Natural nano-hydroxyapatite(HA)was extracted from Pacific cod(Gadus macrocephalus)bone with a thermostable col-lagenolytic protease in the present study.Conditions for the enzymatic reaction were optimized to be 60℃a...Natural nano-hydroxyapatite(HA)was extracted from Pacific cod(Gadus macrocephalus)bone with a thermostable col-lagenolytic protease in the present study.Conditions for the enzymatic reaction were optimized to be 60℃and pH 7.0,and a desir-able extraction efficiency was achieved by using the crude collagenolytic protease.Dynamic light scattering,transmission electron microscopy and energy-dispersive X-ray analysis revealed that nano-HA are anionic spherical(about 110nm)particles mainly com-prised of calcium and phosphorus at an approximate ratio of 5:3.As evaluated with the mouse ex vivo intestinal segments,the extracted nano-HA displayed comparable level of intestinal bioavailability to the positive control CaCl_(2).By treating with inhibitors(NaN3,ami-loride)and low temperature(4℃),clathrin-mediated endocytosis was assumed to involve the intestinal absorption of nano-HA.Over-all,the application of thermostable collagenolytic protease is proved to be a promising alternative method for nano-HA extraction from natural resource with improved ecological and biological value.展开更多
A xylanase-producing bacterium, isolated from deep sea sediments, was identified as the cold-adapted marine species Acinetobacter Johnsonii. A cold-adapted marine species Acinetobacter Johnsonii could grow at 4 ℃. Th...A xylanase-producing bacterium, isolated from deep sea sediments, was identified as the cold-adapted marine species Acinetobacter Johnsonii. A cold-adapted marine species Acinetobacter Johnsonii could grow at 4 ℃. The optimum temperature and pH of xylanase from a cold-adapted marine species Acinetobacter Johnsonii were 55 ℃ and pH 6.0. Xylanase from a cold-adapted marine species Acinetobacter Johnsonii remained at 80% activity after incubation for 1 h at 65 ℃. The xylanase activity was 1.2-fold higher in 4% ethanol solution than in ethanol free solution. Gibbs free energy of denaturation, ΔG, was higher in 4% ethanol solution than in ethanol free solution. Thermostable ethanol tolerant xylanase was valuable for bioethanol production by simultaneous saccharification and fermentation process with xylan as a carbon source.展开更多
A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular...A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular size of the purified chitinase was estimated to be 48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55℃. The enzyme was stable at 50℃, and its half-life time at 65℃ was 25 rain. The thermostable chitinase was obtained with 60% of the full activity, when it was incubated in the buffer (pH 2.5). The enzyme showed the unique properties for thermostability and pH stability since it was one of the most thermostable chitinases so far isolated in fungi. Ca^2+, Ba^2+, Na^+, and K^+ enhanced the enzyme activity, whereas Fe^2+, Ag^+, Hg^2+, and ethylene diamine tetraacetic acid caused obvious inhibition. The N-terminal amino acids were AQGYLSVQYFVNWAI. Degenerate primers based on the N-terminal sequences of purified chitinase and a cDNA fragment encoding the chitinase gene were obtained through reverse transcriptase-polymerase chain reaction amplication. The RACE was used to generate full-length cDNA clones. The cDNA of chit contained an open reading frame of 1 326 bp encoding 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The alignment results of putative amino acid sequence showed the lower similarity to other chitinases in family-18 except for the catalytic domain containing two conserved motifs related with catalytic activity of chitinase.展开更多
With the aim of identifying novel thermostable esterases, comprehensive sequence databases and cloned fosmid libraries of metagenomes derived from an offshore oil reservoir on the Norwegian Continental Shelf were scre...With the aim of identifying novel thermostable esterases, comprehensive sequence databases and cloned fosmid libraries of metagenomes derived from an offshore oil reservoir on the Norwegian Continental Shelf were screened for enzyme candidates using both sequence-and function-based screening. From several candidates identified in both approaches, one enzyme discovered by the functional approach was verified as a novel esterase and subjected to a deeper characterization. The enzyme was successfully over-produced in Escherichia coli and was shown to be thermostable up to 90°C, with the highest esterase activity on short-chain ester substrates and with tolerance to solvents and metal ions. The fact that the thermostable enzyme was solely found by functional screening of the oil reservoir metagenomes illustrates the importance of this approach as a complement to purely sequence-based screening, in which the enzyme candidate was not detected. In addition, this example indicates the large potential of deep-sub-surface oil reservoir metagenomes as a source of novel, thermostable enzymes of potential relevance for industrial applications.展开更多
