Proteolytic stability and aggregation in a key metabolic enzyme of bacteria.

Proteins that are kinetically stable are thought to be less prone to both aggregation and proteolysis. We demonstrate that the classical lac system of Escherichia coli can be leveraged as a model system to study this relation. ß-galactosidase (LacZ) plays a critical role in lactose metabolism and is an extremely stable protein that can persist in growing cells for multiple generations after expression has stopped. By attaching degradation tags to the LacZ protein, we find that LacZ can be transiently degraded during lac operon expression but once expression has stopped functional LacZ is protected from degradation. We reversibly destabilize its tetrameric assembly using α-complementation, and show that unassembled LacZ monomers and dimers can either be degraded or lead to formation of aggregates within cells, while the tetrameric state protects against proteolysis and aggregation. We show that the presence of aggregates is associated with cell death, and that these proteotoxic stress phenotypes can be alleviated by attaching an ssrA tag to LacZ monomers which leads to their degradation. We unify our findings using a biophysical model that enables the interplay of protein assembly, degradation, and aggregation to be studied quantitatively in vivo. This work may yield approaches to reversing and preventing protein-misfolding disease states, while elucidating the functions of proteolytic stability in constant and fluctuating environments.

Development of porcine skeletal muscle extracellular matrix-derived hydrogels with improved properties and low immunogenicity.

Hydrogels derived from decellularized extracellular matrices (ECM) of animal origin show immense potential for regenerative applications due to their excellent cytocompatibility and biomimetic properties. Despite these benefits, the impact of decellularization protocols on the properties and immunogenicity of these hydrogels remains relatively unexplored. In this study, porcine skeletal muscle ECM (smECM) underwent decellularization using mechanical disruption (MD) and two commonly employed decellularization detergents, sodium deoxycholate (SDC) or Triton X-100. To mitigate immunogenicity associated with animal-derived ECM, all decellularized tissues were enzymatically treated with α-galactosidase to cleave the primary xenoantigen-the α-Gal antigen. Subsequently, the impact of the different decellularization protocols on the resultant hydrogels was thoroughly investigated. All methods significantly reduced total DNA content in hydrogels. Moreover, α-galactosidase treatment was crucial for cleaving α-Gal antigens, suggesting that conventional decellularization methods alone are insufficient. MD preserved total protein, collagen, sulfated glycosaminoglycan, laminin, fibronectin, and growth factors more efficiently than other protocols. The decellularization method impacted hydrogel gelation kinetics and ultrastructure, as confirmed by turbidimetric and scanning electron microscopy analyses. MD hydrogels demonstrated high cytocompatibility, supporting satellite stem cell recruitment, growth, and differentiation into multinucleated myofibers. In contrast, the SDC and Triton X-100 protocols exhibited cytotoxicity. Comprehensive in vivo immunogenicity assessments in a subcutaneous xenotransplantation model revealed MD hydrogels' biocompatibility and low immunogenicity. These findings highlight the significant influence of the decellularization protocol on hydrogel properties. Our results suggest that combining MD with α-galactosidase treatment is an efficient method for preparing low-immunogenic smECM-derived hydrogels with enhanced properties for skeletal muscle regenerative engineering and clinical applications.

A near-infrared fluorescent probe with a substantial Stokes shift designed for the detection and imaging of ß-galactosidase within living cells and animals.

ß-Galactosidase serves as a pivotal biomarker for both cancer and cellular aging. The advancement of fluorescent sensors for tracking ß-galactosidase activity is imperative in the realm of cancer diagnosis. We have designed a near-infrared fluorescent probe (PTA-gal) for the detection of ß-galactosidase in living systems with large Stokes shifts. PTA-gal exhibits remarkable sensitivity and selectivity in detecting ß-galactosidase, producing near-infrared fluorescent signals with a remarkably low detection limit (2.2 × 10-5 U/mL) and a high quantum yield (0.30). Moreover, PTA-gal demonstrates biocompatibility and can effectively detect ß-galactosidase in cancer cells as well as within living animals.

Determinants of raffinose family oligosaccharide use in Bacteroides species.

