桦褶孔菌多糖的分离纯化及清除自由基活性

利用水提醇沉法对桦褶孔菌Lenzites betulina粗多糖HZKJc进行提取,随后经Sevage法和酶法联合脱蛋白,再经DEAE‐Sephadex A‐25,G‐100柱层析纯化后得到多糖纯品HZKJv。通过高效液相色谱(HPLC)测定上述纯化多糖的纯度和相对分子量分布;经纸层析(PC)、气相色谱(GC)、红外光谱(IR)进行单糖组成分析;并研究其清除自由基活性。分离纯化后得到的HZKJv为纯多糖,相对分子质量约为11 687。当HZKJv溶液浓度为3.0mg/m L时,其DPPH自由基清除率可达82%;浓度为2.0mg/m L时,对?OH自由基的清除率可达85%。表明HZKJv对?OH与DPPH自由基存在很好的清除作用。     

Beta proteobacterium sp.T1菌株产壳聚糖酶的分离纯化

目的:分离纯化Beta proteobacterium sp.T1菌株所产的壳聚糖酶.方法:采用(NH4)2SO4(20%～70%)分级盐析、阴离子交换树脂DEAE Cellulose 52柱层析、SephadexG-100柱层析技术进行分离纯化,采用SDS-PAGE鉴定酶的纯度、分子量.结果:经DEAE Cellulose 52柱层析,壳聚糖酶纯化了13.19倍;经Sephadex G-100柱层析,壳聚糖酶纯化了26.32倍.结论:纯化后的酶经SDS-PAGE鉴定已达到电泳纯,分子量29.5kDa.     

丹皮多糖PSM2b的纯化及其理化性质研究

从中药丹皮(MoutanCortex)蒸馏水浸提液得到粗多糖,经DEAE Cellulose 52柱层析分离得到降血糖组分PSM2b,Surperdex200柱层析进一步纯化得到PSM2b A和PSM2b B两个组分,快速层析纯化系统(FPLC)和电泳鉴定为均一的多糖蛋白复合物。FPLC法测定A和B两组分的分子量分别为1.16×105、1.30×104,苯酚 硫酸法测得其总糖含量分别为76.91%、32.00%,Folin 酚法测得其蛋白含量分别为9.90%、42.60%。气相色谱分析PSM2b A的单糖组成为:L 鼠李糖、L 岩藻糖、L 阿拉伯糖、D 木糖、D 甘露糖、D 葡萄糖、D 半乳糖,摩尔比依次为1.00∶0.18∶4.30∶0.50∶1.30∶2.41∶6.97,PSM2b B的单糖组成为:L 鼠李糖、L 岩藻糖、L 阿拉伯糖、D 葡萄糖、D 半乳糖,摩尔比依次为1.00∶1.17∶0.183∶2.34∶4.18。两者的红外光谱呈现多糖吸收特征峰,含有吡喃糖苷键。β消去反应初步证明所提取的两种糖蛋白不存在O 型糖肽键。     

视黄醇结合蛋白的分离纯化及其抗血清的制备

建立了经硫酸铵分级、DEAE-Sephadex A25柱、Sephadex G200柱、Ultrogel ACA44柱和 Sephadex G100柱层析分离纯化人血浆视黄醇结合蛋白的方法.经SDS-聚丙烯酰胺凝胶电泳鉴定,其纯度达到电泳纯.以此电泳纯的视黄醇结合蛋白免疫家兔得到了高效价的抗血清.     

维生素A棕榈酸酯的分离纯化与分析

本文对脂肪酶利用维生素A醋酸酯作为底物催化合成维生素A棕榈酸酯的分离纯化作了研究。确定了维生素A棕榈酸酯的萃取条件:萃取体系为乙醇/水和正己烷,乙醇浓度80%,乙醇/水与正己烷的体积比为5:1,萃取温度-20℃,萃取级数为5次,最终维生素A棕榈酸酯质量分数达到96.4%。萃取后,利用硅胶柱层析进一步纯化,条件为:反应液1mL上硅胶柱层析(硅胶200~300目,柱20mm×280mm),流动相为乙酸乙酯/石油醚(1:9,V/V),流速为68.5mL/h,5mL/管收集洗脱液,并用高效液相色谱(HPLC)鉴定,结果表明硅胶柱层析可以完全分离维生素A醋酸酯和维生素A棕榈酸酯。     

