Degradation Of Mimosine Research Articles

Heterologous Expression and Characterization of Mimosinase from Leucaena leucocephala.

Heterologous expression of eukaryotic genes in bacterial system is an important method in synthetic biology to characterize proteins. It is a widely used method, which can be sometimes quite challenging, as a number of factors that act along the path of expression of a transgene to mRNA, and mRNA to protein, can potentially affect the expression of a transgene in a heterologous system. Here, we describe a method for successful cloning and expression of mimosinase-encoding gene from Leucaena leucocephala (leucaena) in E. coli as the heterologous host. Mimosinase is an important enzyme especially in the context of metabolic engineering of plant secondary metabolite as it catalyzes the degradation of mimosine, which is a toxic secondary metabolite found in all Leucaena and Mimosa species. We also describe the methods used for characterization of the recombinant mimosinase.

Degrading Mimosine and Tannins of <i>Leucaena l</i><i>e</i><i>ucocephala</i> by Ensiling

Lucaena (Leucaena leucocephala) is a valuable fodder tree, but it contains high contents of anti-nutritional factors, such as mimosine and tannins. The present study investigated the silage fermentation characteristics and ensiling effects on the degradation of mimosine and tannins of leucaena. Mimosine and tannins gradually reduced during ensiling, and sucrose and lactic acid bacteria (LAB) addition promoted their degradation. The highest degradation rate of mimosine was 49.0% in sucrose treatment and the lowest was 25.5% in the control. The highest degradation rate of tannins was 54.7% in the combined treatment of sucrose and LAB and the lowest was 40% in the control. The degradation rates of mimosine and tannins had negative correlation with the pH value and had positive correlation with lactic acid content in silage.

MidD-encoded ‘rhizomimosinase’ from Rhizobium sp. strain TAL1145 is a C–N lyase that catabolizes L-mimosine into 3-hydroxy-4-pyridone, pyruvate and ammonia

Rhizobium sp. strain TAL1145 catabolizes mimosine, which is a toxic non-protein amino acid present in Leucaena leucocephala (leucaena). The objective of this investigation was to study the biochemical and catalytic properties of the enzyme encoded by midD, one of the TAL1145 genes involved in mimosine degradation. The midD-encoded enzyme, MidD, was expressed in Escherichia coli, purified and used for biochemical and catalytic studies using mimosine as the substrate. The reaction products in the enzyme assay were analyzed by HPLC and mass spectrometry. MidD has a molecular mass of ~45 kDa and its catalytic activity was found to be optimal at 37 °C and pH 8.5. The major product formed in the reaction had the same retention time as that of synthetic 3-hydroxy-4-pyridone (3H4P). It was confirmed to be 3H4P by MS/MS analysis of the HPLC-purified product. The K m, V max and K cat of MidD were 1.27 × 10(-4) mol, 4.96 × 10(-5) mol s(-1) mg(-1), and 2,256.05 s(-1), respectively. Although MidD has sequence similarities with aminotransferases, it is not an aminotransferase because it does not require a keto acid as the co-substrate in the degradation reaction. It is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and the addition of 50 μM hydroxylamine completely inhibited the reaction. However, the supplementation of the reaction with 0.1 μM PLP restored the catalytic activity of MidD in the reaction containing 50 μM hydroxylamine. The catalytic activity of MidD was found to be specific to mimosine, and the presence of its structural analogs including L-tyrosine, L-tryptophan and L-phenylalanine did not show any competitive inhibition. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products in equimolar quantities of the substrate used. The degradation of mimosine into a ring compound, 3H4P with the release of ammonia indicates that MidD of Rhizobium sp. strain TAL1145 is a C-N lyase.

Within—country variation in the ability of ruminants to degrade DHP following the ingestion of Leucaena leucocephala—a Thailand experience

Goats fed Leucaena leucocephala (leucaena) at an experimental site in Thailand were shown to be excreting DHP in their urine. This was unexpected as earlier results from another site had shown that goats and cattle fed leucaena did not excrete DHP and so possessed DHP-degrading bacteria. Goats sampled near the earlier sample site excreted no DHP in their urine. Rumen fluid taken from these goats was successfully used to transfer DHP--degrading ability to the goats at the Experimental site some 350 km away that did not show the presence of DHP-degrading bacteria. Degradation of mimosine in-vitro and excretion of DHP in the urine ceased 72 hr after addition of rumen fluid and infusion with rumen fluid from protected goats, respectively. The situation in Thailand may not be unique. Countries where leucaena is fed should check that animals are protected. Fortunately, the ferric chloride urine test is simple to use and effective in detecting the problem and also the recovery after transfer of rumen fluid from protected animals.

