Development of a strictly regulated xylose-induced expression system in Streptomyces.

BACKGROUND: Genetic tools including constitutive and inducible promoters have been developed over the last few decades for strain engineering in Streptomyces. Inducible promoters are useful for controlling gene expression, however only a limited number are applicable to Streptomyces. The aim of this study is to develop a controllable protein expression system based on an inducible promoter using sugar inducer, which has not yet been widely applied in Streptomyces. RESULTS: To determine a candidate promoter, inducible protein expression was first examined in Streptomyces avermitilis MA-4680 using various carbon sources. Xylose isomerase (xylA) promoter derived from xylose (xyl) operon was selected due to strong expression of xylose isomerase (XylA) in the presence of D-xylose. Next, a xylose-inducible protein expression system was constructed by investigating heterologous protein expression (chitobiase as a model protein) driven by the xylA promoter in Streptomyces lividans. Chitobiase activity was detected at high levels in S. lividans strain harboring an expression vector with xylA promoter (pXC), under both xylose-induced and non-induced conditions. Thus, S. avermitilis xylR gene, which encodes a putative repressor of xyl operon, was introduced into constructed vectors in order to control protein expression by D-xylose. Among strains constructed in the study, XCPR strain harboring pXCPR vector exhibited strict regulation of protein expression. Chitobiase activity in the XCPR strain was observed to be 24 times higher under xylose-induced conditions than that under non-induced conditions. CONCLUSION: In this study, a strictly regulated protein expression system was developed based on a xylose-induced system. As far as we could ascertain, this is the first report of engineered inducible protein expression in Streptomyces by means of a xylose-induced system. This system might be applicable for controllable expression of toxic products or in the field of synthetic biology using Streptomyces strains.

Alteration of a recombinant protein N-glycan structure in silkworms by partial suppression of N-acetylglucosaminidase gene expression.

OBJECTIVE: To synthesize complex type N-glycans in silkworms, shRNAs against the fused lobe from Bombyx mori (BmFDL), which codes N-acetylglucosaminidase (GlcNAcase) in the Golgi, was expressed by recombinant B. mori nucleopolyhedrovirus (BmNPV) in silkworm larvae. RESULTS: Expression was under the control of the actin promoter of B. mori or the U6-2 and i.e.-2 promoters from Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). The reduction of specific GlcNAcase activity was observed in Bm5 cells and silkworm larvae using the U6-2 promoter. In silkworm larvae, the partial suppression of BmFDL gene expression was observed. When shRNA against BmFDL was expressed under the control of U6-2 promoter, the Man3GlcNAc(Fuc)GlcNAc structure appeared in a main N-glycans of recombinant human IgG. These results suggested that the control of BmFDL expression by its shRNA in silkworms caused the modification of its N-glycan synthetic pathway, which may lead to the alteration of N-glycans in the expressed recombinant proteins. CONCLUSIONS: Suppression of BmFDL gene expression by shRNA is not sufficient to synthesize complex N-glycans in silkworm larvae but can modify the N-glycan synthetic pathway.

Corynebacterium glutamicum possesses ß-N-acetylglucosaminidase.

BACKGROUND: In Gram-positive Corynebacterium glutamicum and other members of the suborder Corynebacterianeae, which includes mycobacteria, cell elongation and peptidoglycan biosynthesis is mainly due to polar growth. C. glutamicum lacks an uptake system for the peptidoglycan constituent N-acetylglucosamine (GlcNAc), but is able to catabolize GlcNAc-6-phosphate. Due to its importance in white biotechnology and in order to ensure more sustainable processes based on non-food renewables and to reduce feedstock costs, C. glutamicum strains have previously been engineered to produce amino acids from GlcNAc. GlcNAc also is a constituent of chitin, but it is unknown if C. glutamicum possesses chitinolytic enzymes. RESULTS: Chitin was shown here not to be growth substrate for C. glutamicum. However, its genome encodes a putative N-acetylglucosaminidase. The nagA 2 gene product was active as ß-N-acetylglucosaminidase with 0.27 mM 4-nitrophenyl N,N'-diacetyl-ß-D-chitobioside as substrate supporting half-maximal activity. NagA2 was secreted into the culture medium when overproduced with TAT and Sec dependent signal peptides, while it remained cytoplasmic when overproduced without signal peptide. Heterologous expression of exochitinase gene chiB from Serratia marcescens resulted in chitinolytic activity and ChiB secretion was enhanced when a signal peptide from C. glutamicum was used. Colloidal chitin did not support growth of a strain secreting exochitinase ChiB and ß-N-acetylglucosaminidase NagA2. CONCLUSIONS: C. glutamicum possesses ß-N-acetylglucosaminidase. In the wild type, ß-N-acetylglucosaminidase activity was too low to be detected. However, overproduction of the enzyme fused to TAT or Sec signal peptides led to secretion of active ß-N-acetylglucosaminidase. The finding that concomitant secretion of endogenous NagA2 and exochitinase ChiB from S. marcescens did not entail growth with colloidal chitin as sole or combined carbon source, may indicate the requirement for higher or additional enzyme activities such as processive chitinase or endochitinase activities.

