枯草芽孢杆菌中性β-甘露聚糖酶的产生及性质

由土壤中分离出一株产中性β甘露聚糖酶的枯草芽孢杆菌(Bacillus subtilis),编号BM9602。该菌在液体培养条件下,产生中性β甘露聚糖酶。多糖能作为碳源,而单糖不能作为碳源;有机氮源优于无机氮源。产酶最适培养基组成：魔芋粉4%,牛肉蛋白胨和酵母膏各1%。产酶最适培养条件：培养基起始pH85,35℃,振荡培养36〖KG*3]h。以槐豆胶为底物,培养滤液中性β甘露聚糖酶活力为96IU/mL。酶在pH50～100和50℃下稳定;作用最适条件为pH60和50℃;水解魔芋粉和槐豆胶均产生寡聚糖。     

培养因子对艾西丝南瓜芽增殖及不定根形成的影响

以艾西丝南瓜带芽茎段为外植体,研究了基本培养基、激素、糖、光照、培养基支持物等因子对芽增殖及不定根形成的影响。结果表明：艾西丝南瓜芽增殖的最佳培养条件为：MS+BA 0.5～1.0 mg/L+IAA 0.1～0.5 mg/L+食用白糖30 g/L,芽的月[KG(*9]增殖系数稳定在10左右;不定根诱导的适宜条件是：[KG)]1/2MS＋食用白糖20 g/L,生根率达86%;且自然散射光条件(1000～5000Lx)优于灯光(1000～2000Lx);以脱脂棉作生根培养基支持物效果优于琼脂,其芽增殖系数和生根率分别提高26%和7%。     

轮状病毒VP4*高聚体的制备及其免疫保护性评价

在前期工作中发现,截短的轮状病毒VP4~*蛋白(aa26–476)在大肠杆菌中能够以可溶形式表达,且在小鼠模型中具有较高的免疫原性和免疫保护性。本研究通过颗粒化进一步提高VP4~*蛋白的免疫保护性。通过37℃水浴加热处理24h使VP4~*蛋白多聚化,通过高效液相色谱、透射电镜、分析超离等分析VP4~*蛋白颗粒化程度,通过酶联免疫吸附试验分析颗粒化对VP4~*蛋白与中和抗体反应性的影响;通过差示量热法分析VP4~*高聚体的热稳定性;最后,通过小鼠母传抗体模型研究颗粒化对VP4~*免疫原性和免疫保护性的影响。结果表明,VP4~*蛋白高聚体结构均一,并且相比三聚体,具有更高热稳定性和中和抗体结合活性;在内毒素20 EU/mg的条件下,与铝佐剂混合,刺激小鼠产生更高滴度的中和抗体;对轮状病毒导致的腹泻具有更高的免疫保护性。综上所述,VP4~*高聚体的研究为轮状病毒基因工程亚单位疫苗的研制提供了更广阔的思路。     

毕赤酵母发酵生产T4溶菌酶的中试研究

研究了T4溶菌酶的中试发酵放大生产。在200L发酵罐中毕赤酵母诱导表达T4溶菌酶蛋白,发酵液蛋白表达量达到1.69g/L,酶活达到192U/mg;制得冻干酶粉211.1g。经测定粗酶粉中T4溶菌酶的含量达到68.9%,酶活为182U/mg,总收率达到66%,成功建立了一条T4溶菌酶的中试发酵生产工艺线路。     

一种高效制备人乳酸脱氢酶LDH_5参考物质的方法及应用

[目的]研究高效、廉价制备人乳酸脱氢酶LDH5参考物质的方法。[方法]选取人乳酸脱氢酶基因(LDHA)CDS序列进行分析及优化,合成优化后的基因,克隆至表达载体pRSF-Duet中构建重组乳酸脱氢酶LDH5表达载体。使用大肠杆菌E.coli BL21(DE3)菌株对目的基因进行表达,异丙基硫代半乳糖苷酶(IPTG)诱导,镍离子亲和层析纯化,SDS-PAGE鉴定酶纯度,BCA法分析酶浓度,全自动生化仪分析酶活性、稳定性。[结果]经优化后的LDHA基因的大肠杆菌密码子适应指数为1;纯化蛋白达到电泳纯,比活性达19.01 U/mg,在4℃及25℃可稳定保存8 d。[结论]重组乳酸脱氢酶的表达效率为100 mg/L,酶活性、稳定性、纯度达到临床生化检测的参考物质的条件,可以作为血清乳酸脱氢酶检测的标准物质。     

