Metabolism of hydrogen cyanide by higher plants

A survey has been made of the occurrence and distribution of three enzymes which metabolize cyanide in a variety of higher plants including both cyanogenic and non-cyanogenic species. The enzymes investigated were β-cyanoalanine synthase, rhodanese and formamide hydrolyase. β-Cyanoalanine synthase was found to be present in every higher plant tested whereas rhodanese was found to occur far less commonly in plants. Formamide hydrolyase activity was not detected in any of the higher plants tested.     

Biodegradation of cyanide by Trichoderma spp. and Fusarium spp.

The rate of degradation of cyanide by certain strains of the Trichoderma spp. was evaluated. For comparison two Fusarium spp., which had previously been demonstrated to degrade metallocyanides were also studied. Studies were carried out to assess the rate of degradation using cyanide as the sole source of carbon or in the presence of glucose. Biodegradation was observed in flask cultures using cyanide as the sole carbon source. Strong evidence of cyanide biodegradation and co-metabolism emerged from studies with flask cultures where glucose was provided as a co-substrate. The rate of degradation of 2000 ppm CN⁻ was enhanced almost three times in the presence of glucose. A concomitant increase in fungal biomass was also observed in all the strains over the experimental period. Growth yield calculations performed provided values that were comparable to those reported in literature for one-carbon substrates.     

Hydrolysis of xylan and fermentation of xylose to ethanol

The focus in the development of pulping processes has usually been exclusively on cellulose. However, hemicellulose could serve as a valuable source of hexose and pentose sugars. Consequently, it should not be destroyed in a process designed for very high cellulose fibre yields. Novel procedures developed for production of ethanol by the fermentation of pentoses as well as hexoses provide new possibilities of hemicellulose utilization.

Many fungi produce extracellular hemicellulases. In the present work the production of xylanase and β-xylosidase with strains of Aspergillus and Trichoderma was studied. The enzymes were used for the hydrolysis of xylan. Xylose was fermented to ethanol by the mold Fusarium oxysporum.     

Cytochrome P450(11β): Structure-function relationship of the enzyme and its involvement in blood pressure regulation

Cytochrome P450(11β) is deeply involved in the final steps of biosynthesis of mineralocorticoids. This paper deals with following issues about this enzyme. (1) The structure and function of the enzymes of various animal species are discussed. By making alignment of amino acid sequences of the enzymes, we identified peptide domains essential for the enzyme actions such as a putative steroid binding domain and a heme binding region. Estimates of molecular similarity among the P450(11β) family enzymes suggested that the enzymes having both 11β-hydroxylation activity and aldosterone (ALDO) synthetic activity of certain animals such as frog, cattle and pig are more similar to the ALDO synthases of the other animals, such as rat, mouse and human, than the 11β-hydroxylases of these animals. (2) The molecular nature of the P450(11β) family enzymes of genetically hypertensive rats as well as adrenal regeneration hypertension (ARH) rats is examined. (i) Mutation was found in the P450(11β) gene of Dahl's salt-resistant normotensive rat. Steroidogenic activity expressed by the mutated gene accounted well for abnormal plasma levels of steroid hormones in this rat. (ii) 11β-, 18- and 19-Hydroxylation activities of adrenal mitochondria prepared from spontaneously hypertensive rat (SHR), Wistar-Kyoto rat (WKY), and stroke-prone (SP)-SHR were not significantly different from each other. Levels of mRNA of ALDO synthase in adrenal glands of 50-week-old SHR was significantly lower than those of 10-week-old SHR, WKY and SHR-SP. (iii) No significant difference in 19-hydroxylation activity was found between adrenal mitochondria prepared from ARH rat and those from control rat. The level of message of ALDO synthase was lower in adrenal glands of ARH rat.     

Genome mining of cyanide-degrading nitrilases from filamentous fungi

A variety of fungal species are known to degrade cyanide through the action of cyanide hydratases, a specialized subset of nitrilases which hydrolyze cyanide to formamide. In this paper, we report on two previously unknown and uncharacterized cyanide hydratases from Neurospora crassa and Aspergillus nidulans. Recombinant forms of four cyanide hydratases from N. crassa, A. nidulans, Gibberella zeae, and Gloeocercospora sorghi were prepared after their genes were cloned with N-terminal hexahistidine purification tags, expressed in Escherichia coli, and purified using immobilized metal affinity chromatography. These enzymes were compared according to their relative specific activity, pH activity profiles, thermal stability, and ability to remediate cyanide contaminated waste water from silver and copper electroplating baths. Although all four were similar, the N. crassa cyanide hydratase (CHT) has the greatest thermal stability and widest pH range of >50% activity. N. crassa also demonstrated the highest rate of cyanide degradation in the presence of both heavy metals. The CHT of A. nidulans has the highest reaction rate of the four fungal nitrilases evaluated in this work. These data will help determine optimization procedures for the possible use of these enzymes in the bioremediation of cyanide-containing waste. Similar to known plant pathogenic fungi, both N. crassa and A. nidulans were induced to express CHT by growth in the presence of KCN.     

