Objective To construct four recombinant Lactococcus lactis strains exhibiting high β-galactosidase activity in fusion or non-fusion ways, and to study the influence factors for their protein expression and secretion....Objective To construct four recombinant Lactococcus lactis strains exhibiting high β-galactosidase activity in fusion or non-fusion ways, and to study the influence factors for their protein expression and secretion. Methods The gene fragments encoding β-galactosidase from two strains of Loctobacillus bulgaricus, wch9901 isolated from yogurt and 1.1480 purchased from the Chinese Academy of Sciences, were amplified and inserted into lactococcal expression vector pMG36e. For fusion expression, the open reading frame of the β-galactosidase gene was amplified, while for non-fusion expression, the open reading frame of the β-galactosidase gene was amplified with its native Shine-Dalgarno sequence upstream. The start codon of the β-galactosidase gene partially overlapped with the stop codon of vector origin open reading frame. Then, the recombinant plasmids were transformed into Escherichia coli DH5α and Lactococcus lactis subsp, lactis MG1363 and confirmed by determining β-galactosidase activities. Results The non-fusion expression plasmids showed a significantly higher β-galactosidase activity in transformed strains than the fusion expression plasmids. The highest enzyme activity was observed in Lactococcus lactis transformed with the non-fusion expression plasmids which were inserted into the β-galactosidase gene from Lactobacillus bulgaricus wch9901. The β-galactosidase activity was 2.75 times as high as that of the native counterpart. In addition, β-galactosidase expressed by recombinant plasmids in Lactococcus lactis could be secreted into the culture medium. The highest secretion rate （27.1%） was observed when the culture medium contained 20 g/L of lactose. Conclusion Different properties of the native bacteria may have some effects on the protein expression of recombinant plasmids. Non-fusion expression shows a higher enzyme activity in host bacteria. There may be a host-related weak secretion signal peptide gene within the structure gene of Lb. bulgaricus β-galactosidase, and its translation product may introduce the enzyme secretion out of cells in special hosts.展开更多
Objective This study is to examine the secretion effects of β-galactosidase in Lactococcus lactis.Methods The usp45 and β-galactosidase genes were cloned and inserted into plasmid pMG36e to obtain the recombinant pl...Objective This study is to examine the secretion effects of β-galactosidase in Lactococcus lactis.Methods The usp45 and β-galactosidase genes were cloned and inserted into plasmid pMG36e to obtain the recombinant plasmid pMG36e-usp-lacZ.This recombinant plasmid was transformed into both Escherichia coli DH5α and L.lactis MG1363.The enzyme activity,gene sequencing,SDS-PAGE and hereditary stability were assessed and studied.Results The lacZ gene inserted into plasmids pMG36e-usp-lacZ was 99.37% similar to the GenBank sequence,and SDS-PAGE revealed an evident idio-strap at 116 KDa between L.lactis MG1363/pMG36eusp-lacZ in both supernatant and cell samples.β-Galactosidase activity measured 0.225 U/mL in L.lactis pMG36e-usp-lacZ transformants,and its secretion rate was 10%.The plasmid pMG36e-usp-lacZ appeared more stable in MG1363.Conclusion The authors concluded that these new recombinant bacteria well expressed and secreted β-galactosidase,indicating that the β-galactosidase expression system was successfully constructed,and this might provide a new solution for management of lactose intolerance specifically and promote the use of gene-modified organisms as part of the food-grade plasmid in general.展开更多
Aging-related diseases are gradually becoming a major problem with the rapid development of aged population in human society.Although many fluorescent probes have been employed to diagnosis senescence via imaging sene...Aging-related diseases are gradually becoming a major problem with the rapid development of aged population in human society.Although many fluorescent probes have been employed to diagnosis senescence via imaging senescence-associatedβ-galactosidase(SA-β-Gal),which is proved to be closely associated with senescent cells,the similar catalytic effectiveness of enzymatic reaction of ovarian cancer-associatedβ-Gal(OA-β-Gal)will interfere with imaging accuracy.Herein,a near-infrared(NIR)hemicyanine based fluorescent probe HCyXA-βGal was designed for light-up imaging of live cells containingβ-Gal.With the organelle-targeting morpholinyl and positive charge moieties,HCyxA-βGal was successfully applicated to image the difference of enzymatic location in senescent cells and ovarian cancer cells.Furthermore,inspired by the fast response performance,fast and precise imaging of the two cell lines was realized via covering another dimension of fluorescence signal:time-dependent intensity.展开更多
Detection and visualization ofβ-galactosidase(β-gal)is essential to reffect its physiological and pathological effects on human health and disease,but it is still challenging to precisely trackβ-gal in vivo owing t...Detection and visualization ofβ-galactosidase(β-gal)is essential to reffect its physiological and pathological effects on human health and disease,but it is still challenging to precisely trackβ-gal in vivo owing to the limitation of current analytical methods.In our work,we reported a photoacoustic(PA)nanoprobe for selective imaging of the endogenousβ-gal in vivo.Our nanoprobe Cy7-β-gal-LP was constructed by encapsulation of a near-infra red(NIR)dye Cy7-β-gal within a liposome(LP,DSPE-PEG2000-COOH).The dye Cy7-β-gal was synthesized based on a dye Cy-OH where the hydroxyl group was replaced by aβ-D-galactopyranoside residue,which can be recognized byβ-gal as an enzyme hydrolytic site.With the addition ofβ-gal,the absorbance of Cy7-β-gal exhibited a significant red shift with the absorption peak moved from 600 nm to 680 nm,which should generate a switch-on PA signal at 680 nm in the presence ofβ-gal.In addition,as theffuorescence of the dye was totally quenched due to aggregation within the liposome,Cy7-β-gal-LP exhibited high PA conversion efficiency.With the nanoprobe,we achieved the selective PA detection and imaging ofβ-gal in the tumor-bearing mice.展开更多