人血清载脂蛋白AⅡ的分离纯化及其抗血清的制备

本文应用超迷离心,盐酸胍处理、DEAE-Sepharose CL-6B离子交换柱层析分离纯化了人血清载脂蛋白AⅡ。经等电聚焦电泳、SDS-聚丙烯酰胺凝胶电泳和免疫电泳等鉴定,其纯度达到电泳纯和免疫纯。以此载脂蛋白AⅡ纯品免疫家兔产生的抗血清,效价较高,特异性强,与载脂蛋白AⅠ、B、CⅠ、CⅡ、CⅢ、E和白蛋白没有交叉反应。     

嗜冷枯草芽孢杆菌低温脂肪酶纯化与酶学性质研究

从筛选出的产低温脂肪酶的菌株发酵液中,经硫铵沉淀、疏水色谱和阴离子交换色谱纯化得到电泳纯酶。酶的最适作用温度为25℃,0℃以下仍可保持25%左右的相对酶活;在pH5.8～8.8的范围内有较高活力,其最适作用pH为7.8;对热很敏感,在60℃保温30min活性即全部丧失,具有典型的低温脂肪酶特征;酶催化不需要金属离子的参与,结构中可能含有二硫键。在25℃,pH8.0测得酶水解反应的Km值为2.65×10-5mol/L,Vmax值为5.21mmol/(L.min)。     

海洋来源真菌Aspergillus sp.SCSIO 41501中含氮化合物

通过多种色谱柱层析(包括凝胶Sephadex LH-20、正相硅胶、中压液相色谱和高效液相色谱)分离手段,从来源于南海柳珊瑚Melitodessquamata的一株共附生真菌Aspergillus sp.SCSIO 41501的发酵液中分离纯化了11个含氮化合物,它们的化学结构通过核磁共振和质谱等技术分别鉴定为13-(S)-赭曲霉素A(1)、13-(S)-甲酯化赭曲霉素A(2)、13-(R)-赭曲霉素A(3)、4-(R)-OH-赭曲霉素A(4)、18-OH-赭曲霉素A(5)、transtorine(6)、quinolactacin C1(7)、marinamide(8)、methyl marinamide(9)、aspergilliamide(10)、anoectochine(11)。所有化合物进行了抗菌实验,其中化合物2对大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌具有较强的活性,其MIC值分别为31.25、7.8、15.6μg/m L,化合物3显示中等抗菌活性,其MIC值分别为62.5、31.25、62.5μg/m L。     

毕赤酵母表面展示南极假丝酵母脂肪酶B全细胞催化合成葡萄糖月桂酸酯

利用表面展示南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)的毕赤酵母细胞为全细胞催化剂,以葡萄糖为酰基受体,月桂酸为酰基供体,在非水相体系中催化合成糖酯。用硅胶柱层析对产物进行初提,再用制备液相色谱进一步分离纯化,并用高效液相色谱-质谱鉴定纯品性质。对该酶法合成糖脂反应体系进行了优化,其中考察了有机溶剂种类、复合溶剂体系中二甲基亚砜(DMSO)体积百分比、酶量、底物摩尔比、水活度和温度等几个影响酯化反应的因素。结果表明:在5mL反应体系中,以叔戊醇/二甲基亚砜(DMSO30%,V/V)为反应介质,添加初始水活度为0.11的全细胞催化剂0.5g,葡萄糖0.5mmol/L,月桂酸1.0mmol/L,60°C下反应72h后,葡萄糖月桂酸单酯的转化率达到48.7%。     

表皮生长因子受体的分离与纯化

建立了一种从胎盘合胞体滋养层微绒毛膜蛋白中简单迅速两步纯化 EGFR 蛋 白的方法.用 Triton X-100 抽提微绒毛膜蛋白,使之两次通过 FPLC(Fast Pro-tein Liquid Chromatography)系统的 Superose 6 色谱柱纯化.SDS 胶电泳后,得到单一的 170000分子量 EGFR 蛋白带.纯化后的受体在 EGF,MnCl₂ 和 (γ-³²P)ATP 条件下,仍然保留胞内所具有的对 EGF 敏感的使受体自身磷酸化的蛋白激酶活性.     