Degradation of cyanide by Trichoderma mutants constructed by restriction enzyme mediated integration (REMI)

REMI technique was used to construct mutants with improved cyanide-degradation ability from biocontrol fungus Trichoderma koningii strain T30. The plasmid pV2 transformation was confirmed by PCR and Southern blot analysis. Out of 21 transformants, 15 single-copied transformants (71.4%) were found. To compare enzyme activities of rhodanese and cyanide hydratase, T. atroviride T23, T. harzianum T21 and their transformants constructed by REMI previously were also included. Transformants TkB6 (0.173 micromols thiocyanate formed min(-1)mg protein(-1)) from T30 and TaK1 (0.174 micromols thiocyanate formed min(-1)mg protein(-1)) from T23 showed higher rhodanese activity than other transformants and their wild strains. TkA9 (5.53 micromols formamide formed h(-1)mg protein(-1)) from T30 and Th64 (5.35 micromols formamide formed h(-1)mg protein(-1)) from T21 had higher cyanide hydratase activity than other transformants and their wild strains.     

Biotransformation of nitriles to amides using soluble and immobilized nitrile hydratase from Rhodococcus erythropolis A4

A semi-purified nitrile hydratase from Rhodococcus erythropolis A4 was applied to biotransformations of 3-oxonitriles 1a–4a, 3-hydroxy-2-methylenenitriles 5a–7a, 4-hydroxy-2-methylenenitriles 8a–9a, 3-hydroxynitriles 10a–12a and 3-acyloxynitrile 13a into amides 1b–13b. Cross-linked enzyme aggregates (CLEAs) with nitrile hydratase and amidase activities (88% and 77% of the initial activities, respectively) were prepared from cell-free extract of this microorganism and used for nitrile hydration in presence of ammonium sulfate, which selectively inhibited amidase activity. The genes nha1 and nha2 coding for and β subunits of nitrile hydratase were cloned and sequenced.     

Comparative study of subunits of phenylalanyl-tRNA synthetase from Escherichia coli and Thermus thermophilus

FPLC separation of - and β-subunits of phenylalanyl-tRNA synthetases from E. coli MRE-600 and Thermus thermophilus HB8 has been carried out in the presence of urea. Native -subunits of both enzymes were primarily ₂-dimers and tended to aggregate. Most E. coli enzyme β-subunits were monomeric and only a small fraction was represented by β₂-dimers. All thermophilic β-subunits were β-dimers. It was shown that monomers and all forms of homologous subunits had no catalytic activity in tRNA^Phe aminoacylation. For the enzymes and their subunits, titration curves were obtained and isoelectric points were determined. The comparison of the relative surface charges indicated similarity of the surfaces of entire enzymes and the corresponding β-subunits. -Subunits displayed a distinctly different pH dependence of the surface charge. A spatial model of the oligomeric structure and a putative mechanism for its formation are discussed.     

Selection and preliminary characterization of β-glycosidases producer Patagonian wild yeasts

Four pre-selected indigenous yeast strains belonging to Candida guilliermondii (V₂ and V₅), Candida pulcherrima (V₆) and Kloeckera apiculata (V₉), were used as β-glucosidase (βGL) and β-xylosidase (βXL) sources. The optimization of yeast culture conditions was carried out and the effects of oenological parameters on β-glycosidase activities were evaluated. C. guilliermondii V₂ and C. pulcherrima V₆ strains were selected. These strains showed intracellular (C. pulcherrima V₆) and parietal (C. guilliermondii V₂) constitutive βGL and βXL. The enzymatic activities were active at pH, glucose, ethanol and SO₂ concentrations usually found in winemaking and they were able to release monoterpenols and alcohols from grape juice glycoside extracts. Additionally, these yeast strains were not able to produce volatile acidity and off flavour. Regional ecological relevance of these species was also discussed. Our results evidence that the selected C. guilliermondii V₂ and C. pulcherrima V₆ strains have interesting oenological characteristics and allow us to think in their potential application in winemaking.     