Bacteroides species are successful colonizers of the human colon and can utilize a wide variety of complex polysaccharides and oligosaccharides that are indigestible by the host. To do this, they use enzymes encoded in polysaccharide utilization loci (PULs). While recent work has uncovered the PULs required for the use of some polysaccharides, how Bacteroides utilize smaller oligosaccharides is less well studied. Raffinose family oligosaccharides (RFOs) are abundant in plants, especially legumes, and consist of variable units of galactose linked by α-1,6 bonds to a sucrose (glucose α-1-ß-2 fructose) moiety. Previous work showed that an α-galactosidase, BT1871, is required for RFO utilization in Bacteroides thetaiotaomicron. Here, we identify two different types of mutations that increase BT1871 mRNA levels and improve B. thetaiotaomicron growth on RFOs. First, a novel spontaneous duplication of BT1872 and BT1871 places these genes under the control of a ribosomal promoter, driving high BT1871 transcription. Second, nonsense mutations in a gene encoding the PUL24 anti-sigma factor likewise increase BT1871 transcription. We then show that hydrolases from PUL22 work together with BT1871 to break down the sucrose moiety of RFOs and determine that the master regulator of carbohydrate utilization (BT4338) plays a role in RFO utilization in B. thetaiotaomicron. Examining the genomes of other Bacteroides species, we found homologs of BT1871 in a subset and showed that representative strains of species with a BT1871 homolog grew better on melibiose than species that lack a BT1871 homolog. Altogether, our findings shed light on how an important gut commensal utilizes an abundant dietary oligosaccharide. IMPORTANCE: The gut microbiome is important in health and disease. The diverse and densely populated environment of the gut makes competition for resources fierce. Hence, it is important to study the strategies employed by microbes for resource usage. Raffinose family oligosaccharides are abundant in plants and are a major source of nutrition for the microbiota in the colon since they remain undigested by the host. Here, we study how the model commensal organism, Bacteroides thetaiotaomicron utilizes raffinose family oligosaccharides. This work highlights how an important member of the microbiota uses an abundant dietary resource.

Podocyte injury at young age causes premature senescence and worsens glomerular aging.

As life expectancy continues to rise, age-related diseases are becoming more prevalent. For example, proteinuric glomerular diseases typified by podocyte injury have worse outcomes in the elderly compared with young patients. However, the reasons are not well understood. We hypothesized that injury to nonaged podocytes induces senescence, which in turn augments their aging processes. In primary cultured human podocytes, injury induced by a cytopathic antipodocyte antibody, adriamycin, or puromycin aminonucleoside increased the senescence-related genes CDKN2A (p16INK4a/p14ARF), CDKN2D (p19INK4d), and CDKN1A (p21). Podocyte injury in human kidney organoids was accompanied by increased expression of CDKN2A, CDKN2D, and CDKN1A. In young mice, experimental focal segmental glomerulosclerosis (FSGS) induced by adriamycin and antipodocyte antibody increased the glomerular expression of p16, p21, and senescence-associated ß-galactosidase (SA-ß-gal). To assess the long-term effects of early podocyte injury-induced senescence, we temporally followed young mice with experimental FSGS through adulthood (12 m of age) and middle age (18 m of age). p16 and Sudan black staining were higher at middle age in mice with earlier FSGS compared with age-matched mice that did not get FSGS when young. This was accompanied by lower podocyte density, reduced canonical podocyte protein expression, and increased glomerular scarring. These results are consistent with injury-induced senescence in young podocytes, leading to increased senescence of podocytes by middle age accompanied by lower podocyte lifespan and health span.NEW & NOTEWORTHY Glomerular function is decreased by aging. However, little is known about the molecular mechanisms involved in age-related glomerular changes and which factors could contribute to a worse glomerular aging process. Here, we reported that podocyte injury in young mice and culture podocytes induced senescence, a marker of aging, and accelerates glomerular aging when compared with healthy aging mice.

Galactomannan utilization by Cellvibrio japonicus relies on a single essential α-galactosidase encoded by the aga27A gene.

