Endo-xylogalacturonan hydrolase, a novel pectinolytic enzyme

We screened an Aspergillus tubingensis expression library constructed in the yeast Kluyveromyces lactis for xylogalacturonan-hydrolyzing activity in microwell plates by using a bicinchoninic acid assay. This assay detects reducing carbohydrate groups when they are released from a carbohydrate by enzymatic activity. Two K. lactis recombinants exhibiting xylogalacturonan-hydrolyzing activity were found among the 3,400 colonies tested. The cDNA insert of these recombinants encoded a 406-amino-acid protein, designated XghA, which was encoded by a single-copy gene, xghA. A multiple-sequence alignment revealed that XghA was similar to both polygalacturonases (PGs) and rhamnogalacturonases. A detailed examination of conserved regions in the sequences of these enzymes revealed that XghA resembled PGs more. High-performance liquid chromatography and matrix-assisted laser desorption ionization-time of flight mass spectrometry of the products of degradation of xylogalacturonan and saponified modified hairy regions of apple pectin by XghA demonstrated that this enzyme uses an endo type of mechanism. XghA activity appeared to be specific for a xylose-substituted galacturonic acid backbone.     

Expression, purification, and characterization of a novel methyl parathion hydrolase

The mpd gene coding for a novel methyl parathion hydrolase (MPH) was previously reported and its putative open reading frame was also identified. To further confirm its coding region, the intact region encoding MPH was obtained by PCR and expressed in Escherichia coli as a hexa-His C-terminal fusion protein. The fusion protein was purified to homogeneity by metal-affinity chromatography. The enzyme activity and zymogram assay showed that the fusion protein was functional in degrading methyl parathion. The amino terminal sequencing of the purified recombinant MPH indicated that a signal peptide of the first 35 amino acids was cleaved from its precursor to form active MPH. A rat polyclonal antiserum was raised against the purified mature fusion protein. The results of Western blot and zymogram demonstrated that mature MPH in native Plesiomonas sp. strain M6 was also processed from its precursor by cleavage of a putative signal peptide at the amino terminus. The production of active MPH in E. coli was greatly improved after the coding region for the signal peptide was deleted. HPLC gel filtration of the purified mature recombinant MPH revealed that the MPH was a monomer.     

Olfactory adenylyl cyclase. Identification and purification of a novel enzyme form

Rat olfactory adenylyl cyclase has been identified by means of a monoclonal antibody BBC-2, which reacts with both Ca2+/calmodulin-sensitive and -insensitive forms of adenylyl cyclase (Mollner, S., and Pfeuffer, T. (1988) Eur. J. Biochem. 171, 265-271). The antibody recognized a 180-kDa polypeptide in olfactory cilia but not in decilitated olfactory epithelial membranes. A protein of the same mobility was observed when olfactory adenylyl cyclase was purified by forskolin-agarose affinity chromatography followed by radioiodination. Its identity was further established by cross-linking to [32P]ADP-ribosylated Gs alpha (GTP-binding protein), to yield a single radiolabeled product of Mr approximately 220. Olfactory adenylyl cyclase has a approximately 3-fold higher turnover number, as assessed from stoichiometric binding of [35S]guanosine 5'-(3-O-thio)triphosphate. Therefore, the considerably higher specific adenylyl cyclase activity in olfactory cilia must be due to a approximately 100-fold higher molar concentration of enzyme in this tissue.     

The purification and characterization of phosphonopyruvate hydrolase,a novel carbon-phosphorus bond cleavage enzyme from Variovorax sp Pal2

