Enzymes for production of whey protein hydrolysates and other value-added products.

Whey is a byproduct of dairy industries, the aqueous portion which separates from cheese during the coagulation of milk. It represents approximately 85-95% of milk's volume and retains much of its nutrients, including functional proteins and peptides, lipids, lactose, minerals, and vitamins. Due to its composition, mainly proteins and lactose, it can be considered a raw material for value-added products. Whey-derived products are often used to supplement food, as they have shown several physiological effects on the body. Whey protein hydrolysates are reported to have different activities, including antihypertensive, antioxidant, antithrombotic, opioid, antimicrobial, cytomodulatory, and immuno-modulatory. On the other hand, galactooligosaccharides obtained from lactose can be used as prebiotic for beneficial microorganisms for the human gastrointestinal tract. All these compounds can be obtained through physicochemical, microbial, or enzymatic treatments. Particularly, enzymatic processes have the advantage of being highly selective, more stable than chemical transformations, and less polluting, making that the global enzyme market grow at accelerated rates. The sources and different products associated with the most used enzymes are particularly highlighted in this review. Moreover, we discuss metagenomics as a tool to identify novel proteolytic enzymes, from both cultivable and uncultivable microorganisms, which are expected to have new interesting activities. Finally enzymes for the transformation of whey sugar are reviewed. In this sense, carbozymes with ß-galactosidase activity are capable of lactose hydrolysis, to obtain free monomers, and transgalactosylation for prebiotics production. KEY POINTS: • Whey can be used to obtain value-added products efficiently through enzymatic treatments • Proteases transform whey proteins into biopeptides with physiological activities • Lactose can be transformed into prebiotic compounds using ß-galactosidases.

Antarctic Rahnella inusitata: A Producer of Cold-Stable ß-Galactosidase Enzymes.

There has been a recent increase in the exploration of cold-active ß-galactosidases, as it offers new alternatives for the dairy industry, mainly in response to the current needs of lactose-intolerant consumers. Since extremophilic microbial compounds might have unique physical and chemical properties, this research aimed to study the capacity of Antarctic bacterial strains to produce cold-active ß-galactosidases. A screening revealed 81 out of 304 strains with ß-galactosidase activity. The strain Se8.10.12 showed the highest enzymatic activity. Morphological, biochemical, and molecular characterization based on whole-genome sequencing confirmed it as the first Rahnella inusitata isolate from the Antarctic, which retained 41-62% of its ß-galactosidase activity in the cold (4 °C-15 °C). Three ß-galactosidases genes were found in the R. inusitata genome, which belong to the glycoside hydrolase families GH2 (LacZ and EbgA) and GH42 (BglY). Based on molecular docking, some of these enzymes exhibited higher lactose predicted affinity than the commercial control enzyme from Aspergillus oryzae. Hence, this work reports a new Rahnella inusitata strain from the Antarctic continent as a prominent cold-active ß-galactosidase producer.

H2A.Z overexpression suppresses senescence and chemosensitivity in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most intractable and devastating malignant tumors. Epigenetic modifications such as DNA methylation and histone modification regulate tumor initiation and progression. However, the contribution of histone variants in PDAC is unknown. Here, we demonstrated that the histone variant H2A.Z is highly expressed in PDAC cell lines and PDAC patients and that its overexpression correlates with poor prognosis. Moreover, all three H2A.Z isoforms (H2A.Z.1, H2A.Z.2.1, and H2A.Z.2.2) are highly expressed in PDAC cell lines and PDAC patients. Knockdown of these H2A.Z isoforms in PDAC cell lines induces a senescent phenotype, cell cycle arrest in phase G2/M, increased expression of cyclin-dependent kinase inhibitor CDKN2A/p16, SA-ß-galactosidase activity and interleukin 8 production. Transcriptome analysis of H2A.Z-depleted PDAC cells showed altered gene expression in fatty acid biosynthesis pathways and those that regulate cell cycle and DNA damage repair. Importantly, depletion of H2A.Z isoforms reduces the tumor size in a mouse xenograft model in vivo and sensitizes PDAC cells to gemcitabine. Overexpression of H2A.Z.1 and H2A.Z.2.1 more than H2A.Z.2.2 partially restores the oncogenic phenotype. Therefore, our data suggest that overexpression of H2A.Z isoforms enables cells to overcome the oncoprotective barrier associated with senescence, favoring PDAC tumor grow and chemoresistance. These results make H2A.Z a potential candidate as a diagnostic biomarker and therapeutic target for PDAC.