Plant mannans are a component of lignocellulose that can have diverse compositions in terms of its backbone and side-chain substitutions. Consequently, the degradation of mannan substrates requires a cadre of enzymes for complete reduction to substituent monosaccharides that can include mannose, galactose, and/or glucose. One bacterium that possesses this suite of enzymes is the Gram-negative saprophyte Cellvibrio japonicus, which has 10 predicted mannanases from the Glycoside Hydrolase (GH) families 5, 26, and 27. Here we describe a systems biology approach to identify and characterize the essential mannan-degrading components in this bacterium. The transcriptomic analysis uncovered significant changes in gene expression for most mannanases, as well as many genes that encode carbohydrate active enzymes (CAZymes) when mannan was actively being degraded. A comprehensive mutational analysis characterized 54 CAZyme-encoding genes in the context of mannan utilization. Growth analysis of the mutant strains found that the man26C, aga27A, and man5D genes, which encode a mannobiohydrolase, α-galactosidase, and mannosidase, respectively, were important for the deconstruction of galactomannan, with Aga27A being essential. Our updated model of mannan degradation in C. japonicus proposes that the removal of galactose sidechains from substituted mannans constitutes a crucial step for the complete degradation of this hemicellulose.

Senolytic Prodrugs: A Promising Approach to Enhancing Senescence-targeting Intervention.

Cellular senescence has emerged as a potential therapeutic target for aging and a wide range of age-related disorders. Despite the encouraging therapeutic impact of senolytic agents on improving lifespan and the outcomes of pharmacological intervention, the senolytic induced side effects pose barriers to clinical application. There is a pressing need for selective ablation of senescent cells (SnCs). The design of senolytic prodrugs has been demonstrated as a promising approach to addressing these issues. These prodrugs are generally designed via modification of senolytics with a cleavable galactose moiety to respond to the senescent biomarker - senescence-associated ß-galactosidase (SA-ß-gal) to restore their therapeutic effects. In this Concept, we summarize the developments by categorizing these prodrugs into two classes: 1) galactose-modified senolytic prodrugs, in which sensing unit galactose is either directly conjugated to the drug or via a self-immolative linker and 2) bioorthogonal activation of senolytic prodrugs. In the bioorthogonal prodrug design, galactose is incorporated into dihydrotetrazine to sense SA-ß-gal for click activation. Notably, in addition to repurposed chemotherapeutics and small molecule inhibitors, PROTACs and photodynamic therapy have been introduced as new senolytics in the prodrug design. It is expected that the senolytic prodrugs would facilitate translating small-molecule senolytics into clinical use.

Cellular senescence of granulosa cells in the pathogenesis of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women of reproductive age, but its pathology has not been fully characterized and the optimal treatment strategy remains unclear. Cellular senescence is a permanent state of cell-cycle arrest that can be induced by multiple stresses. Senescent cells contribute to the pathogenesis of various diseases, owing to an alteration in secretory profile, termed 'senescence-associated secretory phenotype' (SASP), including with respect to pro-inflammatory cytokines. Senolytics, a class of drugs that selectively eliminate senescent cells, are now being used clinically, and a combination of dasatinib and quercetin (DQ) has been extensively used as a senolytic. We aimed to investigate whether cellular senescence is involved in the pathology of PCOS and whether DQ treatment has beneficial effects in patients with PCOS. We obtained ovaries from patients with or without PCOS, and established a mouse model of PCOS by injecting dehydroepiandrosterone. The expression of the senescence markers p16INK4a, p21, p53, γH2AX, and senescence-associated ß-galactosidase and the SASP-related factor interleukin-6 was significantly higher in the ovaries of patients with PCOS and PCOS mice than in controls. To evaluate the effects of hyperandrogenism and DQ on cellular senescence in vitro, we stimulated cultured human granulosa cells (GCs) with testosterone and treated them with DQ. The expression of markers of senescence and a SASP-related factor was increased by testosterone, and DQ reduced this increase. DQ reduced the expression of markers of senescence and a SASP-related factor in the ovaries of PCOS mice and improved their morphology. These results indicate that cellular senescence occurs in PCOS. Hyperandrogenism causes cellular senescence in GCs in PCOS, and senolytic treatment reduces the accumulation of senescent GCs and improves ovarian morphology under hyperandrogenism. Thus, DQ might represent a novel therapy for PCOS.

Assessment of combined α-GAL enzyme activity and lyso-GL3 for Fabry disease screening in women with chronic kidney disease.