Expression, purification, crystallization and preliminary crystallographic analysis of a GH20 ß-N-acetylglucosaminidase from the marine bacterium Vibrio harveyi.

Vibrio harveyi ß-N-acetylglucosaminidase (VhGlcNAcase) is a new member of the GH20 glycoside hydrolase family responsible for the complete degradation of chitin fragments, with N-acetylglucosamine (GlcNAc) monomers as the final products. In this study, the crystallization and preliminary crystallographic data of wild-type VhGlcNAcase and its catalytically inactive mutant D437A in the absence and the presence of substrate are reported. Crystals of wild-type VhGlcNAcase were grown in 0.1 M sodium acetate pH 4.6, 1.4 M sodium malonate, while crystals of the D437A mutant were obtained in 0.1 M bis-tris pH 7.5, 0.1 M sodium acetate, 20% PEG 3350. X-ray data from the wild-type and the mutant crystals were collected at a synchrotron-radiation light source and were complete to a resolution of 2.5 Å. All crystals were composed of the same type of dimer, with the substrate N,N'-diacetylglucosamine (GlcNAc2 or diNAG) used for soaking was cleaved by the active enzyme, leaving only a single GlcNAc molecule bound to the protein.

Recombinant, truncated B. circulans keratanase-II: Description and characterisation of a novel enzyme for use in measuring urinary keratan sulphate levels via LC-MS/MS in Morquio A syndrome.

OBJECTIVE: Morquio A syndrome (mucopolysaccharidosis IVA; MPS IVA) is an autosomal recessive lysosomal storage disorder caused by deficient N-acetylgalactosamine-6-sulphatase (GALNS) activity. Early and accurate diagnosis of this condition is critical for improved patient outcomes, particularly as enzyme replacement therapy has recently become available. An LC-MS/MS assay utilising keratan sulphate (KS) disaccharides derived from keratanase-II digestion provides a sensitive and specific means for quantitation of urinary KS, a screening biomarker for Morquio A (Oguma et al., 2007; Martell et al., 2011). To ensure a reliable supply of keratanase-II, we sought to produce a Bacillus circulans-derived enzyme via a recombinant approach in Escherichia coli. DESIGN AND METHODS: Bioinformatics analysis of the B. circulans keratanase-II enzyme identified likely dispensable C-terminal domains amenable to enhancement via protein engineering. A truncated form of the enzyme was designed to remove the domains predicted to be unnecessary for catalytic activity and detrimental to recombinant expression in E. coli. RESULTS: C-terminally truncated, recombinant B. circulans keratanase-II was purified to >98% homogeneity and extensively characterised, demonstrating desired activity, specificity and utility in LC-MS-based quantitation of urinary KS from Morquio A and control samples, and is functionally indistinguishable from full-length, native B. circulans-derived keratanase-II. CONCLUSIONS: This novel, recombinant keratanase-II meets all performance requirements and can be produced in a rapid and reproducible manner. We speculate that other related bacterial enzymes of biomedical or industrial interest may be amenable to similar engineered enhancements.

High-level expression of endo-ß-N-acetylglucosaminidase H from Streptomyces plicatus in Pichia pastoris and its application for the deglycosylation of glycoproteins.