Production of beta-galactosidase fused to a cellulose-binding domain for application in sustainable industrial processes.

This study aimed to produce and characterize a recombinant Kluyveromyces sp. ß-galactosidase fused to a cellulose-binding domain (CBD) for industrial application. In expression assays, the highest enzymatic activities occurred after 48 h induction on Escherichia coli C41(DE3) strain at 20 °C in Terrific Broth (TB) culture medium, using isopropyl ß-d-1-thiogalactopyranoside (IPTG) 0.5 mM (108.77 U/mL) or lactose 5 g/L (93.10 U/mL) as inducers. Cultures at bioreactor scale indicated that higher product yield values in relation to biomass (2000 U/g) and productivity (0.72 U/mL.h) were obtained in culture media containing higher protein concentration. The recombinant enzyme showed high binding affinity to nanocellulose, reaching both immobilization yield and efficiency values of approximately 70% at pH 7.0 after 10 min reaction. The results of the present study pointed out a strategy for recombinant ß-galactosidase-CBD production and immobilization, aiming toward the application in sustainable industrial processes using low-cost inputs.

Effect of by-products from the dairy industry as alternative inducers of recombinant ß-galactosidase expression.

OBJECTIVE: The aim of the present study was to evaluate the efficiency of lactose derived from cheese whey and cheese whey permeate as inducer of recombinant Kluyveromyces sp. ß-galactosidase enzyme produced in Escherichia coli. Two E. coli strains, BL21(DE3) and Rosetta (DE3), were used in order to produce the recombinant enzyme. Samples were evaluated for enzyme activity, total protein content, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis after induction with isopropyl-ß-D-1-thiogalactoside (IPTG) (0.05 and 1 mM) and lactose, cheese whey, and cheese whey permeate solutions (1, 10, and 20 g/L lactose) at shake-flask cultivation, and whey permeate solution (10 g/L lactose) at bioreactor scale. RESULTS: The highest specific activities obtained with IPTG as inducer (0.05 mM) after 9 h of induction, were 23 and 33 U/mgprotein with BL21(DE3) and Rosetta(DE3) strains, respectively. Inductions performed with lactose and cheese whey permeate (10 and 20 g/L lactose) showed the highest specific activities at the evaluated hours, exhibiting better results than those obtained with IPTG. Specific activity of recombinant ß-galactosidase using whey permeate (10 g/L lactose) showed values of approximately 46 U/mgprotein after 24-h induction at shake-flask study, and approximately 26 U/mgprotein after 16-h induction at bench bioreactor. CONCLUSIONS: The induction with cheese whey permeate was more efficient for recombinant ß-galactosidase expression than the other inducers tested, and thus, represents an alternative form to reduce costs in recombinant protein production.

Loss of Dystroglycan Drives Cellular Senescence via Defective Mitosis-Mediated Genomic Instability.