INTRODUCTION: The spectrum of clinical presentation of Fabry disease (FD) in women is broad and challenging. The aim is to evaluate the effectiveness of an alternative screening method for FD in women. METHODS: A collaborative multicenter cross-sectional study to evaluate the sensitivity and specificity of the combination of two tests (α-GAL enzyme activity assay and lyso-GL3 assay) for the diagnosis of FD in women. We included women with chronic kidney disease (CKD) stages 3 to 5, receiving conservative treatment or on dialysis programs, from different nephrology services in Brazil. RESULTS: We evaluated 1874 patients that underwent blood collection for α-GAL and lyso-GL3 assays. Isolated decreased α-GAL enzyme activity was found in 64 patients (3.5%), while isolated increased lyso-GL3 levels were found in 67 patients (3.6%), with one patient presenting alterations in both tests. All cases with low α-GAL enzyme activity and/or increased lyso-GL3 levels underwent genetic analysis for FD variants (132 performed GLA genetic test). Low α-GAL enzyme activity had higher sensitivity and specificity to detect FD compared to the other measures (elevated lyso-GL3 alone or both altered). The negative predictive value (NPV) of α-GAL activity was 99%, and the positive predictive value (PPV) was 9.2%. For lyso-GL3 assay, the specificity was 99.7% and the PPV was 2.9%, therefore considered inferior to α-GAL assay. Both assays altered, had higher PPV (100%) and higher NPV (99.7%) considered the best method. We found 7 cases of GLA gene variants found, resulting in an initial prevalence of 0.37% for FD in this sample female population. CONCLUSION: This study contributes to the diagnostic value of the biomarkers α-GAL and lyso-GL3 in the context of FD in women with CKD. The combination of these biomarkers was an effective approach for the diagnosis of the disease, with high PPV and NPV.

Usefulness of antibody-drug conjugate as preconditioning for hematopoietic stem cell-targeted gene therapy in wild-type and Fabry disease mouse models.

BACKGROUND: Fabry disease (FD) is characterized by deficient activity of α-galactosidase A (GLA). Consequently, globotriaosylceramide (Gb3) accumulates in various organs, causing cardiac, renal, and cerebrovascular damage. Gene therapies for FD have been investigated in humans. Strong conditioning is required for hematopoietic stem cell-targeted gene therapy (HSC-GT). However, strong conditioning leads to various side effects and should be avoided. In this study, we tested antibody-based conditioning for HSC-GT in wild-type and FD model mice. METHODS: After preconditioning with an antibody-drug conjugate, HSC-GT using a lentiviral vector was performed in wild-type and Fabry model mice. In the wild-type experiment, the EGFP gene was introduced into HSCs and transplanted into preconditioned mice, and donor chimerism and EGFP expression were analyzed. In the FD mouse model, the GLA gene was introduced into HSCs and transplanted into preconditioned Fabry mice. GLA activity and Gb3 accumulation in the organs were analyzed. RESULTS: In the wild-type mouse experiment, when anti-CD45 antibody-drug conjugate was used, the percentage of donor cells at 6 months was 64.5%, and 69.6% of engrafted donor peripheral blood expressed EGFP. When anti-CD117 antibody-drug conjugate and ATG were used, the percentage of donor cells at 6 months was 80.7%, and 73.4% of engrafted donor peripheral blood expressed EGFP. Although large variations in GLA activity among mice were observed in the FD mouse experiment for both preconditioning regimens, Gb3 was significantly reduced in many organs. CONCLUSIONS: Antibody-based preconditioning may be an alternative preconditioning strategy for HSC-GT for treating FD.

Restoration of peripheral neuropathy in Fabry mice via intrathecal administration of an adeno-associated virus vector encoding mGLA cDNA.