Phosphonopyruvate hydrolase, a novel bacterial carbon-phosphorus bond cleavage enzyme, was purified to homogeneity by a series of chromatographic steps from cell extracts of a newly isolated environmental strain of Variovorax sp. Pal2. The enzyme was inducible in the presence of phosphonoalanine or phosphonopyruvate; unusually, its expression was independent of the phosphate status of the cell. The native enzyme had a molecular mass of 63 kDa with a subunit mass of 31.2 kDa. Activity of purified phosphonopyruvate hydrolase was Co2+-dependent and showed a pH optimum of 6.7-7.0. The enzyme had a Km of 0.53 mm for its sole substrate, phosphonopyruvate, and was inhibited by the analogues phosphonoformic acid, 3-phosphonopropionic acid, and hydroxymethylphosphonic acid. The nucleotide sequence of the phosphonopyruvate hydrolase structural gene indicated that it is a member of the phosphoenolpyruvate phosphomutase/isocitrate lyase superfamily with 41% identity at the amino acid level to the carbon-to-phosphorus bond-forming enzyme phosphoenolpyruvate phosphomutase from Tetrahymena pyriformis. Thus its apparently ancient evolutionary origins differ from those of each of the two carbon-phosphorus hydrolases that have been reported previously; phosphonoacetaldehyde hydrolase is a member of the haloacetate dehalogenase family, whereas phosphonoacetate hydrolase belongs to the alkaline phosphatase superfamily of zinc-dependent hydrolases. Phosphonopyruvate hydrolase is likely to be of considerable significance in global phosphorus cycling, because phosphonopyruvate is known to be a key intermediate in the formation of all naturally occurring compounds that contain the carbon-phosphorus bond.     

Identification and partial purification of a novel milk clotting enzyme from Onopordum turcicum

Summary Seeds, flowers and leaves of Onopordum turcicum were found to contain proteolytic enzymes able to coagulate milk. Extraction, concentration and identification of the operational parameters affecting the activity of the enzyme complex were followed by partial purification steps involving gel-filtration and ion-exchange chromatography. Milk clotting activity of the enzyme complex was tested in several steps of its purification and an increase of almost 200 fold was obtained. Molecular weight of the proteolytic enzyme fraction having the maximum activity was determined to be about 19000–24000. Isoelectric point (pI) of the enzyme complex with maximum activity was estimated to be in the range 3.3–3.7.     

来源于Rhinocladiella mackenziei的玉米赤霉烯酮水解酶的表达纯化与酶学性质

【背景】玉米赤霉烯酮(Zearalenone,ZEN)是污染最广泛的霉菌毒素之一,对饲料行业和畜牧业造成了巨大的经济损失。目前研究最为广泛的玉米赤霉烯酮降解酶ZHD101因其热稳定性较差,无法满足工业应用上的要求。【目的】为实现玉米赤霉烯酮降解酶在工业上的应用,寻找酶学性质更突出的ZEN降解酶。【方法】基于对Gen Bank数据库的挖掘,发现一个来源于麦氏喙枝孢霉(Rhinocladiella mackenziei CBS 650.93)的Rmzhd基因,构建p ET-46-Rmzhd质粒。利用大肠杆菌表达体系和亲和层析、离子交换纯化体系对蛋白进行表达和纯化,通过高效液相凝胶色谱分析酶学性质。【结果】发现一个新的ZEN水解酶Rm ZHD,RmZHD在pH 8.6和45°C条件下的活性最高,而且具有较高的耐热性。结构分析表明,较高的盐桥数目和溶剂暴露脯氨酸含量可能是造成其高耐热性的原因。【结论】本研究为促进玉米赤霉烯酮降解酶在工业上的应用打下基础。     

Probing the evolution of hydroxyisourate hydrolase into transthyretin through active-site redesign

5-Hydroxyisourate hydrolase (HIUase) and transthyretin (TTR) are closely related phylogenetically and structurally, while performing quite different functions. The former catalyzes the hydrolysis of 5-hydroxyisourate within the urate degradation pathway, and the latter is a carrier protein involved in the extracellular transport of thyroid hormones and in the cotransport of retinol. The evolution of HIUase into TTR represents a remarkable example of adaptation of a new function by active-site modification of an enzyme. On the basis of phylogenetic reconstructions and structural comparison of HIUase and TTR, two mutations (Y116T and I16A) were likely to be crucial events in order to induce, after a gene duplication event, the conversion of the enzyme into a binding protein. By rational reshaping of the active sites of HIUase and functional analyses of its mutant forms, we have provided insights into how its neofunctionalization could be achieved. We show here that the two mutations at the active sites of HIUase open up the two ends of the channel that transverses the entire tetrameric protein, generating two cavities accessible to the thyroxine molecule and abrogating, at the same time, the enzymatic activity. Our data indicate that a small number of critical mutations affecting the active site of an enzyme may be sufficient to generate a drastically different function, while a large number of additional mutations may be required for the fine-tuning of the structural and functional features of new proteins.     