The enzyme β-galactosidase （lactase; EC 3.2.1.23） is a commercially important enzyme due to its various applications in dairy and food industries, which are based on the β-galactosidase-catalysed hydrolysis of lac...The enzyme β-galactosidase （lactase; EC 3.2.1.23） is a commercially important enzyme due to its various applications in dairy and food industries, which are based on the β-galactosidase-catalysed hydrolysis of lactose into glucose and galactose. The objectives of this work were to identify novel and attractive sources of this industrially relevant enzyme, and to study the effect of selected growth parameters （carbon source, lactose concentration, nitrogen source, peptone concentration, initial pH and temperature） on the formation of β-galactosidase. Based on a screening of isolates from Tha Pai hot spring, Mae Hong Son Province, Thailand, strain BI.1 was selected for further studies. Strain BI.1 is a Gram-positive, rod-shaped, catalase-positive bacterium that forms endospores. Based on the sequence of the 16S rDNA determined, this isolate is most closely related to Anoxybacillus sp. and Bacillus sp., and hence the strain is designated as Bacillus sp. B 1. I.β-Galactosidase was produced by this strain with lactose and peptone as carbon and nitrogen sources, respectively. Optimal enzyme production occurred at an initial culture pH of 8.5 and at 45 ℃. Under these optimum culture conditions, maximal volumetric and specific β-galactosidase activity of 0.478 U mL^-1 and 0.338 U mg^-1 protein, respectively, were obtained after 13 h of cultivation in a medium contain 2.5% lactose, 2.0% peptone, 0.3% K2HPO4, 0.1% KH2PO4 and 0.05% MgSOa·7H2O.展开更多
β-galactosidases (EC 3.2.1.23) constitute a widespread family of glycosyl hydrolases in plants and are thought to be involved in metabolism of cell wall polysaccharides. A cDNA of the cotton (Gossypium hirsutum) β-g...β-galactosidases (EC 3.2.1.23) constitute a widespread family of glycosyl hydrolases in plants and are thought to be involved in metabolism of cell wall polysaccharides. A cDNA of the cotton (Gossypium hirsutum) β-galactosidase gene, designated GhGal1, has previously been identified and its transcripts are highly abundant at the elongation stage of the cotton fiber. To examine the temporal and spatial control of GhGal1 expression, a transcriptional fusion of the GhGal1 promoter region (1770 bp) with the β-glucuronidase (GUS) reporter gene was introduced into tobacco plants by the Agrobacterium infection method. The resulting transgenic plants showed higher GUS activity of fruit in the transgenic plants than that in the negative and positive controls. Histochemical localization of GUS activity demonstrated that the expression of the GUS gene could be found in the meristem zones of roots, cotyledons, vascular tissues, fruit and trichomes in transgenic tobacco plants. Additionally, se-quence analysis of the regulatory region also revealed several conserved motifs among which some were shared with previously reported fruit/seed-specific elements and the others were related with trichome expression. These results indicated the temporal and spatial expression characterization of the GhGal1 promoter in transgenic tobacco plants and provided an important insight into the roles of GhGal1 in cotton fiber development.展开更多
β-galactosidases (EC 3.2.1.23) constitute a widespread family of enzymes in plants that is thought to be involved in metabolism of cell wall polysaccharides. We reported herein the isolation of a fulllength cDNA enco...β-galactosidases (EC 3.2.1.23) constitute a widespread family of enzymes in plants that is thought to be involved in metabolism of cell wall polysaccharides. We reported herein the isolation of a fulllength cDNA encoding a typical β-galactosidase protein, designated GhGal1 (Gossypium hirsutum L.galactosidase), of 843 amino acids with a predicted molecular mass of nearly 94.8 kDa. In addition to a glycosyl hydrolase family 35 domain and a putative signal peptide, an unusual characteristic of GhGal 1 is that, at the C-terminus of the enzyme, a domain was found that is structurally related to a sea urchin egg lectin (SUEL-lectin) with D-galactose- and L-rhamnose-binding domains. Based on results from Southern blot, we estimated that there would be two copies of the GhGal1 gene per haploid genome of G. hirsutum.The transcripts of GhGal1 were regulated spatially and temporally and were present in very high abundance at the elongation stage of the cotton fiber. The expression pattern suggests that the GhGal1 gene could be involved in metabolism of the primary cell wall.展开更多
[Objective] The research aimed to construct the fusion protein expression vector of α-galactosidase-EGFP （enhanced green fluorescent protein） in cucumber controlled by CaMV35S promoter.[Method] CaMV35S promoter seq...[Objective] The research aimed to construct the fusion protein expression vector of α-galactosidase-EGFP （enhanced green fluorescent protein） in cucumber controlled by CaMV35S promoter.[Method] CaMV35S promoter sequence and the coding region of EGFP were amplified by polymerase chain reactions （PCR） with vector pCambia 1303 as the template.Using reverse transcript PCR technology,with total RNAs of cucumber as template,the coding region of acid α-galactosidase Ⅰ in cucumber was amplified.The above three fragments were inserted into the multiple cloning sites of expression vector pCambia 1381c.The fusion expression vector of α-galactosidase-EGFP located at the C-terminal of the target genes was constructed.[Result] After enzyme digestion and sequencing,the fusion expression of α-galactosidase-EGFP in cucumber was constructed successfully.[Conclusion] The research laid the experimental basis for further study on the subcellular localization of α-galactosidase in cucumber.展开更多
