Measurement of bile salt hydrolase activity from Lactobacillus acidophilus based on disappearance of conjugated bile salts

Bile salt hydrolase activity of Lactobacillus acidophilus was measured based on the disappearance of sodium glycocholate and sodium taurocholate from the reaction mixture using HPLC. The amount of sodium glycocholate and sodium taurocholate that disappeared was proportional to the amount of sodium cholate that appeared in the mixture as detected by HPLC. Sodium glycocholate did not precipitate at the enzyme reaction conditions (37 degrees C and pH 5.4) for determining bile salt hydrolase activity. The bile salt hydrolase assay was insensitive to low oxidation-reduction potential when measuring bile salt hydrolase from L. acidophilus, an intestinal microorganism. However, EDTA and freezing temperatures were necessary to maintain stability of the partially purified enzyme during storage.     

Cloning and nucleotide sequence of carbaryl hydrolase gene (cahA) from Arthrobacter sp. RC100

A carbaryl hydrolase gene (cahA) encoded on the plasmid pRC1 in Arthrobacter sp. RC100 was cloned and sequenced. The entire region of the deduced amino acid sequence was found to be homologous to that of an amidase family. Parts of the consensus sequences of the amidase gene have been identified in CahA from strain RC100. CahA was overexpressed in Escherichia coli JM109, and the enzyme was purified to homogeneity by protamine sulfate treatment, ammonium sulfate precipitation, and hydrophobic and anion-exchange chromatographies. The purified enzyme showed hydrolase activity toward 1-naphthylacetamide and isobutyramide but showed no activity toward 1-naphthylacetate. This is the first report of an amidase that is able to hydrolyze N-methylcarbamate pesticides.     

Molecular cloning and nucleotide sequencing of organophosphorus insecticide hydrolase gene from Arthrobacter sp. strain B-5

The organophosphorus insecticide hydrolase (OPH) gene of Arthrobacter sp. strain B-5, isolated from turf green soil was cloned into Escherichia coli JM109. Three clones, termed EpB511, EpB521 and EpB531, exhibiting OPH activity were obtained. However, these three clones showed lower OP-degrading ability than strain B-5. A 7.7-kb inserted fragment of the plasmid pB521 harbored by EpB521 was subcloned, resulting in construction of a plasmid, pB526, carrying the 2.6-kb inserted fragment with OP-degrading ability. In this sequence, an open reading frame (ORF) that encodes a 43,607 Da polypeptide composed of 415 amino acids was identified. The N-terminal amino acid sequence deduced from the nucleotide sequence was identical to that of purified OPHs. The deduced amino acid sequence was compared with the sequences in the data bank and a 58.1% amino acid identity was found with the aryldialkylphosphatase from Nocardia sp. strain B-1, an enzyme that possesses catalytic functions similar to OPH.     

大豆异黄酮糖苷酶的发酵和纯化

大豆异黄酮糖苷酶可水解大豆异黄酮的糖基使其生成苷元,从而提高大豆异黄酮的生物活性。实验中筛选得到了大豆异黄酮糖苷酶产酶菌株 Absidia sp．R,确定了该菌株发酵产酶培养基。采用硫酸铵沉淀和离子交换法对该酶进行了纯化,并初步确定了其分子质量。     

娃娃菜农残消降组合方法及对品质的影响

以娃娃菜为试验对象,研究了有机磷水解酶、吐温、臭氧3种处理方法及其两两组合方法对蔬菜中7种有机磷农药残留的消降效果及对蔬菜营养、色泽和质地的影响。结果表明:6种消降方法都能有效降解娃娃菜表面的残留农药,吐温+臭氧、吐温+酶制剂组合方法较单一消降方法有明显的增效作用。吐温+臭氧组合处理消降率达65%～81%,吐温+酶制剂组合处理敌敌畏、毒死蜱、马拉硫磷、甲基对硫磷的消降率达65%～99%,但对甲胺磷、乙酰甲胺磷、氧乐果等内吸性农药的消降率只有40%。蔬菜经消降技术处理后,L*值、a*值、b*值与对照相比没有明显差异,还原型Vc仍保留在80%以上,且能保持其固有的感官质量要求。     

农药残留快速检测用酶——胆碱酯酶的筛选

在常见易得的前提下,以猪血、鸡血、鸭血、链鱼和鸡肝为实验材料提取胆碱酯酶,通过比较不同来源、不同动物组织胆碱酯酶的活性,并进行了胆碱酯酶对常见农药的敏感性实验,结果表明:鸭血清BChE酶含量丰富、对常见的有机磷农药及氨基甲酸酯类农药较敏感、且来源广泛、廉价易得。可作为果蔬农药残留的快速检测用酶。     

