溶菌酶的研究进展

对溶菌酶的稳定性的改善、制备、基因工程表达产物的复性处理作了介绍,并且对溶菌酶在医药、食品工业和生物工程上的应用作了概述。     

溶菌酶的研究进展

溶菌酶在自然界中广泛存在,是一种与单核-巨噬细胞系统有关的非特异性防御机制。人溶菌酶在临床上具有潜在的使用价值。由于来源有限,利用基因工程技术从细菌或酶母中生产人溶菌酶实现产业化,是解决其供需矛盾的有效途径。有关溶菌酶抗菌功能以外的其它未知生物学作用,也是当前研究的热点。     

重组人溶菌酶研究进展

与其他来源的溶菌酶相比,人溶菌酶具有独特的优越性和多种多样的药理作用效果,在临床上具有多种重要应用价值。但天然人溶菌酶来源极其困难,利用重组DNA技术进行生产是解决这一难题的有效途径。迄今人们已利用化学合成或从人类细胞组织中制备cDNA等途径获取人溶菌酶基因,并在大肠杆菌、酵母菌和真菌表达系统中进行了表达,最高水平为40mgL,低于人乳中溶菌酶含量(50～250mgL),虽然距离工业化生产仍有一定距离。但重组人溶菌酶发展前景看好。     

溶菌酶的结晶研究

介绍了溶菌酶结晶的意义,通过对晶核形成、晶体生长和停止生长三个阶段的论述,详细地阐述了溶菌酶的结晶机理;并综述了结晶过程中的各个影响因素:蛋白质浓度、pH值、添加剂、生长温度及重力场、磁场等;展望了溶菌酶结晶研究的发展趋势和发展前景。     

溶菌酶分离纯化方法的研究进展

对近年来溶菌酶分离纯化的方法,如离子交换法、色谱法、膜处理技术、反胶团萃取法、亲和层析等进行了综述,并讨论了分离纯化方法的应用前景。     

反刍动物溶菌酶研究进展

目前,抗生素滥用带来的副作用日益凸显,寻找抗生素的有效替代品显得尤为迫切。溶菌酶能水解细菌细胞壁肽聚糖中的β-1,4糖苷键,具有消化分解细菌、抑制外源微生物生长、增强机体免疫力的作用,在动物尤其是反刍动物体内广泛存在。本文讨论了反刍动物溶菌酶的来源与分布、基因序列、蛋白结构和酶学性质、蛋白功能及活性,对其耐酸分子机理进行了归纳总结;同时阐述了反刍动物溶菌酶基因的进化研究,最后对反刍动物溶菌酶研究进行了展望。研究反刍动物溶菌酶对于基础科学,并应用其转变成现实生产力意义重大。     

动物源溶菌酶研究进展

动物源溶菌酶是一种动物体内广泛存在的酶类,它可以水解细菌细胞壁肽聚糖中的β-1,4糖苷键,具有消化分解细菌、抑制外源微生物生长、增强机体免疫力的作用.目前溶菌酶已被用作研究蛋白功能、性质以及分子进化的模型.首先介绍了溶菌酶及其分子的晶体结构,溶菌酶基因及其蛋白研究进展,其次介绍了动物源溶菌酶的功能,包括溶菌酶生物学功能和重组蛋白功能活性,重点介绍了溶菌酶基因在转基因工程中的应用研究,最后对动物源溶菌酶研究进行了展望.研究动物源溶菌酶对于基础科学,并应用其转变成现实生产力具有重要的指导意义.     

溶菌酶晶体及其交联技术的研究进展

在结晶蛋白中,溶菌酶成为一种典范,是一种模型蛋白.在不同的环境中,通过调变各项反应条件,可以实现对不同晶系溶菌酶晶体形貌、结构的控制并进行最优化,然后对其进行交联,讨论其反应机理.所采用的方法还能扩展到其他类型蛋白质晶体中,为设计和制备具有优良应用性能的不同形貌的蛋白晶体及交联晶体材料奠定基础,必将大大促进蛋白晶体材料在我国生物材料应用方面的研究.     