Identification of a novel chondroitin hydrolase in Caenorhabditis elegans

Hyaluronidases have been postulated to be the enzyme acting at the initial step of chondroitin sulfate (CS) catabolism in vivo. Since chondroitin (Chn) but not hyaluronic acid (HA) has been detected in Caenorhabditis elegans, the nematode is a good model for elucidating the mechanism of the degradation of CS/Chn in vivo. Here we cloned the homolog of human hyaluronidase in C. elegans, T22C8.2. The Chn-degrading activity in vitro was first demonstrated when it was expressed in COS-7 cells. The enzyme cleaved preferentially Chn. CS-A and CS-C were also depolymerized but to lesser extents, and HA was hardly degraded. In order of preference, the substrates ranked Chn > CS-A > CS-C > HA. The products of the degradation of Chn by the enzyme were characterized by anion-exchange high performance liquid chromatography and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The structure of the major component in the digest was determined as GlcUAbeta1-3GalNAcbeta1-4GlcUAbeta1-3GalNAc, where GlcUA and GalNAc represent D-glucuronic acid and N-acetyl-D-galactosamine, respectively, indicating that this enzyme is a Chn hydrolase, an endo-beta-galactosaminidase specific for Chn. Investigation of the effects of pH on the activity revealed the optimum pH of Chn hydrolase to be 6.0. Since Chn in C. elegans has been demonstrated to play critical roles in cell division, Chn hydrolase possibly regulates the function of Chn in vivo. This is the first demonstration of a Chn hydrolase in an animal.     

A familiar motif in a new context: the catalytic mechanism of hydroxyisourate hydrolase

Hydroxyisourate hydrolase is a recently discovered enzyme that participates in the ureide pathway in soybeans. Its role is to catalyze the hydrolysis of 5-hydroxyisourate, the product of the urate oxidase reaction. There is extensive sequence homology between hydroxyisourate hydrolase and retaining glycosidases; in particular, the conserved active site glutamate residues found in retaining glycosidases are present in hydroxyisourate hydrolase as Glu 199 and Glu 408. However, experimental investigation of their roles, as well as the catalytic mechanism of the enzyme, have been precluded by the instability of 5-hydroxyisourate. Here, we report that diaminouracil serves as a slow, alternative substrate and can be used to investigate catalysis by hydroxyisourate hydrolase. The activity of the E199A protein was reduced 400-fold relative to wild-type, and no activity could be detected with the E408A mutant. Steady-state kinetic studies of the wild-type protein revealed that the pH-dependence of V(max) and V/K describe bell-shaped curves, consistent with the hypothesis that catalysis requires two ionizable groups in opposite protonation states. Addition of 100 mM azide accelerated the reaction catalyzed by the wild-type enzyme 8-fold and the E199A mutant 20-fold but had no effect on the E408A mutant. These data suggest that Glu 408 acts as a nucleophile toward the substrate forming a covalent anhydride intermediate, and Glu 199 facilitates formation of the intermediate by serving as a general acid and then activates water for hydrolysis of the intermediate. Thus, the mechanism of hydroxyisourate hydrolase is strikingly similar to that of retaining glycosidases, even though it catalyzes hydrolysis of an amide bond.     

Cloning, expression, purification, and characterization of a novel epoxide hydrolase from Aspergillus niger SQ-6