The Effects of One Amino Acid Substitutions at the C-Terminal Region of Thermostable L2 Lipase by Computational and Experimental Approach

The substitutions of the amino acid at the predetermined critical point at the C-terminal of L2 lipase may increase its thermostability and enzymatic activity, or even otherwise speed up the unfolding of the protein structure. The C-terminal of most proteins is often flexible and disordered. However, some protein functions are directly related to flexibility and play significant role in enzyme reaction. The critical point for mutation of L2 lipase structure was predicted at the position 385 of the L2 sequence, and the best three mutants were determined based on I-Mutant2.0 software. The best three mutants were S385E, S385I and S385V. The effects of the substitution of the amino acids at the critical point were analysed with molecular dynamics simulation by using Yet Another Scientific Artificial Reality Application software. The predicted mutant L2 lipases were found to have lower root mean square deviation value as compared to L2 lipase. It was indicated that all the three mutants had higher compactness in the structure, consequently enhanced the stability. Root mean square fluctuation analysis showed that the flexibility of L2 lipase was reduced by mutations. Purified S385E lipase had an optimum temperature of 80 °C in Tris–HCl pH 8. The highest enzymatic activity of purified S385E lipase was obtained at 80 °C temperature in Tris–HCl pH 8, while for L2 lipase it was at 70 °C in Glycine–NaOH pH 9. The thermal stability of S385V lipase was enhanced as compared to other protein since that the melting point (T _m) value was at 85.96 °C. S385I lipase was more thermostable compared to recombinant L2 lipase and other mutants at temperature 60 °C within 16 h preincubation.     

Sesquiterpene farnesol as a competitive inhibitor of lipase activity of Staphylococcus aureus

Staphylococcus aureus lipase (SAL) is known to possess broad substrate specificity for triacylglycerides. We found that a sub-minimum inhibitory concentration of farnesol (1000 mg L(-1)) inhibits this lipase activity on a Mueller-Hinton agar containing 1% Tween substrates. A quantitative lipase assay using p-nitrophenyl palmitate (pNPP) revealed that the inhibitory action of farnesol appears to be the result of the inhibition of lipase activity rather than of its secretion into the culture medium. The inhibition was observed in all the tested 8 methicillin-susceptible S. aureus and 31 methicillin-resistant S. aureus clinical isolates. Using homogeneous lipase purified by hydrophobic interaction chromatography, it was revealed that farnesol could competitively inhibit the lipase activity against the substrate pNPP.     

Regulation of lipoprotein lipase synthesis in 3T3-L1 adipocytes by cachectin. Further proof for identity with tumour necrosis factor.

We investigated the mechanism by which the endotoxin-induced macrophage secretory protein cachectin is able to suppress the activity of lipoprotein lipase in 3T3-L1 adipocytes. The loss in activity results from an effect on the synthesis of the enzyme, as determined by a decreased incorporation of [35S]methionine into immunoprecipitable lipoprotein lipase. The results were nearly identical whether crude conditioned medium or a highly purified preparation was utilized as a source of cachectin. [35S]Methionine incorporation into acid-precipitable protein was minimally affected by purified cachectin, suggesting that the suppression of the lipoprotein lipase was not due to a general suppression of protein synthesis. These results, taken together with our previous work, provide additional evidence that cachectin and tumour necrosis factor are functionally identical.     

Quantitative spectrophotometric assay for staphylococcal lipase.

