In the present study, the cellulose binding proteins（CBPs） secreted by a putative cellulolytic microbial consortium were isolated and purified by affinity digestion. The purified CBPs were subsequently separated by ...In the present study, the cellulose binding proteins（CBPs） secreted by a putative cellulolytic microbial consortium were isolated and purified by affinity digestion. The purified CBPs were subsequently separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis（SDS-PAGE）. Using mass spectrometric analyses, eight CBPs were identified and annotated to be similar to known proteins secreted by Clostridium clariflavum DSM 19732 and Paenibacillus sp. W-61. In addition, in combination with dilution-to-extinction approach and zymogram analysis technique, CBPs 6（97 k Da） and 12（52 k Da） were confirmed to be the key functional proteins that influence cellulolytic activities. Moreover, structural domain analyses and enzymatic activity detection indicated that CBPs 6 and 12 contained glycoside hydrolase families（GH） 9 and 48 catalytic modules, which both revealed endoglucandase and xylanase activities. It was suggested that the coexistence of GH9 and GH48 catalytic domains present in these two proteins could synergistically promote the efficient degradation of cellulose.展开更多
The global demand for large-scale and cost-effective production of high-quality protein has become increasingly urgent,with microbial protein derived from methanol being recognized as a promising solution.Among 50 met...The global demand for large-scale and cost-effective production of high-quality protein has become increasingly urgent,with microbial protein derived from methanol being recognized as a promising solution.Among 50 methylotrophic strains,Methylophilus sp.HN238 was selected for its capability to utilize methanol as the sole carbon.Through optimization of the medium composition,a 387.30%increase in protein yield was achieved during shake-flask fermentation.Subsequent scale-up to a 5 L bioreactor resulted in a protein content of 57.30%.Amino acid composition analysis revealed that 18 amino acids were quantitatively detectable in the protein,with essential amino acids accounting for 44.10%of the total composition,thereby demonstrating compliance with the World Health Organization(WHO)standards for high-quality protein.Tran-scriptomic differential analysis was conducted to investigate the metabolic response of Methylophilus sp.HN238 to methanol concentrations(10 g/L vs.50 g/L).It was observed that high methanol concentrations promoted the upregulation of methanol dehydrogenase(MDH)encoded by the maxI gene,while formaldehyde dehydrogenase(FLD)and cytochrome c(cyt c)were downregulated.This regulatory imbalance was associated with intracellular formaldehyde accumulation,impaired electron transport chain efficiency,oxidative stress,and subsequent inhibition of cellular growth.The study not only validated the potential of Methylophilus sp.HN238 for protein production with high methanol concentrations but also provided critical insights into metabolic engineering strategies to enhance methanol-to-biomass conversion efficiency.展开更多
The intensifying global warming may increase the impact of heat stress on the dairy industry. Our previous study showed that chromium yeast(CY) alleviated the negative effects of heat stress and improved the lactation...The intensifying global warming may increase the impact of heat stress on the dairy industry. Our previous study showed that chromium yeast(CY) alleviated the negative effects of heat stress and improved the lactation performance by increasing milk protein content and yield in mid-lactation dairy cows. This study further investigated whether the increased milk protein after CY supplementation results from the promotion of microbial crude protein(MCP) synthesis by regulating rumen microorganisms and amino acid metabolites. Twelve heat-stressed dairy cows were divided into two treatment groups: one with CY supplementation(0.36 mg Cr/kg DM) and the other without CY supplementation.Samples were collected after eight weeks of formal experiment in a hot summer with the mean temperature-humidity index of 79.0 ± 3.13. Dietary CY supplementation did not affect rumen pH, total volatile fatty acid, acetate, propionate, isobutyrate, butyrate, isovalerate, and valerate, but increased ruminal MCP concentration(P < 0.05). Simultaneously, the alpha or beta diversity of rumen microbial bacteria were not influenced by CY supplementation. At genus level, supplementation with CY increased the relative abundances of Olsenella, Lachnospiraceae_UCG-002, and Shuttleworthia(P < 0.05) and decreased those of Enterobacter, Escherichia-Shigella, Oribacterium, and Bacteroidetes_BD2-2(P < 0.05).There were 17 up-regulated and 57 down-regulated differential metabolites in the CON and CY groups.The partial least-squares discriminant analysis(PLS-DA) and orthogonal partial least-squares discriminant analysis(OPLS-DA) scores clearly distinguished the two groups. Chromium yeast supplementation reduced the concentrations of D-(+)-proline, DL-glutamic acid, DL-lysine, Gly-l-pro, L-(-)-serine, L-(+)-alanine, and L-(+)-aspartic acid(P < 0.05) in the ruminal fluid, which were involved in arginine biosynthesis(P = 0.029), glutathione metabolism(P = 0.047), lysine degradation(P = 0.069), and Damino acid metabolism(P = 0.084). Spearman correlation analysis showed that milk protein content was positively correlated with MCP and negatively correlated with amino acid concentrations in the ruminal fluid(P < 0.05). Collectively, CY supplementation promoted the utilization of amino acids by rumen microorganisms to synthesize MCP, thereby increasing milk protein content and yield in heat-stressed dairy cows.展开更多