Endo-ß-N-acetylglucosaminidase H (Endo H, EC3.2.1.96) is a glycohydrolase that is widely used in the study of glycoproteins. The present study aimed to assess the effect of high-level endo-ß-N-acetylglucosaminidase H expression in Pichia pastoris. The DNA coding sequence of this enzyme was optimized based on the codon usage bias of Pichia pastoris and synthesized through overlapping PCR. This novel gene was cloned into a pHBM905A vector and introduced into Pichia pastoris GS115 for secretary expression. The yield of the target protein reached approximately 397 mg/l after a 6-d induction with 1% (v/v) methanol in shake flasks, which is much higher than that observed upon heterologous expression in Escherichia coli and silkworm. This recombinant enzyme was purified and its enzymatic features were studied. Its specific activity was 461573 U/mg. Its optimum pH and temperature were pH 5.5 and 37°C, respectively. Moreover, our study showed that the N-linked glycan side-chains of several recombinant proteins expressed in Pichia pastoris can be efficiently removed through either the co-fermentation of this recombinant strain with strains expressing substrates or by mixing the cell culture supernatants of the endo-ß-N-acetylglucosaminidase H expressing strain with strains expressing substrates after fermentation. This is the first report of high-level endo-ß-N-acetylglucosaminidase H expression in Pichia pastoris and the application of this enzyme in the deglycosylation of raw glycoproteins heterologously expressed in Pichia pastoris using simplified methods.

Antibacterial metabolites and bacteriolytic enzymes produced by Bacillus pumilus during bacteriolysis of Arthrobacter citreus.

The marine isolate Bacillus pumilus SBUG 1800 is able to lyse living cells of Arthrobacter citreus on solid media as well as pasteurized A. citreus cells in liquid mineral salt medium. The cultivation of B. pumilus in the presence of pasteurized A. citreus is accompanied by an enhanced production of 2,5-diketopiperazines (DKPs). DKPs inhibit bacterial growth, but do not seem to cause bacteriolysis. This study shows that B. pumilus also lyses living cells of A. citreus in co-culture experiments as an intraguild predator, even if the inoculum of B. pumilus is low. In order to characterize the bacteriolytic process, more precisely changes in the extracellular metabolome and proteome have been analyzed under different culture conditions. Besides the known DKPs, a number of different pumilacidins and bacteriolytic enzymes are produced. Two lipopeptides with [M + H](+) = 1008 and [M + H](+) = 1022 were detected and are proposed to be pumilacidin H and I. While the lipopeptides lyse living bacterial cells in lysis test assays, a set of extracellular enzymes degrades the dead cell material. Two of the cell wall hydrolases involved have been identified as N-acetylmuramoyl-L-alanine amidase and beta-N-acetylglucosaminidase. These findings together with electron microscopic and cell growth monitoring during co-culture experiments give a detailed view on the bacteriolytic process.

Chitinase but N-acetyl-ß-D-glucosaminidase production correlates to the biomass decline in Penicillium and Aspergillus species.

Hydrolytic enzyme production is typical of the autolysis in filamentous fungi; however, less attention has been given to the physiological role of the enzymes. Here, the aim was to investigate the possible relation of the chitinolytic enzymes to the changes in the biomass in some filamentous fungi of high importance for pharmaceutical or food industry. In Penicillium and Aspergillus filamentous fungi, which showed different characteristics in submerged cultures, the growth and biomass decline rates were calculated and correlated to the chitinase and N-acetyl-ß-D-glucosaminidase enzyme productions. Correlation was found between the biomass decrease rate and the chitinase level at the stationary growth phase; while chitinase production covariates negatively with N-acetyl-ß-D-glucosaminidase activities. The chitinase production and the intensive autolysis hindered the production of N-acetyl-ß-D-glucosaminidase and, therefore, could hinder the cell death in the cultures.

Insulin-like growth factor II peptide fusion enables uptake and lysosomal delivery of α-N-acetylglucosaminidase to mucopolysaccharidosis type IIIB fibroblasts.

