Microbial cell disruption methods for efficient release of enzyme L-asparaginase

Abstract

The efficacy of a simple laboratory method for cell disruption based on the glass bead stirring, sonication, osmotic shock, freezing and grinding, or use of solvents and detergents was assessed in this study, via measurements of the release of total protein and L-asparaginase activity. Three different microbial sources of L-asparaginase were used: Escherichia coli BL21 (DE3), Leucosporidium muscorum, and Aspergillus terreus (CCT 7693). This study adjusted and identified the best procedure for each kind of microorganism. Sonication and glass bead stirring led to obtaining filamentous fungus cell-free extracts containing high concentrations of soluble proteins and specific activity; however, sonication was the best since it obtained 4.61?±?0.12?IU?mg^?1 after 3?min of operation time. Mechanical methods were also the most effective for yeast cell disruption, but sonication was the technique which yielded a higher efficiency releasing 7.3 IUtotal compared to glass bead stirring releasing 2.7 IUtotal at the same operation time. For bacterium, sonication proved to be the best procedure due to getting the highest specific activity (9.01?IU?mg^?1) and total enzyme activity (61.7?IU). The data presented lead to conclude that the mechanical methods appeared to be the most effective for the disintegration of the all microbial cells studies. This is the first report related to the experimental comparison of L-ASNase extraction procedures from different microorganisms, which can also be used for extracting periplasm located enzymes from other organisms.     

Polysaccharide-hydrolyzing enzymes ofFrankia (Actinomycetales)

Studies were made of the polysaccharide-hydrolyzing activity inFrankia (Actinomycetales) grown in synthetic media using modifications of three standard assay procedures. In screening five different strains ofFrankia for cellulase activity, based on the method of utilization of cellulose in liquid culture, only one strain, CcI3, degraded filter paper cellulose to complete disintegration and only under very specific conditions of pH and primary carbon source. When carboxymethylcellulose (CMC) at 1% was used as substrate, all five strains showed the capacity to produce reducing sugars as hydrolytic products. Microcystalline cellulose, xylans and gum arabic were hydrolyzed to a lesser extent. Optimum activity depended upon pH and primary carbon source with pH 5.0 and pyruvate or propionate producing highest activities. In fractionation studies of culturedFrankia, assays for hydrolysis of 1% CMC in liquid medium showed that highest activity was in the enzyme preparation supernatant with lesser activity in the cell-free extract and cell wall fractions.Frankia strain CpI1 showed the greatest total hydrolytic activity against CMC after 2 weeks of culture. Strains ArI3 and CcI3 also showed good activity. The agar plate method for direct dye-polysaccharide interaction proved to be the least sensitive assay method with only ArI3 showing significant activity using CMC as substrate. It appears that theFranka strains grown in synthetic media all showed hydrolytic activity but the degree of hydrolysis of polysaccharides to reducing sugars depends upon strain of bacteria and very specific cultural conditions.     

Isolation and characterization of a nitrile hydrolysing acidotolerant black yeast—<Emphasis Type="Italic">Exophiala oligosperma</Emphasis> R1

Different nitriles were used as sole sources of nitrogen in a series of enrichments under acidic conditions to isolate acidotolerant nitriles hydrolysing microorganisms. From an enrichment in Na–citrate–phosphate buffer at pH 4 with glucose as carbon source and phenylacetonitrile as sole source of nitrogen, a black yeast (strain R1) was obtained which was identified by subsequent 18S rRNA gene sequencing as Exophiala oligosperma. The growth conditions of the organism were optimized for the production of cell material and the induction of the nitrile converting activity. Resting cell experiments demonstrated that phenylacetonitrile was converted via phenylacetic acid and 2-hydroxyphenylacetic acid. The organism could grow at pH 4 with phenylacetonitrile as sole source of carbon, nitrogen, and energy. The nitriles hydrolysing activity was also detected in cell-free extracts and indications for a nitrilase activity were found. The cell-free extracts converted, in addition to phenylacetonitrile, also different substituted phenylacetonitriles. Whole cells of E. oligosperma R1 converted phenylacetonitrile with almost the same reaction rates in the pH range from pH 1.5–pH 9.     