This study investigated the effects of recycled poultry bedding(RPB) on performance and protozoa population, microbial enzyme activity and microbial protein synthesis(MPS) in rumen contents of fattening lambs. Thirty-...This study investigated the effects of recycled poultry bedding(RPB) on performance and protozoa population, microbial enzyme activity and microbial protein synthesis(MPS) in rumen contents of fattening lambs. Thirty-six male Moghani lambs(31.4 ± 3.2 kg body weight) were fed iso-energetic and iso-nitrogenous diets containing 0, 70,140 or 210 g/kg dry matter(DM) RPB in a balanced randomized design(9 lambs per treatment). Results showed that final body weight, DM intake, average daily gain and feed conversion ratio were unchanged(P > 0.05) by RPB inclusion. Total protozoa population and subfamily of Entoniniinae and Diplodiniiae were linearly decreased by RPB(L, P < 0.05). For rumen fibrolytic enzymes including carboxymethyl-cellulase, microcrystalline-cellulase and filter paper degrading activity, the extra cellular, cellular and total(extra cellular plus cellular fraction) activity were similar(P > 0.05) by feeding the experimental diets. Inclusion of RPB in the diet linearly decreased extra cellular and total a-amylase activity(L, P < 0.05), while cellular activity was unchanged(P > 0.05). The extra cellular activity of proteases tended to increase(L, P=0.07) and their total and cellular activity increased(P > 0.05) in lambs fed RPB. Incorporation of RPB into the diet had no effect(L, P > 0.05) on urinary purine derivative excretion and MPS. In conclusion, inclusion of RPB up to 210 g/kg DM had no negative impact on performance, ruminal fibrolytic enzyme activity and MPS, while it increased rumen protease activity and decreased protozoa population in fattening Moghani lambs.展开更多
基金supported by the National Natural Science Foundation of China (No. 31260024)the Science and Technology Support Program of Jiangxi Province (No. 20151BBF60029)+1 种基金the Jiangxi Province Science Foundation (Nos. 20122BAB214012, 20151BAB204001)Science and Technology Project Founded by the Education Department of Jiangxi Province (No. GJJ14297)
文摘In the present study, the cellulose binding proteins（CBPs） secreted by a putative cellulolytic microbial consortium were isolated and purified by affinity digestion. The purified CBPs were subsequently separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis（SDS-PAGE）. Using mass spectrometric analyses, eight CBPs were identified and annotated to be similar to known proteins secreted by Clostridium clariflavum DSM 19732 and Paenibacillus sp. W-61. In addition, in combination with dilution-to-extinction approach and zymogram analysis technique, CBPs 6（97 k Da） and 12（52 k Da） were confirmed to be the key functional proteins that influence cellulolytic activities. Moreover, structural domain analyses and enzymatic activity detection indicated that CBPs 6 and 12 contained glycoside hydrolase families（GH） 9 and 48 catalytic modules, which both revealed endoglucandase and xylanase activities. It was suggested that the coexistence of GH9 and GH48 catalytic domains present in these two proteins could synergistically promote the efficient degradation of cellulose.
基金Supported by the Strategic Priority Research Program of the Chinese Academy of Sciences,Grant No.XDC 0110300The Youth Project of Hebei Province Natural Science Foundation(C20211407039).
文摘The global demand for large-scale and cost-effective production of high-quality protein has become increasingly urgent,with microbial protein derived from methanol being recognized as a promising solution.Among 50 methylotrophic strains,Methylophilus sp.HN238 was selected for its capability to utilize methanol as the sole carbon.Through optimization of the medium composition,a 387.30%increase in protein yield was achieved during shake-flask fermentation.Subsequent scale-up to a 5 L bioreactor resulted in a protein content of 57.30%.Amino acid composition analysis revealed that 18 amino acids were quantitatively detectable in the protein,with essential amino acids accounting for 44.10%of the total composition,thereby demonstrating compliance with the World Health Organization(WHO)standards for high-quality protein.Tran-scriptomic differential analysis was conducted to investigate the metabolic response of Methylophilus sp.HN238 to methanol concentrations(10 g/L vs.50 g/L).It was observed that high methanol concentrations promoted the upregulation of methanol dehydrogenase(MDH)encoded by the maxI gene,while formaldehyde dehydrogenase(FLD)and cytochrome c(cyt c)were downregulated.This regulatory imbalance was associated with intracellular formaldehyde accumulation,impaired electron transport chain efficiency,oxidative stress,and subsequent inhibition of cellular growth.The study not only validated the potential of Methylophilus sp.HN238 for protein production with high methanol concentrations but also provided critical insights into metabolic engineering strategies to enhance methanol-to-biomass conversion efficiency.
