β-Galactosidase的应用研究

本文综述了β-galactosidase的国内外研究概况、作用机理以及来源，着重论述了β-galactosidase在畜牧生产、食品工业、制药工业等领域的应用。

Non-Fusion and Fusion Expression of β-Galactosidase from Lactobacillus bulgaricus in Lactococcus lactis

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.

Photoacoustic nanoprobe forβ-galactosidase activity detection and imaging in vivo

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.

A near-infrared fluorescent probe for fast and precise imaging of senescent cells and ovarian cancer cells via trackingβ-galactosidase

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.

载基因微泡介导β-galactosidase质粒转染心肌细胞的对照研究

目的 观察不同的载基因微泡介导体外培养心肌细胞报告基因转染与表达,探讨微泡性质 与浓度在超声破裂微泡介导基因转染过程中的效应。方法 以β galactosidase质粒为报告基因,制备两 种不同性质的载基因微泡,利用诊断性超声破裂微泡进行体外心肌细胞基因转染,测定β galactosidase表 达水平并进行细胞活性检测。结果 超声载基因破裂氟烷气体微泡(PESDA)转染组心肌细胞β galactosidase表达约为单纯质粒转染组80倍(P<0.01),并在一定范围内随微泡浓度增加而增高。超声 破裂载基因PESDA转染组基因表达水平明显高于超声破裂载基因声振白蛋白微泡(ASDA)转染组(P< 0.05),而两组对细胞活性影响却无明显差异(P>0.05)。结论 超声微泡性质及浓度对超声破裂微泡介 导基因转染效率具有重要影响,低能量诊断性超声破裂载基因PESDA较超声破裂载基因ASDA能更为 有效地介导基因转染,是一种安全高效的基因传输方法。

Isolation of the promoter of a cotton β-galactosidase gene (GhGal1) and its expression in transgenic tobacco plants

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.

Molecular Cloning and Characterization of a β-Galactosidase Gene Expressed Preferentially in Cotton Fibers

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.

SELECTION OF RECOMBINANT VACCINIA VIRUSES (TIAN TAN STRAIN) EXPRESSING HEPATITIS B VIRUS SURFACE ANTIGEN BY USING β-GALACTOSIDASE AS A MARKER*

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.

Development of Non-Labeled QCM Biosensor for the Detection of <i>β</i>-Galactosidase: A Comparative Study of Gold and Polystyrene Nanoparticles

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.

A Novel Thermostable β-Galactosidase from Geobacillus kaustophilus HTA42

A novel thermostable β-galactosidase gene, designated as GkGallA, from the thermophilic bacterium Geobacillus kaustophilus HTA426 was cloned and heterologously overexpressed in Escherichia coli（E, coli）. Based on the sequence analysis, GkGallA belongs to the glycosyl hydrolase family 1 that was the first β-galactosidase of bacterial origins expressed by us in this family. The apparent molecular weight of GkGallA determined by sodium deodecyl sulfate-polyacrylamide gel electrophoresis is 52000. It exhibited the highest activity toward p-nitrophenyl-β-D-galactopyranoside at pH 7.8 and 70℃ and displayed high thermal stability, Divalent cations are prerequisite for the activity of GKGallA, with the highest activity in the presence of Mn2＋. Moreover, the three-dimensional structure of GkGaI1A was modeled to speculate the structure of the catalytic residues and the reac- tion mechanism. The catalytic residues consisting of Glu166 and Glu355 were verified by site-directed mutagenesis.

Gene Clone and Characterization of a Novel Thermostable β-Galactosidase with Transglycosylation Activity from Thermotoga naphthophila RUK-10

We cloned and expressed a new recombinant β-galactosidase（TN0949） from Thermotoga naphthophila RKU-10 with the pET28a（＋） vector system in Escherichia coli BL21（DE3）, and determined its catalytic capability to synthesize alkyl glucosides. The recombinant enzyme was purified to a single band via heat treatment and Ni2＋-NTA affinity chromatography. The molecular mass of the recombinant enzyme was estimated to be 79 kDa with sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）. TN0949 can hydrolyze o-nitrophenylβ-D- galactopyranoside at the optimum pH and temperature of 6.5 and 80 ℃, respectively. TN0949 can also hydrolyze lactose at the optimum pH and temperature of 5.2 and 80 ℃, respectively. The Km values for the hydrolyses of o-nitrophenyl fl-D-galactopyranoside and lactose were 0.82 and 83.65 mmol/L, respectively. TN0949 was stable over a wide range of pH（3.0 to 7.0） after 24 h of incubation. The half-lives of TN0949 at 75, 80 and 85 ℃ were 22, 6 and 1.33 h, respectively. The enzyme displayed the capability to use lactose as the transglycosylation substrate to synthesize butyl galactopyranoside and hexyl galactopyranoside, indicating its suitability as a candidate industrial biocatalyst.

Kinetic Studies on <i>β</i>-Galactosidase Isolated from Apricots (<i>Prunus armeniaca kaisa</i>)

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.

Construction of a Food-Grade Expression Vector Based on pMG36e by Using an α-Galactosidase Gene as a Selectable Marker

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.

A quick and reliable image-based AI algorithm for evaluating cellular senescence of gastric organoids

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.

