Characterization of an Alkali- and Halide-Resistant Laccase Expressed in E. coli: CotA from <i>Bacillus clausii</i>

The limitations of fungal laccases at higher pH and salt concentrations have intensified the search for new extremophilic bacterial laccases. We report the cloning, expression, and characterization of the bacterial cotA from Bacillus clausii, a supposed alkalophilic ortholog of cotA from B. subtilis. Both laccases were expressed in E. coli strain BL21(DE3) and characterized fully in parallel for strict benchmarking. We report activity on ABTS, SGZ, DMP, caffeic acid, promazine, phenyl hydrazine, tannic acid, and bilirubin at variable pH. Whereas ABTS, promazine, and phenyl hydrazine activities vs. pH were similar, the activity of B. clausii cotA was shifted upwards by ~0.5-2 pH units for the simple phenolic substrates DMP, SGZ, and caffeic acid. This shift is not due to substrate affinity (K(M)) but to pH dependence of catalytic turnover: The k(cat) of B. clausii cotA was 1 s⁻¹ at pH 6 and 5 s⁻¹ at pH 8 in contrast to 6 s⁻¹ at pH 6 and 2 s⁻¹ at pH 8 for of B. subtilis cotA. Overall, k(cat)/K(M) was 10-fold higher for B. subtilis cotA at pH(opt). While both proteins were heat activated, activation increased with pH and was larger in cotA from B. clausii. NaCl inhibited activity at acidic pH, but not up to 500-700 mM NaCl in alkaline pH, a further advantage of the alkali regime in laccase applications. The B. clausii cotA had ~20 minutes half-life at 80°C, less than the ~50 minutes at 80°C for cotA from B. subtilis. While cotA from B. subtilis had optimal stability at pH~8, the cotA from B. clausii displayed higher combined salt- and alkali-resistance. This resistance is possibly caused by two substitutions (S427Q and V110E) that could repel anions to reduce anion-copper interactions at the expense of catalytic proficiency, a trade-off of potential relevance to laccase optimization

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Not AvailableFour month feeding trial was conducted to evaluate the effect of a vitamin-mineral based nu- traceutical conglomerate on Labeo rohita fingerlings (Hamilton, 1822) exposed to different stressors like crowding (normal stocking density NSD, 10 number of fish in 75 L water or high stocking density, HSD, 20 number of fish in 75 L water) and feed restriction (satiation feeding, SF or restricted feeding, RF, at 1% of body weight). One thousand and eighty (1080) fingerlings of Labeo rohita with an average size of 8.5 g ± 0.5 were randomly distributed in 6 distinct experi- mental groups, viz, THR0 (HSD, RF and 0% nutraceutical), THR1 (HSD; RF; 0.1 % nutraceutical), THR5 (HSD, RF, 0.5 % nutraceutical), TNS0 (NSD, SF and 0% nutraceutical), TNS1 (NSD; SF; 0.1 % nutraceutical) and TNS5 (NSD; SF; 0.5 % nutraceutical). Feeding of nutraceuticals improved the overall growth of the fish both in stress exposed and non-exposed groups. The hepatic anti- oxidant genes (superoxide dismutase-1, SOD-1; glutathione peroxidase-1, GPx-1) and enzymes (SOD and GPx) showed significantly higher values in stress exposed group (THR0) during the experimental trial in all the four months while catalase gene and enzyme showed a higher activity from 2nd month onwards. Reduced glutathione was found lower at the end of 3- and 4-months trial. Serum total antioxidant status (TAS) was significantly reduced due to stress at the end of each trial period, whereas feeding of 0.1 % nutraceutical increased the TAS in stress exposed groups. Hence, it can be concluded that feeding of 0.1 % nutraceutical conglomerate containing zinc, selenium, vitamin E and methionine could mitigate the crowding and feed restriction- mediated oxidative stress in Labeo rohita fingerlings at the end of 3- and 4-months period. The result of this study will be highly applicable for the production of stunted carp fingerlings for short duration crop in seasonal ponds or water bodies.Not Availabl

