎｉｓｐ的突变株。此菌株能分泌完整的ｎｉｓｉｎ 前体肽链,却不能分泌有活性的ｎｉｓｉｎ。经生物活性、ＳＤＳ—ＰＡＧＥ和ＲＰ—ＨＰＬＣ等技术测定,ｎｉｓｉｎ 前体Ｎ—末端连接有先导序列,用胰酶或产活性ｎｉｓｉｎ的菌细胞膜蛋白处理ｎｉｓｉｎ前体,使之失去先导链,成为有活性的成熟的ｎｉｓｉｎ肽分子。此研究结果证明ｎｉｓｐ基因在ｎｉｓｉｎ生物合成最后阶段的成熟过程起决定作用。     

Simple one-step purification of nisin Z from unclarified culture broth of Lactococcus lactis subsp. lactis A164 using expanded bed ion exchange chromatography

A simple one-step purification method, using expanded bed, ion-exchange chromatography, for the fractionation of nisin Z produced by Lactococcus lactis subsp. lactis A164 was developed. The highest dynamic binding capacity (0.92) of the adsorbent was obtained at a superficial velocity of 367 cm h(-1), resulting in approx. 2.7-fold bed expansion. The range of pH for the maximum adsorption was 3-4. The isocratic elution with 0.15 M NaCl led to approx. >90% recovery. Single-step purification of nisin Z from unclarified A164 culture broth resulted in 31-fold purification with a 90% yield.     

Complete nisin A gene cluster from Lactococcus lactis M78 (HM219853) — obtaining the nucleic acid sequence and comparing it to other published nisin sequences

Nisin is an antimicrobial peptide produced by Lactococcus lactis. It has a long history of safe use, mainly in food production. This bacteriocin has been studied from many aspects of genetics, biosynthesis, immunity, regulation and mode of action. The strain Lac. lactis M78 has already been described in previous studies as a good nisin A producer with equally good potential to be used in food production. The main objective of the present study was to determine the complete nucleic acid sequence of the nisin A gene cluster from this strain. This is the first time that all 11 genes that form the nisin A gene cluster were sequenced. The obtained sequence (GenBank: HM219853) was compared to other known nucleic acid sequences of bacteriocin nisin. The results of the comparison showed certain differences in sequences that might influence the structure and function of proteins involved in nisin biosynthesis, immunity and regulation.     

Extractive cultivation of Lactococcus lactis using a polyethylene glycol/MgSO4 · 7H2O aqueous two-phase system to produce nisin

To relieve lactic acid inhibition, an aqueous two-phase system (ATPS) was used to grow Lactococcus lactis. Its composition was 11% (w/v) PEG 20000/3.5% (w/v) MgSO₄ 7H₂O. In this ATPS medium, the cells were completely partitioned in the bottom phase, and lactic acid had the biggest partition coefficient of the eight ATPS media tested. The cell biomass in this medium was 0.64 mg ml^–1, only 60% of that of the control medium, but nisin production (803 IU ml^–1) was enhanced by 33%. The increase in nisin was explained as a result of extraction of lactic acid from the bottom phase to the top one. The changes of tie-line length and phase volume ratio for the identical tie line could affect cell growth and nisin accumulation.     

Pfk基因过表达对乳酸乳球菌N8产nisin速率的影响

【目的】通过基因工程手段增加糖酵解途径中编码限速酶6-磷酸果糖激酶基因Pfk在乳酸链球菌素(nisin)产生菌Lactococcus lactis N8中的表达,增快nisin的产生,从而提高单位时间内nisin的产量,缩短发酵周期。【方法】将pfk基因及编码以c AMP为依赖的蛋白激酶催化亚基基因pka C克隆到表达质粒p MG36e上,将共表达重组质粒转入L.lactis N8中,使Pfk-pka C基因过量表达,得到重组菌株L.lactis N8-p MG36epfk-pka C,并比较该重组菌株与野生菌的生长曲线、胞内6-磷酸果糖激酶活性、发酵上清液的抑菌活性及效价,并从转录水平分析两株菌nis A及pfk-pka C的转录差异,比较野生菌与重组菌在不同葡萄糖含量下培养产nisin的变化。【结果】Pfk基因与pka C基因的过表达对重组菌的生长速度没有明显的影响,却能提高重组菌产nisin的速度,在发酵10 h时nisin的产量比野生菌提高了20%,使得发酵周期缩短近2 h。野生菌及重组菌在不同葡萄糖含量下培养发酵上清液的nisin效价没有明显的变化。【结论】糖酵解途径中6-磷酸果糖激酶基因Pfk的过表达可以加快乳酸乳球菌N8产nisin的速率,缩短发酵周期。     