Isolation,identification and characterization of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> ZJB-063, a versatile nitrile-converting bacterium

Strain ZJB-063, a versatile nitrile-amide-degrading strain, was newly isolated from soil in this study. Based on morphology, physiological tests, Biolog and the 16S rDNA sequence, strain ZJB-063 was identified as Bacillus subtilis. ZJB-063 exhibited nitrilase activity without addition of inducers, indicating that the nitrilase in B. subtilis ZJB-063 is constitutive. Interestingly, the strain exhibited nitrile hydratase and amidase activity with the addition of ɛ-caprolactam. Moreover, the substrate spectrum altered with the alteration of enzyme systems due to the addition of ɛ-caprolactam. The constitutive nitrilase was highly specific for arylacetonitriles, while the nitrile hydratase/amidase in B. subtilis ZJB-063 could not only hydrolyze arylacetonitriles but also other nitriles including some aliphatic nitriles and heterocyclic nitriles. Despite comparatively low activity, the amidase of hydratase/amidase system was effective in converting amides to acids. The versatility of this strain in the hydrolysis of various nitriles and amides makes it a potential biocatalyst in organic synthesis.     

Optimization in the recovery of a labile intracellular enzyme

A high molecular weight intracellular enzyme of Bacillus brevis ATCC 9999 is released when the organism is disrupted by sonication of homogenization. However, both processes also degrade the enzyme. Assays for protein release and specific enzymatic activity of the released protein indicate that both release and degradation can be represented by first-order kinetic models. Utilization of the difference between the kinetics of release and degradation allows optimization in the recovery of this enzyme for both the sonication and homogenization processes.     

Characterization and purification of hydrolytic enzymes in Sinorhizobium fredii CCRC15769

Rhizobial symbiosis provides the nitrogen for the leguminous plant through fixation of the gaseous nitrogen component of air. For the bacteria in the plant root-hair wall, carboxymethylcellulase (CM-cellulase, EC 3.2.1.4) may be the key enzyme in this symbiotic process, with polygalacturonase (pectinase, EC 3.2.1.15) another critical enzyme involved early in the mechanism of nitrogen supply. The precise cytosolic location, function and expression of CM-cellulase are still uncertain, however. To detect the relevant enzyme activity in Sinorhizobium fredii CCRC15769, various assay methods were used including double-layer plate assay and quantitation of reducing sugar products. After sonication of the cell pellet, ammonium sulphate precipitation, gel filtration, and ion-exchange chromatography are the preferred methods for derivation of the purified protein, with CM-cellulase characterized as follows: purification ratio, 33.35; recovery, 10.8%; and specific activity, 0.053 U mg^–1. The endoglucanase in the purified samples was resolved using native and sodium dodecyl sulphate-polyacrylamide-gel electrophoresis; it was then assayed with an ultrathin CM-cellulose overlay stained with Congo Red. Two CM-cellulase isozymes were determined by native activity stain assay, with gel-filtration revealing molecular weights of approximately 196 and 30 kD; the SDS-PAGE activity gel resolved four enzyme subunits of 94, 67, 37, and 30 kD. It is suggested that the CM-cellulase in S.fredii CCRC15769 is a two-isozyme form, one a trimer of 196 kD (94, 67 and 37 kD), and the other a 30 kD monomer.     