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Not AvailableOne thousand and eighty rohu fingerlings with an average size of 8.5 ± 0.5 g were randomly distributed in 6 distinct experimental groups in triplicates, where the first three groups were exposed to high stocking density (HSD; 20 nos/ 75 L water) with restricted feeding (RF), viz. THR0 (HSD, RF, 0% nutraceutical), THR1 (HSD, RF, 0.1% nutraceutical) and THR5 (HSD, RF, 0.5% nutraceutical), and second three groups were exposed to normal stocking density (NSD; 10 nos /75 L water) with satiation feeding (SF) and designated as TNS0 (NSD, SF, 0% nutraceutical), TNS1 (NSD, SF, 0.1% nutraceutical) and TNS5 (NSD, SF, 0.5% nutraceutical). The weight gain (WG) %, specific growth rate (SGR), protein efficiency ratio (PER) and IGF-1 expression were significantly lower, whereas feed conversion ratio (FCR), serum superoxide dismutase (SOD) and catalase activities were significantly higher in fish exposed to HSD with RF at monthly sampling. Feeding of 0.1% dietary nutraceutical resulted in a significantly higher WG%, SGR, PER and IGF-1 expression and lower FCR, SOD and catalase activ-ities at the end of the third and fourth month of feeding in HSD with RF groups. The present study concluded that 3-month feeding of 0.1% nutraceutical could improve growth and reduce stress in fish.Not Availabl

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Not AvailableA feeding trial was conducted for 4 months to study the effect of a nutraceutical conglomerate at different levels (0, 0.1, and 0.5%) on stress-exposed (high stocking density, H and feed restriction, R) Labeo rohita fingerlings. Six isonitrogenous diets with a crude protein of 35% were prepared for the different treatments, viz. THR0, THR1, THR5, TNS0, TNS1 and TNS5. Stress significantly reduced the weight gain and feeding of 0.1% nutraceutical improved it in both stress-exposed and non-exposed groups from 3 months onwards. Two-and three-month stress-exposed groups exhibited lower (p < 0.05) protease activity, while amylase activity was significantly higher in 2-month stress-exposed groups. Higher lipase activity was found in stress-exposed groups irrespective of the duration of stress. Higher serum glutathione peroxidase (GPx), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and hepatic and muscle lactate dehydrogenase (LDH) activities were observed in the THR0 groups; however, THR1 showed a lower activity of these enzymes. Increased malate dehydrogenase (MDH) and glucose-6-phosphate dehydrogenase (G6PDH) activity was recorded in the 1st and 2nd months stress-exposed groups. However, THR1 group exhibited a reduced MDH and G6PDH activity compared to THR0 group in 1st and 2nd months. Hence, it can be concluded that the duration of multiple stress exposure adversely affected the digestive and metabolic enzymes activities and feeding of 0.1% nutraceutical conglomerates could restore the activities of digestive and metabolic enzymes in rohu fingerlings.Not Availabl

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Not AvailableA 60-day feeding trial was conducted to evaluate the nutritional potential of fermented Jatropha kernel meal (FJKM) in the diets of rohu, Labeo rohita, fingerlings. Four iso-nitrogenous (336 g kg-1) and iso-energetic (20 MJ kg-1) diets containing 0, 100, 200, and 300 g kg-1 FJKM in replacement of 0, 33.3, 66.7, and 100% soybean meal protein, respectively, were prepared and designated as control, T1, T2, and T3, respectively. A total of 180 acclimatized fish (average weight 6.05 ± 0.04 g) were equally distributed into four experimental groups in triplicates, and were fed their respective diets to satiation twice daily at 10:00 h and 18:00 h. Higher percent weight gain (%WG); specific growth rate (SGR); protein efficiency ratio (PER); protease, aspartate amino transferase (AST), and alanine amino transferase (ALT) activities; and lower feed conversion ratio (FCR) were recorded in T2 and T3 groups compared to other groups. Whereas hepatosomatic index (HSI), intestinal somatic index (ISI), and amylase, lactate dehydrogenase (LDH), malate dehydrogenase (MDH), superoxide dismutase (SOD), and catalase activities among the various dietary groups did not vary significantly (P > 0.05). Whole body composition of fish analyzed at the end of the feeding trial exhibited significantly (P < 0.05) higher ether extract and lower crude protein in the control group compared to the FJKM-fed groups. These results indicated that rohu fingerlings can efficiently utilize FJKM without any detrimental effect on growth performance, nutrient utilization, and metabolic response.Not Availabl

