温度和pH对白斑狗鱼消化酶活性的影响

目的:通过改变酶的反应温度和pH,离体分析白斑狗鱼体内淀粉酶、蛋白酶和脂肪酶活性变化.方法:分别采用Folin酚法、DNS法和氢氧化钠滴定法测定蛋白酶、淀粉酶和脂肪酶活性.结果:在白斑狗鱼肝胰脏、胃二部位,淀粉酶的最适温度均为30℃,肠道淀粉酶的最适温度为40℃;最适pH值分别为4.0、2.0、7.0.肝胰脏、胃二部位脂肪酶的最适温度均为40℃,肠道脂肪晦的最适温度为50℃;最适pH值分别为5.0,4.0、5.0.肝胰脏、胃、肠道蛋白酶的最适温度均为50℃;最适pH值分别3.0、3.0、9.0.结论:在各自最适温度下,脂肪酶、淀粉酶、蛋白酶比活力均为:肠道＞胃＞肝胰脏.     

一株耐热纤维素酶产生菌的筛选及酶学特性

从辽宁鞍山汤岗子温泉附近土样中分离得到能产生纤维素酶的真菌,通过形态观察和18S rRNA序列分析,该菌株为蒙昧的散囊菌纲（Uncultured Eurotiomycetes）。实验中对酶学性质进行了检验,测定得出该菌株产生的纤维素酶最适温度为65℃,在温度高达75℃仍能保持70%的酶活力,它的最适pH值为6.5,pH在5～8的范围内酶活力保持稳定。实验表明该菌株所产纤维素酶具有较高的pH稳定性和温度稳定性,值得对该酶进行进一步的研究。     

豇豆几丁质酶部分酶学特性的研究

该文测定了纯化的豇豆几丁质酶部分酶学特性。结果表明,该酶在pH5-8,温度低于60℃的范围内稳定性较好,酶活力最适pH为65,最适温度为50℃。10mmol/L浓度的Hg2 、Mn2 、Mg2 、Co2 等金属离子对酶活力有一定抑制作用,其中Hg2 离子抑制率最高(6883%)。Km(胶状几丁质)值为1662mg/ml;以SDS-PAGE电泳和SephadexG-100柱层析两种方法分别测得分子量为34kD、325kD;IEF电泳测得等电点为83。     

链霉菌G4溶菌酶的产生条件及特性

对链霉菌G4的产酶发酵条件和溶菌特性进行研究结果表明:蔗糖30 g/L、大豆蛋白胨12.5 g/L、牛肉膏2 g/L,对产酶最为有利;G4溶菌酶最适培养温度33 ℃,培养时间72 h,培养基初始pH 8.G4溶菌酶的最适作用温度和最适作用pH分别是55 ℃和6.5,多数金属离子会抑制G4溶菌酶的活性,其中Zn2+、Cu2+、Fe2+、 Pb2+几乎可以使其完全失活;对几种细菌、酵母菌的研究表明,G4溶菌酶对卵清溶菌酶不能作用的变形链球菌和金黄色葡萄球菌有很强的溶解活性.     

虎纹蛙消化道淀粉酶和脂肪酶活力研究

以酶学分析方法研究了虎纹蛙消化道淀粉酶和脂肪酶的分布以及pH和温度对这两种消化酶活力的影响。结果表明：在各自生理pH值条件下,虎纹蛙消化道不同部位淀粉酶活力大小顺序依次为前肠〉中肠〉后肠〉食道〉胃,胃和肠淀粉酶最适pH值分别为8．6和7．0,最适温度分别为35℃和40℃。脂肪酶活力大小顺序依次为中肠〉后肠〉前肠〉胃〉食道,各部位之间差异显著（P〈0．05）,胃和肠脂肪酶的最适pH值均为9．0,最适温度分别为50℃和55℃。     

菜心溶菌酶的提纯及酶学性质

菜心叶子高速捣碎后,滤液经酸碱处理,硫酸铵分步沉淀,凝胶柱层析等步聚分离纯化溶菌酶,酶比活力达３４１４．６Ｕ／ｍｇ,纯化倍数为１９７．４。菜心溶菌酶在较宽的温度或ｐＨ值范围均有活性,最适温度６０℃,最适ｐＨ值为５．８,底物Ｋｍ值为８７μｇ／ｍＬ。该酶对热和酸碱的稳定性较高,巯基和酪氨酸残基不是该酶活性中心的必需基团。     

木霉菌株T6木聚糖酶固态发酵条件和酶学性质研究*

研究了碳源和氮源、起始pH、接种量及温度等条件对一野生型木霉Trichoderma sp.T6菌株固态发酵产木聚糖酶的影响。在28℃培养4d后,酶活力可达1918IU/g干培养物。酶的最适反应温度为50℃,最适反应pH4.5。不同温度保温1h后,测定酶的半失活温度为47.7℃,酶的pH稳定性也进行了研究。     