A comparative analysis of karyotypes of three species of Macleaya—producers of a complex of isoquinoline alkaloids

Using a set of methods (C-banding, DAPI-staining, fluorescence hybridization in situ (FISH) with probes of 26S and 5S rDNA, and analysis of meiosis), the first comparative cytogenetic study of three species of Macleaya, producers of complex isoquinoline alkaloids, cordate Macleaya cordata (Willd.) R. Br. (2n = 20), small-fruited Macleaya microcarpa (Maxim.) Fedde (2n = 20) and Macleaya kewensis Turrill (2n = 20), was first carried out. On the basis of morphometric analysis, formulas of karyotypes were made for each species. Species ideograms for M. cordata, M. microcarpa, and M. kewensis were constructed taking into account the polymorphic variants of the C-banding patterns and indicating the location of 26S and 5S rDNA sites. A comparative study revealed that the karyotypes of M. microcarpa and M. kewensis have more in common with each other than with M. cordata. Analysis of meiotic chromosomes suggests of genetic stability of Macleaya genomes. The results of chromosome analysis were used to confirm the close relationship of Macleaya and to clarify their phylogenetic relationships.     

Effect of γ-Irradiation on Development of Lachrymator of Onion

A thermosensitive uracil requiring mutant of Bacillus subtilis Marburg 168 thy trp₂ ts42 was examined as to the colony forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in the modified Woese’s (MW) medium. However, the cells retained viability when sodium succinate or potassium chloride was added to the medium at that temperature although uracil deficiency was unchanged. A little but significant incorporation of adenine-8-¹⁴C into RNA still continued even after the incorporation of N-acetyl-³H-d-glucosamine into acid insoluble fraction of the cells terminated in the MW medium at 48°C. Both incorporations as well as increase of absorbance were slowed down in the presence of sodium succinate at 48°C. This mutant, ts42, was more sensitive to deoxycholate (DOC) than the parent strain. The restoration of colony forming ability after the temperature shift back from 48 to 37°C was suppressed by the addition of DOC to the medium. However, the cell became resistant to DOC when uracil was added to the medium prior to the temperature shift.     

Mechanisms of action of the α-amylase of Micromonospora melanosporea

The -amylase of Micromonospora melanosporea was produced extracellularly during batch fermentation in a 5.0-1 fermentor. The absence of an organic nitrogen source in its growth medium facilitated subsequent purification of the enzyme by ammonium sulphate fractionation and two consecutive Superose-12 gel-filtration steps. The enzyme exhibited maxima for activity at pH 7.0 and 55° C and was 72% stable at pH 6.0–12.0 for 30 min at 40° C. It had a relative molecular mass of 45 000 and an isoelectric point at pH 7.6. The enzyme catalyses the conversion of starch to maltose (53%, w/w) as the predominant final end-product. Initial hydrolysis of this substrate, however, gave rise to the formation of maltooligosaccharides in the range maltotriose to maltohexaose. Maximum yields of these intermediate sugars accumulated to between 31 and 42% (w/w) as the reaction proceeded. The action of the M. melanosporea amylase on high concentrations of saccharides larger than maltotriose resulted in the formation of mainly maltose and maltotriose without concomitant glucose production. A combination of hydrolytic and transfer events is postulated to be responsible for this phenomenon and for the high maltose levels achieved. Correspondence to: C. T. Kelly     

Selection of optimal variants of Gō-like models of proteins through studies of stretching

The Gō-like models of proteins are constructed based on the knowledge of the native conformation. However, there are many possible choices of a Hamiltonian for which the ground state coincides with the native state. Here, we propose to use experimental data on protein stretching to determine what choices are most adequate physically. This criterion is motivated by the fact that stretching processes usually start with the native structure, in the vicinity of which the Gō-like models should work the best. Our selection procedure is applied to 62 different versions of the Gō model and is based on 28 proteins. We consider different potentials, contact maps, local stiffness energies, and energy scales—uniform and nonuniform. In the latter case, the strength of the nonuniformity was governed either by specificity or by properties related to positioning of the side groups. Among them is the simplest variant: uniform couplings with no i, i + 2 contacts. This choice also leads to good folding properties in most cases. We elucidate relationship between the local stiffness described by a potential which involves local chirality and the one which involves dihedral and bond angles. The latter stiffness improves folding but there is little difference between them when it comes to stretching.     