In the present work, for the first time on the basis ofpoly (vinyl alcohol) (PVA), 2- (4-dimethylaminostyryl)-l-ethylquinolinium iodide (quinaldine red (QR)) and trisodium (4E)-5-oxo- 1-(4-sulfonatophenyl...In the present work, for the first time on the basis ofpoly (vinyl alcohol) (PVA), 2- (4-dimethylaminostyryl)-l-ethylquinolinium iodide (quinaldine red (QR)) and trisodium (4E)-5-oxo- 1-(4-sulfonatophenyl)-4-[(4-sulfonatophenyl)hydrazono]-3 pyrazolecarboxylate (tartrazine (T)), thermostable polarizing film in a wide range of spectra (λmax=394-511 nm) with polarization efficiency (PE) = 98% in absorption maximum and stretching degree (Rs) = 3.5 was developed. The basic spectral-polarization parameters (polarization efficiency and transmittance) of oriented colored PVA-films were measured and discussed. During the work it was found that oriented PVA-films are the phenomenon of anisotropy of thermal conductivity (λ|/λ⊥). It is a very important parameter for the development of thermostable PVA-polarizing films. For the first time quantum-chemical calculations using density functional theory (DFT) approach for structural analysis and electronic spectrum of the QR were carried out via the B3LYP/dgdzvp and TDB3LYP/dgdzvp methods. Interpretation of absorption strips in visible region of spectrum was also reported. The excitation energies, electronic transitions and oscillator strengths for the studied structures have also been calculated (B3LYP/dgdzvp). The NBO analysis and Mulliken atomic charges of the QR were carried out.展开更多
A thermostable superoxide dismutase (SOD) from the inshore thermophile Thermus sp. JM1 was purified to homogeneity by steps of fractional ammonium sulfate precipitation, DEAE-Sepharose chromatography and Phenyl-Seph...A thermostable superoxide dismutase (SOD) from the inshore thermophile Thermus sp. JM1 was purified to homogeneity by steps of fractional ammonium sulfate precipitation, DEAE-Sepharose chromatography and Phenyl-Sepharose chromatography. The specific activity of the purified native enzyme was 1 656 U/mg. A sod gene from this strain was cloned and overexpressed in Escherichia coli (E. coli). The prepared apo-enzyme of the purified recombinant SOD (rSOD) was reconstituted with either Fe or Mn by means of incubation with appropriate metal salts. As a result, only Mn 2+ - reconstituted rSOD (Mn-rSOD) exhibited the specific activity of 1 598 U/mg. SOD from Thermus sp. JM1 was Mn-SOD, judging by the specific activities analysis of Fe or Mn reconstituted rSODs and the insensitivity of the native SOD to both cyanide and H 2 O 2 . Both the native SOD and Mn- rSOD were determined to be homotetramers with monomeric molecular mass of 26 kDa and 27.5 kDa, respectively. They had high thermostability at 50 ° C and 60 ° C, and showed striking stability across a wide pH span from 4.0 to 11.0.展开更多
An agarase gene containing 1 302 bp was cloned from Microbulbifer sp. AG1. It encoded a mature protein of 413 amino acids plus a 20-residue signal peptide. The recombinant enzyme without the signal peptide was express...An agarase gene containing 1 302 bp was cloned from Microbulbifer sp. AG1. It encoded a mature protein of 413 amino acids plus a 20-residue signal peptide. The recombinant enzyme without the signal peptide was expressed and purified from Escherichia coli BL21(DE3). When agarose was used as a substrate, the optimal temperature and pH for the enzyme were 60℃ and 7.5, respectively. The recombinant agarase showed excellent thermostability with 67% and 19% of residual activities after incubation at 50℃ and 60℃ for 1 h, respectively.Except SDS, the recombinant agarase had a relatively good resistance against the detected inhibitors, detergents and urea denaturant. Thin layer chromatography analysis and enzyme assay using p-nitrophenyl-α/β-Dgalactopyranoside revealed that the recombinant agarase was a β-agarase that degraded agarose into neoagarotetraose as the main end product. The enzymatic hydrolysis products with different degree of polymerization exhibited the antioxidant activities.展开更多
Production of alkaline protease from Bacillus subtilis SHS-04 was investigated under different fermentation conditions involving low-cost substrates with the aim of optimizing yield of enzyme. Maximum enzyme productio...Production of alkaline protease from Bacillus subtilis SHS-04 was investigated under different fermentation conditions involving low-cost substrates with the aim of optimizing yield of enzyme. Maximum enzyme production (1616.21 U/mL) was achieved using groundnut meal (0.75%) as nitrogen source and 0.5% glucose as carbon source at 48 h cultivation period, pH 9, 45 ° C and 200 rpm. The yield was 348% increase over comparable control samples. The alkaline protease had optimum temperature of 60 ° C and remarkably exhibited 80% relative activity at 70 ° C. It was highly thermostable showing 98.7% residual activity at 60 ° C after 60 minutes of incubation at pH 9.0 and was stable in the presence of organic solvents studied. These properties indicate the viability of the protease for biotechnological and industrial applications. The optimized yield of enzyme achieved in this study establishes groundnut meal as potential low-cost substrate for alkaline protease production by B. subtilis SHS-04.展开更多
A new extracellular κ-carrageenase, namely CgkP, 34.0 kDa in molecular weight, was purified from Pseudoalteromonas sp. QY203. CgkP showed relatively high activity at acidities ranging from pH6.0 to pH9.0 and temperat...A new extracellular κ-carrageenase, namely CgkP, 34.0 kDa in molecular weight, was purified from Pseudoalteromonas sp. QY203. CgkP showed relatively high activity at acidities ranging from pH6.0 to pH9.0 and temperatures ranging from 30℃ to 50℃ with the highest activity at 45℃ and pH7.2. Sodium chloride increased its activity markedly, and KCl increased its activity slightly. The divalent and trivalent metal ions including Cu2+ , Ni2+ , Zn2+ , Mn2+ , Al3+ and Fe3+ significantly inhibited its activity, while Mg2+ did not. CgkP remained 70% of original activity after being incubated at 40℃ for 48 h, and remained 80% of the activity after being incubated at 45℃ for 1 h. It exhibited endo-κ-carrageenase activity, mainly depolymerizing the κ-carrageenan into disaccharide and tetrasaccharide. CgkP was more thermostable than most of previously reported κ-carrageenases with a potential of being used in industry.展开更多