Enzyme replacement therapy in two patients with classic Fabry disease from the same family tree:Two case reports

BACKGROUND The pathophysiology of Fabry disease(FD)-induced progressive vital organ damage is irreversible.Disease progression can be delayed using enzyme replacement therapy(ERT).In patients with classic FD,sporadic accumulation of globotriaosylceramide(GL-3)in the heart and kidney begins in utero;however,until childhood,GL-3 accumulation is mild and reversible and can be restored by ERT.The current consensus is that ERT initiation during early childhood is paramount.Nonetheless,complete recovery of organs in patients with advanced FD is challenging.CASE SUMMARY Two related male patients,an uncle(patient 1)and nephew(patient 2),presented with classic FD.Both patients were treated by us.Patient 1 was in his 50s,and ERT was initiated following end-organ damage;this was subsequently ineffective.He developed cerebral infarction and died of sudden cardiac arrest.Patient 2 was in his mid-30s,and ERT was initiated when the patient was diagnosed with FD,during which the damage to vital organs was not overtly apparent.Although he had left ventricular hypertrophy at the beginning of this treatment,the degree of hypertrophy progression was limited to a minimal range after>18 years of ERT.CONCLUSION We obtained discouraging ERT outcomes for older patients but encouraging outcomes for younger adults with classic FD.

The Diagnostic and Therapeutic Challenges of Fabry Nephropathy—A Review of the Literature, Illustrated by a Clinical Case

Fabry Disease (FD) is a rare lysosomal storage disorder characterized by α-galactosidase A (α-Gal A) enzyme deficiency, resulting in glycosphingolipid accumulation. Its clinical spectrum ranges from severe classical to milder nonclassical or late-onset phenotypes. Renal involvement, termed Fabry Nephropathy (FN), can vary from mild proteinuria to kidney failure. FN diagnosis, especially in nonclassical cases with a genetic Variant of Unknown Significance (VUS) in the GLA gene, poses challenges. Measurement of plasma lyso-Gb3 levels is gaining importance in FN diagnosis, while renal biopsy with electron microscopy remains the gold standard in equivocal cases. Treatment options include Enzyme Replacement Therapy (ERT) and chaperone therapy, demanding careful candidate selection due to high treatment costs. Research has predominantly focused on classical FD, revealing modest treatment benefits. However, evidence for treating patients, especially females, with milder nonclassical or late-onset phenotypes is scarce, emphasizing the necessity for placebo-controlled clinical trials in these subgroups. Meanwhile, participation in global FD registries can improve our understanding of disease management. Case Presentation: A woman in her late sixties presented with moderate chronic kidney disease, mild proteinuria, and microscopic hematuria. Her family history included a prevalence of renal, cardiac and cerebrovascular diseases. Kidney biopsy revealed characteristic myelin figures and zebra bodies in podocytes, strongly suggestive of FN. Genetic analysis identified a VUS in the GLA gene (c.655A > C, p.Ile219Leu), introducing diagnostic uncertainty. Further investigations revealed severe cardiac involvement. Considering the recurring difficulty presented by the finding of a VUS in the GLA gene during FN assessments, along with the uncertainty regarding the need for treatment in nonclassical or late-onset FD phenotypes, especially in women, this case becomes a central focus for a thorough review of the literature. This review aims to propose a practical algorithm that integrates clinical, biochemical, and genetic markers for FN screening and diagnosis. Additionally, it explores treatment benefits in nonclassical or late-onset FD phenotypes, with a focus on female patients.

苜蓿中华根瘤菌(Sinorhizobium meliloti)LuxR家族转录因子ExpR调节motC操纵子的表达

目前已知苜蓿中华根瘤菌(S.meliloti)Rm1021 ExpR+突变导致胞外多糖Ⅱ(EPSⅡ)的过量表达,而胞外多糖是根瘤菌成功侵染宿主植物形成有效根瘤必需的物质。软琼脂板实验发现ExpR+突变株运动能力有缺陷。但是鞭毛染色实验并没有检测到突变株的鞭毛与野生型有什么不同。通过启动子-lacZ融合子进一步研究突变株中基因表达的差异发现,ExpR以细胞密度依赖的方式调节motC操纵子的表达。由此可见,在苜蓿中华根瘤菌中,ExpR同时参与了胞外多糖Ⅱ的合成和细胞运动能力的调节。

Robust temporal changes of cellular senescence and proliferation after sciatic nerve injury

Cellular senescence and proliferation are essential for wound healing and tissue remodeling.However,senescence-proliferation cell fate after peripheral nerve injury has not been clearly revealed.Here,post-injury gene expression patterns in rat sciatic nerve stumps(SRP113121)and L4–5 dorsal root ganglia(SRP200823)obtained from the National Center for Biotechnology Information were analyzed to decipher cellular senescence and proliferation-associated genetic changes.We first constructed a rat sciatic nerve crush model.Then,β-galactosidase activities were determined to indicate the existence of cellular senescence in the injured sciatic nerve.Ki67 and EdU immunostaining was performed to indicate cellular proliferation in the injured sciatic nerve.Both cellular senescence and proliferation were less vigorous in the dorsal root ganglia than in sciatic nerve stumps.These results reveal the dynamic changes of injury-induced cellular senescence and proliferation from both genetic and morphological aspects,and thus extend our understanding of the biological processes following peripheral nerve injury.The study was approved by the Animal Ethics Committee of Nantong University,China(approval No.20190226-001)on February 26,2019.

High Efficiency Gene Transfer to Autologous Rabbit Jugular Vein Grafts Using Recombinant Adenovirus─Transferrin／Polylysine─D

Thesedatashowthathigh-efficientgenetransductioncanbeachievedinautologousveingraftsusingadenovirus-transferin/polylysine-DNAcomplexes.Thismethodoferssomeadvantagesoverrecombinantretrovirusofadenovirusvectors.Exvivogenetherapyofveingraftsmayreducegraftocclusionbytheintroductionandhighlevelexpresionofantithromboticand/orantiproliferativegenes.