一株产木聚糖酶的黑曲霉固态发酵产酶性质的研究

目的：选育产木聚糖酶活力高的黑曲霉菌株,对其产酶条件进行优化,并研究其酶学性质。方法：通过木聚糖酶解木聚糖产生透明圈的方法,筛选产木聚糖酶菌株,测定固体发酵培养基中玉米芯与麸皮的比例、培养温度、培养时间、添加氮源对产酶的影响。进行了作用温度、pH值、金属离子对酶活力的影响试验,以及酶不同温度下的热稳定性的试验。结果：从自然界筛选得到一株产木聚糖酶的黑曲霉菌株,通过对固态发酵培养条件优化,最终产酶水平达到了5500u／g固体干曲。酶的最适作用温度是45℃、最适作用pH值4．8,是一种偏酸性酶。该酶在45℃以上的温度保存会使酶活力迅速丧失,Mg^2＋、Zn^2＋对该酶有激活作用,而Mn^2＋、Cu^2＋、Hg^2＋则完全抑制酶的活性。结论：选育的黑曲霉菌株产木聚糖酶活力较高,培养条件简单。     

温度和pH对橄榄蚶消化酶活性的影响

采用酶学分析方法,测定了温度和pH对橄榄蚶蛋白酶、淀粉酶、纤维素酶活力的影响。结果表明:温度和pH显著影响橄榄蚶蛋白酶、淀粉酶和纤维素酶的活力;蛋白酶、淀粉酶和纤维素酶的最适温度分别为60℃、40℃和30℃～40℃,最适pH分别为3.6、5.2和6.0。橄榄蚶3种消化酶活性大小为淀粉酶>蛋白酶>纤维素酶。     

木霉菌株T6木聚糖酶固态发酵条件和酶学性质研究

研究了碳源和氮源、起始pH、接种量及温度等条件对一野生型木霉Trichoderma sp.T6菌株固态发酵产木聚糖酶的影响。在28℃培养4d后,酶活力可达1918IU/g干培养物。酶的最适反应温度为50℃,最适反应pH4.5。不同温度保温1h后,测定酶的半失活温度为47.7℃,酶的pH稳定性也进行了研究。     

The disulfide bridges of duck egg-white lysozyme II

The disposition of the disulfide bonds of duck egg-white lysozyme II was established in detail by the study of cystine containing peptides obtained by enzymic digestions. A comparison of some optical properties of duck II and duck III lysozymes suggests the same disposition of the disulfide bonds in both enzymes.     

Process evaluations and economic analyses of recombinant human lysozyme and hen egg-white lysozyme purifications

Human lysozyme and hen egg-white lysozyme have antibacterial, antiviral, and antifungal properties with numerous potential commercial applications. Currently, hen egg-white lysozyme dominates low cost applications but the recent high-level expression of human lysozyme in rice could provide an economical source of lysozyme. This work compares human lysozyme and hen egg-white lysozyme adsorption to the cation exchange resin, SP-Sepharose FF, and the effect of rice extract components on lysozyme purification. With one exception, the dynamic binding capacities of human lysozyme were lower than those of hen egg-white at pH 4.5, 6, and 7.5 with ionic strengths ranging from 0 to 100 mM (5-20 mS). Ionic strength and pH had a similar effect on the adsorption capacities, but human lysozyme was more sensitive to these two factors than hen egg-white lysozyme. In the presence of rice extract, the dynamic binding capacities of human and hen egg-white lysozymes were reduced by 20-30% and by 32-39% at pH 6. Hen egg-white lysozyme was used as a benchmark to compare the effectiveness of human lysozyme purification from transgenic rice extract. Process simulation and cost analyses for human lysozyme purification from rice and hen egg-white lysozyme purification from egg-white resulted in similar unit production costs at 1 ton per year scale.     

Immunoregulation of lysozyme-specific suppression. III. Epitope-specific amplification of immunosuppression induced by monoclonal suppressor-T-cell products