Cyanide Metabolism in Higher Plants: Cyanoalanine Hydratase is a NIT4 Homolog

Cyanoalanine hydratase (E.C. 4.2.1.65) is an enzyme involved in the cyanide detoxification pathway of higher plants and catalyzes the hydrolysis of β-cyano-l-alanine to asparagine. We have isolated the enzyme from seedlings of blue lupine (Lupinus angustifolius) to obtain protein sequence information for molecular cloning. In contrast to earlier reports, extracts of blue lupine cotyledons were found also to contain cyanoalanine-nitrilase (E.C. 3.5.5.4) activity, resulting in aspartic acid production. Both activities co-elute during isolation of cyanoalanine hydratase and are co-precipitated by an antibody directed against Arabidopsis thaliana nitrilase 4 (NIT4). The isolated cyanoalanine hydratase was sequenced by nanospray-MS/MS and shown to be a homolog of Arabidopsis thaliana and Nicotiana tabacum NIT4. Full-length cDNA sequences for two NIT4 homologs from blue lupine were obtained by PCR using degenerate primers and RACE-experiments. The recombinant LaNIT4 enzymes, like Arabidopsis NIT4, hydrolyze cyanoalanine to asparagine and aspartic acid but show a much higher cyanoalanine-hydratase activity. The two nitrilase genes displayed differential but overlapping expression. Taken together these data show that the so-called ‘cyanoalanine hydratase’ of plants is not a bacterial type nitrile hydratase enzyme but a nitrilase enzyme which can have a remarkably high nitrile-hydratase activity.     

Purification and characterization of an enantioselective arylacetonitrilase from Pseudomonas putida

The highly enantioselective arylacetonitrilase of Pseudomonas putida was purified to homogeneity using a combination of (NH₄)₂SO₄ fractionation and different chromatographic techniques. The enzyme has a molecular weight of 412 kDa and consisted of approximately nine to ten identical subunits (43 kDa). The purified enzyme exhibited a pH optimum of 7.0 and temperature optimum of 40°C. The nitrilase was highly susceptible to thiol-specific reagents and metal ions and also required a reducing environment for its activity. These reflected the presence of a catalytically essential thiol group for enzyme activity which is in accordance with the proposed mechanism for nitrilase-catalyzed reaction. The enzyme was highly specific for arylacetonitriles with phenylacetonitrile and its derivatives being the most preferred substrates. Higher specificity constant (k _cat/K _m) values for phenylacetonitrile compared to mandelonitrile also revealed the same. Faster reaction rate achieved with this nitrilase for mandelonitrile hydrolysis was possibly due to the low activation energy required by the protein. Incorporation of low concentration (<5%) of organic solvent increased the enzyme activity by increasing the availability of the substrate. Higher stability of the enzyme at slightly alkaline pH and ambient temperature provides an excellent opportunity to establish a dynamic kinetic resolution process for the production of (R)-(−)-mandelic acid from readily available mandelonitrile.     

Hyperinduction of nitrilases in filamentous fungi

2-Cyanopyridine proved to act as a powerful nitrilase inducer in Aspergillus niger K10, Fusarium solani O1, Fusarium oxysporum CCF 1414, Fusarium oxysporum CCF 483 and Penicillium multicolor CCF 2244. Valeronitrile also enhanced the nitrilase activity in most of the strains. The highest nitrilase activities were produced by fungi cultivated in a Czapek-Dox medium with both 2-cyanopyridine and valeronitrile. The specific nitrilase activities of these cultures were two to three orders of magnitude higher than those of cultures grown on other nitriles such as 3-cyanopyridine or 4-cyanopyridine.     

Production of a fibrinolytic enzyme by thermophilic Streptomyces species

A strong fibrin-specific fibrinolytic enzyme was purified from the cell-free spent culture broth of a thermophilic organism, Streptomyces megasporus SD5. The strain could produce 150 mg crude protein per litre of spent broth, with a specific activity of 80 IU (Plough units) per milligram, within 18 h of incubation at 55 °C in glucose yeast/extract/peptone (GYP) medium, pH 8.0. For production of the enzyme, the strain could utilize different carbon and nitrogen sources with a C:N ratio of ∼ 1:2. The enzyme was stable at a broad range of pH ranging from 5 to 9, and highly thermostable with 50% activity after storage at 60 °C for 6 months. The enzyme belonged to the serine endopeptidase group. In vitro clot lysis revealed that the enzyme was active at 37 °C. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification and characterization of a nitrilase from Aspergillus niger K10