Nuclear ß-dystroglycan (ß-DG) is involved in the maintenance of nuclear architecture and function. Nonetheless, its relevance in defined nuclear processes remains to be determined. In this study we generated a C2C12 cell-based DG-null model using CRISPR-Cas9 technology to provide insights into the role of ß-DG on nuclear processes. Since DG-null cells exhibited decreased levels of lamin B1, we aimed to elucidate the contribution of DG to senescence, owing to the central role of lamin B1 in this pathway. Remarkably, the lack of DG enables C2C12 cells to acquire senescent features, including cell-cycle arrest, increased senescence-associated-ß-galactosidase activity, heterochromatin loss, aberrant nuclear morphology and nucleolar disruption. We demonstrated that genomic instability is one driving cause of the senescent phenotype in DG-null cells via the activation of a DNA-damage response associated with mitotic failure, as shown by the presence of multipolar mitotic spindles, which in turn induced the formation of micronuclei and γH2AX foci (DNA-damage marker), telomere shortening and p53/p21 upregulation. Altogether, these events might ultimately lead to premature senescence, impeding the replication of the damaged genome. In summary, we present evidence supporting a role for DG in protecting against senescence, through the maintenance of proper lamin B1 expression/localization and proper mitotic spindle organization.

Microbial ß-Galactosidases of industrial importance: Computational studies on the effects of point mutations on the lactose hydrolysis reaction.

Hydrolysis efficiency of ß-galactosidases is affected due to a strong inhibition by galactose, hampering the complete lactose hydrolysis. One alternative to reduce this inhibition is to perform mutations in the enzyme's active site. The aim of this study was to evaluate the effect of point mutations on the active site of different microbial ß-galactosidases, using computational techniques. The enzymes of Aspergillus niger (AnßGal), Aspergillus oryzae (AoßGal), Bacillus circulans (BcßGal), Bifidobacterium bifidum (BbßGal), and Kluyveromyces lactis (KlßGal) were used. The mutations were carried out in all residues that were up to 4.5 Å from the galactose/lactose molecules and binding energy was computed. The mutants Tyr96Ala (AnßGal), Asn140Ala and Asn199Ala (AoßGal), Arg111Ala and Glu355Ala (BcßGal), Arg122Ala and Phe358Ala (BbßGal), Tyr523Ala, Phe620Ala, and Trp582Ala (KlßGal) had the best results, with higher effect on galactose binding energy and lower effect on lactose affinity. To maximize enzyme reactions by reducing galactose affinity, double mutations were proposed for BcßGal, BbßGal, and KlßGal. The double mutations in BcßGal and BbßGal caused the highest reduction in galactose affinity, while no satisfactory results were observed to KlßGal. Using computational tools, mutants that reduced galactose affinity without significantly affecting lactose binding were proposed. The mutations proposed can be used to reduce the negative feedback process, improving the catalytic characteristics of ß-galactosidases and rendering them promising for industrial applications.

The Clinical and Molecular Spectrum of GM1 Gangliosidosis.

OBJECTIVE: To evaluate the clinical presentation of patients with GM1 gangliosidosis and to determine whether specific clinical or biochemical signs could lead to a prompt diagnosis. STUDY DESIGN: We retrospectively analyzed clinical, biochemical, and genetic data of 22 patients with GM1 gangliosidosis from 5 metabolic centers in Germany and Austria. RESULTS: Eight patients were classified as infantile, 11 as late-infantile, and 3 as juvenile form. Delay of diagnosis was 6 ± 2.6 months in the infantile, 2.6 ± 3.79 years in the late-infantile, and 14 ± 3.48 years in the juvenile form. Coarse facial features, cherry red spots, and visceromegaly occurred only in patients with the infantile form. Patients with the late-infantile and juvenile forms presented with variable neurologic symptoms. Seventeen patients presented with dystonia and 14 with dysphagia. Laboratory analysis revealed an increased ASAT concentration (13/20), chitotriosidase activity (12/15), and pathologic urinary oligosaccharides (10/19). Genotype analyses revealed 23 causative or likely causative mutations in 19 patients, 7 of them being novel variants. In the majority, a clear genotype-phenotype correlation was found. CONCLUSIONS: Diagnosis of GM1 gangliosidosis often is delayed, especially in patients with milder forms of the disease. GM1 gangliosidosis should be considered in patients with progressive neurodegeneration and spastic-dystonic movement disorders, even in the absence of visceral symptoms or cherry red spots. ASAT serum concentrations and chitotriosidase activity may be of value in screening for GM1 gangliosidosis.