Mechanism of action of α-galactosidase

The effect ofpH on K_m and V_max values of coconut α-galactosidase indicates the involvement of two ionizing groups with pK_a values of 3.5 and 6.5 in catalysis. Chemical modification has indicated the presence of two carboxyl groups, a tryptophan and a tyrosine, at or near the active site of α-galactosidase. Based on these facts a new mechanism of action for α-galactosidase is proposed in which the ionizing group with a pK_a of 3.5 is a carboxyl group involved in stabilizing a carbonium ion intermediate and the ionizing group with a pK_a of 6.5 is a carboxyl group perturbed due to the presence of a hydrophobic residues in its vicinity which donates a H⁺ ion in catalysis.     

Chemistry of the “molecular trap” of protease-catalyzed splicing reaction of complementary segments of α-subunit of hemoglobin A

The complementary fragments of human Hb α, α_1–30, and α_31–141 are spliced together by V8 protease in the presence of 30%n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated by the organic cosolvent induced α-helical conformation of product acting as the “molecular trap” of the splicing reaction. The segments α_24–30 and α_31–40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) α_24–40 has been manipulated by engineering the amino acid replacements to the positions α²⁷ and α³¹ to delineate the structural basis of the molecular trap. The location of Glu²⁷ and Arg³¹ residues in the contiguous segment α_24–40 (as well as in other larger segments) is ideal to generate (i, i+4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at α²⁷ and α³¹ facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu³⁰-Arg³¹ peptide bond. The second structural aspect is a site-specific event, ani, i+4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven “molecular traps” of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.     

Association of the subunits of the calcium-independent receptor of α-latrotoxin

CIRL-1 also called latrophilin 1 or CL belongs to the family of adhesion G protein-coupled receptors (GPCRs). As all members of adhesion GPSR family CIRL-1 consists of two heterologous subunits, extracellular hydrophilic p120 and heptahelical membrane protein p85. Both CIRL-1 subunits are encoded by one gene but as a result of intracellular proteolysis of precursor, mature receptor has two-subunit structure. It was also shown that a minor portion of the CIRL-1 receptor complexes dissociates, producing the soluble receptor ectodomain, and this dissociation is due to the second cleavage at the site between the site of primary proteolysis and the first transmembrane domain. Recently model of independent localization p120 and p85 on the cell surface was proposed. In this article we evaluated the amount of p120-p85 complex still presented on the cellular membrane and confirmed that on cell surface major amount of mature CIRL-1 presented as a p120-p85 subunit complex.     

Kinetics of renaturation and self-assembly of intermediates on the pathway of folding of the β2-subunit of Escherichia coli tryptophan-synthetase

The kinetics of renaturation of the β₂-subunit of Escherichia coli tryptophan-synthetase (l-serine hydrolyase (adding indole) E.C. 4.2.1.20) and those of its two proteolytic fragments F₁ and F₂ are studied and compared. Steps corresponding to the refolding of F₁, to the association of the folded F₁ and F₂ fragments, and to an isomerization of the associated protein are identified. These steps are ordered on the pathway of renaturation and some of their kinetic parameters are determined. This leads to a tentative kinetic model for the renaturation of nicked-β₂ starting from the denatured F₁ and F₂ fragments.The step corresponding to the refolding of the F₁ domain, as well as that corresponding to the last rate-limiting isomerization leading to the native protein, is shown to be the same in the refolding of the entire, uncleaved β₂-protein. It is concluded that the refolded F₁ fragment corresponds to a folding intermediate on the pathway of renaturation of the β₂-subunit.     

Determination of the number of degrees of freedom of the trapeziometacarpal joint–An in vitro study

ObjectiveMost of the studies about trapeziometacarpal joint assume that it exhibits only two independent degrees of freedom, but the experimental or theoretical support for considering a two-degrees of freedom model is not always clear.Materials and methodsTherefore, an in vitro kinematic study has been designed to demonstrate, from experimental data, that only two of the trapeziometacarpal degrees of freedom (i.e., flexion/extension and adduction/abduction) are non-null and independent. Several movements of maximal amplitude in flexion, abduction and circumduction have been realized and the relative position and orientation of the segment coordinate system embedded on the first metacarpal with respect to that embedded on the trapezium have been collected using electromagnetic sensors. The trapeziometacarpal rotations have been described using a joint coordinate system and the joint displacements have been evaluated on the axes of this coordinate system.ResultsThe root mean square (RMS) values of the joint displacement components have been found small enough to assume that the trapeziometacarpal joint has no translation degrees of freedom. A paraboloid coupling equation has been found between the internal/external rotation angle and the two other, flexion/extension and adduction/abduction, angles.ConclusionThus, this study demonstrates that the trapeziometacarpal joint has only two independent rotational degrees of freedom, and further, the described methodology could also be used to determine the coupling laws between degrees of freedom of various joints.