Regional and Subcellular Distribution of Cyanide Metabolizing Enzymes in the Central Nervous System

Activities of cyanide metabolizing enzymes were measured in various subcellular fractions and regions in the central nervous system. Brain rhodanese and liver beta-mercaptopyruvate sulfurtransferase showed a slight decrease in activity after death. The activity of beta-mercaptopyruvate sulfurtransferase was negligible in the rat brain, compared with that of rhodanese. A small amount of thiocyanate was produced from cyanide and beta-mercaptopyruvate in the human brain, probably due to contamination with red blood cells. Rhodanese activity was widely distributed in all the areas of nervous tissue examined. In the rat the olfactory bulb showed the highest rhodanese activity, and high activity was also observed served in the thalamus, septum, hippocampus, and dorsal part of the midbrain. Rhodanese activity was low in various parts of the cerebral cortex. The distribution pattern of rhodanese in post-mortem human brain was essentially similar to that in rat brain. The thalamus, amygdala, centrum semiovale, colliculus superior, and cerebellar cortex showed high rhodanese activity in the human brain. Rhodanese activity was detected in the spinal cord. Anterior horn showed the highest rhodanese activity in the cervical, thoracic, and lumbar cord. Most rhodanese activity in the rat brain was recovered in the mitochondrial fraction with the highest specific activity. Rhodanese activity was lower in spinal cords obtained from autopsied cases with amyotrophic lateral sclerosis than in those of control subjects. A significant decrease in rhodanese was observed in the posterior column of the cervical or thoracic cord, but the activity in the anterior horn did not differ significantly between the two groups.     

Preparation of both enantiomers of methyl 3-benzoyloxypentanoate by enzyme-catalysed hydrolysis of corresponding racemic nitrile and amide

Rhodococcus rhodochrous IFO 15564 enantioselectively hydrolysed racemic 3-benzoyloxypentanenitrile and 3-benzoyloxypentanamide to afford (R)-amide and (S)-car☐ylic acid with high enantiomeric excess (> 90%). In this reaction, both enantiomers of the starting nitrile were converted to the amide by nitrile hydratase, and amidase-catalysed enantioselective hydrolysis of the amide was responsible for the kinetic resolution. The lack of enantioselectivity of the nittile hydratase toward the racemic nitrile forms a marked contrast to the case of previously reported highly enantioselective conversion of prochiral 3-benzoyloxypentanedinitrile by this enzyme. since (R)-amide could be hydrolysed chemically to (R)-car☐ylic acid without any loss of its ee, the present microbial kinetic resolution serves as an effective method for preparing both enantiomers of synthetically useful 3-hydroxypentanoic acid derivatives.     

昆虫肠道木霉及其对芒果炭疽病菌拮抗作用

为获得优良生防木霉菌株,本研究以昆虫肠道为样本,从中分离鉴定木霉菌株,并以芒果炭疽病菌盘长孢状刺盘孢为靶标菌,通过对峙培养、挥发性物质和非挥发性物质筛选拮抗效果最优的木霉菌株,测定其孢子悬浮液对芒果炭疽病的室内防效研究.结果 显示,从105份昆虫肠道中共分离获得10株木霉,通过形态学特征和Tef1-Rpb2双基因联合建...     

Characterisation and application of glycanases secreted by Aspergillus terreus CCMI 498 and Trichoderma viride CCMI 84 for enzymatic deinking of mixed office wastepaper

Two enzymatic extracts obtained from xylan-grown Aspergillus terreus CCMI 498 and cellulose-grown Trichoderma viride CCMI 84 were characterised for different glycanase activities. Both strains produce extracellular endoxylanase and endoglucanase enzymes. The enzymes optimal activity was found in the temperature range of 45–60 °C. Endoglucanase systems show identical activity profiles towards temperature, regardless of the strain and inducing substrate. Conversely, the endoxylanases produced by both strains showed maximal activity at different pH values (from 4.5 to 5.5), being the more acidic xylanase produced by T. viride grown on cellulose. The endoglucanase activities have an optimum pH at 4.5–5.0. The endoxylanase and endoglucanase activities exhibited high stability at 50 °C and pH 5.0. Mannanase, β-xylosidase, and amylase activities were also found, being the first two activities only present for T. viride extract. These two enzymatic extracts were used for mixed office wastepaper (MOW) deinking. When the enzymatic extract from T. viride was used, a further increase of 24% in ink removal was obtained by comparison with the control. Both enzymes contributed to the improvement of the paper strength properties and the obtained results clearly indicate that the effective use of enzymes for deinking can also contribute to the pulp and paper properties improvement.     