Expression of Rhizobium pydA-pydB fusion gene in Nicotiana tabacum confers resistance to the toxic aromatic compound 3-hydroxy-4-pyridone

The toxic aromatic compound 3-hydroxy-4-pyridone (HP) is an intermediate in both synthesis and degradation of mimosine, which is produced by the tree legume Leucaena leucocephala. The L. leucocephala root-nodule symbiont Rhizobium TAL1145 contains a dioxygenase (pydA) and a hydrolase (pydB) gene that produce enzymes necessary for the degradation of HP. In order to coordinately express both genes in plant tissues under a single promoter, three different pydA-pydB fusion constructs (G0, G3, and G7) with varying glycine linkers between the two genes were developed. Prior to transferring the fusion constructs into L. leucocephala, which is highly recalcitrant to genetic transformation, we tested the expression and activity of the hybrid proteins in Nicotiana tabacum, a model plant system that can be easily transformed and analyzed. Seven independent transgenic tobacco lines were generated by Agrobacterium-mediated transformation, and stable integration and expression of pydA-pydB in these transgenic lines were confirmed by polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR) and Western analysis. Only one of the fusion constructs, G3, containing a 9-nucleotide linker between pydA and pydB, provided significant levels of resistance to 3 mM HP, indicating that the hybrid protein produced by this fusion construct could degrade HP.

The pydA–pydB fusion gene produces an active dioxygenase–hydrolase that degrades 3-hydroxy-4-pyridone, an intermediate of mimosine metabolism

The objective of this research was to construct a pydA-pydB hybrid gene that encodes a functional dioxygenase-hydrolase (PydA-PydB) fusion protein for degradation of 3-hydroxy-4-pyridone (HP). HP is an intermediate in both synthesis and degradation of mimosine, a toxic amino acid produced by the tree legume Leucaena leucocephala. Computer-generated models of the fusion proteins suggested that joining of PydA and PydB with 0, 3, or 7 glycine residues as a linker should produce a functional PydA-PydB fusion protein. Accordingly, three hybrid genes, G0, G3, and G7, were constructed in which pydA and pydB were connected with 0, 9, and 21 nucleotides, respectively, encoding the glycine residues of the linker region. When these hybrid genes were expressed in Rhizobium and Escherichia coli, only one of them, G3, produced a functional PydA-PydB fusion protein, having both the dioxygenase and hydrolase activities. The G3 hybrid gene could complement both pydA and pydB mutants of Rhizobium, and E. coli lysate containing the overexpressed G3 protein was able to degrade HP. This hybrid gene may be useful for developing mimosine-free L. leucocephala plants in the future.

Induction, isolation and purification of mimosine degradation enzyme from newly isolated Pseudomonas putida STM 905

A bacterial isolate STM 905, having high mimosine degradation ability, was isolated from the rhizosphere of Leucaena leucocephala. The cells of this strain were rod shaped, Gram negative, motile, could grow in pH range of 5.0–11.0 and temperature range 15–42 °C. On 16S rDNA sequence analysis based identification the strain STM 905 was found to be closest to Pseudomonas putida strain BH. Growth of this bacterium in the presence of mimosine, as the sole source of carbon and nitrogen, was correlated with the induction of an enzyme capable of mimosine degradation. This enzyme catalyses the degradation of mimosine to their corresponding 3-hydroxy-4(1 H)-pyridone (DHP), pyruvic acid and ammonia. The maximum induction of mimosine degradation enzyme activity has been observed at 15-h growth of culture. The presence of glucose in the culture medium inhibited the mimosine degradation enzyme induction by mimosine, while the presence of NH 4Cl in the growth medium had no significant effect of inhibition on mimosine degradation enzyme induction. The mimosine degradation enzyme was purified with 47% recovery in three steps, ammonium sulphate fractionation, ion exchange and size exclusion-chromatography, with overall 34.59-fold purification. The purified native form had an estimated molecular mass of 280 kDa and was found to composed of four subunits each of molecular mass ∼70 kDa as determined by SDS-PAGE. Km for mimosine was found to be 1.3 mM. The purified enzyme showed sharp pH optima at 7.2.