Aspergillus niger K10 cultivated on 2-cyanopyridine produced high levels of an intracellular nitrilase, which was partially purified (18.6-fold) with a 24% yield. The N-terminal amino acid sequence of the enzyme was highly homologous with that of a putative nitrilase from Aspergillus fumigatus Af293. The enzyme was copurified with two proteins, the N-terminal amino acid sequences of which revealed high homology with those of hsp60 and an ubiquitin-conjugating enzyme. The nitrilase exhibited maximum activity (91.6 U mg^-1) at 45°C and pH 8.0. Its preferred substrates, in the descending order, were 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thiophen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. Formation of amides as by-products was most intensive, in the descending order, for 2-cyanopyridine, 4-chlorobenzonitrile, 4-cyanopyridine, and 1,4-dicyanobenzene. The enzyme stability was markedly improved in the presence of d-sorbitol or xylitol (20% w/v each). p-Hydroxymercuribenzoate and heavy metal ions were the most powerful inhibitors of the enzyme.     

木质素含量对四倍体刺槐嫩枝插穗扦插生根的影响

以四倍体刺槐1年生嫩枝插条为试验材料,分析插穗木质素含量与其横截面剪切强度、相关酶活性和激素关系,并探究不同木质素含量的插穗扦插生根性状的效应,为四倍体刺槐扦插选择合适插穗提供理论参考。结果表明:(1)插穗的木质素含量与其横截面剪切强度呈极显著正相关关系(相关系数为0.99),根据剪切强度可以间接地估算其木质素含量。(2)插穗的木质素含量与其POD和PPO活性呈极显著正相关关系(POD相关系数为0.98,PPO相关系数为0.92),也与激素ABA含量呈极显著的正相关关系(相关系数为0.97),而同IAA、IBA呈不显著的相关性,根据POD和PPO活性以及激素ABA含量对生根的影响可以推测木质素含量对生根有一定的影响。(3)不同木质素含量的插穗生根性状差异显著,木质素含量为19.47%时插穗生根能力最强,其插穗生根率为60.39%,平均每株生根量为9.70个,根长为4.85cm;木质素含量为10.60%时插穗生根能力最差。(4)生根性状最佳时的木质素含量为19.47%,其对应的剪切强度范围为40～50kg。     

Studies on lactate dehydrogenase of Lactobacillus plantarum spp. involved in lactic acid biosynthesis using permeabilized cells

Lactate dehydrogenase (LDH, EC 1.1.1.27) catalyses the reduction of pyruvate to lactate in facultative anaerobes. Whole cells of Lactobacillus plantarum NCIM 2084 showed low levels of LDH activity but permeabilization of cells by treatment with organic solvents toluene, chloroform and diethyl ether increased the measurable LDH activities, ether treated cells showing the highest increase. The maximum intracellular activity was obtained upon treating the cells with ether (1%) at 28°C for 1 min. The LDH activity in permeabilized cells was nearly three-fold higher than that in the cell-free extract prepared by sonication. The kinetic properties of LDH in the permeabilized cells were comparable to that of cell-free extract, indicating that catalytically it functions similar to the isolated enzyme.     

Significantly enhanced production of recombinant nitrilase by optimization of culture conditions and glycerol feeding

The production of a recombinant nitrilase expressed in Escherichia coli JM109/pNLE was optimized in the present work. Various culture conditions and process parameters, including medium composition, inducer, induction condition, pH and temperature, were systematically examined. The results showed that nitrilase production in E. coli JM109/pNLE was greatly affected by the pH condition and the temperature in batch culture, and the highest nitrilase production was obtained when the fermentation was carried out at 37°C, initial pH 7.0 without control and E. coli was induced with 0.2 mM isopropyl-β-d-thiogalactoside at 4.0 h. Furthermore, enzyme production could be significantly enhanced by adopting the glycerol feeding strategy with lower flow rate. The enzyme expression was also authenticated by sodium dodecyl phosphate polyacrylamide gel electrophoresis analysis. Finally, under the optimized conditions for fed-batch culture, cell growth, specific activity and nitrilase production of the recombinant E. coli were increased by 9.0-, 5.5-, and 50-fold, respectively.     