A novel epoxide hydrolase from Aspergillus niger SQ-6 has now been cloned by inverse PCR. Its gene shows eight exons including a non-coding exon at its 5'-terminal (GenBank Accession No. AY966486). Phylogenetic analysis using deduced amino acid sequence (395 aa) confirms it as an epoxide hydrolase and shares 58.3% identity with that of A. niger LCP521 (GenBank Accession No. AF238460). The predicted catalytic triad is composed of Asp(191), His(369) and Glu(343). Active recombinant epoxide hydrolase has been successfully expressed in Escherichia coli as protein fusions with a poly-His tail. Scale-up fermentation can yield 2.5g/L of recombinant protein. The electrophoretic pure recombinant protein, which shows similar characterization as natural enzyme purified from A. niger SQ-6, can be easily purified by Ni(2+)-chelated affinity and gel-filtration chromatography. Optimal pH and temperature for purified enzyme are pH 7.5 and 37 degrees C, respectively. The K(m), k(cat) and maximal velocity (V(max)) for p-nitrostyrene oxide are determined to be 1.02mM, 172s(-1) and 231micromol min(-1)mg(-1), respectively. The enzyme can be inhibited by oxidant (H(2)O(2)), solvent (Tetrahydrofuran) and several metal ions including Hg(2+), Fe(2+) and Co(2+). This (R)-stereospecific epoxide hydrolase exhibits high enantioselectivity (enantiomeric excess value, 99%) for the less hindered carbon atom of epoxide. It may be an industrial biocatalyst for the preparation of enantiopure epoxides or vicinal diols.     

Identification of neutral cholesterol ester hydrolase, a key enzyme removing cholesterol from macrophages

Unstable lipid-rich plaques in atherosclerosis are characterized by the accumulation of macrophage foam cells loaded with cholesterol ester (CE). Although hormone-sensitive lipase and cholesteryl ester hydrolase (CEH) have been proposed to mediate the hydrolysis of CE in macrophages, circumstantial evidence suggests the presence of other enzymes with neutral cholesterol ester hydrolase (nCEH) activity. Here we show that the murine orthologue of KIAA1363, designated as neutral cholesterol ester hydrolase (NCEH), is a microsomal nCEH with high expression in murine and human macrophages. The effect of various concentrations of NaCl on its nCEH activity resembles that on endogenous nCEH activity of macrophages. RNA silencing of NCEH decreases nCEH activity at least by 50%; conversely, its overexpression inhibits the CE formation in macrophages. Immunohistochemistry reveals that NCEH is expressed in macrophage foam cells in atherosclerotic lesions. These data indicate that NCEH is responsible for a major part of nCEH activity in macrophages and may be a potential therapeutic target for the prevention of atherosclerosis.     

Identification of a novel peptidoglycan hydrolase CwlM in Mycobacterium tuberculosis

Mycobacterium tuberculosis is a major global pathogen whose threat has increased with the emergence of multidrug-resistant strains. The cell wall of M. tuberculosis is thick, rigid, and hydrophobic, which serves to protect the organism from the environment and makes it highly impermeable to conventional antimicrobial agents. There is little known about cell wall autolysins (also referred to as peptidoglycan hydrolases) of mycobacteria. We identified an open reading frame (Rv3915) in the M. tuberculosis genome designated cwlM that appeared consistent with a peptidoglycan hydrolase. The 1218-bp gene was amplified by PCR, cloned and expressed in E. coli strain HMS174(DE-3), and its gene product, a 47-kDa recombinant protein, was purified and partially characterized. Purified CwlM was able to lyse whole mycobacteria, release peptidoglycan from the cell wall of Micrococcus luteus and Mycobacterium smegmatis, and cleave N-acetylmuramoyl-L-alanyl-D-isoglutamine, releasing free N-acetylmuramic acid. These results indicate that CwlM is a novel autolysin and identify cwlM as the first, to our knowledge, autolysin gene identified and cloned from M. tuberculosis. CwlM offers a new target for a unique class of drugs that could alter the permeability of the mycobacterial cell wall and enhance the effectiveness of treatments for tuberculosis.     

一株产纤溶酶芽孢杆菌的鉴定及纤溶酶的分离纯化与性质分析

摘要：【目的】从假蕈状芽孢杆菌B-60菌株中纯化具有纤溶活性的单一组分,测定它的N-端氨基酸序列进行比对,并对单一组分的性质进行分析。【方法】利用纤维蛋白平板法检测纤溶酶活性,利用硫酸氨分级沉淀和阴离子交换色谱从假蕈状芽孢杆菌B-60菌株中纯化纤溶酶。【结果】从该菌株的发酵液中获得了一组纤溶酶单一组分（BpFE）,它的表观分子量为34 kDa。它在4℃~50℃活性较稳定,50℃以上活性急剧下降;作用最适pH值为pH5~6,在pH5~10活性较稳定,在pH3.0,活性几乎丧失;金属离子Ca2+,Mg2+,M     

Purification and characterization of a novel enzyme, alpha-neoagarooligosaccharide hydrolase (alpha-NAOS hydrolase), from a marine bacterium, Vibrio sp. strain JT0107.