Enzyme replacement therapy for MPS IIIB (mucopolysaccharidosis type IIIB; also known as Sanfilippo B syndrome) has been hindered by inadequate mannose 6 phosphorylation and cellular uptake of rhNAGLU (recombinant human α-N-acetylglucosaminidase). We expressed and characterized a modified rhNAGLU fused to the receptor-binding motif of IGF-II (insulin-like growth factor 2) (rhNAGLU-IGF-II) to enhance its ability to enter cells using the cation-independent mannose 6-phosphate receptor, which is also the receptor for IGF-II (at a different binding site). RhNAGLU-IGF-II was stably expressed in CHO (Chinese-hamster ovary) cells, secreted and purified to apparent homogeneity. The Km and pH optimum of the fusion enzyme was similar to those reported for rhNAGLU. Both intracellular uptake and confocal microscopy suggested that MPS IIIB fibroblasts readily take up the fusion enzyme via receptor-mediated endocytosis that was inhibited significantly (P<0.001) by the monomeric IGF-II peptide. Glycosaminoglycan storage was reduced by 60% (P<0.001) to near background levels in MPS IIIB cells after treatment with rhNAGLU-IGF-II, with half-maximal correction at concentrations of 3-12 pM. A similar cellular uptake mechanism via the IGF-II receptor was also demonstrated in two different brain tumour-derived cell lines. Fusion of rhNAGLU to IGF-II enhanced its cellular uptake while maintaining enzymatic activity, supporting its potential as a therapeutic candidate for treating MPS IIIB.

Chitinases biosynthesis by immobilized Aeromonas hydrophila SBK1 by prawn shells valorization and application of enzyme cocktail for fungal protoplast preparation.

Production and optimization of ß-N-acetyl glucosaminidase and chitinase by Ca-alginate immobilized Aeromonas hydrophila SBK1 was carried out using prawn shell as cost-effective substrate. Beads prepared with 5.0% Na-alginate (containing 2.0% colloidal chitin) and 1.0 M CaCl2 showed considerable beads integrity and supported maximum production of chitinolytic enzymes. Bead diameter, 3 mm; temperature, 35°C; pH 7.0; agitation, 90 rpm were found ideal for the maximum production of the enzymes. The fermentation and thermodynamic indices revealed the feasibility of immobilized cells over free cells for enzymes production. Reasonable amount of chitosaccharides (degree of polymerization; 1-6) accumulated in the production media which have paramount antioxidant activity. Scale up experiment was successfully carried out in 5 L fermentor. In immobilized state, the chitosaccharides yield and antioxidant activity increased about 44.76% and 22.22%, whereas specific productivity of ß-N-acetyl glucosaminidase and chitinase increased by 22.86% and 33.37% over free state. The cell entrapped beads can be reused upto ten cycles without marked loss of its biocatalytic efficiency. High level of protoplast of Aspergillus niger was generated by treating mycelia with 10 U/ml of crude chitinase after 4 h at pH 7.0 and in the temperature 35-40°C, and 67% of the protoplasts were found to be regenerated.

O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation) in primary and metastatic colorectal cancer clones and effect of N-acetyl-ß-D-glucosaminidase silencing on cell phenotype and transcriptome.

O-linked ß-N-acetylglucosamine (O-GlcNAc) glycosylation is a regulatory post-translational modification occurring on the serine or threonine residues of nucleocytoplasmic proteins. O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase and O-GlcNAcase (OGA), which are responsible for O-GlcNAc addition and removal, respectively. Although O-GlcNAcylation was found to play a significant role in several pathologies such as type II diabetes and neurodegenerative diseases, the role of O-GlcNAcylation in the etiology and progression of cancer remains vague. Here, we followed O-GlcNAcylation and its catalytic machinery in metastatic clones of human colorectal cancer and the effect of OGA knockdown on cellular phenotype and on the transcriptome. The colorectal cancer SW620 metastatic clone exhibited increased O-GlcNAcylation and decreased OGA expression compared with its primary clone, SW480. O-GlcNAcylation elevation in SW620 cells, through RNA interference of OGA, resulted in phenotypic alterations that included acquisition of a fibroblast-like morphology, which coincides with epithelial metastatic progression and growth retardation. Microarray analysis revealed that OGA silencing altered the expression of about 1300 genes, mostly involved in cell movement and growth, and specifically affected metabolic pathways of lipids and carbohydrates. These findings support the involvement of O-GlcNAcylation in various aspects of tumor cell physiology and suggest that this modification may serve as a link between metabolic changes and cancer.