Thermostable α-amylases hold a very important place in commercial industrial applications in Sri Lanka. Therefore, the main aim of this study was to identify superior Bacillus strain and optimize growth conditions th...Thermostable α-amylases hold a very important place in commercial industrial applications in Sri Lanka. Therefore, the main aim of this study was to identify superior Bacillus strain and optimize growth conditions that could yield high α-amylase production. Three Bacillus strains, B. amyloliquefaciens ATCC 23350, B. licheniformis ATCC 14580 and B. megaterium ATCC 14581 were used for the study. Shake flask culture experiments were conducted to identify the effect of various fermentation conditions such as growth temperature, incubation period, carbon source, nitrogen source, initial pH and carbon concentration on extracellular α-amylase production. DNSA assay was carried out to determine the enzyme activity. The highest temperature for enzyme activity was reported by B. licheniformis at 85°C, followed by B. amyloliquefaciens at 75°C and B. megaterium at 45°C. Both B. amyloliquefaciens and B. licheniformis were able to give their optimum enzyme production at 37°C, while B. megaterium at 30°C in 150 rpm with initial pH of 7. B. licheniformis and B. amyloliquefaciens gave their optimum yield of the enzyme after 48 h of incubation while B. megaterium gave after 24 h of incubation. Among the carbon sources tested cassava starch was able to give the highest enzyme production. For B. amyloliquefaciens, the highest yield of the enzyme was obtained with 2% of starch, tryptone as a nitrogen source and initial pH of 7. Maximum enzyme production for B. licheniformis was obtained with 1.5% of starch, KNO<sub>3</sub> as a nitrogen source and initial pH of 6. For B. megaterium 1% of starch, tryptone and pH 7.5 induced the optimum α-amylase production. According to the results obtained, B. amyloliquefaciens is the highest thermostable alpha amylase producer. However, according to the industrial requirement, B. licheniformis can also be used as an enzyme producer due to its stability in higher temperatures.展开更多
AIM To assess the effects of hepatitis B virus(HBV) on the expression of host α-1,2-mannosidases and determine the underlying mechanisms.METHODS We measured the expression levels of MAN1A1, MAN1A2, MAN1B1, and MAN1C1...AIM To assess the effects of hepatitis B virus(HBV) on the expression of host α-1,2-mannosidases and determine the underlying mechanisms.METHODS We measured the expression levels of MAN1A1, MAN1A2, MAN1B1, and MAN1C1 in cell lines HepG 2.2.15, HepN 10, HepA D38 and Hep G2 by Western blot. Viral antigens(HBs Ag and HBe Ag) in the culture medium were measured using the chemiluminescence method. HBV DNA quantification assays were performed using a commercial real-time PCR kit. Protein levels of human liver tissue α-1,2-mannosidases were also evaluated by Western blot. Plasmids containing seven individual viral genes of HBV(PTT22-HBx, PTT22-HBs, PTT22-pre S2, PTT22-pre S1, PTT22-HBc, PTT22-HBe, and PTT22-HBp) or control plasmids(PTT22-vector) were transfected into Hep G2 cells. MK886(PPARα) and GW9662(PPARγ) inhibitors were used to explore the effects of HBV on α-1,2-mannosidase expression after the PPARα and PPARγ pathways were blocked.RESULTS We showed that the expression of α-1,2-mannosidases was higher in stably transfected HBV cells than in controls. The expression levels of α-1,2-mannosidase were higher in AD38 cells than those in ND10 cells, which were in turn greater than those in G2.2.15 cells, and positively correlated with the expression of HBsA gin all the cell lines. Levels of α-1,2-mannosidase in nontumorous liver tissues of HBV-related HCC patients were also higher than in the tissues from non-HBVrelated HCC patients. Moreover, transfecting Hep G2 cells with a component of the HBV viral envelope also increased the expression of α-1,2-mannosidases. However, this envelope protein component could not induce MAN1C1 expression in the presence of a PPARα inhibitor, MK886. We also found that MK886 did not affect the expression of MAN1C1 in AD38 cells without tetracycline in the culture medium. This phenomenon was not observed in the case of GW9662.CONCLUSION Our results indicate that HBV increases the expression of α-mannosidases both in vitro and in vivo via activation of the PPARα pathway by its envelope protein.展开更多
Serralysin inhibitors have been proposed as potent drugs against many diseases and may help to prevent further development of antibiotic-resistant pathogenic bacteria. In this study, a novel serralysin inhibitor gene,...Serralysin inhibitors have been proposed as potent drugs against many diseases and may help to prevent further development of antibiotic-resistant pathogenic bacteria. In this study, a novel serralysin inhibitor gene, l up I, was cloned from the marine bacterium F lavobacterium sp. YS-80-122 and expressed in Escherichia coli. The deduced serralysin inhibitor, Lup I, shows <40% amino acid identity to other reported serralysin inhibitors. Multiple sequence alignment and phylogenetic analysis of Lup I with other serralysin inhibitors indicated that Lup I was a novel type of serralysin inhibitor. The inhibitory constant for Lup I towards its target metalloprotease was 0.64 μmol/L. Lup I was thermostable at high temperature, in which 35.6%–90.7% of its inhibitory activity was recovered after treatment at 100°C for 1–60 min followed by incubation at 0°C. This novel inhibitor may represent a candidate drug for the treatment of serralysin-related infections.展开更多