双表达解毒酶基因工程菌的酶活性分析及其应用

羧酸酯酶和有机磷水解酶可以水解不同的底物,为了获得降解谱广的解毒酶系,将两种酶共表达在同一载体。采用正交法对有机磷水解酶酶活性的分析条件进行了优化,以对硫磷为底物,底物浓度2×10-4mol/L、反应温度25℃、反应时间10min、pH8.0是测定酶活性最佳条件。在pH7.0～8.0的体系中存放一年活力可以保持50%以上。用于蚊子解毒实验,95%以上的对硫磷中毒蚊幼虫可以救活。在生物传感器等其它领域也都有应用。     

Purification and characterization of a lysine-p-nitroanilide hydrolase, a broad specificity aminopeptidase, from the cytoplasm of Lactococcus lactis subsp. cremoris AM2.

A hydrolase activity that cleaves lysyl-p-nitroanilide (Lys-pNA) has been purified from the cytoplasm of Lactococcus lactis subsp. cremoris AM2 by chromatography on DE52, DEAE Affi-Gel Blue Gel, Hydroxyapatite Bio-Gel HTP and Phenyl Sepharose. The purified aminopeptidase was found to have a native M(r) of 50,000-55,000 by gel filtration chromatography and by FPLC gel filtration on Superose 12 and to be composed of a single polypeptide chain following SDS-PAGE. Enzyme activity was almost completely inhibited by EDTA, amastatin, puromycin and bestatin, while the sulphydryl-reactive agents p-chloromercuribenzoate and iodoacetamide were inhibitory. The enzyme was found to be very unstable during the purification procedures at 4 degrees C and its stability was greatly improved when 10 ml glycerol/l and 2 mM-dithiothreitol were included in the purification buffers. The purified enzyme was found to hydrolyse a wide range of dipeptides, tripeptides and longer peptides provided that proline was not present in the penultimate position from the N-terminus or that a pyroglutamyl residue was not present at the N-terminus. While neither Asp-pNA nor Pro-pNA was hydrolysed by the purified enzyme, the release of N-terminal acidic residues from peptides was observed in addition to the release of N-terminal proline from Pro-Leu-Gly-NH2, Pro-Leu-Gly-Gly and Pro-His-Pro-Phe-His-Leu-Phe-Val-Tyr. This ability of Lys-pNA hydrolase to release N-terminal proline residues was employed in concert with a purified aminopeptidase P preparation to release alternate N-terminal amino acids from Tyr-Pro-Phe-Pro-Gly. The complementary action of these enzymes represents an alternative mechanism to that of post-proline dipeptidyl aminopeptidase for metabolism of proline-containing peptides.     

Purification and characterization of a novel extracellular beta-1,3-glucanase produced by Bacillus clausii NM-1 isolated from ezo abalone Haliotis discus hannai

A novel extracellular alkaline stable beta-1,3-glucanase produced by Bacillus clausii NM-1 isolated from the ezo abalone Haliotis discus hannai was purified by ammonium sulfate precipitation, DEAE-Sepharose FF ion exchange chromatography and Sephacryl S-200HR gel filtration. The molecular weight of the purified enzyme was estimated to be 71 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was very stable at pH 5.3 to 11.5 but unstable at pH 4.0 to 4.5. The optimum temperature and thermostability of the enzyme increased in the presence of CaC1, The enzyme hydrolyzed R-1,3-glucan from marine organisms, but did not show activity against any other beta-1,3-glucans. The major hydrolysis products of beta-1,3-glucan from Laminaria digitata and Eisenia bicyclis were laminaritriose and laminaritetraose, respectively. The N-terminal amino acid sequence of the purified enzyme was similar to that of several beta-1,3-glucanases in the glycoside hydrolase family 16.     

猪肝中提取纯化动物酯酶的研究

本实验探索了一种从猪肝中提取纯化动物酯酶的有效方法,从而为农药残留的动物酯酶检测提供了一种廉价酶源。将所制动物酯酶液用于检测毒死蜱的结果表明,其检测效果良好,所制动物酯酶具有实用价值。     