Expression of ß-1,4-galactosyltransferase and suppression of ß-N-acetylglucosaminidase to aid synthesis of complex N-glycans in insect Drosophila S2 cells.

Previously, we have shown that simple paucimannosidic N-glycan structures in insect Drosophila S2 cells arise mainly because of ß-N-acetylglucosaminidase (GlcNAcase) action. Thus, in an earlier report, we suppressed GlcNAcase activity and clearly demonstrated that more complex N-glycans with two terminal N-acetylglucosamine (GlcNAc) residues were then synthesized. In the present work, we investigated the synergistic effects of ß-1,4-galactosyltransferase (GalT) expression and GlcNAcase suppression on N-glycan patterns. We found that the N-glycan pattern of human erythropoietin secreted by engineered S2 cells expressing GalT but not GlcNAcase was complete, even in small portion, except for sialylation; the N-glycan structures had two terminal galactose (Gal) residues. When GalT was expressed but GlcNAcase was not inhibited, N-glycan with GlcNAc and Gal at only one branch end was synthesized. Therefore, it will be possible to express a complete functional human glycoprotein in engineered Drosophila S2 cells by suppressing GlcNAcase and co-expressing additional glycosyltransferases of N-glycosylation pathway.

O-GlcNAc-selective-N-acetyl-beta-D-glucosaminidase activity and mRNA expression in muscle is related to glucosamine-induced insulin resistance.

Glucosamine (GlcN)-induced insulin resistance is associated with an increase in O-linked-N-acetylglucosaminylated modified proteins (O-GlcNAcylated proteins). The role played by O-GlcNAc-selective-N-acetyl-beta-D-glucosaminidase (O-GlcNAcase), which removes O-N-acetyl-glucosamine residues from O-GlcNAcylated proteins, has not yet been demonstrated. We investigated whether GlcN-induced whole-body insulin resistance is related to tissue O-GlcNAcase activity and mRNA expression. GlcN (30 mumol/kg/min) or physiological saline (control) was intravenously infused into Sprague-Dawley rats for 2 h. After GlcN treatment, rats were subjected to the following: intravenous glucose tolerance test, insulin tolerance test or removal of the liver, muscle and pancreas. GlcN was found to provoke hyperglycemia compared to control (8.6 +/- 0.41 vs. 4.82 +/- 0.17 mM, p < 0.001). The insulin resistance index (HOMA-IR) increased (15.76 +/- 1.47 vs. 10.14 +/- 1.41, p < 0.001) and the beta-cell function index (HOMA-beta) diminished (182.69 +/- 22.37 vs. 592.01 +/- 103, p < 0.001). Liver glucose concentration was higher in the GlcN group than in the control group (0.37 +/- 0.04 vs. 0.24 +/- 0.038 mmol/g dry weight, p < 0.001). Insulin release index (insulin/glucose) was less in the GlcN group than in the control (2.2 +/- 0.1 vs. 8 +/- 0.8 at 120 min, p < 0.001). In the GlcN group, muscle O-GlcNAcase activity diminished (0.28 +/- 0.019 vs. 0.36 +/- 0.018 nmol of p-nitrophenyl/mg protein/min, p < 0.001), and K(m) increased (1.51 +/- 0.11 vs. 1.12 +/- 0.1 mM, p < 0.001) compared to the control. In the GlcN group, O-GlcNAcase activity/mRNA expression was altered (0.6 +/- 0.07 vs. 1 +/- 0.09 of control, p < 0.05). In conclusion, O-GlcNAcase activity is posttranslationally inhibited during GlcN-induced insulin resistance.

A molecular biomarker for disruption of crustacean molting: the N-acetyl-beta-glucosaminidase mRNA in the epidermis of the fiddler crab.

Several environmentally persistent chemicals have been found to be capable of disrupting crustacean molting. Considering the importance of molting in the life of crustaceans, there is a need to develop a molecular biomarker that can reflect the disrupting effects of contaminants on ecdysteroid signaling in crustaceans. N-acetyl-beta-glucosaminidase (NAG) is a chitinolytic enzyme found in crustacean epidermis. The results of the present investigation show that the transcription of NAG gene in the epidermis of the fiddler crab, Uca pugilator, is inducible by the molting hormone 20-hydroxyecdysone, which validates the use of NAG mRNA as a biomarker for molt-disrupting effects of xenobiotics.