乳酸克鲁维酵母乳糖酶的可溶性表达及优化

[目的]实现乳酸克鲁维酵母乳糖酶的可溶性表达,并初步研究其酶学性质。[方法]首先克隆了来源于乳酸克鲁维酵母的乳糖酶基因KLLAC,构建pET-KLLAC重组表达载体,并采用蛋白质复性及与pKJE7、pG-KJE8、pGro7、pG-Tf2和p Tf-16伴侣蛋白共表达等方式拟提高其可溶性表达;并优化产酶条件,进一步提高其可溶性;采用ONPG法测定其酶学性质。[结果]在5种伴侣蛋白中pGro7与KLLAC共表达时可溶性最高;产酶最优条件为:阿拉伯糖浓度0. 5 mg/m L,IPTG浓度0. 1 mmol/L,诱导温度20℃;在最优条件下,重组KLLAC与伴侣蛋白p Gro7共表达时,表达量及酶活最高;经纯化后,乳糖酶KLLAC比酶活最高为102. 36 U/mg。该酶的最适温度30℃,最适p H 7. 0。[结论]KLLAC与伴侣蛋白的共表达以及诱导条件的优化,有效提高了该酶的可溶性表达水平、酶活性及稳定性。     

Cd、Pb对蟾蜍肝脏超氧化物歧化酶活性及其同工酶的影响

以腹腔注射法分别对蟾蜍(Bufobufogargarizans)给Cd和Pb (按镉计0 . 2、0 . 4、0 . 8、1 .6mg/kg体重;按铅计2、4、8、16mg/kg体重) ,连续染毒7d后,观察不同浓度Cd、Pb染毒条件下的蟾蜍肝超氧化物歧化酶(SOD)活性及其同工酶的变化。结果表明:在0 . 2mg/kg、0 4mg/kgCd和4mg/kg、8mg/kg和16mg/kgPb染毒,下蟾蜍肝SOD活性被显著诱导(P <0 . 0 5 ) ;前者酶带2 (Rf=0 . 4 3)活性增强,且均比对照增加1条新酶带(Rf=0 . 38) ;后者只表现为酶带1(Rf=0 . 5 1)和酶带2 (Rf=0 . 4 3)活性的增强,无新酶带的出现。     

人胱硫醚β-合酶及其截短型片段的表达、纯化和活性测定

将人胱硫醚β-合酶(CBS)基因克隆至质粒pGEX-4T-1中,获得的重组质粒pGEX-4T-1-CBS转入大肠杆菌E.coli Rosetta (DE3)菌株,构建了高效表达CBS的重组菌E.coli Rosetta (pGEX4T-1-CBS)。重组菌在0.1mmol/L的IPTG于30℃诱导16h,可溶性CBS表达量达到28mg/L培养基。将重组菌破碎后上清液经GSTrap Fast Flow亲和层析一步纯化得到CBS融合蛋白,在凝血酶柱上切割缓冲液中加入3%甘油和0.1%CHAPS可以有效抑制酶切后CBS聚沉,酶活性回收率为54.8%,蛋白质产率为15.2mg/L培养基,纯度达到95%,单位酶活为143U/mg,终浓度为1mmol/L的S-腺苷甲硫氨酸(AdoMet)可使CBS单位酶活提高5.1倍,达到735U/mg。同时构建了表达CBS_1-413(删除了CBS羧基端调控域138个氨基酸残基)的重组菌E.coli Rosetta (pETDuet-1-CBS_1-413),经过一步HisTrap Fast Flow亲和层析,酶活性回收率为74.3%,蛋白质产率为12.8mg/L培养基,纯度达到95%,单位酶活为965U/mg; 还表达和纯化了胱硫醚β-裂解酶(CBL),并在此基础上建立了一种新的CBL偶联的CBS酶活性测定方法。     