海刺参i型溶菌酶基因的重组表达及抑菌谱分析

将本实验室已分离到的海刺参 (Stichopus japonicus) i型溶菌酶的基因(GenBank Accession No. EF036468)亚克隆进原核表达载体pET32a(+)中, 构建重组质粒pET32a(+)-SjLys, 转化至大肠杆菌BL21(DE3)pLysS。阳性克隆子经诱导表达、亲和纯化和透析复性, 得到了酶活力为19.2 U/mg的重组SjLys (rSjLys), 并对rSjLys进行了抑菌谱测定。结果表明, rSjLys对革兰氏阳性菌和阴性菌均有抑制作用, 尤其对常见的海洋致病菌副溶血弧菌和铜绿假单胞菌具有很强的抑菌活性。更为显著的结果是, rSjLys经100oC、40 min加热处理后, 失活的rSjLys对革兰氏阳性菌和阴性菌的抑菌能力高于rSjLys的9%~25%。上述结果表明, 海刺参溶菌酶是一种具有糖苷酶活性和非酶抑菌活性的特殊的i型溶菌酶, 且具有很广的抑菌活性, 在海刺参机体先天免疫系统中是一个重要的效应分子。     

七鳃鳗g型溶菌酶的分子特征和抗菌活性

溶菌酶是先天免疫系统中对抗细菌病原体感染的一种关键蛋白．本研究从七鳃鳗中克隆g型溶菌酶基因. 其酶基因cDNA为701 bp（GenBank 序列号KP204854）,开放阅读框为555 bp,编码由184个氨基酸组成的多肽,理论分子质量为20.24 kD,等电点为5.48,含有1个半胱氨酸残基,无信号肽．实时荧光定量PCR分析表明,七鳃鳗g型溶菌酶基因在各组织中广泛表达,其中在肠中表达量最高．脂多糖（LPS）体内刺激七鳃鳗后发现,溶菌酶在口腔腺和头肾表达量显著升高．以溶壁微球菌和哈维弧菌为底物检测重组g型溶菌酶的活性时,均表现出抗菌活性,最适pH为7.5,最适温度为35℃．扫描电镜分析表明,重组酶能够使溶壁微球菌破裂．以上结果均表明,g型溶菌酶在七鳃鳗的先天免疫系统防御病菌感染中起到重要作用．     

Effect on catalysis by replacement of catalytic residue from hen egg white lysozyme to Venerupis philippinarum lysozyme*

Asn46Asp/Asp52Ser or Asn46Glu/Asp52Ser hen egg white lysozyme (HEL) mutant was designed by introducing the substituted catalytic residue Asp46 or Glu46, respectively, based on Venerupis philippinarum (Vp) lysozyme structure as a representative of invertebrate‐type (i‐type) lyzozyme. These mutations restored the bell‐shaped pH‐dependency of the enzyme activity from the sigmoidal pH‐dependency observed for the Asp52Ser mutant. Furthermore both lysozyme mutants possessed retaining mechanisms like Vp lysozyme and HEL. The Asn46Glu/Asp52Ser mutant, which has a shorter distance between two catalytic residues, formed a glycosyl adduct in the reaction with the N‐acetylglucosamine oligomer. Furthermore, we found the accelerated turnover through its glycosyl adduct formation and decomposition. The turnover rate estimated from the glycosyl formation and decomposition rates was only 20% of the observed hydrolysis rate of the substrate. Based on these results, we discussed the catalytic mechanism of lysozymes.     

Irreversible lysozyme inactivation and aggregation induced by stirring: kinetic study and aggregates characterisation

Stirring strongly enhanced irreversible inactivation and aggregation of lysozyme being studied as a model enzyme. From 0 to 740 rpm (equivalent to impeller tip speeds from 0 to 0.77 m s^–1), the inactivation kinetic constant was proportional to the power imparted by the impeller. Collisions between inactive and native molecules induced inactivation of the latter and led to lysozyme aggregation. These fractal aggregates of lysozyme were made of monomers, dimers and trimers.     

A theoretical DFT investigation of the lysozyme mechanism: computational evidence for a covalent intermediate pathway

To test the occurrence of local particularities during the unfolding of Ca2+-loaded goat alpha-lactalbumin (GLA) we replaced Trp60 and -118, either one or both, by Phe. In contrast with alternative studies, our recombinant alpha-lactalbumins are expressed in Pichia pastoris and do not contain the extra N-terminal methionine. The substitution of Trp60 leads to a reduction of the global stability. The effect of the Trp118Phe substitution on the conformation and stability of the mutant, however, is negligible. Comparison of the fluorescence spectra of these mutants makes clear that Trp60 and -118 are strongly quenched in the native state. They both contribute to the quenching of Trp26 and -104 emission. By the interplay of these quenching effects, the fluorescence intensity changes upon thermal unfolding of the mutants behave very differently. This is the reason for a discrepancy of the apparent transition temperatures derived from the shift of the emission maxima (Tm,Fl lambda) and those derived from DSC (Tm,DSC). However, the transition temperatures derived from fluorescence intensity (Tm,Fl int) and from DSC (Tm,DSC), respectively, are quite similar, and thus, no local rearrangements are observed upon heat-induced unfolding. At room temperature, the occurrence of specific local rearrangements upon GdnHCl-induced denaturation of the different mutants is deduced from the apparent free energies of their transition state obtained from stopped-flow fluorescence measurements. By phi-value analysis it appears that, while the surroundings of Trp118 are exposed in the kinetic transition state, the surroundings of Trp60 remain native.     