A fused lobes gene encodes the processing beta-N-acetylglucosaminidase in Sf9 cells.

Manalpha6(Manalpha3)Manbeta4GlcNAcbeta4GlcNAc-R is the core structure of the major processed protein N-glycans produced by insect cells. Ultimately, this paucimannose type structure is produced by an unusual beta-N-acetylglucosaminidase, which removes the terminal N-acetylglucosamine residue from the upstream intermediate, Manalpha6(GlcNAcbeta2Manalpha3)Manbeta4GlcNAcbeta4GlcNAc-R. Because the N-glycan processing pathways leading to the production of this intermediate are probably identical in insects and higher eukaryotes, the presence or absence of this specific, processing beta-N-acetylglucosaminidase is a key factor distinguishing the processing pathways in these two different types of organisms. Recent studies have shown that the fused lobes (fdl) gene encodes the specific, processing beta-N-acetylglucosaminidase of Drosophila melanogaster. However, there are conflicting reports on the identity of the gene encoding this enzyme in the lepidopteran insect, Spodoptera frugiperda. One has suggested that a gene alternatively designated SfGlcNAcase-3 or SfHex encodes this function, whereas another has suggested that this gene encodes a broad-spectrum beta-N-acetylglucosaminidase that functions in glycan and chitin degradation. In this study we resolved this conflict by molecularly cloning an S. frugiperda fdl ortholog (Sf-fdl) and demonstrating that it encodes a product with the substrate specificity expected of the processing beta-N-acetylglucosaminidase. Moreover, we showed that the endogenous levels of specific, processing beta-N-acetylglucosaminidase activity were significantly reduced in S. frugiperda cells engineered to express a double-stranded RNA derived from the Sf-fdl gene. These results indicate that Sf-fdl encodes the specific, processing beta-N-acetylglucosaminidase of S. frugiperda and validate our previous suggestion that the broad-spectrum beta-N-acetylglucosaminidase encoded by the SfGlcNAcase-3/SfHex gene is more likely to be involved in N-glycan and/or chitin degradation.

Protective effect of CpG-DNA against mastitis induced by Escherichia coli infection in a rat model.

A mastitis model in rats, induced by Escherichia coli infection, was established and the protective effect of Cytosine-phosphate-Guanosine (CpG)-DNA was determined. An E. coli suspension containing either 2 x 10(3) colony forming units (CFU)mL(-1)(EL group), 2 x 10(5)CFU mL(-1) (EH group), or (as controls) 100 microL phosphate buffer saline (CON group), was inoculated into the mammary glands 72 h after parturition. The rats were euthanased 24 h post-infection. The histopathological changes in mammary tissue in the EL group were mild, whereas the structural changes in the EH group were severe and polymorphonuclear leukocytes (PMNs) had accumulated in the mammary alveoli. Interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha and N-acetyl-beta-d-glucosaminidase (NAGase) were significantly increased in the mammary tissue from the EH group but not significantly changed in the EL group. On the basis of these findings, the potential protective effect of CpG-DNA on mammary glands was tested using a 2 x 10(5)CFU mL(-1) suspension. An intramuscular injection of either CpG-DNA (200 microg) or PBS (100 microL) was given immediately after parturition. At 72 h post-partum, 2 x 10(5)CFU mL(-1)E. coli (100 microL) were inoculated into the mammary glands of all rats. At pre-infection (0 h), and 8, 16, 24, 48 and 72 h after inoculation six rats were euthanased. CpG-DNA induced more rapid migration of PMNs from the blood to mammary tissue at the initial stage of infection, stimulated the secretion of IL-6 and TNF-alpha at different time points, reduced viable E. coli in mammary tissues and decreased the activity of NAGase. CpG-DNA also promoted the expression of its specific receptor TLR-9 mRNA in mammary tissue. The study showed that CpG-DNA protected against E. coli mastitis in this rat model.

Molecular cloning and expression of two novel beta-N-acetylglucosaminidases from silkworm Bombyx mori.