Calcium chloride treatment modifies cell wall metabolism and activates defense responses in strawberry fruit (Fragaria × ananassa, Duch).

BACKGROUND: Fruit dips in calcium ions solutions have been shown as an effective treatment to extend strawberries (Fragaria × ananassa, Duch) quality during storage. In the present work, strawberry fruit were treated with 10 g L-1 calcium chloride solution and treatment effects on cell wall enzymes activities and the expression of encoding genes, as well as enzymes involved in fruit defense responses were investigated. RESULTS: Calcium treatment enhanced pectin methylesterase activity while inhibited those corresponding to pectin hydrolases as polygalacturonase and ß-galactosidase. The expression of key genes for strawberry pectin metabolism was up-regulated (for FaPME1) and down-regulated (for FaPG1, FaPLB, FaPLC, FaßGal1 and FaAra1) by calcium dips. In agreement, a higher firmness level and ionically-bound pectins (IBPs) amount were detected in calcium-treated fruit compared with controls. The in vitro and in vivo growth rate of fungal pathogen Botrytis cinerea was limited by calcium treatment. Moreover, the activities of polyphenol oxidases, chitinases, peroxidases and ß-1,3-glucanases were enhanced by calcium ion dips. CONCLUSION: News insights concerning the biochemical and molecular basis of cell wall preservation and resistance to fungal pathogens on calcium-treated strawberries are provided. © 2019 Society of Chemical Industry.

ß-Galactosidase from Exiguobacterium acetylicum: Cloning, expression, purification and characterization.

The main goal of this work was to evaluate the performance of ß-galactosidase from Exiguobacterium acetylicum MF03 in both hydrolysis and transgalactosylation reactions from different substrates. The enzyme gene was expressed in Escherichia coli BL21 (DE3), sequenced, and subjected to bioinformatic and kinetic assessment. Results showed that the enzyme was able to hydrolyze lactulose and o-nitrophenyl-ß-d-galactopyranoside, but unable to hydrolyze lactose, o-nitrophenyl-ß-d-glucopyranoside, butyl- and pentyl-ß-d-galactosides. This unique and novel substrate specificity converts the E. acetylicum MF03 ß-galactosidase into an ideal catalyst for the formulation of an enzymatic kit for lactulose quantification in thermally processed milk. This is because costly steps to eliminate glucose (resulting from hydrolysis of lactose when a customary ß-galactosidase is used) can be avoided.

Superactive ß-galactosidase inclusion bodies.

Bacterial inclusion bodies (IBs) were historically considered one of the major obstacles in protein production through recombinant DNA techniques and conceived as amorphous deposits formed by passive and rather unspecific structures of unfolded proteins aggregates. Subsequent studies demonstrated that IBs contained an important quantity of active protein. In this work, we proved that recombinant ß-galactosidase inclusion bodies (IBß-Gal) are functional aggregates. Moreover, they exhibit particular features distinct to the soluble version of the enzyme. The particulate enzyme was highly active against lactose in physiological and in acid pH and also retained its activity upon a pre-incubation at high temperature. IBß-Gal washing or dilution induced the spontaneous release of active enzymes from the supramolecular aggregates. Along this process, we observed a continuous change in the values of several kinetic parameters, including specific activity and Michaelis-Menten constant, measured in the IBß-Gal suspensions. Simultaneously, IBß-Gal turned into a more heterogeneous population where smaller particles appeared. The released protein exhibited secondary structure features more similar to those of the soluble species than to the aggregated enzyme. Concluding, IBß-Gal represents a reservoir and packed source of highly active and stable enzyme.

Characterization of a ß-galactosidase from Bacillus subtilis with transgalactosylation activity.