Effect of Transinoculation of Goat Rumen Liquor on Degradation and Metabolism of Mimosine in Sheep Fed with Leucaena leucocephala Leaves

The effect of transinoculation of goat rumen liquor into sheep rumen on mimosine toxicity was studied. One adult Kutchi male goat having higher mimosine degradation capacity than sheep was gradually adapted to Leucaena leucocephala (Leucaena) leaves by feeding increasing level of eucaena leaves supplementation for 1 month. Six Bharat Merino rams (12-18 months of age) were divided into two equal groups with (group I) or without (group II) infusion of 200 ml of goat rumen liquor per animal. The mimosine degradation in groups I and II were 3.04 and 2.31; 3.90 and 3.73 mg per day per 10 ml rumen liquor respectively after 1 and 2 weeks of leucaena feeding leaves. Total rumen bacterial population in RGCA medium and in a selective medium containing iron showed an increasing trend in both groups, while the bacterial population growing in the presence of cellulose showed a decreasing trend. Animal performance data did not show any adverse effect. Results revealed that transinoculation of rumen liquor from leucaena leaves adapted goat to sheep rumen did not help to improve mimosine degradation in the sheep. The sheep transinoculated with goat rumen liquor displayed no in vivo improvements in nutrient utilization vis-a-vis mimosine metabolism.

The mid genes of Rhizobium sp. strain TAL1145 are required for degradation of mimosine into 3-hydroxy-4-pyridone and are inducible by mimosine

Mimosine is a toxin present in the tree-legume leucaena (Leucaena leucocephala), including its root nodules and the root exudates. The leucaena-nodulating Rhizobium sp. strain TAL1145 degrades mimosine (Mid(+)) and utilizes it as a source of carbon and nitrogen. Twelve TAL1145 mutants defective in mimosine degradation (Mid(-)) were made through Tn3Hogus, TnphoA or kanamycin-resistance-cassette insertions. A 5.0 kb PstI fragment of TAL1145, subcloned from a cosmid clone containing mid genes for mimosine degradation, complemented most of the Mid(-) mutants. Sequencing this fragment and the adjacent 0.9 kb PstI fragment identified five genes, midA, midB, midC, midD and midR, of which the first three genes encode ABC transporter proteins involved in mimosine uptake, while midD encodes an aminotransferase required for degrading mimosine into 3-hydroxy-4-pyridone, and midR is a regulatory gene encoding a LysR-type transcriptional activator. The location of MidA in the periplasm was shown by making two midA : : phoA fusions, which made active alkaline phosphatase in the periplasm. The various mid : : gus and midA : : phoA fusions were inducible by mimosine, and a midD : : gus fusion mutant showed beta-glucuronidase activity in the leucaena nodules, indicating that midD is expressed in the nodules. Similarly, a midA : : phoA fusion expressed alkaline phosphatase activity in the leucaena nodules, indicating that mimosine induces midA transcription in the bacteroids. mid genes are specific for the Mid(+) strains of leucaena Rhizobium and are absent in strains of other Rhizobium, Sinorhizobium and Bradyrhizobium spp.

Influence of gradual adaptation of cattle to Leucaena leucocephala leaf meal on biodegradation of mimosine and 3- hydroxy-4(1H)-pyridone (3,4 DHP) in rumen, their levels in blood, fate and influence of absorbed DHP on thyroid hormones and liver enzymes