The hen egg-white lysozyme (HEL)-specific suppression induced by soluble molecules produced by a monoclonal T-cell lymphoma line (LH8-105) obtained from HEL-specific suppressor T lymphocytes has been examined. Injection of I-J+ molecules from LH8-105 cell culture supernatant (TsFa) in HEL-primed mice during the afferent phase of the response induced Lyt-2+ second order suppressor T (Ts) cells which, upon transfer into HEL-CFA-primed syngeneic recipients, inhibit the delayed-type hypersensitivity (DTH) response to HEL. Transfer of spleen cells from TsFa-injected mice primed with HEL or human lysozyme suppresses the DTH response to HEL in recipient mice whereas this response is not affected by cell transfer from ring-necked pheasant egg-white lysozyme (REL)-primed and TsFa-injected mice, indicating that induction of second order Ts by TsFa is specific for a lysozyme epitope including phenylalanine at position 3. Fine antigenic specificity of second order Ts-cell induction is confirmed by similar results obtained upon injection of TsFa in mice primed with HEL N-terminal synthetic peptide or with an analog in which, as in REL, phenylalanine has been substituted by tyrosine at position 3. The same fine antigenic specificity observed in the induction of second order Ts cells is also present in the expression of TsFe suppressive activity. The similar antigenic specificity of Tsa and Tse suggests that Tse cells could result from amplification of the Tsa cell population or these two cell subsets could reflect different maturation stages of the same cell type rather than distinct T-cell populations activated in cascade.     

Conversion of Trp 62 of hen egg-white lysozyme to Tyr by site-directed mutagenesis

An expression plasmid for hen egg-white lysozyme in Saccharomyces cerevisiae was constructed by inserting almost full-length cDNA (about 600 base pairs) encoding hen egg-white pre-lysozyme into a yeast expression vector, pAM 82. The hen lysozyme was expressed under the control of the repressible acid phosphatase promoter of pAM 82 in S. cerevisiae. About half of the expressed lysozyme was secreted in the yeast growth medium as a precise mature protein which exhibited specific activity consistent with that of authentic hen egg-white lysozyme. The replacement of Trp 62 of hen egg-white lysozyme with a tyrosine residue was performed by site-directed mutagenesis using a 19-mer oligodeoxyribonucleotide. The mutant lysozyme with Tyr 62 was found to exhibit enhanced bacteriolytic activity.     

Thermal stability and enzymatic activity of a smaller lysozyme from silk moth (Bombyx mori)

Bombyx mori lysozyme is 10 amino acids shorter than hen egg-white lysozyme, which is a typical c-type lysozyme. It was expressed by using the methylotrophic yeast Pichia pastoris. The thermal stability and the enzymatic activity of the Bombyx mori lysozyme were estimated and compared with those of human and hen egg-white lysozymes. The denaturation temperature was 17-26°C lower than those of human and hen egg-white lysozymes. Further, the enthalpy change and the heat capacity change for unfolding were smaller than those of human lysozyme. It was also confirmed that the stability against guanidine hydrochloride was lower than those of the other two lysozymes. The enzymatic activity toward a simple synthetic substrate was measured and compared with those of human and hen egg-white lysozymes. The B-F binding mode was obviously dominant, although the A-E binding mode was preferred in human and hen egg-white lysozymes.     

Catalytic reaction mechanism of goose egg-white lysozyme by molecular modelling of enzyme-substrate complex

Despite the low similarity between their amino acid sequences, the core structures of the fold between chicken-type and goose-type lysozymes are conserved. However, their enzymatic activities are quite different. Both of them exhibit hydrolytic activities, but the goose-type lysozyme does not exhibit transglycosylation activity. The chicken-type lysozyme has a retaining-type reaction mechanism, while the reaction mechanism of the goose-type lysozyme has not been clarified. To clarify the latter mechanism, goose egg-white lysozyme (GEL)-N-acetyl-D-glucosamine (GlcNAc)6 complexes were modelled and compared with hen egg-white lysozyme (HEL)-(GlcNAc)6 complexes. By systematic conformational search, 48 GEL-(GlcNAc)6 complexes were modelled. The right and left side, and the amino acid residues in subsites E-G were identified in GEL. The GlcNAc residue D could bind towards the right side without distortion and there was enough room for a water molecule to attack the C1 carbon of GlcNAc residue D from alpha-side in the right side and not for acceptor molecule. The result of molecular dynamics simulation suggests that GEL would be an inverting enzyme, and Asp97 would act as a second carboxylate and that the narrow space of the binding cleft at subsites E-G in GEL may prohibit the sugar chain to bind alternative site that might be essential for transglycosylation.     

Amino acid residues in subsites e and f responsible for the characteristic enzymatic activity of duck egg-white lysozyme