Microbial ß-galactosidases (EC 3.1.2.23) have applications in the production of galacto-oligosaccharides, which are established prebiotic food ingredients. The ß-galactosidase from Bacillus subtilis (YesZ) was expressed as a heterologous protein in Escherichia coli, and presented an optimum activity at pH 6.5 and 40 °C. The catalytic constants Km and Vmax of the enzyme were 8.26 mM and 1.42 µmol·min-1·mg-1 against pNP-ß-d-galactopyranoside, respectively. Structural characterization revealed that YesZ is a homotrimer in solution, and homology modeling suggested that the YesZ conserves a Cys cluster zinc binding site. Flame photometry experiments confirmed the presence of bound zinc in the recombinant enzyme, and YesZ activity was inhibited by 1 mM zinc, copper and silver ions. Transgalactosylation activity of YesZ was observed with the synthetic substrate p-NP-ßGal in the presence of a d-xylose acceptor, producing a ß-d-galactopyranosyl-(1 → 4)-d-xylopyranose disaccharide. Analysis of this disaccharide by MALDI-ToF-MS/MS suggested a ß-1,4 glycosidic linkage between a non-reducing galactose residue and the xylose. The ß-galactosidase YesZ from B. subtilis is a candidate for enzymatic synthesis showing favorable thermostability (with residual activity of 50% after incubation at 30 °C for 25 h) and transgalactosylation activity.

Biochemical characterization of three Aspergillus niger ß-galactosidases

Background: ß-Galactosidases catalyze both hydrolytic and transgalactosylation reactions and therefore have many applications in food, medical, and biotechnological fields. Aspergillus niger has been a main source of ß-galactosidase, but the properties of this enzyme are incompletely studied. Results: Three new ß-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned, expressed, and biochemically characterized. In addition to the known activity of LacA encoded by lacA, three putative ß-galactosidases, designated as LacB, LacC, and LacE encoded by the genes lacB, lacC, and lacE, respectively, were successfully cloned, sequenced, and expressed and secreted by Pichia pastoris. These three proteins and LacA have N-terminal signal sequences and are therefore predicted to be extracellular enzymes. They have the typical structure of fungal ß-galactosidases with defined hydrolytic and transgalactosylation activities on lactose. However, their activity properties differed. In particular, LacB and lacE displayed maximum hydrolytic activity at pH 4­5 and 50°C, while LacC exhibited maximum activity at pH 3.5 and 60°C. All ß-galactosidases performed transgalactosylation activity optimally in an acidic environment. Conclusions: Three new ß-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned and biochemically characterized. In addition to the known LacA, A. niger has at least three ß-galactosidase family members with remarkably different biochemical properties.

Human umbilical cord-derived mesenchymal stromal cells protect against premature renal senescence resulting from oxidative stress in rats with acute kidney injury.

BACKGROUND: Mesenchymal stromal cells (MSCs) represent an option for the treatment of acute kidney injury (AKI). It is known that young stem cells are better than are aged stem cells at reducing the incidence of the senescent phenotype in the kidneys. The objective of this study was to determine whether AKI leads to premature, stress-induced senescence, as well as whether human umbilical cord-derived MSCs (huMSCs) can prevent ischaemia/reperfusion injury (IRI)-induced renal senescence in rats. METHODS: By clamping both renal arteries for 45 min, we induced IRI in male rats. Six hours later, some rats received 1 × 106 huMSCs or human adipose-derived MSCs (aMSCs) intraperitoneally. Rats were euthanised and studied on post-IRI days 2, 7 and 49. RESULTS: On post-IRI day 2, the kidneys of huMSC-treated rats showed improved glomerular filtration, better tubular function and higher expression of aquaporin 2, as well as less macrophage infiltration. Senescence-related proteins (ß-galactosidase, p21Waf1/Cip1, p16INK4a and transforming growth factor beta 1) and microRNAs (miR-29a and miR-34a) were overexpressed after IRI and subsequently downregulated by the treatment. The IRI-induced pro-oxidative state and reduction in Klotho expression were both reversed by the treatment. In comparison with huMSC treatment, the treatment with aMSCs improved renal function to a lesser degree, as well as resulting in a less pronounced increase in the renal expression of Klotho and manganese superoxide dismutase. Treatment with huMSCs ameliorated long-term kidney function after IRI, minimised renal fibrosis, decreased ß-galactosidase expression and increased the expression of Klotho. CONCLUSIONS: Our data demonstrate that huMSCs attenuate the inflammatory and oxidative stress responses occurring in AKI, as well as reducing the expression of senescence-related proteins and microRNAs. Our findings broaden perspectives for the treatment of AKI.