Three rumen fistulated Karan Fries crossbred (Holstein X Tharparkar) calves were fed increasing dry matter (DM) levels of 25%, 50%, 75% and 100% through leucaena leaf meal (LLM) starting at week 1, 2, 3 and 4, respectively. The mimosine, 3,4 DHP and 2,3 DHP levels were determined in strained rumen liquor (SRL) and serum at 0, 1, 2, 4, 8, 12 and 24 h postfeeding on days 1, 8, 15, 22, 29 and 42. LLM was incubated for 24 h with SRL in vitro on days 0, 7, 14, 21, 28 and 41 to study mimosine and dihydroxypyridone (DHP) biodegradation. On day 43, 1–1.5 l of rumen liquor was transferred to another set of three unadapted calves which were fed 50% LLM after transfer of inoculum. DM intake was 1.78%, 2.13%, 2.27%, 1.66%, 1.54% and 1.35% of live weight during the 1st through 5th week, respectively. Both in vitro and in vivo studies showed extensive degradation of mimosine to 3,4 DHP and 2,3 DHP from first day of LLM feeding. The overall in vitro DHP degradation was nil, 28.6%, 43.3% and 40.1% on day upto 15, 21, 28 and 42 of LLM feeding. No mimosine was found in serum on any day of sampling. The 3,4 DHP detected (56.94±31.65 μg ml −1 serum) one hour post feeding on day 1 exhibited a decline from day 22 onwards. The serum also contained 2,3 DHP on days 8, 15, 22, 42. The faecal and urinary excretion of 3,4 DHP and 2,3 DHP as percent of mimosine intake declined from first week (76.3±2.8) to 4th week (42.1±4.1). The feeding of LLM resulted in reduced level of T 3 and T 4 within a week of LLM feeding. The level of T 3 improved to normal by 6th week while that of T 4 remained low. The SGOT and SGPT activities were within normal range. The gradual adaptation to LLM feeding caused Karan Fries calves to acquire DHP degrading ability to nontoxic compounds and this ability was transferred through transfer of rumen liquor from such calves to other unadapted calves at as early as 9th day of LLM feeding. The results revealed the possibility of two types of microbes degrading mimosine and 3,4 DHP to 2,3 DHP. One type of 2,3 DHP degrading microbes may be inhibited in the presence of 3,4 DHP whereas the other type may be active.

Influence of ferric chloride treated Leucaena leucocephala on metabolism of mimosine and 3-hydroxy 4(1H)-pyridone in growing rabbits

Twenty-five 42-days old New Zealand white rabbits were weaned and accustomed to a control ration in the 1st week and randomly allotted to five groups of five rabbits each. They were offered the control ration (G-1), and in other groups a portion of the control ration was replaced by Leucaena leaf meal (LLM) treated with 1.2% FeCl 3 or untreated i.e. 25% LLM (G-2), 50% LLM (G-3), 25% treated LLM (G-4), and 50% treated LLM (G-5) ration in pelleted form in a 8 weeks feeding cum metabolism trial. Average intake of mimosine and 3,4 DHP (dihydroxypyridone) was 304.6 and 129.5; 680.2 and 212.3; 279.6 and 147.6; and 643.1 and 239.9 mg day −1 in G-2–G-5, respectively. Mimosine and 2,3 DHP were not detected in faeces. The faecal excretion of 3,4 DHP (as % intake of mimosine plus 3,4 DHP) in the rabbits of groups G-4 (43.5) and G-5 (40.6) was significantly ( P<0.05) higher due to FeCl 3 treatment as compared to excretion in groups G-2 (30.1) and G-3 (21.4) fed untreated LLM. GOT (Glutamic oxalacetic transaminase), GPT (Glutamic-pyruvic transaminase), T 3 (tri-iodothyronine) and T 4 (thyroxine) levels in blood were within normal physiological range. Mimosine 3,4 DHP and 2,3 DHP, all were excreted through urine. The urinary excretion of 3,4 DHP was significantly lower ( P<0.05) in G-4 and G-5. The overall excretion of DHP (2,3 and 3,4 DHP) was similar in all the groups. Severe hepatic and kidney damage occured in G-2 and G-3, while, in G-4 and G-5 very mild or no damage to liver and kidney was recorded. All tissues were devoid of mimosine, but DHP was present in liver, kidney and lungs. The maximum DHP in liver indicated as the primary site of DHP metabolism. In vitro incubation of LLM with caecal contents revealed 72.68–100% microbial degradation of mimosine. The overall DHP degradation ranged from 7.10% to 37.81% being the highest in G-3. The results indicated that, FeCl 3 treated leucaena could be used in commercial meat rabbit rations.