Proteases due to their wide range of applications in biotechnological processes have been the??focus of intense research for many decades. However, from industrial?application view point most of the available protease...Proteases due to their wide range of applications in biotechnological processes have been the??focus of intense research for many decades. However, from industrial?application view point most of the available proteases lack desired properties;?therefore, search for better and efficient thermostable alkaline proteases are?always on.?Bacillus pumilus?D-6, isolated from dairy plant soil sample, in the?current study produced protease which showed activity and stability at high?alkaline?pH (8 - 12) and high?temperatures (70。C- 100。C). Enzyme activity remained unfazed even in presence?of inhibitors like Pb2+and Hg2+which are considered?universal inhibitors of enzyme activity. Besides, the organism successfully?utilized crude agriculture based substrates as carbon and nitrogen source and?produced substantial enzyme titre.展开更多
The field trial of a candidate thermostable Peste des petits ruminants (PPR) vaccine was carried out in flocks of sheep and goats under the extensive system of management. The immune response of vaccinated animals was...The field trial of a candidate thermostable Peste des petits ruminants (PPR) vaccine was carried out in flocks of sheep and goats under the extensive system of management. The immune response of vaccinated animals was determined using the neutralisation test to detect PPR virus specific antibody. Vaccinated animals seroconverted and a four-fold or more rise in antibody titre were observed between pre-vaccination and post-vaccination antibodies. The vaccine elicited significant antibody response in goats through the different routes of administration (intramuscular, intranasal, intraocular, subcutaneous and orally), but was poorly transmitted between the vaccinees and in-contact animals. The sheep responded poorly to the vaccine administered through most of the routes, except for those vaccinated through intramuscular and subcutaneous routes that seroconverted significantly (≥4 fold rise). The vaccine retained a potent titre of 3.1 log10 TCID50 for more than 8 hours after reconstitution in PBS at room temperature. Based on the response of goats to oral vaccination, it is suggested that the vaccine could be administered on the field through the oral routes and has the potential to be adapted to a feed-based administration for wider application to the scattered livestock populations under the extensive system of management.展开更多
The effect of Oxytropis glabra DC. on α-mannosidase( AMA) expression in mice brain tissue was explored to reveal the toxicity mechanism of O. glabra. Forty mice were randomly divided into four groups,namely control g...The effect of Oxytropis glabra DC. on α-mannosidase( AMA) expression in mice brain tissue was explored to reveal the toxicity mechanism of O. glabra. Forty mice were randomly divided into four groups,namely control group,experimental group I,experimental group II and experimental group III. The mice in three experimental groups were fed with O. glabra at the doses of 1,5 and 10 g per kilogram weight,respectively. After challenge for 63 d,mice brains were collected to detect changes in distribution and expression of AMA in different brain regions. The results showed that O. glabra poisoning led to declined AMA mRNA expression in mice brain tissue,but the mice in experimental group I had no significant difference with those in control group( P > 0. 05). The AMA mRNA expression in cerebellum,cerebrum and thalamus of mice in experimental groups II and III were significantly lower than that in control group( P < 0. 05),but the AMA mRNA expression in hippocampus and brainstem in three experimental groups had no significant difference with that in control group( P > 0. 05). AMA had very weak expression in hippocampus and brainstem,but it had expressions in other regions,and the expression was positively correlated with the number of neurons and granulosa cells. The results showed that different doses of O. glabra reduced AMA mRNA expression in mice brain tissue,while cerebellum,cerebrum and thalamus were the main target function areas.展开更多
2,6-bis(picrylamino)-3,5-dinitropyridine(PYX)has excellent thermostability,which makes its thermal decomposition mechanism receive much attention.In this paper,the mechanism of PYX thermal decomposition was investigat...2,6-bis(picrylamino)-3,5-dinitropyridine(PYX)has excellent thermostability,which makes its thermal decomposition mechanism receive much attention.In this paper,the mechanism of PYX thermal decomposition was investigated thoroughly by the ReaxFF-lg force field combined with DFT-B3LYP(6-311++G)method.The detailed decomposition mechanism,small-molecule product evolution,and cluster evolution of PYX were mainly analyzed.In the initial stage of decomposition,the intramolecular hydrogen transfer reaction and the formation of dimerized clusters are earlier than the denitration reaction.With the progress of the reaction,one side of the bitter amino group is removed from the pyridine ring,and then the pyridine ring is cleaved.The final products produced in the thermal decomposition process are CO_(2),H_(2)O,N_(2),and H_(2).Among them,H_(2)O has the earliest generation time,and the reaction rate constant(k_(3))is the largest.Many clusters are formed during the decomposition of PYX,and the formation,aggregation,and decomposition of these clusters are strongly affected by temperature.At low temperatures(2500 K-2750 K),many clusters are formed.At high temperatures(2750 K-3250 K),the clusters aggregate to form larger clusters.At 3500 K,the large clusters decompose and become small.In the late stage of the reaction,H and N in the clusters escaped almost entirely,but more O was trapped in the clusters,which affected the auto-oxidation process of PYX.PYX's initial decomposition activation energy(E_(a))was calculated to be 126.58 kJ/mol.This work contributes to a theoretical understanding of PYX's entire thermal decomposition process.展开更多
基金supported by the Natural Science Foundation of Shandong Province(No.ZR202102270334)the National Key Research and Development Program of China(No.2020YFD0901004).