Purification and characterization of a novel nitrilase of Rhodococcus rhodochrous K22 that acts on aliphatic nitriles.

A novel nitrilase that preferentially catalyzes the hydrolysis of aliphatic nitriles to the corresponding carboxylic acids and ammonia was found in the cells of a facultative crotononitrile-utilizing actinomycete isolated from soil. The strain was taxonomically studied and identified as Rhodococcus rhodochrous. The nitrilase was purified, with 9.08% overall recovery, through five steps from a cell extract of the stain. After the last step, the purified enzyme appeared to be homogeneous, as judged by polyacrylamide gel electrophoresis, analytical centrifugation, and double immunodiffusion in agarose. The relative molecular weight values for the native enzyme, estimated from the ultracentrifugal equilibrium and by high-performance liquid chromatography, were approximately 604,000 +/- 30,000 and 650,000, respectively, and the enzyme consisted of 15 to 16 subunits identical in molecular weight (41,000). The enzyme acted on aliphatic olefinic nitriles such as crotononitrile and acrylonitrile as the most suitable substrates. The apparent Km values for crotononitrile and acrylonitrile were 18.9 and 1.14 mM, respectively. The nitrilase also catalyzed the direct hydrolysis of saturated aliphatic nitriles, such as valeronitrile, 4-chlorobutyronitrile, and glutaronitrile, to the corresponding acids without the formation of amide intermediates. Hence, the R. rhodochrous K22 nitrilase is a new type distinct from all other nitrilases that act on aromatic and related nitriles.     

编码腈水解酶的大豆疫霉基因PsNIA的克隆与转录分析

包括大豆在内的许多植物都可以产生氰化物,对侵染的病原菌产生毒害作用而阻碍其进一步扩展。采用抑制性差减杂交(suppression subtractive hybridization,SSH)的方法,筛选到一个在大豆疫霉侵染早期上调表达的、编码腈水解酶的cDNA片段;克隆了该基因的全长序列,命名为PsNIA。Southern杂交结果显示,PsNIA在大豆疫霉基因组中只有1个拷贝。系统发育分析表明,PsNIA与绿脓杆菌Pseudomonas aeruginosa的腈水解酶的序列同源性最高,且该基因编码的氨基酸序列具有腈水解酶的保守结构域。RT-PCR分析表明,该基因在大豆疫霉侵染大豆12h时可以检测到转录。     

Cell-free metabolic engineering promotes high-level production of bioactive Gaussia princeps luciferase

Due to its small size and intense luminescent signal, Gaussia princeps luciferase (GLuc) is attractive as a potential imaging agent in both cell culture and small animal research models. However, recombinant GLuc production using in vivo techniques has only produced small quantities of active luciferase, likely due to five disulfide bonds being required for full activity. Cell-free biology provides the freedom to control both the catalyst and chemical compositions in biological reactions, and we capitalized on this to produce large amounts of highly active GLuc in cell-free reactions. Active yields were improved by mutating the cell extract source strain to reduce proteolysis, adjusting reaction conditions to enhance oxidative protein folding, further activating energy metabolism, and encouraging post-translational activation. This cell-free protein synthesis procedure produced 412 μg/mL of purified GLuc, relative to 5 μg/mL isolated for intracellular Escherichia coli expression. The cell-free product had a specific activity of 4.2×10²⁴ photons/s/mol, the highest reported activity for any characterized luciferase.     

