ENGase Activity Research Articles

Construction of tomato plants with suppressed endo-β-N-acetylglucosaminidase activity using CRISPR-Cas9 mediated genome editing

High mannose-type free N-glycans with a single N-acetyl-D-glucosamine (GlcNAc) residue at the reducing end (GN1-HMT-FNGs) are produced by cytosolic endo-β-N-acetylglucosaminidase (EC:3.2.1.96) (ENGase) and are ubiquitous in differentiating and growing plant cells. To elucidate the physiological functions of HMT-FNGs in plants, we identified the ENGase gene in tomato (Solyc06g050930) and detected ENGase activity and increased production of GN1-HMT-FNGs during tomato fruit maturation. However, the precise role of GN1-HMT-FNGs in fruit maturation remains unclear. In this study, we established tomato ENGase mutants with suppressed ENGase activity via CRISPR/Cas9 genome editing technology. DNA sequencing of the Δeng mutants (T0 and T1 generations) revealed that they had the same mutations in the genomic DNA around the target sequences. Three null CRISPR/Cas9 segregant plants of the T1 generation (Δeng1-2, −22, and −26) were used to measure ENGase activity and analyze the structural features of HMT-FNGs in the leaves. The Δeng mutants did not exhibit ENGase activity and produced GN2-HMT-FNGs bearing tow GlcNAc residues at the reducing end side instead of GN1-HMT-FNGs. The Δeng mutants lack the N-terminal region of ENGase, indicating that the N-terminal region is important for full ENGase activity. The fruits of Δeng mutants (T2 generation) also showed loss of ENGase activity and similar structural features of HMT-FNGs of the T1 generation. However, there was no significant difference in fruit maturation between the T2 generation of the Δeng mutants and the wild type. The Δeng mutants rich in GN2-HMT-FNGs could be offered as a new tomato that is different from wild type containing GN1-HMT-FNGs.

Cytosolic Free N-Glycans Are Retro-Transported Into the Endoplasmic Reticulum in Plant Cells.

During endoplasmic reticulum (ER)-associated degradation, free N-glycans (FNGs) are produced from misfolded nascent glycoproteins via the combination of the cytosolic peptide N-glycanase (cPNGase) and endo-β-N-acetylglucosaminidase (ENGase) in the plant cytosol. The resulting high-mannose type (HMT)-FNGs, which carry one GlcNAc residue at the reducing end (GN1-FNGs), are ubiquitously found in developing plant cells. In a previous study, we found that HMT-FNGs assisted in protein folding and inhibited β-amyloid fibril formation, suggesting a possible biofunction of FNGs involved in the protein folding system. However, whether these HMT-FNGs occur in the ER, an organelle involved in protein folding, remained unclear. On the contrary, we also reported the presence of plant complex type (PCT)-GN1-FNGs, which carry the Lewisa epitope at the non-reducing end, indicating that these FNGs had been fully processed in the Golgi apparatus. Since plant ENGase was active toward HMT-N-glycans but not PCT-N-glycans that carry β1-2xylosyl and/or α1-3 fucosyl residue(s), these PCT-GN1-FNGs did not appear to be produced from fully processed glycoproteins that harbored PCT-N-glycans via ENGase activity. Interestingly, PCT-GN1-FNGs were found in the extracellular space, suggesting that HMT-GN1-FNGs formed in the cytosol might be transported back to the ER and processed in the Golgi apparatus through the protein secretion pathway. As the first step in elucidating the production mechanism of PCT-GN1-FNGs, we analyzed the structures of free oligosaccharides in plant microsomes and proved that HMT-FNGs (Man9-7GlcNAc1 and Man9-8GlcNAc2) could be found in microsomes, which almost consist of the ER compartments.

Draft Genome Sequence of Sphingobacterium sp. Strain HMA12, Which Encodes Endo-β-N-Acetylglucosaminidases and Can Specifically Hydrolyze Fucose-Containing Oligosaccharides.

ABSTRACTThe genome sequence of the soil bacterium Sphingobacterium sp. strain HMA12, the culture supernatant of which exhibited endo-β-N-acetylglucosaminidase (ENGase) activity, was examined for ENGase-encoding genes. Here, we report the characterization of new genes of ENGases, obtained by whole-genome shotgun sequencing, that are capable of specifically hydrolyzing fucose-containing oligosaccharides.