文摘Natural nano-hydroxyapatite(HA)was extracted from Pacific cod(Gadus macrocephalus)bone with a thermostable col-lagenolytic protease in the present study.Conditions for the enzymatic reaction were optimized to be 60℃and pH 7.0,and a desir-able extraction efficiency was achieved by using the crude collagenolytic protease.Dynamic light scattering,transmission electron microscopy and energy-dispersive X-ray analysis revealed that nano-HA are anionic spherical(about 110nm)particles mainly com-prised of calcium and phosphorus at an approximate ratio of 5:3.As evaluated with the mouse ex vivo intestinal segments,the extracted nano-HA displayed comparable level of intestinal bioavailability to the positive control CaCl_(2).By treating with inhibitors(NaN3,ami-loride)and low temperature(4℃),clathrin-mediated endocytosis was assumed to involve the intestinal absorption of nano-HA.Over-all,the application of thermostable collagenolytic protease is proved to be a promising alternative method for nano-HA extraction from natural resource with improved ecological and biological value.
基金Supported by the Science and Technology Research Project of Hubei Provincial Department of Education(B2017041)
文摘A xylanase-producing bacterium, isolated from deep sea sediments, was identified as the cold-adapted marine species Acinetobacter Johnsonii. A cold-adapted marine species Acinetobacter Johnsonii could grow at 4 ℃. The optimum temperature and pH of xylanase from a cold-adapted marine species Acinetobacter Johnsonii were 55 ℃ and pH 6.0. Xylanase from a cold-adapted marine species Acinetobacter Johnsonii remained at 80% activity after incubation for 1 h at 65 ℃. The xylanase activity was 1.2-fold higher in 4% ethanol solution than in ethanol free solution. Gibbs free energy of denaturation, ΔG, was higher in 4% ethanol solution than in ethanol free solution. Thermostable ethanol tolerant xylanase was valuable for bioethanol production by simultaneous saccharification and fermentation process with xylan as a carbon source.
基金the Science Technology Plan Foundation of Hebei Province, China (07225533)the Doctor Foundation from Agricultural University of Hebei (050031)
文摘A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular size of the purified chitinase was estimated to be 48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55℃. The enzyme was stable at 50℃, and its half-life time at 65℃ was 25 rain. The thermostable chitinase was obtained with 60% of the full activity, when it was incubated in the buffer (pH 2.5). The enzyme showed the unique properties for thermostability and pH stability since it was one of the most thermostable chitinases so far isolated in fungi. Ca^2+, Ba^2+, Na^+, and K^+ enhanced the enzyme activity, whereas Fe^2+, Ag^+, Hg^2+, and ethylene diamine tetraacetic acid caused obvious inhibition. The N-terminal amino acids were AQGYLSVQYFVNWAI. Degenerate primers based on the N-terminal sequences of purified chitinase and a cDNA fragment encoding the chitinase gene were obtained through reverse transcriptase-polymerase chain reaction amplication. The RACE was used to generate full-length cDNA clones. The cDNA of chit contained an open reading frame of 1 326 bp encoding 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The alignment results of putative amino acid sequence showed the lower similarity to other chitinases in family-18 except for the catalytic domain containing two conserved motifs related with catalytic activity of chitinase.
文摘With the aim of identifying novel thermostable esterases, comprehensive sequence databases and cloned fosmid libraries of metagenomes derived from an offshore oil reservoir on the Norwegian Continental Shelf were screened for enzyme candidates using both sequence-and function-based screening. From several candidates identified in both approaches, one enzyme discovered by the functional approach was verified as a novel esterase and subjected to a deeper characterization. The enzyme was successfully over-produced in Escherichia coli and was shown to be thermostable up to 90°C, with the highest esterase activity on short-chain ester substrates and with tolerance to solvents and metal ions. The fact that the thermostable enzyme was solely found by functional screening of the oil reservoir metagenomes illustrates the importance of this approach as a complement to purely sequence-based screening, in which the enzyme candidate was not detected. In addition, this example indicates the large potential of deep-sub-surface oil reservoir metagenomes as a source of novel, thermostable enzymes of potential relevance for industrial applications.