We constructed a plasmid that contains a small piece of DNA with two vaccinis promoters running in opposite directions——a promoter from a late gene encoding an 11 K polypeptide (P11) and a promoter from an early gen...We constructed a plasmid that contains a small piece of DNA with two vaccinis promoters running in opposite directions——a promoter from a late gene encoding an 11 K polypeptide (P11) and a promoter from an early gene encoding 25K(P25). These promoters were isolated from the Tian Tan strain of vaccinia virus and were flanked by the thymidine kinase (TK) sequence of the same virus. Genes encoding the hepatitis B virus surface antigen (HBsAg) and the Rscherichia coli β-galactosidase (LacZ) were inserted downstream of the 11 K and 25 K promoters respectively so that coexpression plasmids were constructed. Recombinant vaccinis Viruses were selected directly by picking blue plaques formed under overlaying agarose medium containing X-gal. HBsAg was expressed to high level by these recombinant viruses. These recombinant viruses showed reduced virulence on rabbit skin and induced anti-HBs after intrsdermal inoculation of rabbits.展开更多
The performance of gold and polystyrene nanoparticles was investigated using quartz crystal microbalance (QCM) as sensor platform;β-Galactosidase antibody with corresponding antigen was utilized for the immunoreactio...The performance of gold and polystyrene nanoparticles was investigated using quartz crystal microbalance (QCM) as sensor platform;β-Galactosidase antibody with corresponding antigen was utilized for the immunoreaction. The development of the immunosensor included: 1) formation of self assembled monolayers on quartz crystals;2a) immobilization of p-aminothiophenol functionalized gold nanoparticles on carboxyl-terminated self assembled monolayer, or 2b) immobilization of polystyrene nanoparticles on crystals modified with p-aminothiophenol self assembled monolayer;3) attachment of monoclonal anti β-Gal on nanoparticles;and 4) detection of target analyte. The nanoparticles used were synthesized in house and characterized by transmission electron microscopy and infrared spectroscopy. The results revealed that antibodies were strongly attached to functionalized gold nanoparticles;the weaker immobilization of antibodies to polystyrene nanoparticles provoked their detachment during antigen detection. When cross reactivity of polystyrene nanoparticles was checked using a different antigen (Brucella), displacement of antibody was not recorded, demonstrating specificity of the reaction. To the best of our knowledge this is the first direct comparison between behaviors of biosensors developed with two commonly used nanoparticles. The results showed that both nanoparticles produced biosensors capable to detect β-Gal. Nevertheless biosensors developed using polystyrene nanoparticles are simpler, cheaper and more eco-friendly than those developed using gold nanoparticles.展开更多
β-galactosidase was extracted from apricots (Prunus armeniaca kaisa) and characterized biochemically. Three isoenzymes (β-gal I, β-gal II and β-gal III) were obtained by salt fractionation and ionexchange and Seph...β-galactosidase was extracted from apricots (Prunus armeniaca kaisa) and characterized biochemically. Three isoenzymes (β-gal I, β-gal II and β-gal III) were obtained by salt fractionation and ionexchange and Sephadex G-100 column chromatography. β-galactosidase II showed a high ability to hy-drolyze the substrate p-nitrophenyl β-D-galactopyranoside than that of β-galactosidase I and III. The individual peaks showed charge homogeneity as revealed by single band on polyacrylamide gel. The molecular weight of β-gal I, β-gal II and β-gal III as determined by gel filtration was found to be 44.15, 34.70 and 23.71 KDa respectively. The optimum pH for the activity different isozymes was found between 4 and 6. The isoenzymes were determined to be thermally stable upto 40?C. The Km value for β-gal I was 1.85 mM which was higher than that of β-gal II (Km = 1.7), and β-gal III (Km = 1.19). The Vmax value for β-gal I, β-gal II and β-gal III was found to be 0.52, 0.70 and 0.38 μmole/min respectively.展开更多
The work is intended to achieve optimum culture conditions of α-galactosidase production by a mutant strain ,Aspergillusfoetidus ZU-GI in solid-state fermentation （SSF）. Certain fermentation parameters involving mo...The work is intended to achieve optimum culture conditions of α-galactosidase production by a mutant strain ,Aspergillusfoetidus ZU-GI in solid-state fermentation （SSF）. Certain fermentation parameters involving moisture content, incubation temperature, cultivation period of seed, inoculum volume, initial pH value, layers of pledget, load size of medium and period of cultivation were investigated separately. The optimal cultivating conditions of α-galactosidase production in SSF were 60% initial moisture of medium, 28 ℃ incubation temperature, 18^h cultivation period of seed, 10% inoculum volume, 5.0-6.0 initial pH of medium, 6 layers of pledget and 10 g dry matter loadage. Under the optimized cultivation conditions, the maximum α-galactosidase production was 2037.51 U/g dry matter near the 144th hour of fermentation.展开更多