A New Fluorogenic Probe for the Detection of endo-β-N-Acetylglucosaminidase.

We developed a fluorescence-quenching-based assay system to determine the hydrolysis activity of endo-β-N-acetylglucosaminidases (ENGases). The pentasaccharide derivative 1 was labeled with an N-methylanthraniloyl group as a reporter dye at the non-reducing end and with a 2,4-dinitrophenyl group as a quencher molecule at the reducing end. This derivative is hydrolyzed by ENGase, resulting in an increase in fluorescence intensity. Thus, the fluorescence signal is directly proportional to the amount of the tetrasaccharide derivative, hence allowing ENGase activity to be evaluated easily and quantitatively. Using this system, we succeeded in measuring the hydrolysis activities of ENGases and thus the inhibitory activities of known inhibitors. We confirmed that this assay system is suitable for high-throughput screening for potential inhibitors of human ENGase that might serve as therapeutic agents for the treatment of N-glycanase 1 (NGLY1) deficiency.

Characterization of novel endo-\u03b2-N-acetylglucosaminidases from Sphingobacterium species, Beauveria bassiana and Cordyceps militaris that specifically hydrolyze fucose-containing oligosaccharides and human IgG

Endo-β-N-acetylglucosaminidase (ENGase) catalyzes hydrolysis of N-linked oligosaccharides. Although many ENGases have been characterized from various organisms, so far no fucose-containing oligosaccharides-specific ENGase has been identified in any organism. Here, we screened soil samples, using dansyl chloride (Dns)-labeled sialylglycan (Dns-SG) as a substrate, and discovered a strain that exhibits ENGase activity in the culture supernatant; this strain, named here as strain HMA12, was identified as a Sphingobacterium species by 16S ribosomal RNA gene analysis. By draft genome sequencing, five candidate ENGase encoding genes were identified in the genome of this strain. Among them, a recombinant protein purified from Escherichia coli expressing the candidate gene ORF1188 exhibited fucose-containing oligosaccharides-specific ENGase activity. The ENGase exhibited optimum activities at very acidic pHs (between pH 2.3–2.5). A BLAST search using the sequence of ORF1188 identified two fungal homologs, one in Beauveria bassiana and the other in Cordyceps militaris. Recombinant ORF1188, Beauveria and Cordyceps ENGases released the fucose-containing oligosaccharides residues from rituximab (immunoglobulin G) but not the high-mannose-containing oligosaccharides residues from RNase B, a result that not only confirmed the substrate specificity of these novel ENGases but also suggested that natural glycoproteins could be their substrates.

Repurposing of Proton Pump Inhibitors as first identified small molecule inhibitors of endo-β-N-acetylglucosaminidase (ENGase) for the treatment of NGLY1 deficiency, a rare genetic disease

N-Glycanase deficiency, or NGLY1 deficiency, is an extremely rare human genetic disease. N-Glycanase, encoded by the gene NGLY1, is an important enzyme involved in protein deglycosylation of misfolded proteins. Deglycosylation of misfolded proteins precedes the endoplasmic reticulum (ER)-associated degradation (ERAD) process. NGLY1 patients produce little or no N-glycanase (Ngly1), and the symptoms include global developmental delay, frequent seizures, complex hyperkinetic movement disorder, difficulty in swallowing/aspiration, liver dysfunction, and a lack of tears. Unfortunately, there has not been any therapeutic option available for this rare disease so far. Recently, a proposed molecular mechanism for NGLY1 deficiency suggested that endo-β-N-acetylglucosaminidase (ENGase) inhibitors may be promising therapeutics for NGLY1 patients. Herein, we performed structure-based virtual screening utilizing FDA-approved drug database on this ENGase target to enable repurposing of existing drugs. Several Proton Pump Inhibitors (PPIs), a series of substituted 1H-benzo [d] imidazole, and 1H-imidazo [4,5-b] pyridines, among other scaffolds, have been identified as potent ENGase inhibitors. An electrophoretic mobility shift assay was employed to assess the inhibition of ENGase activity by these PPIs. Our efforts led to the discovery of Rabeprazole Sodium as the most promising hit with an IC50 of 4.47±0.44μM. This is the first report that describes the discovery of small molecule ENGase inhibitors, which can potentially be used for the treatment of human NGLY1 deficiency.