基金supported by the National Academy of Sciences of Belarus
文摘In the present work, for the first time on the basis ofpoly (vinyl alcohol) (PVA), 2- (4-dimethylaminostyryl)-l-ethylquinolinium iodide (quinaldine red (QR)) and trisodium (4E)-5-oxo- 1-(4-sulfonatophenyl)-4-[(4-sulfonatophenyl)hydrazono]-3 pyrazolecarboxylate (tartrazine (T)), thermostable polarizing film in a wide range of spectra (λmax=394-511 nm) with polarization efficiency (PE) = 98% in absorption maximum and stretching degree (Rs) = 3.5 was developed. The basic spectral-polarization parameters (polarization efficiency and transmittance) of oriented colored PVA-films were measured and discussed. During the work it was found that oriented PVA-films are the phenomenon of anisotropy of thermal conductivity (λ|/λ⊥). It is a very important parameter for the development of thermostable PVA-polarizing films. For the first time quantum-chemical calculations using density functional theory (DFT) approach for structural analysis and electronic spectrum of the QR were carried out via the B3LYP/dgdzvp and TDB3LYP/dgdzvp methods. Interpretation of absorption strips in visible region of spectrum was also reported. The excitation energies, electronic transitions and oscillator strengths for the studied structures have also been calculated (B3LYP/dgdzvp). The NBO analysis and Mulliken atomic charges of the QR were carried out.
基金The Natural Science Foundation of Fujian Province,China under contract Nos 2008J0067 and 2009J01033the Program for New Century Excellent Talents in Fujian Province University under contract No.NCETFJ-2007the Foundation for Innovative Research Team of Jimei University under contract No.2010A005
文摘A thermostable superoxide dismutase (SOD) from the inshore thermophile Thermus sp. JM1 was purified to homogeneity by steps of fractional ammonium sulfate precipitation, DEAE-Sepharose chromatography and Phenyl-Sepharose chromatography. The specific activity of the purified native enzyme was 1 656 U/mg. A sod gene from this strain was cloned and overexpressed in Escherichia coli (E. coli). The prepared apo-enzyme of the purified recombinant SOD (rSOD) was reconstituted with either Fe or Mn by means of incubation with appropriate metal salts. As a result, only Mn 2+ - reconstituted rSOD (Mn-rSOD) exhibited the specific activity of 1 598 U/mg. SOD from Thermus sp. JM1 was Mn-SOD, judging by the specific activities analysis of Fe or Mn reconstituted rSODs and the insensitivity of the native SOD to both cyanide and H 2 O 2 . Both the native SOD and Mn- rSOD were determined to be homotetramers with monomeric molecular mass of 26 kDa and 27.5 kDa, respectively. They had high thermostability at 50 ° C and 60 ° C, and showed striking stability across a wide pH span from 4.0 to 11.0.
基金The Natural Science Foundation of Fujian Province of China under contract No.2016J01162the Program for New Century Excellent Talents in Fujian Province University,China under contract No.B15139
文摘An agarase gene containing 1 302 bp was cloned from Microbulbifer sp. AG1. It encoded a mature protein of 413 amino acids plus a 20-residue signal peptide. The recombinant enzyme without the signal peptide was expressed and purified from Escherichia coli BL21(DE3). When agarose was used as a substrate, the optimal temperature and pH for the enzyme were 60℃ and 7.5, respectively. The recombinant agarase showed excellent thermostability with 67% and 19% of residual activities after incubation at 50℃ and 60℃ for 1 h, respectively.Except SDS, the recombinant agarase had a relatively good resistance against the detected inhibitors, detergents and urea denaturant. Thin layer chromatography analysis and enzyme assay using p-nitrophenyl-α/β-Dgalactopyranoside revealed that the recombinant agarase was a β-agarase that degraded agarose into neoagarotetraose as the main end product. The enzymatic hydrolysis products with different degree of polymerization exhibited the antioxidant activities.
文摘Production of alkaline protease from Bacillus subtilis SHS-04 was investigated under different fermentation conditions involving low-cost substrates with the aim of optimizing yield of enzyme. Maximum enzyme production (1616.21 U/mL) was achieved using groundnut meal (0.75%) as nitrogen source and 0.5% glucose as carbon source at 48 h cultivation period, pH 9, 45 ° C and 200 rpm. The yield was 348% increase over comparable control samples. The alkaline protease had optimum temperature of 60 ° C and remarkably exhibited 80% relative activity at 70 ° C. It was highly thermostable showing 98.7% residual activity at 60 ° C after 60 minutes of incubation at pH 9.0 and was stable in the presence of organic solvents studied. These properties indicate the viability of the protease for biotechnological and industrial applications. The optimized yield of enzyme achieved in this study establishes groundnut meal as potential low-cost substrate for alkaline protease production by B. subtilis SHS-04.
基金supported by National Science Foundation of China (31000361 and 31070712)Program for Changjiang Scholars and Innovative Research Team in University (IRT0944)+1 种基金Special Fund for Marine Scientific Research in the Public Interest (201005024)the Fundamental Research Funds for the Central Universities(201013008)
文摘A new extracellular κ-carrageenase, namely CgkP, 34.0 kDa in molecular weight, was purified from Pseudoalteromonas sp. QY203. CgkP showed relatively high activity at acidities ranging from pH6.0 to pH9.0 and temperatures ranging from 30℃ to 50℃ with the highest activity at 45℃ and pH7.2. Sodium chloride increased its activity markedly, and KCl increased its activity slightly. The divalent and trivalent metal ions including Cu2+ , Ni2+ , Zn2+ , Mn2+ , Al3+ and Fe3+ significantly inhibited its activity, while Mg2+ did not. CgkP remained 70% of original activity after being incubated at 40℃ for 48 h, and remained 80% of the activity after being incubated at 45℃ for 1 h. It exhibited endo-κ-carrageenase activity, mainly depolymerizing the κ-carrageenan into disaccharide and tetrasaccharide. CgkP was more thermostable than most of previously reported κ-carrageenases with a potential of being used in industry.