Construction of a food-grade expression vector for application to lactic acid bacteria（LAB） is of importance for dairy fermentation system. An α-galactosidase（aga） gene encoding an enzyme degrading melibiose was ...Construction of a food-grade expression vector for application to lactic acid bacteria（LAB） is of importance for dairy fermentation system. An α-galactosidase（aga） gene encoding an enzyme degrading melibiose was amplified by PCR from the plasmid p RAF800 of Lactococcus lactis NZ9000. The aga gene was introduced into pMG36 e to substitute the p rimary antibiotic selectable marker of pMG36 e, resulting in construction of a new food-grade expression vector pMG36-aga. To testify the expression efficiency of exogenous gene in pMG36-aga, a 1.5 kb long α-amylase（amy） gene from Ba cillus li cheniformis was cloned by PCR and introduced into the plasmid pMG36-aga. The resultant plasimd pMG36-aga-amy was transformed into L. lactis ML23 by electroporation. The positive clones were selected with the medium containing melibiose as the sole carbon source. Th e selection efficiency of aga was 8.71×103 CFU with a standard deviation of 9.1×102 CFU ？g-1 DNA of pMG36-aga. Furthermore, the SDS-PAGE analysis showed that the pMG36-aga-amy expressed a 56.4 kDa protein which was the same as the putati ve molecular weight of α-amylase. The starch plate assay also indicated that L. lactis ML23 displayed high activity of α-amylase by expressing of amy gene of pMG36-aga-amy.展开更多
Humanβ-galactosidase(β-gal)is recognized as a crucial biomarker for evaluating senescence at the cellular and tissue levels in humans.However,tools to precisely track the endogenousβ-gal are still limited.Herein,we...Humanβ-galactosidase(β-gal)is recognized as a crucial biomarker for evaluating senescence at the cellular and tissue levels in humans.However,tools to precisely track the endogenousβ-gal are still limited.Herein,we present two novel self-calibratingβ-gal probes 7a and 7b which were constructed on a unique green/red dual-emissive fluorescence platform.The two probes inherently exhibited a stable green fluorescence signal impervious toβ-gal activity,serving as a reliable internal reference.They also displayed a progressively diminishing red fluorescence signal with the increasing ofβ-gal expression levels.The dual behavior endows them with self-calibration capacity and then renders excellently selective and sensitive for precisely monitoringβ-gal activity.Notably,compared with E.coliβ-gal,the two probes are more effectively response to A.oryzaeβ-gal homologous to humanβ-gal,indicating their unique species-selectivity.Furthermore,7a was validated for its effectiveness in determining senescenceassociatedβ-galactosidase(SA-β-gal)expression in senescent NRK-52E and HepG2 cells,underscoring its practical applicability in senescence research.展开更多
A retroviral vector pINC-lacZ containing an E coli β-galactosidase(β-gal)gene was constructed and introduced into the MESPU-13 cells by electroporation.Four G418-resistant colonies were obtained from 1×107,elec...A retroviral vector pINC-lacZ containing an E coli β-galactosidase(β-gal)gene was constructed and introduced into the MESPU-13 cells by electroporation.Four G418-resistant colonies were obtained from 1×107,electroporated MESPU-13 cells. Histochemical staining for β-gal activity showed that lacZ gene was expressed in at least three of the four colonies.Southern analysis proved that one copy of foreign gene was integrated in the chromosome of one of the transformed lines(MC15).These results showed that the expression of lacZ gene driven by SiCMVIE promoter can be detected in the transformed MESPU-13 cells.展开更多
Objective:Organoids are a powerful tool with broad application prospects in biomedicine.Notably,they provide alternatives to animal models for testing potential drugs before clinical trials.However,the number of passa...Objective:Organoids are a powerful tool with broad application prospects in biomedicine.Notably,they provide alternatives to animal models for testing potential drugs before clinical trials.However,the number of passages for which organoids maintain cellular vitality ex vivo remains unclear.Methods:Herein,we constructed 55 gastric organoids from 35 individuals,serially passaged the organoids,and captured microscopic images for phenotypic evaluation.Senescence-associatedβ-galactosidase(SA-β-Gal),cell diameter in suspension,and gene expression reflecting cell cycle regulation were examined.The YOLOv3 object detection algorithm integrated with a convolutional block attention module(CBAM)was used to evaluate organoid vitality.Results:SA-β-Gal staining intensity;single-cell diameter;and expression of p15,p16,p21,CCNA2,CCNE2,and LMNB1 reflected the progression of aging in organoids during passaging.The CBAM-YOLOv3 algorithm precisely evaluated aging organoids on the basis of organoid average diameter,organoid number,and number×diameter,and the findings positively correlated with SA-β-Gal staining and single-cell diameter.Organoids derived from normal gastric mucosa had limited passaging ability(passages 1–5),before aging,whereas tumor organoids showed unlimited passaging potential for more than 45 passages(511 days)without showing clear senescence.Conclusions:Given the lack of indicators for evaluating organoid growth status,we established a reliable approach for integrated analysis of phenotypic parameters that uses an artificial intelligence algorithm to indicate organoid vitality.This method enables precise evaluation of organoid status in biomedical studies and monitoring of living biobanks.展开更多