Functional analysis of glycoside hydrolase family 18 and 20 genes in Neurospora crassa

Glycoside hydrolase family 18 contains hydrolytic enzymes with chitinase or endo-N-acetyl-β-d-glucosaminidase (ENGase) activity, while glycoside hydrolase family 20 contains enzymes with β-N-acetylhexosaminidase (NAGase) activity. Chitinases and NAGases are involved in chitin degradation. Chitinases are phylogenetically divided into three main groups (A, B and C), each further divided into subgroups. In this study, we investigated the functional role of 10 Neurospora crassa genes that encode chitinases, 2 genes that encode ENGases and 1 gene that encode a NAGase, using gene deletion and gene expression techniques. No phenotypic effects were detected for any of the studied group A chitinase gene deletions. Deletion of the B group member chit-1 resulted in reduced growth rate compared with the wild type (WT) strain. In combination with the presence of a predicted glycosylphosphatidylinositol anchor motif in the C-terminal of chit-1, indicating cell wall localization, these data suggest a role in cell wall remodeling during hyphal growth for chit-1. Deletion of the ENGase gene gh18-10 resulted in reduced growth rate compared with WT, increased conidiation, and increased abiotic stress tolerance. In addition, Δgh18-10 strains displayed lower secretion of extracellular proteins compared to WT and reduced levels of extracellular protease activity. The connection between gh18-10 ENGase activity and the endoplasmic reticulum associated protein degradation process, a stringent quality control of glycoprotein maturation, is discussed. N. crassa group C chitinase genes gh18-6 and gh18-8 were both induced during fungal–fungal interactions. However, gh18-6 was only induced during interspecific interactions, while gh18-8 displayed the highest induction levels during self–self interactions. These results provide new information on functional differentiation of fungal chitinases.

Disruption of the Eng18B ENGase Gene in the Fungal Biocontrol Agent Trichoderma atroviride Affects Growth, Conidiation and Antagonistic Ability

The recently identified phylogenetic subgroup B5 of fungal glycoside hydrolase family 18 genes encodes enzymes with mannosyl glycoprotein endo-N-acetyl-β-D-glucosaminidase (ENGase)-type activity. Intracellular ENGase activity is associated with the endoplasmic reticulum associated protein degradation pathway (ERAD) of misfolded glycoproteins, although the biological relevance in filamentous fungi is not known. Trichoderma atroviride is a mycoparasitic fungus that is used for biological control of plant pathogenic fungi. The present work is a functional study of the T. atroviride B5-group gene Eng18B, with emphasis on its role in fungal growth and antagonism. A homology model of T. atroviride Eng18B structure predicts a typical glycoside hydrolase family 18 (αβ)8 barrel architecture. Gene expression analysis shows that Eng18B is induced in dual cultures with the fungal plant pathogens Botrytis cinerea and Rhizoctonia solani, although a basal expression is observed in all growth conditions tested. Eng18B disruption strains had significantly reduced growth rates but higher conidiation rates compared to the wild-type strain. However, growth rates on abiotic stress media were significantly higher in Eng18B disruption strains compared to the wild-type strain. No difference in spore germination, germ-tube morphology or in hyphal branching was detected. Disruption strains produced less biomass in liquid cultures than the wild-type strain when grown with chitin as the sole carbon source. In addition, we determined that Eng18B is required for the antagonistic ability of T. atroviride against the grey mould fungus B. cinerea in dual cultures and that this reduction in antagonistic ability is partly connected to a secreted factor. The phenotypes were recovered by re-introduction of an intact Eng18B gene fragment in mutant strains. A putative role of Eng18B ENGase activity in the endoplasmic reticulum associated protein degradation pathway of endogenous glycoproteins in T. atroviride is discussed in relation to the observed phenotypes.