文摘Thermostable α-amylases hold a very important place in commercial industrial applications in Sri Lanka. Therefore, the main aim of this study was to identify superior Bacillus strain and optimize growth conditions that could yield high α-amylase production. Three Bacillus strains, B. amyloliquefaciens ATCC 23350, B. licheniformis ATCC 14580 and B. megaterium ATCC 14581 were used for the study. Shake flask culture experiments were conducted to identify the effect of various fermentation conditions such as growth temperature, incubation period, carbon source, nitrogen source, initial pH and carbon concentration on extracellular α-amylase production. DNSA assay was carried out to determine the enzyme activity. The highest temperature for enzyme activity was reported by B. licheniformis at 85°C, followed by B. amyloliquefaciens at 75°C and B. megaterium at 45°C. Both B. amyloliquefaciens and B. licheniformis were able to give their optimum enzyme production at 37°C, while B. megaterium at 30°C in 150 rpm with initial pH of 7. B. licheniformis and B. amyloliquefaciens gave their optimum yield of the enzyme after 48 h of incubation while B. megaterium gave after 24 h of incubation. Among the carbon sources tested cassava starch was able to give the highest enzyme production. For B. amyloliquefaciens, the highest yield of the enzyme was obtained with 2% of starch, tryptone as a nitrogen source and initial pH of 7. Maximum enzyme production for B. licheniformis was obtained with 1.5% of starch, KNO<sub>3</sub> as a nitrogen source and initial pH of 6. For B. megaterium 1% of starch, tryptone and pH 7.5 induced the optimum α-amylase production. According to the results obtained, B. amyloliquefaciens is the highest thermostable alpha amylase producer. However, according to the industrial requirement, B. licheniformis can also be used as an enzyme producer due to its stability in higher temperatures.
基金Supported by the National Natural Science Foundation of China,No.81171559
文摘AIM To assess the effects of hepatitis B virus(HBV) on the expression of host α-1,2-mannosidases and determine the underlying mechanisms.METHODS We measured the expression levels of MAN1A1, MAN1A2, MAN1B1, and MAN1C1 in cell lines HepG 2.2.15, HepN 10, HepA D38 and Hep G2 by Western blot. Viral antigens(HBs Ag and HBe Ag) in the culture medium were measured using the chemiluminescence method. HBV DNA quantification assays were performed using a commercial real-time PCR kit. Protein levels of human liver tissue α-1,2-mannosidases were also evaluated by Western blot. Plasmids containing seven individual viral genes of HBV(PTT22-HBx, PTT22-HBs, PTT22-pre S2, PTT22-pre S1, PTT22-HBc, PTT22-HBe, and PTT22-HBp) or control plasmids(PTT22-vector) were transfected into Hep G2 cells. MK886(PPARα) and GW9662(PPARγ) inhibitors were used to explore the effects of HBV on α-1,2-mannosidase expression after the PPARα and PPARγ pathways were blocked.RESULTS We showed that the expression of α-1,2-mannosidases was higher in stably transfected HBV cells than in controls. The expression levels of α-1,2-mannosidase were higher in AD38 cells than those in ND10 cells, which were in turn greater than those in G2.2.15 cells, and positively correlated with the expression of HBsA gin all the cell lines. Levels of α-1,2-mannosidase in nontumorous liver tissues of HBV-related HCC patients were also higher than in the tissues from non-HBVrelated HCC patients. Moreover, transfecting Hep G2 cells with a component of the HBV viral envelope also increased the expression of α-1,2-mannosidases. However, this envelope protein component could not induce MAN1C1 expression in the presence of a PPARα inhibitor, MK886. We also found that MK886 did not affect the expression of MAN1C1 in AD38 cells without tetracycline in the culture medium. This phenomenon was not observed in the case of GW9662.CONCLUSION Our results indicate that HBV increases the expression of α-mannosidases both in vitro and in vivo via activation of the PPARα pathway by its envelope protein.
基金Supported by the National Natural Science Foundation of China(No.41376175)the Joint Funds of the National Natural Science Foundation of China(No.U1406402-5)+3 种基金the International S&T Cooperation Program of China(No.2014DFG30890)the Qingdao Science and Technology Plan Project(No.14-2-4-11-jch)the National Science Foundation for PostDoctoral Scientists of China(No.2016M590673)the Application Foundation of Qingdao for Post-Doctoral Scientists of China(No.Q51201601)
文摘Serralysin inhibitors have been proposed as potent drugs against many diseases and may help to prevent further development of antibiotic-resistant pathogenic bacteria. In this study, a novel serralysin inhibitor gene, l up I, was cloned from the marine bacterium F lavobacterium sp. YS-80-122 and expressed in Escherichia coli. The deduced serralysin inhibitor, Lup I, shows <40% amino acid identity to other reported serralysin inhibitors. Multiple sequence alignment and phylogenetic analysis of Lup I with other serralysin inhibitors indicated that Lup I was a novel type of serralysin inhibitor. The inhibitory constant for Lup I towards its target metalloprotease was 0.64 μmol/L. Lup I was thermostable at high temperature, in which 35.6%–90.7% of its inhibitory activity was recovered after treatment at 100°C for 1–60 min followed by incubation at 0°C. This novel inhibitor may represent a candidate drug for the treatment of serralysin-related infections.