基金supported by the National Key Research and Development Program of China(2022YFD1300505,2022YFD1301101)the earmarked fund for the China Agriculture Research System(CARS-37)the Agricultural Science and Technology Innovation Program(cxgc-ias-07,Beijing,China).
文摘The intensifying global warming may increase the impact of heat stress on the dairy industry. Our previous study showed that chromium yeast(CY) alleviated the negative effects of heat stress and improved the lactation performance by increasing milk protein content and yield in mid-lactation dairy cows. This study further investigated whether the increased milk protein after CY supplementation results from the promotion of microbial crude protein(MCP) synthesis by regulating rumen microorganisms and amino acid metabolites. Twelve heat-stressed dairy cows were divided into two treatment groups: one with CY supplementation(0.36 mg Cr/kg DM) and the other without CY supplementation.Samples were collected after eight weeks of formal experiment in a hot summer with the mean temperature-humidity index of 79.0 ± 3.13. Dietary CY supplementation did not affect rumen pH, total volatile fatty acid, acetate, propionate, isobutyrate, butyrate, isovalerate, and valerate, but increased ruminal MCP concentration(P < 0.05). Simultaneously, the alpha or beta diversity of rumen microbial bacteria were not influenced by CY supplementation. At genus level, supplementation with CY increased the relative abundances of Olsenella, Lachnospiraceae_UCG-002, and Shuttleworthia(P < 0.05) and decreased those of Enterobacter, Escherichia-Shigella, Oribacterium, and Bacteroidetes_BD2-2(P < 0.05).There were 17 up-regulated and 57 down-regulated differential metabolites in the CON and CY groups.The partial least-squares discriminant analysis(PLS-DA) and orthogonal partial least-squares discriminant analysis(OPLS-DA) scores clearly distinguished the two groups. Chromium yeast supplementation reduced the concentrations of D-(+)-proline, DL-glutamic acid, DL-lysine, Gly-l-pro, L-(-)-serine, L-(+)-alanine, and L-(+)-aspartic acid(P < 0.05) in the ruminal fluid, which were involved in arginine biosynthesis(P = 0.029), glutathione metabolism(P = 0.047), lysine degradation(P = 0.069), and Damino acid metabolism(P = 0.084). Spearman correlation analysis showed that milk protein content was positively correlated with MCP and negatively correlated with amino acid concentrations in the ruminal fluid(P < 0.05). Collectively, CY supplementation promoted the utilization of amino acids by rumen microorganisms to synthesize MCP, thereby increasing milk protein content and yield in heat-stressed dairy cows.
文摘This study investigated the effects of recycled poultry bedding(RPB) on performance and protozoa population, microbial enzyme activity and microbial protein synthesis(MPS) in rumen contents of fattening lambs. Thirty-six male Moghani lambs(31.4 ± 3.2 kg body weight) were fed iso-energetic and iso-nitrogenous diets containing 0, 70,140 or 210 g/kg dry matter(DM) RPB in a balanced randomized design(9 lambs per treatment). Results showed that final body weight, DM intake, average daily gain and feed conversion ratio were unchanged(P > 0.05) by RPB inclusion. Total protozoa population and subfamily of Entoniniinae and Diplodiniiae were linearly decreased by RPB(L, P < 0.05). For rumen fibrolytic enzymes including carboxymethyl-cellulase, microcrystalline-cellulase and filter paper degrading activity, the extra cellular, cellular and total(extra cellular plus cellular fraction) activity were similar(P > 0.05) by feeding the experimental diets. Inclusion of RPB in the diet linearly decreased extra cellular and total a-amylase activity(L, P < 0.05), while cellular activity was unchanged(P > 0.05). The extra cellular activity of proteases tended to increase(L, P=0.07) and their total and cellular activity increased(P > 0.05) in lambs fed RPB. Incorporation of RPB into the diet had no effect(L, P > 0.05) on urinary purine derivative excretion and MPS. In conclusion, inclusion of RPB up to 210 g/kg DM had no negative impact on performance, ruminal fibrolytic enzyme activity and MPS, while it increased rumen protease activity and decreased protozoa population in fattening Moghani lambs.