Anderson-Fabry disease (FD) is an X-linked lysosomal storage disorder caused by a pathological variant of the α-galactosidase A (GLA) gene that results in deficient GLA activity. GLA deficiency leads to the accumulation of globotriaosylceramide (Gb3) and lyso-Gb3 in many tissues. A certain number of FD patients have burning pain or acroparesthesia in the feet and hands since childhood. Enzyme replacement therapy (ERT) is available for FD patients. However, ERT does not dramatically improve these FD-related peripheral neuropathic pain. We generated an adeno-associated virus serotype PHP.eB (AAV-PHP.eB) vector encoding mouse GLA cDNA, which was administered to FD mice intrathecally (it) or intravenously (iv). In the it-administered AAV (it-AAV) FD mice, the GLA enzyme activity in the lumbar dorsal root ganglion (DRG) was significantly greater than that in the untreated (NT) FD mice, and the level of activity was similar to that in wild-type (WT) B6 mice. However, in iv-administered AAV (iv-AAV) FD mice, GLA activity in the DRG did not increase compared to that in NT FD mice. Gb3 storage in the DRG of it-AAV FD mice was reduced compared to that in the DRG of NT FD mice. However, compared with NT FD mice, iv-AAV FD mice did not exhibit a significant reduction in the expression of the Gb3 substrate. Compared with WT mice, FD mice were thermally hyposensitive at 52 °C according to the hot plate test. The it-AAV FD mice showed significant recovery from thermal hyposensitivity. However, the iv-AAV FD mice did not exhibit significant improvement in thermal hyposensitivity. These results suggest that the intrathecal delivery of AAV-PHP.eB-mGLA may be a valuable tool for the treatment of FD-related peripheral neuropathic pain.

Raffinose catabolism enhances maize waterlogging tolerance by stimulating adventitious root growth and development.

Raffinose mitigates plant heat, drought, and cold stresses; however, whether raffinose contributes to plant waterlogging tolerance is unknown. The maize raffinose synthase mutant zmrafs-1 had seedlings that lack raffinose, generated fewer and shorter adventitious roots, and were more sensitive to waterlogging stress, while overexpression of the raffinose synthase gene, ZmRAFS, increased raffinose content, stimulated adventitious root formation, and enhanced waterlogging tolerance of maize seedlings. Transcriptome analysis of null segregant seedlings compared with zmrafs-1, particularly when waterlogged, revealed that the expression of genes related to galactose metabolism and the auxin biosynthetic pathway were up-regulated by raffinose. Additionally, indole-3-acetic acid content was significantly decreased in zmrafs-1 seedlings and increased in ZmRAFS-overexpressing seedlings. Inhibition of the hydrolysis of raffinose by 1-deoxygalactonojirimycin decreased the waterlogging tolerance of maize seedlings, the expression of genes encoding proteins related to auxin transport-related genes, and the indole-3-acetic acid level in the seedlings, indicating that the hydrolysis of raffinose is necessary for maize waterlogging tolerance. These data demonstrate that raffinose catabolism stimulates adventitious root formation via the auxin signaling pathway to enhance maize waterlogging tolerance.

Development of a thermostable Cre/lox-based gene disruption system and in vivo manipulations of the megaplasmid pTT27 in Thermus thermophilus HB27.

We previously reported the development of a Cre/lox-based gene disruption system for multiple markerless gene disruption in Thermus thermophilus; however, it was a time-consuming method because it functioned at 50 °C, the minimum growth temperature of T. thermophilus HB27. In the present study, we improved this system by introducing random mutations into the cre-expressing plasmid, pSH-Cre. One of the resulting mutant plasmids, pSH-CreFM allowed us to remove selection marker genes by Cre-mediated recombination at temperatures up to 70 °C. By using the thermostable Cre/lox system with pSH-CreFM, we successfully constructed two valuable pTT27 megaplasmid mutant strains, a plasmid-free strain and ß-galactosidase gene deletion strain, which were produced by different methods. The thermostable Cre/lox system improved the time-consuming nature of the original Cre/lox system, but it was not suitable for multiple markerless gene disruption in T. thermophilus because of its highly efficient induction of Cre-mediated recombination even at 70 °C. However, in vivo megaplasmid manipulations performed at 65 °C were faster and easier than with the original Cre/lox system. Collectively, these results indicate that this system is a powerful tool for engineering T. thermophilus megaplasmids.

Nutrient limitation and oxidative stress induce the promoter of acetate operon in Salmonella Typhimurium.