PP333、B9和CCC对脱毒马铃薯试管繁殖的影响

以MS0为增殖培养基,1/2MS(大量元素减半) 6-BA 0.1 mg*L-1 NAA 0.1 mg*L-1为生根培养基,分别附加不同浓度PP333、B9和CCC,结果表明:20 mg*L-1 B9或20～50 mg*L-1 CCC对脱毒马铃薯试管苗兼有促进增殖和复壮的双重效果;0.05～0.5 mg*L-1 PP333及50 ～100 mg*L-1CCC适合于试管苗的保存;而0.05～0.1 mg*L-1 PP333及20～100 mg*L-1CCC有利于根的发生及移栽,其中尤以0.1 mg*L-1 PP333效果最佳,其试管苗生根率和移栽成活率均为100%,且移栽后缓苗期短,生长旺盛.     

影响大白菜高效离体培养再生的因素

大白菜的4日龄子叶和6日龄下胚轴外植体在MS 4～6 mg*L-1 6-BA 0.5 mg*L-1 NAA 5～10 mg*L-1 AgNO3培养基上3～4周后分化出不定芽,带柄子叶和子叶圆片最高再生频率均可达到80%～90%,下胚轴再生频率为40%～55%,在1/2MS 0.2 mg*L-1 IBA培养基上100%生根,据此建立了大白菜离体培养再生系统.     

人溶菌酶工程菌株培养条件的研究

人溶菌酶在食品工业和医药上具有广泛的用途,最近发现它在癌症的继承性免疫治疗上也有作用。为使人溶菌酶达到工业化生产,我们已成功地人工合成厂人溶菌酶基因,并构建了人溶菌酶基因的重组质粒和工程菌株。为提高该工程菌人溶菌酶的表达水平,我们对影响该工程菌表达人溶菌酶的培养条件进行探讨。     

Spin-label assay for lysozyme.

A spin-label assay for lysozyme, which is based on the enzymatic hydrolysis of spin-labeled peptidoglycan, is described. Hydrolysis of this polymer by lysozyme results in sharpening of the esr spectrum. The rate of spectral sharpening is a function of enzyme concentration. When the activities of hen egg-white and human lysozymes are compared by this method, human lysozyme is 3.5 times as active as the hen enzyme. The pH optima for both enzymes are pH 5.0. At this pH, the maximal activity for the hen egg-white lysozyme is observed at an ionic strength of 0.09. This assay is suitable for measuring lysozyme levels in biological fluids. It is a sensitive, continuous assay that measures muramidase activity on a defined substrate.     

Factors influencing recombinant human lysozyme extraction and cation exchange adsorption

Human lysozyme has numerous potential therapeutic applications to a broad spectrum of human diseases. This glycosidic enzyme is present in tears, saliva, nasal secretions, and milk--sources not amendable for commercial development. Recently, a high expression level of recombinant human lysozyme (0.5% dry weight) was achieved in transgenic rice seed. This paper evaluates the effects of pH and ionic strength on rice protein and lysozyme extractability, as well as their interactions with the strong cation-exchange resin, SP-Sepharose FF. The extraction conditions that maximized lysozyme yield and the ratio of extracted human lysozyme to native rice protein were not optimal for lysozyme adsorption. The conditions that gave the highest extracted lysozyme to native protein ratio were pH 4.5 and 100 mM NaCl in 50 mM sodium acetate buffer. At pH 4.5, salt concentrations above 100 mM NaCl reduced the lysozyme-to-protein ratio. The best conditions for lysozyme adsorption were pH 4.5 and 50 mM sodium acetate buffer. Lysozyme extraction and subsequent adsorption at pH 4.5 and 50 mM NaCl was an acceptable compromise between lysozyme extractability, adsorption, and purity. The primary recovery of human lysozyme from pH 6 extracts, irrespective of ionic strength, was inferior to that using pH 4.5 with unacceptably low saturation capacities and lysozyme purity. High purity was achieved with a single chromatography step by adjusting the pH 4.5 extract to pH 6 before adsorption. The disadvantage of this approach was the drastically lower saturation capacity compared to adsorption at pH 4.5.     