嗜酸乳杆菌胆盐水解酶的分离纯化及酶学性质

对嗜酸乳杆菌La-XH1产生的胆盐水解酶进行分离纯化,并对其部分酶学性质进行研究。结果表明：嗜酸乳杆菌La-XH1胆盐水解酶的粗酶液经硫酸铵沉淀、DEAE-Sepharose CL-6B柱层析后的酶比活力分别为47.82 U/mg和115.85 U/mg,纯化倍数分别为4.46 倍和10.82 倍,酶的回收率分别为59.89%和25.11%;通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecylsulfate polyacrylamide gel electrophoresis,SDS-PAGE）电泳分析,该酶的分子质量约为43 kD,最适温度为40 ℃,最适pH值为6.0,Fe3+、Ca2+、Mg2+、Mn2+、Zn2+对该酶有激活作用,其中Fe3+的激活作用最强,Na+、K+对该酶几乎无作用,而Cu2+、Ba2+对该酶有很强的抑制作用。     

有机磷农药降解酶基因Oph2的克隆及甲基对硫磷降解效果研究

摘要：目的 挖掘可高效表达农残降解酶的基因,并构建含有该表达基因的工程菌,以此提供一种高效去除有机磷农残的新方法。方法 利用基因克隆获得高效表达农残降解酶基因Oph2,采用基因工程手段将其转化至毕赤酵母表达系统,对毕赤酵母的发酵产物进行SDS-PAGE定性研究和酶活定量研究,最后通过与目标底物甲基对硫磷反应,分析工程菌表达降解酶对有机磷农残的去除效果。结果 本研究成功克隆得到了高效表达有机磷降解酶基因Oph2,基因全长1000bp,编码256个氨基酸,酶蛋白分子量为37KD,并利用表达载体pPIC9K成功将其转化至毕赤酵母GS115。构建的工程菌GS115-pPIC9K-Oph2表达有机磷降解酶活性为11.57 U/mL,对6.8 mg/L的甲基对硫磷降解效率为90%以上。结论 本研究得到的工程菌GS115-pPIC9K-Oph2可高效表达有机磷降解酶,该酶活性高,对有机磷农残降解能力强,可有效应用于有机磷农残的降解。     

Purification, characterization, and steady-state kinetics of a meta-cleavage compound hydrolase from Pseudomonas fluorescens IPO1

2-Hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl-HODA) hydrolase (CumD), an enzyme of the cumene biodegradation pathway encoded by the cumD gene of Pseudomonas fluorescens IP01, was purified to homogeneity from an overexpressing Escherichia coli strain. SDS-polyacrylamide gel electrophoresis and gel filtration demonstrated that it is a dimeric enzyme with a subunit molecular mass of 32 kDa. The pH optima for activity and stability were 8.0 and 7.0-9.0, respectively. The enzyme exhibited a biphasic Arrhenius plot of catalysis with two characteristic energies of activation with a break point at 20 degrees C. The enzyme has a K(m) of 7.3 microM and a k(cat) of 21 s(-1) for 6-isopropyl-HODA (150 mM phosphate, pH 7.5, 25 degrees C), and its substrate specificity covers larger C6 substituents compared with another monoalkylbenzene hydrolase, TodR Unlike TodF, CumD could slightly hydrolyze 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA). A mutant enzyme as to a putative active site residue, S103A, had 10(5)-fold lower activity than that of the wild-type enzyme.     

Purification and molecular cloning of an intracellular 3-hydroxybutyrate-oligomer hydrolase from Paucimonas lemoignei

An intracellular 3-hydroxybutyrate-oligomer hydrolase was purified from a poly(3-hydroxybutyrate)-degrading bacterium, Paucimonas lemoignei. It hydrolyzed the 3-hydroxybutyrate dimer with the highest specific activity of any of the enzymes reported so far. The gene was cloned and sequenced. The deduced amino acid sequence showed that the enzyme is a homolog of the PhaZc of Ralstonia eutropha H16.     

Nomilin Acetyl-Lyase, a Bacterial Enzyme for Nomilin Debittering of Citrus Juices

Nomilin acetyl-lyase, an enzyme responsible for the conversion of bitter nomilin to nonbitter obacunone was purified from cell-free extracts of Corynebacterium fuscians by (NH₄)₂SO₄ precipitation, followed by HPLC gel filtration and ion exchange chromatography. The electrophoretically pure preparation represented a 1050-fold increase in specific activity with a recovery of 38%. The enzyme, (MW 90,000) required sulfhydryl groups for its catalysis, had a pH optimum of 8.5, a K_m of 38.1 PM, and a V_max of 4.83 μmoles/min. Obacunone A-ring lactone hydrolase, which catalyzes the conversion of obacunone to obacunoate, was also purified from the extract by HPLC gel filtration.     