The two endo-β-N-acetylglucosaminidase genes from Arabidopsis thaliana encode cytoplasmic enzymes controlling free N-glycan levels

Endo-β-N-acetylglucosaminidases (ENGases) cleave N-glycans from proteins and/or peptides by hydrolyzing the O-glycosidic linkage between the two core-N-acetylglucosamine (GlcNAc) residues. Although, two homologous genes potentially encoding ENGases have been identified in Arabidopsis thaliana, their respective substrate specificity, their subcellular and their organ specific localization was hitherto unknown. In order to investigate the role of ENGases in this model plant species, we transiently expressed the two A. thaliana genes in Nicotiana benthamiana and determined the substrate specificities, as well as the Km values, of the purified recombinant enzymes. The assumed predominantly cytosolic localisation of both enzymes, here referred to as AtENGase85A and AtENGase85B, was determined by confocal microscopy of plant leaves expressing the respective GFP-fusion constructs. For the individual characterization of the two enzymes expression patterns in planta, single knock-out plants were selected for both genes. Although both enzymes are present in most organs, only AtENGase85A (At5g05460) was expressed in stems and no ENGase activity was detected in siliques. A double knock-out was generated by crossing but-like single knock-out plants-no apparent phenotype was observed. In contrast, in this double knock-out, free N-glycans carrying a single GlcNAc at the reducing end are completely absent and their counterparts with two GlcNAc-visible only at a trace level in wild type-accumulated dramatically.

Double-Knockout of Putative Endo-β-N-acetylglucosaminidase (ENGase) Genes inArabidopsis thaliana: Loss of ENGase Activity Induced Accumulation of High-Mannose Type FreeN-Glycans BearingN,N′-Acetylchitobiosyl Unit

Endo-β-N-acetylglucosaminidase (ENGase) is involved in the production of high-mannose type free N-glycans during plant development and fruit maturation. In a previous study (K. Nakamura et al. Biosci. Biotechnol. Biochem., 73, 461-464 (2009)), we identified the tomato ENGase gene and found that gene expression remained relatively constant. In the present study, we constructed an Arabidopsis thaliana mutant in which the expression of two putative ENGase genes was suppressed. The mutant showed no ENGase activity, but produced high-mannose type free N-glycans carrying the N,N'-acetylchitobiosyl unit that is produced by peptide:N-glycanase, indicating that both these genes encode Arabidopsis ENGase.

Identification of Roles for Peptide: N-Glycanase and Endo-β-N-Acetylglucosaminidase (Engase1p) during Protein N-Glycosylation in Human HepG2 Cells

BackgroundDuring mammalian protein N-glycosylation, 20% of all dolichol-linked oligosaccharides (LLO) appear as free oligosaccharides (fOS) bearing the di-N-acetylchitobiose (fOSGN2), or a single N-acetylglucosamine (fOSGN), moiety at their reducing termini. After sequential trimming by cytosolic endo β-N-acetylglucosaminidase (ENGase) and Man2c1 mannosidase, cytosolic fOS are transported into lysosomes. Why mammalian cells generate such large quantities of fOS remains unexplored, but fOSGN2 could be liberated from LLO by oligosaccharyltransferase, or from glycoproteins by NGLY1-encoded Peptide-N-Glycanase (PNGase). Also, in addition to converting fOSGN2 to fOSGN, the ENGASE-encoded cytosolic ENGase of poorly defined function could potentially deglycosylate glycoproteins. Here, the roles of Ngly1p and Engase1p during fOS metabolism were investigated in HepG2 cells.Methods/Principal FindingsDuring metabolic radiolabeling and chase incubations, RNAi-mediated Engase1p down regulation delays fOSGN2-to-fOSGN conversion, and it is shown that Engase1p and Man2c1p are necessary for efficient clearance of cytosolic fOS into lysosomes. Saccharomyces cerevisiae does not possess ENGase activity and expression of human Engase1p in the png1Δ deletion mutant, in which fOS are reduced by over 98%, partially restored fOS generation. In metabolically radiolabeled HepG2 cells evidence was obtained for a small but significant Engase1p-mediated generation of fOS in 1 h chase but not 30 min pulse incubations. Ngly1p down regulation revealed an Ngly1p-independent fOSGN2 pool comprising mainly Man8GlcNAc2, corresponding to ∼70% of total fOS, and an Ngly1p-dependent fOSGN2 pool enriched in Glc1Man9GlcNAc2 and Man9GlcNAc2 that corresponds to ∼30% of total fOS.Conclusions/SignificanceAs the generation of the bulk of fOS is unaffected by co-down regulation of Ngly1p and Engase1p, alternative quantitatively important mechanisms must underlie the liberation of these fOS from either LLO or glycoproteins during protein N-glycosylation. The fully mannosylated structures that occur in the Ngly1p-dependent fOSGN2 pool indicate an ERAD process that does not require N-glycan trimming.