Production of human lysozyme in biofilm reactor and optimization of growth parameters of Kluyveromyces lactis K7

Lysozyme (1,4-β-N-acetylmuramidase) is a lytic enzyme, which degrades the bacterial cell wall. Lysozyme has been of interest in medicine, cosmetics, and food industries because of its anti-bactericidal effect. Kluyveromyces lactis K7 is a genetically modified organism that expresses human lysozyme. There is a need to improve the human lysozyme production by K. lactis K7 to make the human lysozyme more affordable. Biofilm reactor provides high biomass by including a solid support, which microorganisms grow around and within. Therefore, the aim of this study was to produce the human lysozyme in biofilm reactor and optimize the growth conditions of K. lactis K7 for the human lysozyme production in biofilm reactor with plastic composite support (PCS). The PCS, which includes polypropylene, soybean hull, soybean flour, bovine albumin, and salts, was selected based on biofilm formation on PCS (CFU/g), human lysozyme production (U/ml), and absorption of lysozyme inside the support. To find the optimum combination of growth parameters, a three-factor Box–Behnken design of response surface method was used. The results suggested that the optimum conditions for biomass and lysozyme productions were different (27 °C, pH 6, 1.33 vvm for biomass production; 25 °C, pH 4, no aeration for lysozyme production). Then, different pH and aeration shift strategies were tested to increase the biomass at the first step and then secrete the lysozyme after the shift. As a result, the lysozyme production amount (141 U/ml) at 25 °C without pH and aeration control was significantly higher than the lysozyme amount at evaluated pH and aeration shift conditions (p?<?0.05).     

Engineering of human lysozyme as a polyelectrolyte by the alteration of molecular surface charge

The surface positive charges of human lysozyme were either increased or decreased to alter the electrostatic interaction between enzyme and substrate in the lytic action of human lysozyme using site-directed mutagenesis. The amino acid substitutions accompanying either the addition or the removal of two units of positive charge have shifted the optimal ionic strength (NaCl concentration in 10 mM Mes buffer, pH 6.2) for the lysis of Micrococcus lysodeikticus cell from 0.04 M to 0.1 M and from 0.04 M to 0.02 M respectively. In addition to the change in ionic strength-activity profile, the pH-activity profile and the effect of a polycationic electrolyte, poly-L-Lys-HCl, on the lytic activity were significantly changed. Owing to the shifts in both ionic strength profiles and pH profiles the Arg74/Arg126 mutant has become a better catalyst than wild-type enzyme under the conditions of high ionic strength and high pH, and the Gln41/Ser101 mutant has become a better catalyst under the conditions of low ionic strength and low pH.     

Quantitative recovery, selective removal and one-step purification of human parotid and leukemic lysozymes by immunoadsorption

Immunoadsorption affinity chromatography was used to isolate and purify human lysozyme. The immunoadsorbent was prepared by coupling sheep anti-(human leukemic lysozyme) IgG to epoxy-activated Sepharose 6B. Lyophilized parotid saliva (21) was resuspended in distilled water (325 ml, 50 mg/ml, w/v) and applied to a column which had a capacity to bind 4.25 mg human enzyme. Non-adsorbed material did not contain lysozyme, as determined by enzymatic and immunological analyses. All lysozyme activity present in the applied sample (1.97 mg) bound to and was desorbed from the column by elution with 0.2 M sodium acetate HCl buffer, pH 1.8. The isolated material was homogeneous as determined by cationic and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, ultracentrifugation, amino acid and amino-terminal analyses, and immunoelectrophoretic analysis. The one-step purification procedure yielded a 1370-fold increase in specific activity. Human lysozyme was also selectively purified by this method from an ammonium sulfate precipitate of the urine of a patient with chronic monocytic leukemia. Amino acid and polyacrylamide gel electrophoretic analyses indicated that the purified enzyme was identical to human lysozyme isolated from leukemic urine by classical biochemical techniques.     