Mimosine degradation in calves fed a sole diet of Leucaena leucocephala in India.

Five Karan Swiss crossbred (Sahiwal x Brown Swiss) calves were abruptly switched over from a diet of concentrate and maize fodder to ad libitum air dried Leucaena leucocephala leaves plus twigs. After 17 days on the L. leucocephala diet, 3 of the calves were supplemented with copper sulphate (10 mg/kg DM L. leucocephala) for 12 days. Thereafter all the calves were taken off the L. leucocephala diet and returned to the pre-experimental concentrate and maize fodder diet. While on the L. leucocephala diet, the average DM intake/d of L. leucocephala declined to 497 g within 3 weeks and all calves lost weight. This weight loss was reversed in the 3 calves that received copper sulphate, and all calves gained weight when they resumed the concentrate and maize fodder diet. The toxic effects of L. leucocephala feeding for 24 days were characterised by poor growth, emaciation, alopecia, loss of hair from the tail switch, ear and eye lesions, ulceration of the mouth region, drooling viscid saliva and vomiting of thick green saliva in one of the calves. Mean levels of 3,4 dihydroxypyridone (DHP) (mg/100 ml) were 30.35 +/- 13.52 and 55.57 +/- 13.77 on days 2 and 4 respectively in rumen liquor and up to 136.01 +/- 80.18 in urine. The mean ratios of mimosine: DHP of 3.14, 0.12 and 0.04 in feed, faeces and urine respectively revealed extensive degradation of mimosine to DHP in the calves fed the L. leucocephala diet and it was concluded the calves were unable to tolerate a diet consisting solely of L. leucocephala.

Degradation of mimosine, 2,3-dihydroxy pyridine and 3-hydroxy-4(1H)-pyridine by bacteria from the rumen of sheep in Venezuela

The addition of Leucaena leucocephala herbage did not diet of sheep in Venezuela did not affect the concentration of volatile fatty acids (VFA) in the rumen, the degradation of rice straw incubated in sacco, or the numbers of rumen fungi or bacteria. However, feeding Leucaena increased the concentration of ammonia in the rumen. In addition, two products of the degradation of the toxic amino acid mimosine were detected in the rumen when Leucaena was fed. One of these products, 2,3-dihydroxy pyridine (2,3-DHP), was detected at concentrations of up to 1.1 μmol/ml. The other, 3-hydroxy-4(1H)-pyridone (3,4-DHP) was found at concentrations of up to 0.96 μmol/ml. The examination of bacterial cultures isolated from the rumen of the sheep under investigation showed that feeding Leucaena increased the relative proportions of short Gram-negative rods and decreased the proportion of long roads and coccobacilli present. Although the animals fed Leucaena showed a small loss in weight during the feeding trial, no evidence of Leucaena toxicity was seen. A total of 18 cultures capable of degrading 2,3-DHP or 3,4-DHP were isolated from the rumen of the sheep before Leucaena was fed. These included both Gram-positive and Gram-negative bacteria, and a Gram-positive sporeformer. It seems that 2,3-DHP and 3,4-DHP may be degraded by a much wider range of bacteria than has been recognised previously. The detection of these bacteria before Leucaena was fed suggests that they were indigenous members of the rumen microflora of sheep in Venezuela.

Degradation of mimosine, 2,3-dihydroxy pyridine and 3-hydroxy-4(1H)-pyridine by bacteria from the rumen of sheep in Venezuela