Identification of a gene coding for a deglycosylating enzyme inHypocrea jecorina

An enzyme with mannosyl glycoprotein endo-N-acetyl-beta-D-glucosaminidase (ENGase)-type activity was partially purified from the extracellular medium of the mould Hypocrea jecorina (Trichoderma reesei). Internal peptides were generated and used to identify the gene in the T. reesei genome. The active enzyme is processed both at the N- and at the C-terminus. High-mannose-type glycoproteins are good substrates, whereas complex-type glycans are not hydrolysed. The enzyme represents the first fungal member of glycoside hydrolase family 18 with ENGase-type activity. Bacterial ENGases and the fungal chitinases belonging to the same family show very low homology with Endo T. Database searches identify several highly homologous genes in fungi and the activity is also found within other Trichoderma species. This ENGase activity, not coregulated with cellulase production, could be responsible for the extensive N-deglycosylation observed for several T. reesei cellulases.

Changes in Structural Features of FreeN-Glycan and Endoglycosidase Activity during Tomato Fruit Ripening

In developing plants, free N-glycans occur ubiquitously at micromolar concentrations. Such oligosaccharides have been proposed to be signaling molecules in plant development. As a part of a study to elucidate the physiological roles of de-N-glycosylation machinery involved in fruit ripening, we analyzed changes in the amounts and structural features of free N-glycans in tomato fruits at four ripening stages. The amount of high-mannose type free N-glycans increased significantly in accordance with fruit ripening, and the relative amounts of high-molecular size N-glycans, such as Man(8-9)GlcNAc(1), became predominant. These observations suggest that the de-N-glycosylation machinery, including endo-beta-N-acetylglucosaminidase (ENGase) activity, is stimulated in the later stages of fruit ripening. But contrary to expectation, we found that total ENGase activities in the tomato fruits did not vary significantly with the ripening process, suggesting that ENGase activity must be maintained at a certain level, and that the expression of alpha-mannosidase involved in the clearance of free N-glycans decreases during tomato fruit ripening.

Evidence of two enzymes performing the de‐N‐glycosylation of proteins in barley: expression during germination, localization within the grain and set‐up during grain formation

The occurrence of two enzymes performing de‐N‐glycosylation of glycoproteins, namely, endo‐N‐acetyl‐β‐ d‐glucosaminidase (ENGase, EC 3.2.1.96) and peptide‐N4‐(N‐acetyl‐β‐d‐glucosaminyl) asparagine amidase (PNGase, EC 3.5.1.52) was investigated in barley, cv. Plaisant (a winter six rowed variety). The dry grain showed both activities according to the HPLC detection of the hydrolysis of fluorescent resorufin‐labelled substrates. However, PNGase activity was 16‐fold higher than ENGase activity. During germination, both activities increased, PNGase by only 1.5‐fold compared to nearly 4.8‐fold for ENGase over the 4 d following imbibition. The localization of these activities within the grain showed that the major contribution of PNGase was due to the endosperm, typically representing over 90% of the whole grain activity. In contrast, ENGase activity was especially high in the embryo and, later, in the developing plantlet (10‐fold higher than in the endosperm), particularly in the rootlets and scutellum. In developing spikes, PNGase activity was 5.6‐fold higher than in the leaves, but similar ENGase activity was measured in both organs. During grain formation, PNGase activity followed dry matter increase together with endosperm development. In contrast, ENGase activity dropped by 66% at the beginning of grain filling before stabilizing until harvest. The occurrence of de‐N‐glycosylation‐performing enzymes throughout the development of barley raises the question of the nature of their natural substrates. Moreover, the prevalence of one of these enzymes over the other depending on the organ and the developmental stage, could represent the first evidence of specific functions for each enzyme.