We analyzed the enzymatic properties of duck egg-white lysozyme II (DEL), which differs from hen egg-white lysozyme (HEL) in nineteen amino acid substitutions. A substrate binding study showed that DEL binds to the substrate analog at subsites A-C in the same manner as HEL. However, the experimental time-courses of DEL against the substrate N-acetylglucosamine pentamer, (GlcNAc)(5), revealed remarkably enhanced production of (GlcNAc)(2) and reduced production of (GlcNAc)(1) as compared to in the case of HEL. Computer simulation of the DEL-catalyzed reaction suggested that the amino acid substitutions at subsites E and F (Phe34 to Tyr and Asn37 to Ser) caused the great alteration in the time-courses of DEL. Subsequently, the enzymatic reactions of mutants, in which Phe34 and Asn37 in HEL were converted to Tyr and Ser, respectively, were characterized. The time-courses of the F34Y mutant exhibited profiles similar to those of HEL. In contrast, the characteristics of the N37S mutant were different from those of HEL and rather similar to those of DEL; the order of the amounts of (GlcNAc)(1) and (GlcNAc)(2) was reversed in comparison with in the case of HEL. Enhanced production of (GlcNAc)(2) was also observed for the mutant protein, F34Y/N37S, with two substitutions. These results indicated that the substitution of Asn37 with Ser can account, at least in part, for the characteristic time-courses of DEL. Moreover, replacement of Asn37 with Ser reduced the rate constant of transglycosylation. The substitution of the Asn37 residue may affect the transglycosylation activity of HEL.     

Mechanism of lysozyme catalysis: role of ground-state strain in subsite D in hen egg-white and human lysozymes.

The association constants for the binding of various saccharides to hen egg-white lysozyme and human lysozyme have been measured by fluorescence titration. Among these are the oligosaccharides GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-GlcNAc, GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-N-acetyl-D-xylosamine, and GlcNAc-beta(1 leads to 4-GlcNAc-beta(1 leads to 4)-MurNAc, prepared here for the first time. The binding constants for saccharides which must have N-acetylmuramic acid, N-acetyl-D-glucosamine, or N-acetyl-D-xylosamine bound in subsite D indicate that there is no strain involved in the binding of N-acetyl-D-glycosamine in this site, and that the lactyl group of N-acetylmuramic acid (rather than the hydroxymethyl group) is responsible for the apparent strain previously reported for binding at this subsite. For hen egg-white lysozyme, the dependence of saccharide binding on pH or on a saturating concentration of Gd(III) suggests that the conformation of several of the complexes are different from one another and from that proposed for a productive complex. This is supported by fluorescence difference spectra of the various hen egg-white lysozyme-saccharide complexes. Human lysozyme binds most saccharides studied more weakly than the hen egg-white enzyme, but binds GlcNAc-beta(1 leads to 4)-MurNAc-beta(1leads to 4)-GlcNAc-beta(1 leads to 4)-MurNAc more strongly. It is suggested that subsite C of the human enzyme is "looser" than the equivalent site in the hen egg enzyme, so that the rearrangement of a saccharide in this subsite in response to introduction of an N-acetylmuramic acid residue into subsite D destabilizes the saccharide complexes of human lysozyme less than it does the corresponding hen egg-white lysozyme complexes. This difference and the differences in the fluorescence difference spectra of hen egg-white lysozyme and human lysozyme are ascribed mainly to the replacement of Trp-62 in hen egg-white lysozyme by Tyr-63 in the human enzyme. The implications of our findings for the assumption of superposition and additivity of energies of binding in individual subsites, and for the estimation of the role of strain in lysozyme catalysis, are discussed.     

Growth and Bacteriolytic Activity of a Soil Amoeba, Hartmannella glebae

A soil amoeba, Hartmannella glebae, could grow on a variety of gram-positive and gram-negative bacteria, although the rate of growth was faster in the presence of gram-negative bacteria. The amoeba, however, could not use yeasts, molds, or a green alga as a nutritional source. The extract prepared from amoebae grown in the presence of Aerobacter aerogenes and Alcaligenes faecalis could lyse intact cells and cell walls of many gram-positive bacteria at different rates. The spectrum of lytic activity was similar to that of egg-white lysozyme, with the exception that several species and strains of Bacillus, Micrococcus, and Staphylococcus were resistant to lysozyme and susceptible to the extract. The gram-negative bacteria tested were resistant.     

Inhibitory Effect of Some Gaseous Hydrocarbons on the Cell-lysis of Micrococcus lysodeikticus by Egg-white Lysozyme

The cell-lysis of Micrococcus lysodeikticus by egg-white lysozyme was inhibited by some nonpolar hydrocarbons such as n-butane, isobutane or propane. The lysozyme-induced cell-lysis was, however, no longer inhibited when the same hydrocarbons were added after the cell-lysis had already begun. Bubbling air through the cell suspensions, in which the lysozyme-induced cell-lysis had been inhibited in advance by the hydrocarbons, restored the event of cell-lysis to some extents. The Lineweaver-Burk plots indicated the kinetics of competitive inhibition of the lysozyme-induced cell-lysis by these hydrocarbons. From these results, the gaseous hydrocarbons were assumed to exert little influence on the enzymatic activity of egg-white lysozyme itself, that is the hydrolysis of β-1,4-glucosaminide linkage of the mucopeptide chain in the cell wall, but inhibit competitively the binding of lysozyme to the cells.