The addition of Leucaena leucocephala herbage did not diet of sheep in Venezuela did not affect the concentration of volatile fatty acids (VFA) in the rumen, the degradation of rice straw incubated in sacco, or the numbers of rumen fungi or bacteria. However, feeding Leucaena increased the concentration of ammonia in the rumen. In addition, two products of the degradation of the toxic amino acid mimosine were detected in the rumen when Leucaena was fed. One of these products, 2,3-dihydroxy pyridine (2,3-DHP), was detected at concentrations of up to 1.1 μmol/ml. The other, 3-hydroxy-4(1H)-pyridone (3,4-DHP) was found at concentrations of up to 0.96 μmol/ml. The examination of bacterial cultures isolated from the rumen of the sheep under investigation showed that feeding Leucaena increased the relative proportions of short Gram-negative rods and decreased the proportion of long roads and coccobacilli present. Although the animals fed Leucaena showed a small loss in weight during the feeding trial, no evidence of Leucaena toxicity was seen. A total of 18 cultures capable of degrading 2,3-DHP or 3,4-DHP were isolated from the rumen of the sheep before Leucaena was fed. These included both Gram-positive and Gram-negative bacteria, and a Gram-positive sporeformer. It seems that 2,3-DHP and 3,4-DHP may be degraded by a much wider range of bacteria than has been recognised previously. The detection of these bacteria before Leucaena was fed suggests that they were indigenous members of the rumen microflora of sheep in Venezuela.

Effect of post‐harvest treatment on the degradation of mimosine in Leucaena leucocephala leaves

AbstractThe effects of sun drying, soaking at various temperatures and submergence times, and tissue disruption on mimosine removal in Leucaena leucocephala leaves were studied. The rate of mimosine elimination increased with an increase in both temperature and exposure time, but maceration of leaves did not significantly increase the rate of mimosine conversion. Prolonged soaking of leaves was most effective, and virtually all the mimosine was degraded after 48 hours' submergence in water at 30°C.

Isolation of a mimosine degrading enzyme from leucaena leaf

AbstractAn enzyme which catalyses the degradation of mimosine to 3‐hydroxy‐4(1H)‐pyridone(DHP) has been isolated from Leucaena leucocephala leaf by extraction with ammonium sulphate, purification on Sephadex G‐25 and fractionation on Sepharose 6B and gel electrophoresis. The enzyme was found to have a molecular weight of 140000, a Km value of 1.64 mM ml−1 and maximum reaction velocity of 0.3 mmol DHP produced ml−1 min−1. Tyrosine was a competitive inhibitor of the enzyme but DHP did not act as a feedback inhibitor. The enzyme has been termed ‘mimosinase'’.

Degradation of mimosine during ensiling of leucaena

AbstractThe causes of the degradation of the toxic component, mimosine, during ensiling of leucaena leaves were investigated in small‐scale laboratory preparations. Degradation of mimosine to 3‐hydroxy‐4(1H)‐pyridone was shown to be caused almost entirely by endogenous enzymes in the leaf and not by fermentation during ensiling.

Optimisation of conditions for the degradation of mimosine in Leucaena leucocephala leaf

AbstractA study was made of the rate of degradation of mimosine to 3‐hydroxy‐4(1H)‐pyridone in harvested leucaena leaf under various conditions. Optimum rates of degradation of an aqueous leucaena slurry were at pH 8.0 and 45°C with virtual total loss of mimosine in 10 min. Heating the intact leaf also reduced the mimosine content with the maximum rate of degradation at 70°C. The results have implications for reducing the toxicity of leucaena used for stock feed.

Degradation of mimosine by rumen contents: effects of feed composition and Leucaena substrates

Samples of rumen fluid obtained from sheep that had been fed on different diets were fractionated into microorganism and supernatant fractions, and the former divided into bacteria-rich and protozoa-rich fractions. The fractions were evaluated for their ability to degrade purified mimosine during in vitro incubation. The rumen contents of sheep fed on a lucerne-oats mixture produced a more rapid degradation of mimosine than did that from sheep fed on lucerne hay, which was greater than that from a Digitaria pentrii diet. Most activity was in the bacteria-rich fraction for the lucerne-oats diet and in the protozoa-rich fraction for the other diets. The rate of degradation of endogenous mimosine in Leucaena leaf during incubation in ruinen fluid was much greater than for the purified mimosine. The substantial degradation observed when a buffer solution was substituted for rumen fluid was attributed to endogenous leaf enzymes. These enzyme systems were more efficient at degrading mimosine than were the microorganisms in the rumen liquor.

Effect of pH and temperature on the degradation of mimosine and 3-hydroxy-4(1 H)-pyridone

Mimosine and 3-hydroxy-4(1 H)-pyridone were extensively degraded in solutions at pH > 9.5 when held at temperatures of 80–120° for 1–2 hr. Mimosine was also degraded at pH 1.