High sensitive,accurate detection for tumor-associated overexpressed enzyme activity is highly significant for further understanding enzyme function,discovering potential drugs,and early diagnosis and prevention of di...High sensitive,accurate detection for tumor-associated overexpressed enzyme activity is highly significant for further understanding enzyme function,discovering potential drugs,and early diagnosis and prevention of diseases.In this work,we developed a facile,direct and single-step detection platform for primary ovarian cancers related glycosidase activity based on the inner filter effect(IFE)between glycosidase catalytic product and black phosphorus quantum dots(BPQDs).Highly fluorescent BPQDs were successfully synthesized from bulk black phosphorus by a simple liquid exfoliation method.Under the catalysis ofβ-galactosidase,p-nitrophenyl-β-D-galactopyranoside(PNPG)was transformed into pnitrophenol(PNP)andβ-D-galactopyranoside.Meanwhile,the absorption of catalytic product PNP greatly overlapped with the excitation and emission spectra of fluorescent BPQDs,leading to the fluorescence quenching of BPQDs with a high quenching efficiency.The proposed sensing strategy provided a low detection limit of 0.76 U/L,which was 1-2 orders of magnitude lower than most unmodified sensing platforms.D-Galactal was selected as the inhibitor forβ-galactosidase to further assess the feasibility of screening potential inhibitors.The fluorescence recovery of BPQDs suggests that the unmodified sensing platform is feasible to discover potential drugs ofβ-galactosidase.Our work paves a general way in the detection of glycosidase activity with fluorescent BPQDs,which can be promising for glycosidase-related disease diagnosis and pathophysiology elucidation.展开更多
基金a scientific research grant from Health Bureau of Sichuan Province (No. F0201)
文摘Objective To construct four recombinant Lactococcus lactis strains exhibiting high β-galactosidase activity in fusion or non-fusion ways, and to study the influence factors for their protein expression and secretion. Methods The gene fragments encoding β-galactosidase from two strains of Loctobacillus bulgaricus, wch9901 isolated from yogurt and 1.1480 purchased from the Chinese Academy of Sciences, were amplified and inserted into lactococcal expression vector pMG36e. For fusion expression, the open reading frame of the β-galactosidase gene was amplified, while for non-fusion expression, the open reading frame of the β-galactosidase gene was amplified with its native Shine-Dalgarno sequence upstream. The start codon of the β-galactosidase gene partially overlapped with the stop codon of vector origin open reading frame. Then, the recombinant plasmids were transformed into Escherichia coli DH5α and Lactococcus lactis subsp, lactis MG1363 and confirmed by determining β-galactosidase activities. Results The non-fusion expression plasmids showed a significantly higher β-galactosidase activity in transformed strains than the fusion expression plasmids. The highest enzyme activity was observed in Lactococcus lactis transformed with the non-fusion expression plasmids which were inserted into the β-galactosidase gene from Lactobacillus bulgaricus wch9901. The β-galactosidase activity was 2.75 times as high as that of the native counterpart. In addition, β-galactosidase expressed by recombinant plasmids in Lactococcus lactis could be secreted into the culture medium. The highest secretion rate （27.1%） was observed when the culture medium contained 20 g/L of lactose. Conclusion Different properties of the native bacteria may have some effects on the protein expression of recombinant plasmids. Non-fusion expression shows a higher enzyme activity in host bacteria. There may be a host-related weak secretion signal peptide gene within the structure gene of Lb. bulgaricus β-galactosidase, and its translation product may introduce the enzyme secretion out of cells in special hosts.
基金supported by the National Science Foundation of China (NO. 30800910)
文摘Objective This study is to examine the secretion effects of β-galactosidase in Lactococcus lactis.Methods The usp45 and β-galactosidase genes were cloned and inserted into plasmid pMG36e to obtain the recombinant plasmid pMG36e-usp-lacZ.This recombinant plasmid was transformed into both Escherichia coli DH5α and L.lactis MG1363.The enzyme activity,gene sequencing,SDS-PAGE and hereditary stability were assessed and studied.Results The lacZ gene inserted into plasmids pMG36e-usp-lacZ was 99.37% similar to the GenBank sequence,and SDS-PAGE revealed an evident idio-strap at 116 KDa between L.lactis MG1363/pMG36eusp-lacZ in both supernatant and cell samples.β-Galactosidase activity measured 0.225 U/mL in L.lactis pMG36e-usp-lacZ transformants,and its secretion rate was 10%.The plasmid pMG36e-usp-lacZ appeared more stable in MG1363.Conclusion The authors concluded that these new recombinant bacteria well expressed and secreted β-galactosidase,indicating that the β-galactosidase expression system was successfully constructed,and this might provide a new solution for management of lactose intolerance specifically and promote the use of gene-modified organisms as part of the food-grade plasmid in general.
基金supported by National Natural Science Foundation of China(Nos.22122803 and 21788102)the National Natural Science Foundation of Jiangsu Province(No.BK20220644).
文摘Aging-related diseases are gradually becoming a major problem with the rapid development of aged population in human society.Although many fluorescent probes have been employed to diagnosis senescence via imaging senescence-associatedβ-galactosidase(SA-β-Gal),which is proved to be closely associated with senescent cells,the similar catalytic effectiveness of enzymatic reaction of ovarian cancer-associatedβ-Gal(OA-β-Gal)will interfere with imaging accuracy.Herein,a near-infrared(NIR)hemicyanine based fluorescent probe HCyXA-βGal was designed for light-up imaging of live cells containingβ-Gal.With the organelle-targeting morpholinyl and positive charge moieties,HCyxA-βGal was successfully applicated to image the difference of enzymatic location in senescent cells and ovarian cancer cells.Furthermore,inspired by the fast response performance,fast and precise imaging of the two cell lines was realized via covering another dimension of fluorescence signal:time-dependent intensity.
基金This research is supported by the National Natural Science Foundation of China(21771065)the Natural Science Foundation of Guangdong Province,China(2017A020215088)+1 种基金Pearl River Nova Program of Guangzhou,Guangdong Province,China(201806010189)The Scientific and Technological Planning Project of Guangzhou,Guangdong Province,China(201805010002).