Preparations of immobilized lysozyme with reversibly soluble polymer for hydrolysis of microbial cells

Hen egg white lysozyme was immobilized by carbodiimide method to form amide bonds with a polymer (AS-L) showing reversibly soluble-insoluble characteristics with pH change. The immobilized enzyme (LY-AS) was soluble above pH 6 and precipitate below pH 4.5, offering advantages in that it can carry out hydrolysis of microbial cells in a soluble form yet be recovered after precipitation at low pH. The maximum specific activity of LY-AS was 66% of that of free lysozyme with M. lysodeikticus cells as substrate, which is much higher than the values reported in the literature using water-insoluble materials as carriers. The effects of pH and temperature on the activity of LY-AS were studied and compared with those of free lysozyme. With repeated pH cycles between 6.6 and 4.5, the operation half-life of immobilized enzyme activity was nine cycles. Repeated batch lysis of microbial cells could be carried out with intermittent enzyme precipitation and recovery steps. In such an operation the insoluble residual cells should be recovered together with the immobilized enzyme to minimize enzyme loss arising from adsorption to cells.     

Exon grafting yields a "two active-site" lysozyme

The design of enzymes with enhanced stability and activity has long been a goal in protein engineering. We report a strategy to engineer an additional active site for human lysozyme, grafted the entire human lysozyme exon 2, which encodes the catalytically competent domain, into the gene at a position corresponding to an exposed loop region in the translated protein. Exon 2 grafting created a novel lysozyme with twice the activity of the wild type enzyme, equal activity came from each of the two active sites. We dissected the contributions of each active site using site-directed mutagenesis of the catalytic doublets of (E35A/D53A), circular dichroism, fluorescence spectra, and molecular modeling. Temperature and pH stability of the "two active-site" enzyme were similar to those of wild-type lysozyme. Thus, we provide a novel strategy for engineering the active site of enzymes.     

重组大肠杆菌热稳定性过氧化氢酶的纯化及性质研究

将产热稳定性过氧化氢酶的重组大肠杆菌培养后菌体破碎得到的粗酶液经热处理、硫酸铵分级沉淀、DEAE\|Sephadex A\|50离子交换层析、HiPrep16/10 Phenyl疏水作用层析、Superdex200 HR 10/30凝胶层析提纯后得到电泳纯的酶,比酶活达到15629U/mg。此酶的最适温度为70℃,最适pH70,在60℃保温60min酶活力基本不变,在pH3～8的范围内比较稳定。此酶的Km和Vmax分别为775mmol/L和278mmol\5min\+\{-1\}·mg-1。1mmol/L的Zn2+、Ba2+、Mn2+可使该酶完全失活,KCN、NaN\-3、Na\-2S\-2O\-4、巯基乙醇对酶活力有抑制作用,50mmol/L的EDTA不影响酶活性。     

Purification and characterization of goose type lysozyme from cassowary (Casuarius casuarius) egg white

A novel goose-type lysozyme was purified from egg white of cassowary bird (Casuarius casuarius). The purification step was composed of two fractionation steps: pH treatment steps followed by a cation exchange column chromatography. The molecular mass of the purified enzyme was estimated to be 20.8 kDa by SDS-PAGE. This enzyme was composed of 186 amino acid residues and showed similar amino acid composition to reported goose-type lysozymes. The N-terminal amino acid sequencing from transblotted protein found that this protein had no N-terminal. This enzyme showed either lytic or chitinase activities and had some different properties from those reported for goose lysozyme. The optimum pH and temperature on lytic activity of this lysozyme were pH 5 and 30 degrees C at ionic strength of 0.1, respectively. This lysozyme was stable up to 30 degrees C for lytic activity and the activity was completely abolished at 80 degrees C. The chitinase activity against glycol chitin showed dual optimum pH around 4.5 and 11. The optimum temperature for chitinase activity was at 50 degrees C and the enzyme was stable up to 40 degrees C.