人源溶菌酶结构与功能的研究进展

人溶菌酶是一类人体内天然存在的能够溶解细菌细胞壁的碱性蛋白的总称。其作用特征是能够裂解肽聚糖中的N-乙酰氨基葡萄糖与N-乙酰氨基甲酸之间的β-(1,4)-糖苷键。人溶菌酶具有抗菌、抗炎、抗病毒和增强免疫力等多种特性,因此在国内外市场上应用广泛。本文就人溶菌酶的结构特点、表达部位、功能表达以及应用情况进行综述。     

X-ray structure of Glu 53 human lysozyme.

The three-dimensional structure of a modified human lysozyme (HL), Glu 53 HL, in which Asp 53 was replaced by Glu, has been determined at 1.77 A resolution by X-ray analysis. The backbone structure of Glu 53 HL is essentially the same as the structure of wild-type HL. The root mean square difference for the superposition of equivalent C alpha atoms is 0.141 A. Except for the Glu 53 residue, the structure of the active site region is largely conserved between Glu 53 HL and wild-type HL. However, the hydrogen bond network differs because of the small shift or rotation of side chain groups. The carboxyl group of Glu 53 points to the carboxyl group of Glu 35 with a distance of 4.7 A between the nearest carboxyl oxygen atoms. A water molecule links these carboxyl groups by a hydrogen bond bridge. The active site structure explains well the fact that the binding ability for substrates does not significantly differ between Glu 53 HL and wild-type HL. On the other hand, the positional and orientational change of the carboxyl group of the residue 53 caused by the mutation is considered to be responsible for the low catalytic activity (ca. 1%) of Glu 53 HL. The requirement of precise positioning for the carboxyl group suggests the possibility that the Glu 53 residue contributes more than a simple electrostatic stabilization of the intermediate in the catalysis reaction.     

Expression of functional recombinant human lysozyme in transgenic rice cell culture

Using particle bombardment-mediated transformation, a codon-optimized synthetic gene for human lysozyme was introduced into the calli of rice (Oryza sativa) cultivar Taipei 309. The expression levels of recombinant human lysozyme in the transformed rice suspension cell culture approached approximately 4% of total soluble protein. Recombinant human lysozyme was purified to greater than 95% homogeneity using a two-step chromatography process. Amino acid sequencing verified that the N-terminus of the mature recombinant human lysozyme was identical to native human lysozyme. This indicates that the rice RAmy3D signal peptide was correctly cleaved off from the human lysozyme preprotein by endogenous rice signal peptidase. Recombinant human lysozyme was found to have the same molecular mass, isoelectric point and specific activity as native human lysozyme. The bactericidal activity of recombinant human lysozyme was determined by turbidimetric assay using Micrococcus lysodeikticus in 96-well microtiter plates. The bactericidal activity of lysozyme on Gram-negative bacteria was examined by adding purified lysozyme to mid-log phase cultures of E. coli strain JM109. In this study, significant bactericidal activity was observed after E.coli cells were exposed to recombinant human lysozyme for 60min. Both native and recombinant human lysozyme displayed the same thermostability and resistance to degradation by low pH. The potential for using rice-derived lysozyme as an antimicrobial food supplement, particularly for infant formula and baby foods, is discussed.     

Solvent effects on the structure,dynamics and activity of lysozyme

Kuntz and Kauzmann have argued that dehydrating a protein results in conformational changes. In contrast, Rupleyet al. have developed a hydration model which involves no significant change in conformation; the onset of enzyme activity in hen egg-white lysozyme at hydration values of about 0.2 g water/g protein they ascribe rather to a solvation effect. Using a direct difference infra-red technique we can follow specific hydration events as water is added to a dry protein. Conformational studies of lysozyme using laser Raman spectroscopy indicate changes in conformation with hydration that are complete just before measurable activity is found. Parallel nuclear magnetic resonance measurements of exchangeability of the main chain amide hydrogens, as a function of hydration from near dryness, suggest a hydration-related increase in conformational flexibility which occurs before-and is probably necessary for-the Raman-detected conformational changes. Very recent inelastic neutron scattering measurements provides direct evidence of a flexibility change induced by hydration, which is apparently necessary before the enzyme can achieve adequate flexibility for it to begin to function.     

Structural study on hen egg-white lysozyme crystals grown in gravity and microgravity

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