文摘Detection and visualization ofβ-galactosidase(β-gal)is essential to reffect its physiological and pathological effects on human health and disease,but it is still challenging to precisely trackβ-gal in vivo owing to the limitation of current analytical methods.In our work,we reported a photoacoustic(PA)nanoprobe for selective imaging of the endogenousβ-gal in vivo.Our nanoprobe Cy7-β-gal-LP was constructed by encapsulation of a near-infra red(NIR)dye Cy7-β-gal within a liposome(LP,DSPE-PEG2000-COOH).The dye Cy7-β-gal was synthesized based on a dye Cy-OH where the hydroxyl group was replaced by aβ-D-galactopyranoside residue,which can be recognized byβ-gal as an enzyme hydrolytic site.With the addition ofβ-gal,the absorbance of Cy7-β-gal exhibited a significant red shift with the absorption peak moved from 600 nm to 680 nm,which should generate a switch-on PA signal at 680 nm in the presence ofβ-gal.In addition,as theffuorescence of the dye was totally quenched due to aggregation within the liposome,Cy7-β-gal-LP exhibited high PA conversion efficiency.With the nanoprobe,we achieved the selective PA detection and imaging ofβ-gal in the tumor-bearing mice.
文摘The enzyme β-galactosidase （lactase; EC 3.2.1.23） is a commercially important enzyme due to its various applications in dairy and food industries, which are based on the β-galactosidase-catalysed hydrolysis of lactose into glucose and galactose. The objectives of this work were to identify novel and attractive sources of this industrially relevant enzyme, and to study the effect of selected growth parameters （carbon source, lactose concentration, nitrogen source, peptone concentration, initial pH and temperature） on the formation of β-galactosidase. Based on a screening of isolates from Tha Pai hot spring, Mae Hong Son Province, Thailand, strain BI.1 was selected for further studies. Strain BI.1 is a Gram-positive, rod-shaped, catalase-positive bacterium that forms endospores. Based on the sequence of the 16S rDNA determined, this isolate is most closely related to Anoxybacillus sp. and Bacillus sp., and hence the strain is designated as Bacillus sp. B 1. I.β-Galactosidase was produced by this strain with lactose and peptone as carbon and nitrogen sources, respectively. Optimal enzyme production occurred at an initial culture pH of 8.5 and at 45 ℃. Under these optimum culture conditions, maximal volumetric and specific β-galactosidase activity of 0.478 U mL^-1 and 0.338 U mg^-1 protein, respectively, were obtained after 13 h of cultivation in a medium contain 2.5% lactose, 2.0% peptone, 0.3% K2HPO4, 0.1% KH2PO4 and 0.05% MgSOa·7H2O.
文摘β-galactosidases (EC 3.2.1.23) constitute a widespread family of glycosyl hydrolases in plants and are thought to be involved in metabolism of cell wall polysaccharides. A cDNA of the cotton (Gossypium hirsutum) β-galactosidase gene, designated GhGal1, has previously been identified and its transcripts are highly abundant at the elongation stage of the cotton fiber. To examine the temporal and spatial control of GhGal1 expression, a transcriptional fusion of the GhGal1 promoter region (1770 bp) with the β-glucuronidase (GUS) reporter gene was introduced into tobacco plants by the Agrobacterium infection method. The resulting transgenic plants showed higher GUS activity of fruit in the transgenic plants than that in the negative and positive controls. Histochemical localization of GUS activity demonstrated that the expression of the GUS gene could be found in the meristem zones of roots, cotyledons, vascular tissues, fruit and trichomes in transgenic tobacco plants. Additionally, se-quence analysis of the regulatory region also revealed several conserved motifs among which some were shared with previously reported fruit/seed-specific elements and the others were related with trichome expression. These results indicated the temporal and spatial expression characterization of the GhGal1 promoter in transgenic tobacco plants and provided an important insight into the roles of GhGal1 in cotton fiber development.
文摘β-galactosidases (EC 3.2.1.23) constitute a widespread family of enzymes in plants that is thought to be involved in metabolism of cell wall polysaccharides. We reported herein the isolation of a fulllength cDNA encoding a typical β-galactosidase protein, designated GhGal1 (Gossypium hirsutum L.galactosidase), of 843 amino acids with a predicted molecular mass of nearly 94.8 kDa. In addition to a glycosyl hydrolase family 35 domain and a putative signal peptide, an unusual characteristic of GhGal 1 is that, at the C-terminus of the enzyme, a domain was found that is structurally related to a sea urchin egg lectin (SUEL-lectin) with D-galactose- and L-rhamnose-binding domains. Based on results from Southern blot, we estimated that there would be two copies of the GhGal1 gene per haploid genome of G. hirsutum.The transcripts of GhGal1 were regulated spatially and temporally and were present in very high abundance at the elongation stage of the cotton fiber. The expression pattern suggests that the GhGal1 gene could be involved in metabolism of the primary cell wall.
基金Supported by National Basic Research Program of China( 2009CB119000)National Natural Science Foundation(30871721)~~
文摘[Objective] The research aimed to construct the fusion protein expression vector of α-galactosidase-EGFP （enhanced green fluorescent protein） in cucumber controlled by CaMV35S promoter.[Method] CaMV35S promoter sequence and the coding region of EGFP were amplified by polymerase chain reactions （PCR） with vector pCambia 1303 as the template.Using reverse transcript PCR technology,with total RNAs of cucumber as template,the coding region of acid α-galactosidase Ⅰ in cucumber was amplified.The above three fragments were inserted into the multiple cloning sites of expression vector pCambia 1381c.The fusion expression vector of α-galactosidase-EGFP located at the C-terminal of the target genes was constructed.[Result] After enzyme digestion and sequencing,the fusion expression of α-galactosidase-EGFP in cucumber was constructed successfully.[Conclusion] The research laid the experimental basis for further study on the subcellular localization of α-galactosidase in cucumber.