文摘Proteases due to their wide range of applications in biotechnological processes have been the??focus of intense research for many decades. However, from industrial?application view point most of the available proteases lack desired properties;?therefore, search for better and efficient thermostable alkaline proteases are?always on.?Bacillus pumilus?D-6, isolated from dairy plant soil sample, in the?current study produced protease which showed activity and stability at high?alkaline?pH (8 - 12) and high?temperatures (70。C- 100。C). Enzyme activity remained unfazed even in presence?of inhibitors like Pb2+and Hg2+which are considered?universal inhibitors of enzyme activity. Besides, the organism successfully?utilized crude agriculture based substrates as carbon and nitrogen source and?produced substantial enzyme titre.
文摘The field trial of a candidate thermostable Peste des petits ruminants (PPR) vaccine was carried out in flocks of sheep and goats under the extensive system of management. The immune response of vaccinated animals was determined using the neutralisation test to detect PPR virus specific antibody. Vaccinated animals seroconverted and a four-fold or more rise in antibody titre were observed between pre-vaccination and post-vaccination antibodies. The vaccine elicited significant antibody response in goats through the different routes of administration (intramuscular, intranasal, intraocular, subcutaneous and orally), but was poorly transmitted between the vaccinees and in-contact animals. The sheep responded poorly to the vaccine administered through most of the routes, except for those vaccinated through intramuscular and subcutaneous routes that seroconverted significantly (≥4 fold rise). The vaccine retained a potent titre of 3.1 log10 TCID50 for more than 8 hours after reconstitution in PBS at room temperature. Based on the response of goats to oral vaccination, it is suggested that the vaccine could be administered on the field through the oral routes and has the potential to be adapted to a feed-based administration for wider application to the scattered livestock populations under the extensive system of management.
基金Supported by National Natural Science Foundation of China(31460678)Open Project of Key Laboratory of Tarim Animal Husbandry Science and Technology in Xinjiang Production and Construction Corps(HS201409)
文摘The effect of Oxytropis glabra DC. on α-mannosidase( AMA) expression in mice brain tissue was explored to reveal the toxicity mechanism of O. glabra. Forty mice were randomly divided into four groups,namely control group,experimental group I,experimental group II and experimental group III. The mice in three experimental groups were fed with O. glabra at the doses of 1,5 and 10 g per kilogram weight,respectively. After challenge for 63 d,mice brains were collected to detect changes in distribution and expression of AMA in different brain regions. The results showed that O. glabra poisoning led to declined AMA mRNA expression in mice brain tissue,but the mice in experimental group I had no significant difference with those in control group( P > 0. 05). The AMA mRNA expression in cerebellum,cerebrum and thalamus of mice in experimental groups II and III were significantly lower than that in control group( P < 0. 05),but the AMA mRNA expression in hippocampus and brainstem in three experimental groups had no significant difference with that in control group( P > 0. 05). AMA had very weak expression in hippocampus and brainstem,but it had expressions in other regions,and the expression was positively correlated with the number of neurons and granulosa cells. The results showed that different doses of O. glabra reduced AMA mRNA expression in mice brain tissue,while cerebellum,cerebrum and thalamus were the main target function areas.
基金funded by the National Natural Science Foundation of China(Grant No.21975024)。
文摘2,6-bis(picrylamino)-3,5-dinitropyridine(PYX)has excellent thermostability,which makes its thermal decomposition mechanism receive much attention.In this paper,the mechanism of PYX thermal decomposition was investigated thoroughly by the ReaxFF-lg force field combined with DFT-B3LYP(6-311++G)method.The detailed decomposition mechanism,small-molecule product evolution,and cluster evolution of PYX were mainly analyzed.In the initial stage of decomposition,the intramolecular hydrogen transfer reaction and the formation of dimerized clusters are earlier than the denitration reaction.With the progress of the reaction,one side of the bitter amino group is removed from the pyridine ring,and then the pyridine ring is cleaved.The final products produced in the thermal decomposition process are CO_(2),H_(2)O,N_(2),and H_(2).Among them,H_(2)O has the earliest generation time,and the reaction rate constant(k_(3))is the largest.Many clusters are formed during the decomposition of PYX,and the formation,aggregation,and decomposition of these clusters are strongly affected by temperature.At low temperatures(2500 K-2750 K),many clusters are formed.At high temperatures(2750 K-3250 K),the clusters aggregate to form larger clusters.At 3500 K,the large clusters decompose and become small.In the late stage of the reaction,H and N in the clusters escaped almost entirely,but more O was trapped in the clusters,which affected the auto-oxidation process of PYX.PYX's initial decomposition activation energy(E_(a))was calculated to be 126.58 kJ/mol.This work contributes to a theoretical understanding of PYX's entire thermal decomposition process.