A novel enzyme, alpha-neoagarooligosaccharide hydrolase (EC 3.2.1.-), which hydrolyzes the alpha-1,3 linkage of neoagarooligosaccharides to yield agaropentaose (O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-D-galactose], agarotriose [O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro- alpha-L-galactopyranosyl (1-->3)-D-galactose], agarobiose [O-beta-D-galactopyranosyl(1-->4)-3,6-anhydro-L-galactose], 3,6-anhydro-L-galactose, and D-galactose was isolated from the marine bacterium Vibrio sp. strain JT0107 and characterized. This enzyme was purified 383-fold from cultured cells by using a combination of ammonium sulfate precipitation, successive anion-exchange column chromatography, gel filtration, and hydroxyapatite chromatography, gel filtration, and hydroxyapatite chromatography. The purified protein gave a single band (M(r), 42,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the M(r) by the gel filtration method gave a value of 84,000, indicating that the enzyme is dimeric. Amino acid sequence analysis revealed it to have a single N-terminal sequence that has no sequence homology to any other known agarases. The optimum temperature and pH were 30 degrees C and 7.7, respectively. The Km and maximum rate of metabolism for neoagarobiose were 5.37 mM and 92 U/mg of protein, respectively.     

Jejunal brush-border folate hydrolase. A novel enzyme.

Dietary folate, a vitamin required for DNA synthesis and cell regeneration, occurs as pteroylpolyglutamates that are hydrolyzed to pteroylglutamate during the process of intestinal absorption. Studies from our laboratory over the past 15 years have shown that jejunal brush-border folate hydrolase is essential and rate-limiting in folate absorption. Brush-border folate hydrolase activity and pteroylpolyglutamate hydrolysis are inhibited in disease and conditions associated with folate deficiency, including celiac and tropical sprue, the use of sulfasalazine to treat inflammatory bowel disease, and chronic alcoholism. Brush-border folate hydrolase is an exopeptidase located on the jejunal brush-border surface that liberates hydrolytic products of pteroylpolyglutamates in a progressive fashion, with a final release of pteroylglutamate. Subsequent steps in folate absorption include uptake by a brush-border folate-binding-protein receptor and transport across the brush-border membrane into the enterocyte. These steps are probably followed by an intracellular synthesis of pteroylglutamates for folate-dependent reactions and intracellular hydrolysis to pteroylglutamate for transport across the basolateral membrane to the portal circulation. In pigs, the active form of jejunal brush-border folate hydrolase has a molecular weight of 240 kd and is probably a homodimer of the 120-kd protein found after immunoprecipitation with specific antibody. Regulating the synthesis and expression of brush-border folate hydrolase may be critical to the availability of dietary folate.     

Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein.

Autotaxin (ATX) is a potent human motility-stimulating protein that has been identified in the conditioned medium from A2058 melanoma cells. This protein has been purified to homogeneity utilizing a strategy involving five column steps. Homogeneity of ATX was verified by two-dimensional gel electrophoresis. The molecular size of ATX is 125 kDa, and it has an isoelectric point of 7.7 +/- 0.2. Purified ATX was digested with cyanogen bromide and trypsin, and the resulting ATX peptides were purified by reverse-phase high performance liquid chromatography. Eleven peptides were subjected to amino acid sequence analysis, and 114 residues were identified. The partial amino acid sequences and the amino acid composition obtained for ATX show that it does not exhibit any significant homology to known growth factors or previously described motility factors. At picomolar concentrations, ATX stimulates both random and directed migration of human A2058 melanoma cells. Pretreatment of the melanoma cells with pertussis toxin abolishes the response to purified ATX, indicating that ATX stimulates motility through a receptor acting via a pertussis toxin-sensitive G protein.     