基金Project supported by the National Natural Science Foundation of China.
文摘We constructed a plasmid that contains a small piece of DNA with two vaccinis promoters running in opposite directions——a promoter from a late gene encoding an 11 K polypeptide (P11) and a promoter from an early gene encoding 25K(P25). These promoters were isolated from the Tian Tan strain of vaccinia virus and were flanked by the thymidine kinase (TK) sequence of the same virus. Genes encoding the hepatitis B virus surface antigen (HBsAg) and the Rscherichia coli β-galactosidase (LacZ) were inserted downstream of the 11 K and 25 K promoters respectively so that coexpression plasmids were constructed. Recombinant vaccinis Viruses were selected directly by picking blue plaques formed under overlaying agarose medium containing X-gal. HBsAg was expressed to high level by these recombinant viruses. These recombinant viruses showed reduced virulence on rabbit skin and induced anti-HBs after intrsdermal inoculation of rabbits.
文摘The performance of gold and polystyrene nanoparticles was investigated using quartz crystal microbalance (QCM) as sensor platform;β-Galactosidase antibody with corresponding antigen was utilized for the immunoreaction. The development of the immunosensor included: 1) formation of self assembled monolayers on quartz crystals;2a) immobilization of p-aminothiophenol functionalized gold nanoparticles on carboxyl-terminated self assembled monolayer, or 2b) immobilization of polystyrene nanoparticles on crystals modified with p-aminothiophenol self assembled monolayer;3) attachment of monoclonal anti β-Gal on nanoparticles;and 4) detection of target analyte. The nanoparticles used were synthesized in house and characterized by transmission electron microscopy and infrared spectroscopy. The results revealed that antibodies were strongly attached to functionalized gold nanoparticles;the weaker immobilization of antibodies to polystyrene nanoparticles provoked their detachment during antigen detection. When cross reactivity of polystyrene nanoparticles was checked using a different antigen (Brucella), displacement of antibody was not recorded, demonstrating specificity of the reaction. To the best of our knowledge this is the first direct comparison between behaviors of biosensors developed with two commonly used nanoparticles. The results showed that both nanoparticles produced biosensors capable to detect β-Gal. Nevertheless biosensors developed using polystyrene nanoparticles are simpler, cheaper and more eco-friendly than those developed using gold nanoparticles.
文摘β-galactosidase was extracted from apricots (Prunus armeniaca kaisa) and characterized biochemically. Three isoenzymes (β-gal I, β-gal II and β-gal III) were obtained by salt fractionation and ionexchange and Sephadex G-100 column chromatography. β-galactosidase II showed a high ability to hy-drolyze the substrate p-nitrophenyl β-D-galactopyranoside than that of β-galactosidase I and III. The individual peaks showed charge homogeneity as revealed by single band on polyacrylamide gel. The molecular weight of β-gal I, β-gal II and β-gal III as determined by gel filtration was found to be 44.15, 34.70 and 23.71 KDa respectively. The optimum pH for the activity different isozymes was found between 4 and 6. The isoenzymes were determined to be thermally stable upto 40?C. The Km value for β-gal I was 1.85 mM which was higher than that of β-gal II (Km = 1.7), and β-gal III (Km = 1.19). The Vmax value for β-gal I, β-gal II and β-gal III was found to be 0.52, 0.70 and 0.38 μmole/min respectively.
文摘The work is intended to achieve optimum culture conditions of α-galactosidase production by a mutant strain ,Aspergillusfoetidus ZU-GI in solid-state fermentation （SSF）. Certain fermentation parameters involving moisture content, incubation temperature, cultivation period of seed, inoculum volume, initial pH value, layers of pledget, load size of medium and period of cultivation were investigated separately. The optimal cultivating conditions of α-galactosidase production in SSF were 60% initial moisture of medium, 28 ℃ incubation temperature, 18^h cultivation period of seed, 10% inoculum volume, 5.0-6.0 initial pH of medium, 6 layers of pledget and 10 g dry matter loadage. Under the optimized cultivation conditions, the maximum α-galactosidase production was 2037.51 U/g dry matter near the 144th hour of fermentation.
基金supported by the National High-Tech R&D Program of China (863 Program, 2006AA10Z317)
文摘Construction of a food-grade expression vector for application to lactic acid bacteria（LAB） is of importance for dairy fermentation system. An α-galactosidase（aga） gene encoding an enzyme degrading melibiose was amplified by PCR from the plasmid p RAF800 of Lactococcus lactis NZ9000. The aga gene was introduced into pMG36 e to substitute the p rimary antibiotic selectable marker of pMG36 e, resulting in construction of a new food-grade expression vector pMG36-aga. To testify the expression efficiency of exogenous gene in pMG36-aga, a 1.5 kb long α-amylase（amy） gene from Ba cillus li cheniformis was cloned by PCR and introduced into the plasmid pMG36-aga. The resultant plasimd pMG36-aga-amy was transformed into L. lactis ML23 by electroporation. The positive clones were selected with the medium containing melibiose as the sole carbon source. Th e selection efficiency of aga was 8.71×103 CFU with a standard deviation of 9.1×102 CFU ？g-1 DNA of pMG36-aga. Furthermore, the SDS-PAGE analysis showed that the pMG36-aga-amy expressed a 56.4 kDa protein which was the same as the putati ve molecular weight of α-amylase. The starch plate assay also indicated that L. lactis ML23 displayed high activity of α-amylase by expressing of amy gene of pMG36-aga-amy.