Effects of ginsenoside Rg1 on the senescence of vascular smooth muscle cells.

The development of age-related cardiovascular disease is associated with the senescence of vascular cells. This study aimed to investigate the effect of ginsenoside Rg1 on vascular smooth muscle cell (VSMC) senescence. Primary VSMCs were cultured and divided into control, D-galactose (D-gal), Rg1-L, and Rg1-H groups, which were cultured without and with D-gal, and with low- and high-concentrations of Rg1, respectively. D-gal-induced cellular senescence was identified by b-galactosidase staining, and ultrastructural changes within the cells were observed. The expression of p16, p21, and p53 in the four groups of VSMCs was determined by western blotting, and the cell cycle was investigated by flow cytometry. Compared with the control group, there was an obvious change in the ultrastructure of VSMCs in the D-gal group, and the proportion of b-galactosidase-positive cells was significantly increased (P < 0.05). In addition, p16, p21, and p53 expression was significantly increased (P < 0.05) and the cell cycle was arrested in the G0/G1 phase. Compared with the D-gal group, the percentage of positive cells was significantly reduced (P < 0.05) in the Rg1 groups, the expression of p16, p21, and p53 was significantly reduced (P < 0.05), and the number of cells in the G0/G1 phase decreased (P < 0.05). Ginsenoside Rg1 can inhibit VSMC senescence, and the mechanisms may be related to its partial inhibition of the p16INK4a/Rb and p53-p21Cip1/Waf1 signaling pathways during the cell cycle.

Interferon-ß gene transfer induces a strong cytotoxic bystander effect on melanoma cells.

A local gene therapy scheme for the delivery of type I interferons could be an alternative for the treatment of melanoma. We evaluated the cytotoxic effects of interferon-ß (IFNß) gene lipofection on tumor cell lines derived from three human cutaneous and four canine mucosal melanomas. The cytotoxicity of human IFNß gene lipofection resulted higher or equivalent to that of the corresponding addition of the recombinant protein (rhIFNß) to human cells. IFNß gene lipofection was not cytotoxic for only one canine melanoma cell line. When cultured as monolayers, three human and three canine IFNß-lipofected melanoma cell lines displayed a remarkable bystander effect. As spheroids, the same six cell lines were sensitive to IFNß gene transfer, two displaying a significant multicell resistance phenotype. The effects of conditioned IFNß-lipofected canine melanoma cell culture media suggested the release of at least one soluble thermolabile cytotoxic factor that could not be detected in human melanoma cells. By using a secretion signal-free truncated human IFNß, we showed that its intracellular expression was enough to induce cytotoxicity in two human melanoma cell lines. The lower cytoplasmatic levels of reactive oxygen species detected after intracellular IFNß expression could be related to the resistance displayed by one human melanoma cell line. As IFNß gene transfer was effective against most of the assayed melanomas in a way not limited by relatively low lipofection efficiencies, the clinical potential of this approach is strongly supported.

Cell damage detection using Escherichia coli reporter plasmids: fluorescent and colorimetric assays.