Pyrethroid-degrading Sphingobium sp. JZ-2 and the purification and characterization of a novel pyrethroid hydrolase

A pyrethroid-degrading bacterium strain JZ-2 was isolated from activated sludge treating pyrethroid-manufacturing wastewater. Based on the morphological, physiological and biochemical characterization, and phylogenetic analysis of the 16S rRNA gene sequence, the strain was identified as Sphingobium sp. Strain JZ-2 was capable of degrading fenpropathrin, cypermethrin, permethrin, cyhalothrin, deltamethrin, fenvalerate and bifenthrin. This strain degraded fenpropathrin by hydrolysis of the carboxylester linkage to yield 3-phenoxybenzaldehyde and 2,2,3,3-tetramethylcyclopropanecarboxylic acid. 3-Phenoxybenzaldehyde, 3-phenoxybenzoate, protocatechuate and catechol are the intermediates of fenpropathrin degradation. Protocatechuate and catechol were further oxidized by ortho-cleavage pathway. A novel pyrethroid hydrolase from cell-free extract was purified 108.5-fold to apparent homogeneity with a 10.2% overall recovery. It was a monomer with a molecular mass of 31 ± 1 kDa, a pI of 4.85. The optimal pH and temperature were 7.5 and 40 °C, respectively. No cofactors or coenzymes were required for the pyrethroid-hydrolysis activity. The enzyme was strongly inhibited by many irons (Ag⁺, Cu²⁺, Hg²⁺ and Zn²⁺), SDS, p-chloromercuribenzoic acid, phenylmethylsulfonyl fluoride and malathion.     

来源于嗜热氢化杆菌的新型对苯二甲酸双(羟乙)酯水解酶的表达纯化与酶学性质

石化来源的聚对苯二甲酸乙二酯(polyethylene terephthalate,PET)被广泛用于矿泉水瓶、食品包装和纺织品等领域,因其在自然界中不易分解,大量使用后的PET废弃物造成了严重的环境污染与资源浪费。使用生物酶法对PET废弃物进行解聚,并对解聚产物进行升级循环利用是进行塑料污染治理的重要方向之一,其中关键的是PET水解酶的解聚效率。对苯二甲酸双(羟乙基)酯(bis(hydroxyethyl)terephthalate,BHET)是PET生物酶解的中间产物,其累积是限制PET水解酶催化效率的一个重要因素,BHET水解酶和PET水解酶的联用能提升PET的整体水解效率。来源于嗜热氢化杆菌(Hydrogenobacter thermophilus)的双烯内酯酶(HtBHETase)对BHET有显著水解效果,将该酶在大肠杆菌(Escherichia coli)中进行重组表达并纯化后,对其酶学性质进行了研究。结果显示,HtBHETase对短碳链的酯类如对硝基苯酚乙酸酯催化活性较高,HtBHETase以BHET为底物时的最适反应pH值和最适反应温度分别为5.0和55℃;该酶有较好的热稳定性,经80℃的条件处理1 h仍能保持80%以上活性,显示出了良好的热稳定性,HtBHETase有在PET塑料生物解聚中使用的潜力,本研究为推动生物酶法降解PET提供了新的参考。     

trans-Dienelactone hydrolase from Pseudomonas reinekei MT1, a novel zinc-dependent hydrolase

Pseudomonas reinekei MT1 is capable of growing on 4- and 5-chlorosalicylate, involving a pathway with trans-dienelactone hydrolase (trans-DLH) as a key enzyme. It acts on 4-chloromuconolactone formed during cycloisomerization of 3-chloromuconate by hydrolyzing it to maleylacetate. The gene encoding this activity was localized, sequenced and expressed in Escherichia coli. Inductively coupled plasma mass spectrometry showed that both the wild-type as well as recombinant enzymes contained 2 moles of zinc but variable amounts of manganese/mol of protein subunit. The inactive metal-free apoenzyme could be reactivated by Zn²⁺ or Mn²⁺. Thus, trans-DLH is a Zn²⁺-dependent hydrolase using halosubstituted muconolactones and trans-dienelactone as substrates, where Mn²⁺ can substitute for Zn²⁺. It is the first member of COG1878 and PF04199 for which a direct physiological function has been reported.