We report the development of a specific spectrophotometric assay for the quantitative determination of lipase activity in Staphylococcus aureus. The assay is based on the rate of clearance of a tributyrin emulsion, and it can detect as little as 1.0 micrograms of purified Pseudomonas lipase per ml. By comparison with the reaction rates obtained with Pseudomonas lipase, we calculated that S. aureus PS54C and S6C produce approximately 15 and 60 micrograms of extracellular lipase per ml, respectively. Neither PS54, which is lysogenized with the converting bacteriophage L54a and is consequently lipase negative (Lip-), nor KS1905, a Lip- transpositional mutant of strain S6C, was positive in our spectrophotometric assay. The specificity of the spectrophotometric tributyrin assay was confirmed with a triolein plate assay; supernatants from S6C and PS54C hydrolyzed triolein, while supernatants from PS54 and KSI905 did not. In contrast to the results of the spectrophotometric tributyrin assay, all enzyme preparations tested (including commercially purified esterase) were positive when examined by a tributyrin plate assay. The lack of specificity in the tributyrin plate assay emphasizes the need to interpret the results of tributyrin lipolysis kinetically for assessing lipase activity in S. aureus.     

The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase

This paper presents the complete amino acid sequence of the low molecular weight acid phosphatase from bovine liver. This isoenzyme of the acid phosphatase family is located in the cytosol, is not inhibited by L-(+)-tartrate and fluoride ions, but is inhibited by sulfhydryl reagents. The enzyme consists of 157 amino acid residues, has an acetylated NH2 terminus, and has arginine as the COOH-terminal residue. All 8 half-cystine residues are in the free thiol form. The molecular weight calculated from the sequence is 17,953. The sequence was determined by characterizing the peptides purified by reverse-phase high performance liquid chromatography from tryptic, thermolytic, peptic, Staphylococcus aureus protease, and chymotryptic digests of the carboxymethylated protein. No sequence homologies were found with the two known acylphosphatase isoenzymes or the metalloproteins porcine uteroferrin and purple acid phosphatase from bovine spleen (both of which have acid phosphatase activity). Two half-cystines at or near the active site were identified through the reaction of the enzyme with [14C] iodoacetate in the presence or in the absence of a competitive inhibitor (i.e. inorganic phosphate). Ac-A E Q V T K S V L F V C L G N I C R S P I A E A V F R K L V T D Q N I S D N W V I D S G A V S D W N V G R S P N P R A V S C L R N H G I N T A H K A R Q V T K E D F V T F D Y I L C M D E S N L R D L N R K S N Q V K N C R A K I E L L G S Y D P Q K Q L I I E D P Y Y G N D A D F E T V Y Q Q C V R C C R A F L E K V R-OH.     

Quantitative spectrophotometric assay for staphylococcal lipase.

We report the development of a specific spectrophotometric assay for the quantitative determination of lipase activity in Staphylococcus aureus. The assay is based on the rate of clearance of a tributyrin emulsion, and it can detect as little as 1.0 micrograms of purified Pseudomonas lipase per ml. By comparison with the reaction rates obtained with Pseudomonas lipase, we calculated that S. aureus PS54C and S6C produce approximately 15 and 60 micrograms of extracellular lipase per ml, respectively. Neither PS54, which is lysogenized with the converting bacteriophage L54a and is consequently lipase negative (Lip-), nor KS1905, a Lip- transpositional mutant of strain S6C, was positive in our spectrophotometric assay. The specificity of the spectrophotometric tributyrin assay was confirmed with a triolein plate assay; supernatants from S6C and PS54C hydrolyzed triolein, while supernatants from PS54 and KSI905 did not. In contrast to the results of the spectrophotometric tributyrin assay, all enzyme preparations tested (including commercially purified esterase) were positive when examined by a tributyrin plate assay. The lack of specificity in the tributyrin plate assay emphasizes the need to interpret the results of tributyrin lipolysis kinetically for assessing lipase activity in S. aureus.     