Evidence of two enzymes performing the de‐N‐glycosylation of proteins in barley: expression during germination, localization within the grain and set‐up during grain formation

The occurrence of two enzymes performing de-N-glycosylation of glycoproteins, namely, endo-N-acetyl-beta-D-glucosaminidase (ENGase, EC 3.2.1.96) and peptide-N(4)-(N-acetyl-beta-D-glucosaminyl) asparagine amidase (PNGase, EC 3.5.1.52) was investigated in barley, cv. Plaisant (a winter six rowed variety). The dry grain showed both activities according to the HPLC detection of the hydrolysis of fluorescent resorufin-labelled substrates. However, PNGase activity was 16-fold higher than ENGase activity. During germination, both activities increased, PNGase by only 1.5-fold compared to nearly 4.8-fold for ENGase over the 4 d following imbibition. The localization of these activities within the grain showed that the major contribution of PNGase was due to the endosperm, typically representing over 90% of the whole grain activity. In contrast, ENGase activity was especially high in the embryo and, later, in the developing plantlet (10-fold higher than in the endosperm), particularly in the rootlets and scutellum. In developing spikes, PNGase activity was 5.6-fold higher than in the leaves, but similar ENGase activity was measured in both organs. During grain formation, PNGase activity followed dry matter increase together with endosperm development. In contrast, ENGase activity dropped by 66% at the beginning of grain filling before stabilizing until harvest. The occurrence of de-N-glycosylation-performing enzymes throughout the development of barley raises the question of the nature of their natural substrates. Moreover, the prevalence of one of these enzymes over the other depending on the organ and the developmental stage, could represent the first evidence of specific functions for each enzyme.

Regulation of De-N-Glycosylation Enzymes in Germinating Radish Seeds.

The activities of the de-N-glycosylation enzymes endo-N-acetyl- [beta]-D-glucosaminidase (ENGase; EC 3.2.1.96) and peptide-N4- (N-acetyl-[beta]-D-glucosaminyl) asparagine amidase (PNGase; EC 3.5.1.52) were monitored during germination and postgerminative development in radish (Raphanus sativus L. cv Flamboyant). The ENGase activity was detected only during postgermination, whereas the PNGase activity was present at high levels in both stages. When germination was inhibited with abscisic acid or cycloheximide, PNGase activity was detected at a basic level and ENGase activity was not detected at all. PNGase is present as an active protein in dry seeds and is apparently synthesized during seed formation. Conversely, the absence of ENGase in dry seeds suggests that its activity is dependent on the protein synthesis that occurs during and after germination. Treatment with gibberellic acid confirmed the production of both de-N-glycosylation enzymes after germination, and demonstrated a temporal delay between the production of the two enzymes during this period. Our results suggest that the two de-N-glycosylation enzymes are differentially regulated during plant development.

Formula omitted] and peptide- N4-( N-acetyl-glucosaminyl) asparagine amidase activities during germination of Raphanus sativus

Endo-N- acetyl-β- d-glucosaminidase (ENGase, EC 3.2.1.96) and peptide-N 4-(N- acetyl-β- d-glucosaminyl ) asparagine amidase (PNGase, EC 3.5.1.52) activities were monitored during germination and postgerminative development in Raphanus sativus. The PNGase activity was found in dry seeds and its level was constant during germination and postgermination. The ENGase activity was first detected about 18 hr after the start of imbibition (HAI) and displayed a maximum level at 36 HAI. After 36 HAI the production of both enzymes was constant until days 4–5. Both enzymes displayed substrate specificities corresponding to the potential glycoprotein substrates found in plants. They are in agreement (i) with the hypothesis that ENGase and PNGase are at the origin of the production of ‘unconjugated N-glycans’ and (ii) with the possibility that protein activity could be regulated by the removal of N-glycans.

An endo-N-acetyl-beta-D-glucosaminidase, acting on the di-N-acetylchitobiosyl part of N-linked glycans, is secreted during sporulation of Myxococcus xanthus.

After the demonstration that Stigmatella aurantiaca DW4 secretes an endo-N-acetyl-beta-D-glucosaminidase (ENGase), acting on the di-N-acetylchitobiosyl part of N-linked glycans (S. Bourgerie, Y. Karamanos, T. Grard, and R. Julien, J. Bacteriol. 176:6170-6174, 1994), an ENGase activity having the same substrate specificity was also found to be secreted during vegetative growth of Myxococcus xanthus DK1622. The activity decreased in mutants known to secrete less protein than the wild type (Exc +/-). During submerged development, the activity was produced in two steps: the first increase occurred during the aggregation phase, and the second one occurred much later, during spore formation. This production was lower in developmental mutants impairing cell-cell signaling, the late mutants (csg and dsg) being the most deficient. Finally, when sporulation was obtained either by starvation in liquid shake flask culture or by glycerol induction, the activity was produced exclusively by the wild-type cells during the maturation of the coat.