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Not AvailableA 60-day feeding trial was conducted to evaluate the haemato-biochemical, innate immune response, antioxidant capacity and histopathological changes in Labeo rohita fingerlings fed rubber protein isolates (RPI). One hundred and eighty fingerlings (average weight 4.45 ± 0.01 g) were distributed into five experimental groups in triplicate and fed with isonitrogenous and isocaloric diets. Soybean protein isolate (SPI) served as the reference diet (Control), and the treatment diets were formulated as RPI25, RPI50, RPI75 and RPI100 replacing 25, 50, 75 and 100% of SPI protein, respectively. The growth performance indices like final body weight (9.54-10.27 g), net weight gain (5.09-5.84 g), metabolic growth rate (4.54-5.02) and feed efficiency ratio (0.60-0.65) among the various groups were not significantly different (P > 0.05). All the haematological parameters, except red blood cells, showed no significant differences compared with the control group (P > 0.05). The immuno-biochemical parameters like albumin, globulin, total immunoglobulin, respiratory burst and lysozyme activities among the various groups did not differ significantly (P > 0.05). The stress enzyme such as superoxide dismutase (SOD), catalase (CAT) and oxidative stress marker malondialdehyde (MDA) showed no significant difference (P > 0.05). Histopathological examination of the liver revealed no marked changes. In summary, the results showed that RPI was well utilised by the fish and its inclusion did not generate any oxidative-induced stress, thus, RPI may be suggested as a potential replacement for SPI in fish diets without any detrimental effects. Hence, protein isolation offers a unique opportunity for the utilisation of rubber seed meal.Not Availabl

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Not AvailableA 60‐day feeding trial was conducted to evaluate the effects of feeding Houttuynia cordata leaf meal and extract on the growth performance, nutrient utilization and expression of insulin‐like growth factor‐I (IGF‐I) in Labeo rohita fingerlings. Two hun‐dred seventy fingerlings were randomly distributed in six experimental groups in trip‐licates. Six isonitrogenous (350 g/kg CP) and isocaloric (17 MJ/kg DE) diets were prepared with graded levels of leaf meal (LM) and leaf extract (LE), namely C (control, without LE and LM), LE‐0.25 (2.5 g/kg LE), LE‐0.5 (5 g/kg LE), LE‐1.0 (10 g/kg LE), LM‐1.0 (10 g/kg LM) and LM‐2.0 (20 g/kg LM). Significantly (p < 0.05) higher IGF‐I expression, weight gain %, specific growth rate and lower feed conversion ratio were observed in LM‐1.0 followed by LE‐1.0 group compared to control group. Higher pro‐tease activity was observed in LE‐1.0 compared to other experimental groups, while no significant changes were found for amylase and lipase activities. Compared to control, muscle alanine aminotransferase and aspartate aminotransferase activities were significantly higher in treatment groups except for LM‐2.0. These results sug‐gest that either LE‐1.0 or LM‐1.0 can be supplemented in the diet to improve the growth of L. rohita. However, the supplementation of 10 g H. cordata leaf meal/kg feed can be the better option considering the cost of extraction.Not Availabl

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Not AvailableA study was conducted to evaluate the effect of chemical treatment and solid state fermentation on the anti-nutritional factors (ANFs) and proximate composition of defatted Jatropha kernel meal (DJKM). The DJKM samples were treated with alkaline methanol (90% methanol containing 0.2 M NaOH; CT1), alkaline ethanol (80% ethanol containing 0.1 M NaOH; CT-2), highly alkaline methanol (90% methanol and 3% NaOH; CT-3), modified ethanol (4-times extraction using 80% ethanol; CT-4) and modified methanol (4-times extraction using 90% methanol; CT-5), in combination with moist heat treatment in all cases. Another sample was treated by solid state fermentation (SSF) using Aspergillus niger fungus (FT). The untreated sample was considered as control (J0). In comparison to control and the chemical treated samples (CT-1, CT-2, CT-3, CT-4 and CT-5), SSF treated sample contained significantly (P0.05) among the control and chemically treated samples. Though the CP content of FT sample was significantly higher than the control, it was found comparable to all the chemically treated samples. The moisture and ether extract contents of control and treated samples exhibited a reverse trend. Significantly lower and higer GE values were observed in CT-2 and CT-3, respectively; however, it was not affected much due to other treatments. Results indicated that any of the chemical treatments could not result in significant (P>0.05) removal of phytic acid and tannin, whereas FT was able to significantly (P<0.05) remove 100% phytic acid and 65.79% tannin from DJKM. However, all the treatments were equally effective in removal (100%) of trypsin inhibitor from DJKM. It is concluded that among the different treatment methods used, solid state fermentation was found to be the most effective method for removal of ANFs from DJKM.Not Availabl