Identification and characterization of a novel nitrilase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Pf-5

Nitrile groups are catabolized to the corresponding acid and ammonia through one-step reaction involving a nitrilase. Here, we report the use of bioinformatic and biochemical tools to identify and characterize the nitrilase (NitPf5) from Pseudomonas fluorescens Pf-5. The nitPf5 gene was identified via sequence analysis of the whole genome of P. fluorescens Pf-5 and subsequently cloned and overexpressed in Escherichia coli. DNA sequence analysis revealed an open-reading frame of 921 bp, capable of encoding a polypeptide of 307 amino acids residues with a calculated isoelectric point of pH 5.4. The enzyme had an optimal pH and temperature of 7.0°C and 45°C, respectively, with a specific activity of 1.7 and 1.9 μmol min⁻¹ mg protein⁻¹ for succinonitrile and fumaronitrile, respectively. The molecular weight of the nitrilase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography was 33,000 and 138,000 Da, respectively, suggesting that the enzyme is homotetrameric. Among various nitriles, dinitriles were the preferred substrate of NitPf5 with a K _m = 17.9 mM and k _cat/K _m = 0.5 mM⁻¹ s⁻¹ for succinonitrile. Homology modeling and docking studies of dinitrile and mononitrile substrate into the active site of NitPf5 shed light on the substrate specificity of NitPf5. Although nitrilases have been characterized from several other sources, P. fluorescens Pf-5 nitrilase NitPf5 is distinguished from other nitrilases by its high specific activity toward dinitriles, which make P. fluorescens NitPf5 useful for industrial applications, including enzymatic synthesis of various cyanocarboxylic acids.     

Cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus abyssi

Four open reading frames encoding putative nitrilases were identified in the genomes of the hyperthermophilic archaea Pyrococcus abyssi, Pyrococcus horikoshii, Pyrococcus furiosus, and Aeropyrum pernix (growth temperature 90-100 degrees C). The nitrilase encoding genes were cloned and overexpressed in Escherichia coli. Enzymatic activity could only be detected in the case of Py. abyssi. This recombinant nitrilase was purified by heat treatment of E. coli crude extract followed by anion-exchange chromatography with a yield of 88% and a specific activity of 0.14 U/mg. The recombinant enzyme, which represents the first archaeal nitrilase, is a dimer (29.8 kDa/subunit) with an isoelectric point of pI 5.3. The nitrilase is active at a broad temperature (60-90 degrees C) and neutral pH range (pH 6.0-8.0). The recombinant enzyme is highly thermostable with a half-life of 25 h at 70 degrees C, 9 h at 80 degrees C, and 6 h at 90 degrees C. Thermostability measurements by employing circular dichroism spectroscopy and differential scanning microcalorimetry, at neutral pH, have shown that the enzyme unfolds up to 90 degrees C reversibly and has a T(m) of 112.7 degrees C. An inhibition of the enzymatic activity was observed in the presence of acetone and metal ions such as Ag(2+) and Hg(2+). The nitrilase hydrolyzes preferentially aliphatic substrates and the best substrate is malononitrile with a K(m) value of 3.47 mM.     

编码腈水解酶的大豆疫霉基因PsNIA的克隆与转录分析

包括大豆在内的许多植物都可以产生氰化物,对侵染的病原菌产生毒害作用而阻碍其进一步扩展。采用抑制性差减杂交(suppression subtractive hybridization,SSH)的方法,筛选到一个在大豆疫霉侵染早期上调表达的、编码腈水解酶的cDNA片段;克隆了该基因的全长序列,命名为PsNIA。Southern杂交结果显示,PsNIA在大豆疫霉基因组中只有1个拷贝。系统发育分析表明,PsNIA与绿脓杆菌Pseudomonas aeruginosa的腈水解酶的序列同源性最高,且该基因编码的氨基酸序列具有腈水解酶的保守结构域。RT-PCR分析表明,该基因在大豆疫霉侵染大豆12h时可以检测到转录。