Characteristics of chimeric enzymes constructed between Thermotoga maritima and Agrobacterium tumefaciens β-glucosidases: Role of C-terminal domain in catalytic activity

The importance of C-terminal domain of β-glucosidase (family 3 glycosidase) from Thermotoga maritima, a hyper-thermophilic bacterium was investigated by gene shuffling. The amino acid sequences of β-glucosidases from T. maritima and A. tumefaciens share high degree of homology (approximately 40%). However, despite such a high homology, both enzymes exhibited quite distinct characteristics in terms of their pH and temperature profile and substrate specificities. To investigate the functional role of the C-terminal domains of T. maritima and A. tumefaciens β-glucosidases, three chimeric genes were constructed by shuffling at three selected regions. Out of the three chimeric enzymes, only two (Tm533/626At and Tm630/727At) were catalytically active. Parental and the chimeric enzymes were subsequently characterized for the substrate specificities and their response towards pH and temperature. Our results revealed that C-terminal domain was catalytically important. The study clearly establishes the significance of gene shuffling in probing the structure and function relationship in hyper-thermophilic bacterium and evolving enzymes with altered features.     

12-hour phase-shift of mice kidney rhodanese (thiosulfate sulfurtransferase) activity in the first two months of life

This study aimed to investigate and compare variation of renal rhodanese activity at 2^nd, 4^th and 8^th weeks of post-natal development (PND) in mice. The enzyme activity increased with age and was higher in females compared to males in all studied groups. Cosinor analysis revealed significant circadian rhythms (with period τ = 24 h) of enzyme activity in both genders with peak time shift during the PND. At the 2^nd week of PND (pre-weaning time), the circadian rhythm peaked at the beginning of light span, more precisely ≅1 HALO (Hours After Light Onset). A week after weaning (4^th week of PND), the peak time was located at the second half of photophase (≅9 HALO) in both genders. Four to six weeks later, about the 8^th week of PND, the circadian peak time was then recorded at ≅13 HALO. These findings suggest that rhodanese level and rhythm stabilization were age-dependent. Moreover, gender-related differences may stimulate discussions on the relationship between renal rhodanese and cyanide sensitivity.     

Further studies on the NADH oxidase of the cytoplasmic membrane of Mycobacterium tuberculosis

The cytoplasmic membrane of the H₃₇Ra strain of Mycobacterium tuberculosis has been isolated free of cell wall.

These membrane preparations contain very small quantities of cytochromes c, b and cytochrome oxidase. The cytochrome c is not extracted by any method attempted. The cytochrome b is reducible only by dithionite and is believed not to be involved in the direct transfer of electrons during the oxidation of NADH by these preparations. The NADH oxidase activity of the membrane is inhibited by high concentrations of cyanide and also by 2-(n-heptyl)-4-hydroxyquinoline-N-oxide (HQNO). The cytochrome oxidase of the membrane contains both cytochromes a and a₃ and is present in low concentrations relative to cytochrome c. The cytochrome a₃ component was identified by characteristic complexes with both CO and cyanide and shows a γ-band absorption maximum at a slightly lower wavelength than the cytochrome oxidase of mammalian mitochondria (442 nm vs. 445 nm). The functional activity of the cytochrome oxidase is indicated by the inhibition of reoxidation of reduced cytochromes c and a in the presence of cyanide.     

The cyanide hydratase enzyme of Fusarium lateritium also has nitrilase activity

The filamentous fungus Fusarium lateritium produces cyanide hydratase when grown in the presence of cyanide. The cyanide hydratase protein produced at a high level in Escherichia coli shows a low but significant nitrilase activity with acetonitrile, propionitrile and benzonitrile. The nitrilase activity is sufficient for growth of the recombinant strain on acetonitrile, propionitrile or benzonitrile as the sole source of nitrogen. The recombinant enzyme shows highest nitrilase activity with benzonitrile. Site-directed mutagenesis of the F. lateritium cyanide hydratase gene indicates that mutations leading to a loss of cyanide hydratase activity also lead to a loss of nitrilase activity. This suggests that the active site for cyanide hydratase and nitrilase activity in the protein is the same. This is the first evidence of cyanide hydratase having nitrilase activity.