Bacterial reporter assays are powerful tools used to study the effect of different compounds that affect the physiology of cellular processes. Most bacterial reporters are luciferase based and can be monitored in real time. In the present study we designed and implemented two sets of Escherichia coli bacterial reporter assays, using a multicopy plasmid system. Each reporter strain was constructed using either green fluorescent protein or ß-galactosidase (LacZ) proteins. The designed reporter strains are capable of responding in a specific manner to molecules that either oxidative stress, or membrane, protein, or DNA damage. In order to respond to the desired stimulus, promoter sequences from E. coli were used. These sequences correspond to the promoter of the major catalase (KatG) activated with cellular oxidative damage, the promoter of the ß-hydroxydecanoyl-ACP dehydrase (FabA) which is activated with membrane perturbation, the promoter of DNA recombinase (RecA) which is activated by DNA lesions. For protein misfolding, the promoter of the heat-shock responsive chaperon (DnaK) was used. Our constructs displayed activation to damage from specific stimuli, and low response to nonspecific stimuli was detected. Our results suggest that these types of bacterial reporter strains can be used in semiquantitative (fluorometric) and qualitative (ß-galactosidase activity) studies of different xenobiotic substances and pollutants.

The use of lacZ marker in enumeration of Azotobacter chroococcum in carrier based inoculants.

A transconjugant of Azotobacter chroococcum Mac 27 tagged with lac Z(A. chroococcum Mac27 L) was found to possess high levels of ß-galactosidase activity constitutively. Further, the lac Z marker was found to be stably integrated into the chromosome of the A. chroococcum Mac 27 and did not have any adverse effect on growth, nitrogen fixation and excretion of ammonia. A quick method to determine the viable cell number in broth culture and carrier based inoculants has been developed on the basis of ß-galactosidase assay. It was found that there was a direct relationship between the number of cell as determined by standard plate count and intensity of colour that developed upon degradation of ONPG due to ß-galactosidase activity. The method was found to be sensitive enough to determine 1.7 × 10(6) CFU mL(-1) in broth culture as well as carrier based Azotobacter inoculants. Further, it was observed that when A. chroococcum Mac27 L was inoculated on Brassica campestris, it could be detected in the presence of other bacteria capable of growing on Burks agar medium containing X-gal on the basis of lac Z genetic marker.

Physiology of digestion and the molecular characterization of the major digestive enzymes from Periplaneta americana.

Cockroaches are among the first insects to appear in the fossil record. This work is part of ongoing research on insects at critical points in the evolutionary tree to disclose evolutionary trends in the digestive characteristics of insects. A transcriptome (454 Roche platform) of the midgut of Periplanetaamericana was searched for sequences of digestive enzymes. The selected sequences were manually curated. The complete or nearly complete sequences showing all characteristic motifs and highly expressed (reads counting) had their predicted sequences checked by cloning and Sanger sequencing. There are two chitinases (lacking mucin and chitin-binding domains), one amylase, two α- and three ß-glucosidases, one ß-galactosidase, two aminopeptidases (none of the N-group), one chymotrypsin, 5 trypsins, and none ß-glucanase. Electrophoretic and enzymological data agreed with transcriptome data in showing that there is a single ß-galactosidase, two α-glucosidases, one preferring as substrate maltase and the other aryl α-glucoside, and two ß-glucosidases. Chromatographic and enzymological data identified 4 trypsins, one chymotrypsin (also found in the transcriptome), and one non-identified proteinase. The major digestive trypsin is identifiable to a major P. americana allergen (Per a 10). The lack of ß-glucanase expression in midguts was confirmed, thus lending support to claims that those enzymes are salivary. A salivary amylase was molecularly cloned and shown to be different from the one from the midgut. Enzyme distribution showed that most digestion occurs under the action of salivary and midgut enzymes in the foregut and anterior midgut, except the posterior terminal digestion of proteins. A counter-flux of fluid may be functional in the midgut of the cockroach to explain the low excretory rate of digestive enzymes. Ultrastructural and immunocytochemical localization data showed that amylase and trypsin are released by both merocrine and apocrine secretion mainly from gastric caeca. Finally, a discussion on Polyneoptera digestive physiology is provided.