基金the financial support from the National Natural Science Foundation of China(Nos.21977082,22037002and 21472148)the Natural Science Basic Research Program of Shaanxi(No.2020JC-38)。
文摘Humanβ-galactosidase(β-gal)is recognized as a crucial biomarker for evaluating senescence at the cellular and tissue levels in humans.However,tools to precisely track the endogenousβ-gal are still limited.Herein,we present two novel self-calibratingβ-gal probes 7a and 7b which were constructed on a unique green/red dual-emissive fluorescence platform.The two probes inherently exhibited a stable green fluorescence signal impervious toβ-gal activity,serving as a reliable internal reference.They also displayed a progressively diminishing red fluorescence signal with the increasing ofβ-gal expression levels.The dual behavior endows them with self-calibration capacity and then renders excellently selective and sensitive for precisely monitoringβ-gal activity.Notably,compared with E.coliβ-gal,the two probes are more effectively response to A.oryzaeβ-gal homologous to humanβ-gal,indicating their unique species-selectivity.Furthermore,7a was validated for its effectiveness in determining senescenceassociatedβ-galactosidase(SA-β-gal)expression in senescent NRK-52E and HepG2 cells,underscoring its practical applicability in senescence research.
文摘A retroviral vector pINC-lacZ containing an E coli β-galactosidase(β-gal)gene was constructed and introduced into the MESPU-13 cells by electroporation.Four G418-resistant colonies were obtained from 1×107,electroporated MESPU-13 cells. Histochemical staining for β-gal activity showed that lacZ gene was expressed in at least three of the four colonies.Southern analysis proved that one copy of foreign gene was integrated in the chromosome of one of the transformed lines(MC15).These results showed that the expression of lacZ gene driven by SiCMVIE promoter can be detected in the transformed MESPU-13 cells.
基金supported by grants from the National Natural Science Foundation of China(Grant Nos.82072602 and 82173222)the Science and Technology Commission of Shanghai Municipality(Grant Nos.20DZ2201900 and 18411953100)+1 种基金the Innovation Foundation of Translational Medicine of Shanghai Jiaotong University School of Medicine(Grant No.TM202001)the Collaborative Innovation Center for Clinical and Translational Science of the Chinese Ministry of Education&Shanghai(Grant No.CCTS-2022202)。
文摘Objective:Organoids are a powerful tool with broad application prospects in biomedicine.Notably,they provide alternatives to animal models for testing potential drugs before clinical trials.However,the number of passages for which organoids maintain cellular vitality ex vivo remains unclear.Methods:Herein,we constructed 55 gastric organoids from 35 individuals,serially passaged the organoids,and captured microscopic images for phenotypic evaluation.Senescence-associatedβ-galactosidase(SA-β-Gal),cell diameter in suspension,and gene expression reflecting cell cycle regulation were examined.The YOLOv3 object detection algorithm integrated with a convolutional block attention module(CBAM)was used to evaluate organoid vitality.Results:SA-β-Gal staining intensity;single-cell diameter;and expression of p15,p16,p21,CCNA2,CCNE2,and LMNB1 reflected the progression of aging in organoids during passaging.The CBAM-YOLOv3 algorithm precisely evaluated aging organoids on the basis of organoid average diameter,organoid number,and number×diameter,and the findings positively correlated with SA-β-Gal staining and single-cell diameter.Organoids derived from normal gastric mucosa had limited passaging ability(passages 1–5),before aging,whereas tumor organoids showed unlimited passaging potential for more than 45 passages(511 days)without showing clear senescence.Conclusions:Given the lack of indicators for evaluating organoid growth status,we established a reliable approach for integrated analysis of phenotypic parameters that uses an artificial intelligence algorithm to indicate organoid vitality.This method enables precise evaluation of organoid status in biomedical studies and monitoring of living biobanks.
基金supported by the National Key R&D Program of China(No.2017YFA0208000)the National Natural Science Foundation of China(No.21675120)+1 种基金Ten Thousand Talents Program for Young Talents,the Foundation for Innovative Research Groups of the National Nature Science Foundation of China(No.21521063)the Start-up Research Fund for Prof.Q.Yuan(Nos.531107050973,531109010053)
文摘High sensitive,accurate detection for tumor-associated overexpressed enzyme activity is highly significant for further understanding enzyme function,discovering potential drugs,and early diagnosis and prevention of diseases.In this work,we developed a facile,direct and single-step detection platform for primary ovarian cancers related glycosidase activity based on the inner filter effect(IFE)between glycosidase catalytic product and black phosphorus quantum dots(BPQDs).Highly fluorescent BPQDs were successfully synthesized from bulk black phosphorus by a simple liquid exfoliation method.Under the catalysis ofβ-galactosidase,p-nitrophenyl-β-D-galactopyranoside(PNPG)was transformed into pnitrophenol(PNP)andβ-D-galactopyranoside.Meanwhile,the absorption of catalytic product PNP greatly overlapped with the excitation and emission spectra of fluorescent BPQDs,leading to the fluorescence quenching of BPQDs with a high quenching efficiency.The proposed sensing strategy provided a low detection limit of 0.76 U/L,which was 1-2 orders of magnitude lower than most unmodified sensing platforms.D-Galactal was selected as the inhibitor forβ-galactosidase to further assess the feasibility of screening potential inhibitors.The fluorescence recovery of BPQDs suggests that the unmodified sensing platform is feasible to discover potential drugs ofβ-galactosidase.Our work paves a general way in the detection of glycosidase activity with fluorescent BPQDs,which can be promising for glycosidase-related disease diagnosis and pathophysiology elucidation.