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Not AvailableBioenergy generated from plants is one of the best potential energy supplies. Recently, the non-edible Jatropha curcas has been considered one of the world’s most sustainable biofuel crops. Protein rich defatted Jatropha curcas kernel meal (DJKM) is toxic which contains phorbol esters (PEs, 1307 g g−1) as a major toxic component, therefore it was detoxified through physical, chemical and solid-state fermentation treatments. Water soaking and moist heat treatment (autoclaving) had no significant effect on the PEs removal compared to control. Exposure of DJKM to electron beam radiation (2.5, 5, 10, 15 and 20 kGy) had a significant effect on PEs degradation and most efficient was at 25 kGy dose. Solvent extraction with either 90% methanol or 80% ethanol in combination with moist heat treatment was the most efficient method compared to other concentrations of methanol or ethanol. Solvent extraction of kernel meal through 0.2 or 0.3 M NaOH in 90% methanol and 0.1, 0.2 or 0.3 M NaOH in 80% ethanol, followed by moist heat treatment led to the removal of PEs completely. The PEs content in DJKM through highly alkaline methanol plus moist heat treatment (AM3.0) and ferric chloride & hydrogen peroxide plus moist heat treatments significantly ( 0.05) decreased to 13.0 and 53.6 g g−1, respectively. Non-alkaline solvent extraction of DJKM using either 90% methanol (M90H) or 80% ethanol (E80H) in addition to heating while magnetic stirring reduced PEs content significantly ( 0.05) compared to control exhibiting 99.77 and 99.32% removal, respectively. The DJKM was free of PEs when it was treated via solid-state fermentation through Aspergillus niger fungus. Conclusively, chemical detoxification treatments are cumbersome, un-economical, and their incorporation in the diet may affect animal performance, whereas, the fermentation approach is cost economical, environmentally sustainable and offers greaterNot Availabl

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Not AvailableJatropha protein concentrate (JPC) prepared from jatropha seed cake is toxic due to the presence of phorbol ester and antinutritional factors like tannin, trypsin inhibitor, phytate and non-starch polysaccharides. JPC was detoxified by solid state fermentation (SSF) and a feeding trial of 45 days was conducted to study the response of feeding fermented JPC (FJPC) on growth, haemato-immunological and physiological responses in rohu fingerlings. Seven iso-nitrogenous diets such as control (without JPC or FJPC), J5 (5% JPC), J10 (10% JPC), J20 (20% JPC), FJ5 (5% FJPC), FJ10 (10% FJPC) and FJ20 (20% FJPC) were prepared and fed twice daily. The weight gain and specific growth rate values showed an overall, linear and quadratic trend with similar value recorded in the control and FJ fed groups. Feed efficiency also showed an overall significant effect with a higher feed efficiency value recorded in the control (60.50) group which was similar to FJ (54.21–58.37) fed groups, while JPC fed groups registered the lowest value. The haematological studies showed a significantly (p < 0.05) lower red blood cells (RBC) and heamoglobin in all JPC fed groups and 5% FJPC group compared to control and other FJPC groups. The 10% and 20% JPC fed groups showed the highest blood glucose level than any other groups. Serum total protein and albumin followed similar trend as that of RBC and haemoglobin. The highest globulin value were observed in FJ10 group which was significantly different (P < 0.05) to other groups and lowest value was recorded in J20 group. The superoxide dismutase (SOD) activity in liver was higher in J20, while in muscle, J10 and J20 registered the highest value compared to other groups (p < 0.05). Our study revealed that protein concentrate prepared from Jatropha cake cannot be fed directly to rohu without detoxification and solid state fermentation appears to be an ideal approach. Overall, FJPC can be utilized in the diet of rohu at 20% without any detrimental effect on heamato-immunological and physiological response.Not Availabl