塔拉单宁水解酶性质的研究

黑曲霉塔拉单宁水解酶经丙酮初提、葡聚糖凝胶G-150柱层析及PEG-6000浓缩,纯度为纯化前的8.9倍,比活力为6.77 nkat/g。SDS-PAGE电泳分析表明,塔拉单宁水解酶由2个亚基组成,其分子量分别为93.1 ku和39.8 ku。酶学性质显示,其作用的最适pH为4.0,稳定pH为3.5～5.5;最适作用温度为40℃,稳定温度小于50℃;以单宁酸为底物时,单宁酶的米氏常数Km值为1.276 mmol/L。研究结果可为其在实际生产中的应用提供一定的参考。     

Purification and properties of family-10 endo-1,4-beta-xylanase from Penicillium citrinum and structural organization of encoding gene

An extracellular endo-1,4-beta-xylanase was purified from the culture filtrate of a filamentous fungus, Penicillium citrinum strain FERM P-15944, grown on birch-wood xylan. The purified enzyme showed a single band on SDS-PAGE with an apparent M(r) of 31.6 kDa. The xylanase activity was optimal at pH 6.0 and 50 degrees C. Southern blot analysis indicated that the xylanase gene (xynB) was present as a single copy in the genome. The genomic DNA and cDNAs encoding this protein were cloned and sequenced. An open reading frame of xynB was interrupted by nine introns and encoded a presumed prepropeptide of 25 amino acids and a mature protein of 302 amino acids. Sequence alignment and the constructed neighbor-joining tree showed that the P. citrinum enzyme belongs to glycoside hydrolase family 10 and is closely related to several other xylanases from penicillia and black aspergilli. The xynB cDNA was functionally expressed in the methylotrophic yeast Pichia pastoris.     

驴血清胆碱酯酶的生化性质

从不同的动物组织中筛选胆碱酯酶,研究胆碱酯酶的酶学性质和检测有机磷农药残留的条件。实验结果表明:驴血清胆碱酯酶活力较高,确定驴血清为检测有机磷农药的最佳酶源。胆碱酯酶最适反应温度为35℃,最适pH值为7.5～8.0,底物碘化硫代丁酰胆碱对驴血清胆碱酯酶没有过量底物抑制现象,最适浓度为5mmol/L;该酶热稳定性较好,在35℃保温60 min,酶活力基本不变。以有机磷农药—敌敌畏检测为例,对胆碱酯酶的半抑制浓度IC50为0.124 mg/mL,抑制时间为15 min。     

植物源性食品中有机磷农药残留检测前处理技术的研究进展

有机磷农药残留是重要的食品安全问题之一,有必要对食品中的有机磷农药进行监测。但植物源性食品成分复杂,进行有机磷农药检测前必须通过前处理进行提取和纯化。因此选择高效的有机磷农药残留前处理技术是非常重要的环节,已经成为当前研究者关注的重点。本文总结了有机磷农药分析的前处理方法的原理以及应用,包括传统提取法、加速溶剂萃取法、固相萃取法、QuEChERS法,介绍了有机磷农药的快速检测方法,包括分子印迹技术、酶抑制法、纳米材料富集法,并展望了对植物源性有机磷前处理技术的发展趋势。     

Purification,Characterization, and cDNA Cloning of a Prominent β-Glucosidase from the Gut of the Xylophagous Cockroach Panesthia angustipennis spadica

Abstract: In this study, a β-glucosidase (PaBG1b) with high specific activity was purified from gut extracts of the wood-feeding cockroach Panesthia angustipennis spadica using Superdex 75 gel filtration chromatography and High-Trap phenyl hydrophobic chromatography. The protein was purified 14-fold to a single band identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with an apparent molecular mass of 56.7 kDa. The specific activity of the purified enzyme was 708 μmol/min/mg protein using cellobiose as substrate. To the best of our knowledge, this is the highest specific activity reported among β-glucosidases to date. The purified PaBG1b showed optimal activity at pH 5.0 and retained more than 65 % of the activity between pH 4.0 and 6.5. The activity was stable up to 50 °C for 30 min. Kinetic studies on cellobiose revealed that the K_m was 5.3 mM, and the V_max was 1,020 μmol/min/mg. The internal amino acid sequence of PaBG1b was analyzed, and two continuous sequences (a total of 39 amino acids) of the C-terminal region were elucidated. Based on these amino acid sequences, a full-length cDNA (1,552 bp) encoding 502 amino acids was isolated. The encoded protein showed high similarity to β-glucosidases from glycoside hydrolase family 1. Thus, the current study demonstrated the potential of PaBG1b for application in enzymatic biomass-conversion as a donor gene for heterologous recombination of cellulase-producing agents (fungi or bacteria) or an additive enzyme for cellulase products based on the high-performance of PaBG1b as a digestive enzyme in cockroaches.