Beta-N-acetylglucosaminidase is a major glycosidase involved in several physiological processes, such as fertilization, metamorphosis, glycoconjugate degradation, and glycoprotein biosynthesis in insects. A search using the Bombyx mori cDNA database revealed the existence of two putative beta-N-acetylglucosaminidase genes. Their full-length cDNAs were cloned by rapid amplification of cDNA ends and polymerase chain reaction using specific primers, and named BmGlcNAcase1 and BmGlcNAcase2. A BLAST search revealed that BmGlcNAcase1 and BmGlcNAcase2 are homologous to a beta-subunit homolog encoded by Drosophila melanogaster HEXO2 and the Spodoptera frugiperda beta-N-acetylglucosaminidase gene respectively. The recombinant proteins of BmGlcNAcase1 and BmGlcNAcase2 without putative transmembrane domains were expressed in the yeast Pichia pastoris. Both enzymes showed broad substrate specificity, and cleaved terminal N-acetylglucosamine residues from the alpha-3 and alpha-6 branches of a biantennary N-glycan substrate, and also hydrolyzed chitotriose to chitobiose.

Fungal biosynthesis of endochitinase and chitobiase in solid state fermentation and their application for the production of N-acetyl-D-glucosamine from colloidal chitin.

The present study was directed to the production of N-acetyl-D-glucosamine using endochitinase and chitobiase from fungal cultures in solid culturing. Fifteen fungal strains were evaluated for endochitinase and chitobiase production under solid-state fermentation using agro-industrial residues, of which Penicillium aculeatum NRRL 2129 showed maximum endochitinase activity whereas Trichoderma harzianum TUBF 927 showed maximum chitobiase activity. Eleven substrates, alone and in combination with chitin, were evaluated for the enzyme production. Optimization of physico-chemical parameters such as incubation period and initial moisture content, and nutritional parameters such as chitin source, inorganic and organic nitrogen sources, were carried out. Optimization resulted in more than 3-fold increase in endochitinase production (from 3.5 to 12.53 U/g dry weight of substrate) and about 1.5-fold increase in chitobiase production (from 1.6 to 2.25 U/g dry weight of substrate). Studies on the degradation of colloidal chitin to N-acetyl-D-glucosamine showed improved efficiency when endochitinase and chitobiase were used in combination.

A screening system for carbon sources enhancing beta-N-acetylglucosaminidase formation in Hypocrea atroviridis (Trichoderma atroviride).

To identify carbon sources that trigger beta-N-acetylglucosaminidase (NAGase) formation in Hypocrea atroviridis (anamorph Trichoderma atroviride), a screening system was designed that consists of a combination of Biolog Phenotype MicroArray plates, which contain 95 different carbon sources, and specific enzyme activity measurements using a chromogenic substrate. The results revealed growth-dependent kinetics of NAGase formation and it was shown that NAGase activities were enhanced on carbon sources sharing certain structural properties, especially on alpha-glucans (e.g. glycogen, dextrin and maltotriose) and oligosaccharides containing galactose. Enzyme activities were assessed in the wild-type and a H. atroviridis Deltanag1 strain to investigate the influence of the two NAGases, Nag1 and Nag2, on total NAGase activity. Reduction of NAGase levels in the Deltanag1 strain in comparison to the wild-type was strongly carbon-source and growth-phase dependent, indicating the distinct physiological roles of the two proteins. The transcript abundance of nag1 and nag2 was increased on carbon sources with elevated NAGase activity, indicating transcriptional regulation of these genes. The screening method for the identification of carbon sources that induce enzymes or a gene of interest, as presented in this paper, can be adapted for other purposes if appropriate enzyme or reporter assays are available.

Expression of active alpha-N-acetylglucosaminidase/TAT Chimerae in cultured Spodoptera frugiperda cells.

We examined the production and secretion of fusion constructs containing alpha-N-acetylglucosaminidase, the enzyme deficient in Sanfilippo B, and either wildtype TAT or modified TAT in cultured Spodoptera frugiperda cells. All constructs exhibited successful expression of active enzyme, suggesting the future possibility of utilizing TAT/alpha-N-acetylglucosaminidase chimerae in enzyme replacement therapy.