Glyoxylate shunt is an important pathway for microorganisms to survive under multiple stresses. One of its enzymes, malate synthase (encoded by aceB gene), has been widely speculated for its contribution to both the pathogenesis and virulence of various microorganisms. We have previously demonstrated that malate synthase (MS) is required for the growth of Salmonella Typhimurium (S. Typhimurium) under carbon starvation and survival under oxidative stress conditions. The aceB gene is encoded by the acetate operon in S. Typhimurium. We attempted to study the activity of acetate promoter under both the starvation and oxidative stress conditions in a heterologous system. The lac promoter of the pUC19 plasmid was substituted with the putative promoter sequence of the acetate operon of S. Typhimurium upstream to the lacZ gene and transformed the vector construct into E. coli NEBα cells. The transformed cells were subjected to the stress conditions mentioned above. We observed a fourfold increase in the ß-galactosidase activity in these cells resulting from the upregulation of the lacZ gene in the stationary phase of cell growth (nutrient deprived) as compared to the mid-log phase. Following exposure of stationary phase cells to hypochlorite-induced oxidative stress, we further observed a 1.6-fold increase in ß galactosidase activity. These data suggest the induction of promoter activity of the acetate operon under carbon starvation and oxidative stress conditions. Thus, these observations corroborate our previous findings regarding the upregulation of aceB expression under stressful environments.

Oncogene-induced senescence in meningiomas-an immunohistochemical study.

PURPOSE: Meningiomas are tumours originating from meningothelial cells, the majority belonging to grade 1 according to the World Health Organization classification of the tumours of the Central Nervous System. Factors contributing to the progression to the higher grades (grades 2 and 3) have not been elucidated yet. Senescence has been proposed as a potential mechanism constraining the malignant transformation of tumours. Senescence-associated beta-galactosidase (SA-ß-GAL) and inhibitors of cyclin-dependent kinases p16 and p21 have been suggested as senescence markers. METHODS: We analysed 318 meningiomas of total 343 (178 grade 1, 133 grade 2 and 7 grade 3). Tissue microarrays were constructed and stained immunohistochemically, using antibodies for SA-ß-GAL, p16 and p21. RESULTS: The positive correlation of the tumour grade with the expression of p16 (p = 0.016) and SA-ß-GAL (p = 0.002) was observed. The expression of p16 and SA-ß-GAL was significantly higher in meningiomas grade 2 compared to meningiomas grade 1 (p = 0.006 and p = 0.004, respectively). SA-ß-GAL positivity positively correlated with p16 and p21 in the whole cohort. In grade 2 meningiomas, a positive correlation was only between SA-ß-GAL and p16. Correlations of senescence markers in meningiomas grade 2 were not present. CONCLUSION: Our findings suggest the senescence activation in meningiomas grade 2 as a potential mechanism for the restraining of tumour growth and give hope for applying of promising senolytic therapy.

Genetic variants of unknown significance in alpha-galactosidase A: Cellular delineation from Fabry disease.

Fabry disease (FD) is an X-linked multiorgan disorder caused by variants in the alpha-galactosidase A gene (GLA). Depending on the variant, disease phenotypes range from benign to life-threatening. More than 1000 GLA variants are known, but a link between genotype and phenotype in FD has not yet been established for all. p.A143T, p.D313Y, and p.S126G are frequent examples of variants of unknown significance (VUS). We have investigated the potential pathogenicity of these VUS combining clinical data with data obtained in human cellular in vitro systems. We have analyzed four different male subject-derived cell types for alpha-galactosidase A enzyme (GLA) activity and intracellular Gb3 load. Additionally, Gb3 load in skin tissue as well as clinical data were studied for correlates of disease manifestations. A reduction of GLA activity was observed in cells carrying p.A143T compared with controls (p < 0.05). In cells carrying the p.D313Y variant, a reduced GLA activity was found only in endothelial cells (p < 0.01) compared with controls. No pathological changes were observed in cells carrying the p.S126G variant. None of the VUS investigated caused intracellular Gb3 accumulation in any cell type. Our data of aberrant GLA activity in cells of p.A143T hemizygotes and overall normal cellular phenotypes in cells of p.D313Y and p.S126G hemizygotes contribute a basic science perspective to the clinically highly relevant discussion on VUS in GLA.

Recombinant production of Paenibacillus wynnii ß-galactosidase with Komagataella phaffii.

BACKGROUND: The ß-galactosidase from Paenibacillus wynnii (ß-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used ß-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, ß-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce ß-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the ß-gal-Pw in K. phaffii either extracellularly or intracellularly. RESULTS: Firstly, 11 different signal peptides were tested for extracellular production of ß-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of ß-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular ß-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric ß-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for ß-gal-Pw production compared to the AOX1 promoter. After partial purification, a ß-gal-Pw enzyme preparation with a total ß-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter). CONCLUSION: This study showed that the ß-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular ß-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.