基于易错PCR技术的短小芽孢杆菌YZ02脂肪酶基因BpL的定向进化

利用易错PCR技术对短小芽胞杆菌(Bacillus pumilus)YZ02脂肪酶基因BpL进行两轮定向进化研究, 分别获得最佳突变株BpL1-7和BpL2-1369, 其脂肪酶活力比出发酶分别提高了2倍和6倍。序列分析表明, 突变体BpL2-1369有4个碱基发生了突变: T61C/C147T/A334G/T371A, 其中有3个碱基突变导致了氨基酸的改变。通过SWISS-MODEL数据库模拟脂肪酶的结构显示, 3个突变氨基酸分别位于第1个a螺旋的第3个氨基酸、第4和第5个b折叠之间的转角以及第5个b折叠的第1个氨基酸位置。将野生型脂肪酶基因BpL和进化后的基因BpL2-1369的高效表达产物经Ni-Agarose柱和Sephadex-G75纯化后, 酶学性质测定表明: 突变脂肪酶的比活力比野生型脂肪酶提高了1.31倍, Km值由8.24 mmol/L降低至7.17 mmol/L; 在pH>8.0时的稳定性较野生型脂肪酶有所提高。     

The complete amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda

The complete amino acid sequence of coagulogen purified from the hemocytes of the horseshoe crab Carcinoscorpius rotundicauda was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, Staphylococcal aureus protease V8 and trypsin. Upon sequencing the peptides by the automated Edman method, the following sequence was obtained: A D T N A P L C L C D E P G I L G R N Q L V T P E V K E K I E K A V E A V A E E S G V S G R G F S L F S H H P V F R E C G K Y E C R T V R P E H T R C Y N F P P F V H F T S E C P V S T R D C E P V F G Y T V A G E F R V I V Q A P R A G F R Q C V W Q H K C R Y G S N N C G F S G R C T Q Q R S V V R L V T Y N L E K D G F L C E S F R T C C G C P C R N Y Carcinoscorpius coagulogen consists of a single polypeptide chain with a total of 175 amino acid residues and a calculated molecular weight of 19,675. The secondary structure calculated by the method of Chou and Fasman reveals the presence of an alpha-helix region in the peptide C segment (residue Nos. 19 to 46), which is released during the proteolytic conversion of coagulogen to coagulin gel. The beta-sheet structure and the 16 half-cystines found in the molecule appear to yield a compact protein stable to acid and heat. The amino acid sequences of coagulogen of four species of limulus have been compared and the interspecies evolutionary differences are discussed.     

Processing of HEBP1 by cathepsin D gives rise to F2L, the agonist of formyl peptide receptor 3

The peptide F2L was previously characterized as a high-affinity natural agonist for the human formyl peptide receptor (FPR) 3. F2L is an acetylated 21-aa peptide corresponding with the N terminus of the intracellular heme-binding protein 1 (HEBP1). In the current work, we have investigated which proteases were able to generate the F2L peptide from its precursor HEBP1. Structure-function analysis of F2L identified three amino acids, G(3), N(7), and S(8), as the most important for interaction of the peptide with FPR3. We expressed a C-terminally His-tagged form of human HEBP1 in yeast and purified it to homogeneity. The purified protein was used as substrate to identify proteases generating bioactive peptides for FPR3-expressing cells. A conditioned medium from human monocyte-derived macrophages was able to generate bioactivity from HEBP1, and this activity was inhibited by pepstatin A. Cathepsin D was characterized as the protease responsible for HEBP1 processing, and the bioactive product was identified as F2L. We have therefore determined how F2L, the specific agonist of FPR3, is generated from the intracellular protein HEBP1, although it is unknown in which compartment the processing by cathepsin D occurs in vivo.     

Isolation and characterization of proSS1-32, a peptide derived from the N-terminal region of porcine preprosomatostatin

A peptide derived from the N-terminal region of porcine prosomatostatin, proSS1-32, has been purified to homogeneity from extracts of porcine upper intestine. Amino acid analysis revealed that the peptide consists of 32 residues. The complete primary structure was determined as: A P S D P R L R Q F L Q K S L A A A A G K Q E L A K Y F L A E L. This sequence obviously comprises residues 1-32 of porcine prosomatostatin since it is identical to the corresponding sequence in human preprosomatostatin. The postulated cleavage site in porcine prosomatostatin is a Leu-Leu bond between residues 32 and 33, thus confirming previous studies of the processing of the somatostatin precursor in the rat and transgenic mouse.