Secretion of the cytoplasmic and high molecular weight ß-galactosidase of Paenibacillus wynnii with Bacillus subtilis.

BACKGROUND: The gram-positive bacterium Bacillus subtilis is widely used for industrial enzyme production. Its ability to secrete a wide range of enzymes into the extracellular medium especially facilitates downstream processing since cell disruption is avoided. Although various heterologous enzymes have been successfully secreted with B. subtilis, the secretion of cytoplasmic enzymes with high molecular weight is challenging. Only a few studies report on the secretion of cytoplasmic enzymes with a molecular weight > 100 kDa. RESULTS: In this study, the cytoplasmic and 120 kDa ß-galactosidase of Paenibacillus wynnii (ß-gal-Pw) was expressed and secreted with B. subtilis SCK6. Different strategies were focused on to identify the best secretion conditions. Tailormade codon-optimization of the ß-gal-Pw gene led to an increase in extracellular ß-gal-Pw production. Consequently, the optimized gene was used to test four signal peptides and two promoters in different combinations. Differences in extracellular ß-gal-Pw activity between the recombinant B. subtilis strains were observed with the successful secretion being highly dependent on the specific combination of promoter and signal peptide used. Interestingly, signal peptides of both the general secretory- and the twin-arginine translocation pathway mediated secretion. The highest extracellular activity of 55.2 ± 6 µkat/Lculture was reached when secretion was mediated by the PhoD signal peptide and expression was controlled by the PAprE promoter. Production of extracellular ß-gal-Pw was further enhanced 1.4-fold in a bioreactor cultivation to 77.5 ± 10 µkat/Lculture with secretion efficiencies of more than 80%. CONCLUSION: For the first time, the ß-gal-Pw was efficiently secreted with B. subtilis SCK6, demonstrating the potential of this strain for secretory production of cytoplasmic, high molecular weight enzymes.

ß-Galactosidase-guided self-assembled 68Ga nanofibers probe for micro-PET tumor imaging.

ß-galactosidase (ß-gal) has high activity in various malignancies, which is suitable for targeted positron emission tomography (PET) imaging. Meanwhile, ß-gal can successfully guide the formation of nanofibers, which enhances the intensity of imaging and extends the imaging time. Herein, we designed a ß-galactosidase-guided self-assembled PET imaging probe [68Ga]Nap-NOTA-1Gal. We envisage that ß-gal could recognize and cleave the target site, bringing about self-assembling to form nanofibers, thereby enhancing the PET imaging effect. The targeting specificity of [68Ga]Nap-NOTA-1Gal for detecting ß-gal activity was examined using the control probe [68Ga]Nap-NOTA-1. Micro-PET imaging showed that tumor regions of [68Ga]Nap-NOTA-1Gal were visible after injection. And the tumor uptake of [68Ga]Nap-NOTA-1Gal was higher than [68Ga]Nap-NOTA-1 at all-time points. Our results demonstrated that the [68Ga]Nap-NOTA-1Gal can be used for the purpose of a new promising PET probe for helping diagnose cancer with high levels of ß-gal activity.

Visualized Enzyme-Activated Fluorescence Probe for Accurately Detecting ß-Gal in Living Cells and BALB/c Nude Mice.

Colorectal cancer was one of the major malignant tumors threatening human health and ß-Gal was recognized as a principal biomarker for primary colorectal cancer. Thus, designing specific and efficient quantitative detection methods for measuring ß-Gal enzyme activity was of great clinical test significance. Herein, an ultrasensitive detection method based on Turn-on fluorescence probe (CS-ßGal) was reported for visualizing the detection of exogenous and endogenous ß-galactosidase enzyme activity. The test method possessed a series of excellent performances, such as a significant fluorescence enhancement (about 11.3-fold), high selectivity as well as superior sensitivity. Furthermore, under the optimal experimental conditions, a relatively low limit of detection down to 0.024 U/mL was achieved for fluorescence titration experiment. It was thanks to the better biocompatibility and low cytotoxicity, CS-ßGal had been triumphantly employed to visual detect endogenous and exogenous ß-Gal concentration variations in living cells with noteworthy anti-interference performance. More biologically significant was the fact that the application of CS-ßGal in BALB/c nude mice was also achieved successfully for monitoring endogenous ß-Gal enzyme activity.