Chitinase-catalyzed synthesis of alternatingly N-deacetylated chitin: a chitin-chitosan hybrid polysaccharide

A chitin-chitosan hybrid polysaccharide (2) having a beta(1-->4)-linked alternating structure of an N-acetyl-D-glucosamine (GlcNAc) unit and a D-glucosamine (GlcN) unit was synthesized via chitinase-catalyzed polymerization of an oxazoline derivative of a GlcNbeta(1-->4)GlcNAc monomer (1). Monomer 1 was designed as a transition-state analogue substrate (TSAS) monomer for chitinase catalysis, which belongs to the glycoside hydrolase family 18. Monomer 1 was effectively polymerized by the catalysis of enzymes from Bacillus sp., Serratia marcescens and Streptomyces griseus, under weak alkaline conditions, giving rise to a water-soluble hybrid polysaccharide (2) in good yields. Molecular weights of 2 reached 2,020 with using chitinase from Serratia marcescens, which corresponds to 10-12 saccharide units.     

Purification, properties, and partial amino acid sequence of chitinase from a marine Alteromonas sp. strain O-7.

Chitinase (EC 3.2.1.14) was isolated from the culture supernatant of a marine bacterium, Alteromonas sp. strain O-7. The enzyme (Chi-A) was purified by anion-exchange chromatography (DEAE-Toyopearl 650 M) and gel filtration (Sephadex G-100). The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of Chi-A were 70 kDa and 3.9, respectively. The optimum pH and temperature of Chi-A were 8.0 and 50 degrees C, respectively. Chi-A was stable in the range of pH 5-10 up to 40 degrees C. Among the main cations, such as Na+, K+, Mg2+, and Ca2+, contained in seawater, Mg2+ stimulated Chi-A activity. N-Bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide inhibited Chi-A activity. The amino-terminal 27 amino acid residues of Chi-A were sequenced. This enzyme showed sequence homology with chitinases from terrestrial bacteria such as Serratia marcescens QMB1466 and Bacillus circulans WL-12.     

Complete amino acid sequence of endo-beta-N-acetylglucosaminidase from Flavobacterium sp

The complete amino acid sequence of endo-beta-N-acetylglucosaminidase from Flavobacterium sp. has been determined by analysis of peptides after cleavage with lysyl endopeptidase, pepsin and chymotrypsin. The protein consists of a single polypeptide chain consisting of 267 amino acid residues and a molecular mass of 27972 Da. The sequence of Flavobacterium endo-beta-N-acetylglucosaminidase is very close to that of the Streptomyces enzyme (endo-H), having 60% similarity and very similar hydropathy profiles. Similarities were also found between Flavobacterium endo-beta-N-acetylglucosaminidase and chitinases from Bacillus circulans, Serratia marcescens and Phaseolus vulgaris.     

Purification and properties of a thermostable chitinase from Streptomyces thermoviolaceus OPC-520.

A chitinase was purified from the culture filtrate of Streptomyces thermoviolaceus OPC-520. The enzyme showed a high optimum temperature (70 to 80 degrees C), a high optimum pH level (8.0 to 10.0), and heat stability. This enzyme showed high sequence homology with chitinases from Serratia marcescens QMB1466 and Bacillus circulans WL-12.     

Cloning, sequences, and characterization of two chitinase genes from the Antarctic Arthrobacter sp. strain TAD20: isolation and partial characterization of the enzymes

Arthrobacter sp. strain TAD20, a chitinolytic gram-positive organism, was isolated from the sea bottom along the Antarctic ice shell. Arthrobacter sp. strain TAD20 secretes two major chitinases, ChiA and ChiB (ArChiA and ArChiB), in response to chitin induction. A single chromosomal DNA fragment containing the genes coding for both chitinases was cloned in Escherichia coli. DNA sequencing analysis of this fragment revealed two contiguous open reading frames coding for the precursors of ArChiA (881 amino acids [aa]) and ArChiB (578 aa). ArChiA and ArChiB are modular enzymes consisting of a glycosyl-hydrolase family 18 catalytic domain as well as two and one chitin-binding domains, respectively. The catalytic domain of ArChiA exhibits 55% identity with a chitodextrinase from Vibrio furnissii. The ArChiB catalytic domain exhibits 33% identity with chitinase A of Bacillus circulans. The ArChiA chitin-binding domains are homologous to the chitin-binding domain of ArChiB. ArChiA and ArChiB were purified to homogeneity from the native Arthrobacter strain and partially characterized. Thermal unfolding of ArChiA, ArChiB, and chitinase A of Serratia marcescens was studied using differential scanning calorimetry. ArChiA and ArChiB, compared to their mesophilic counterpart, exhibited increased heat lability, similar to other cold-adapted enzymes.     

Enzyme activity determination on macromolecular substrates by isothermal titration calorimetry: application to mesophilic and psychrophilic chitinases

Isothermal titration calorimetry has been applied to the determination of the kinetic parameters of chitinases (EC 3.2.1.14) by monitoring the heat released during the hydrolysis of chitin glycosidic bonds. Experiments were carried out using two different macromolecular substrates: a soluble polymer of N-acetylglucosamine and the insoluble chitin from crab shells. Different experimental temperatures were used in order to compare the thermodependence of the activity of two chitinases from the psychrophile Arthrobacter sp. TAD20 and of chitinase A from the mesophile Serratia marcescens. The method allowed to determine unequivocally the catalytic rate constant k(cat), the activation energy (E(a)) and the thermodynamic activation parameters (DeltaG(#), DeltaH(#), DeltaS(#)) of the chitinolytic reaction on the soluble substrate. The catalytic activity has also been determined on insoluble chitin, which displays an effect of substrate saturation by chitinases. On both substrates, the thermodependence of the activity of the psychrophilic chitinases was lower than that observed with the mesophilic counterpart.     

Effects of Serratia marcescens on the F1 generation of laboratory-reared Heliothis virescens (Lepidoptera: Noctuidae)

The effects of the bacterium Serratia marcescens (Bizio) was investigated on the F1 generation of laboratory-reared Heliothis virescens (F.). There was no difference in adult male or female longevity (i.e., parental generation) for individuals inoculated with S. marcescens as larvae (Serratia treatment) and those that were free of the bacterium (control treatment). However, the number of eggs laid and the prevalence of eclosion of eggs from Serratia treatment adults were reduced relative to control treatment adults. A very low number of F1 Serratia treatment eggs exhibited signs of infection, but a higher prevalence of mortality was observed for F1 larvae (n = 2,888) for the Serratia (3.5-4.6%) than for the control (1.1-1.5%) treatment. No S. marcescens was isolated from dead control larvae; whereas, 48 -54% of dead F1 larvae for the Serratia treatment were positive for the bacterium. However, there was no significant difference in larval weights between treatments. There were also no differences in either mortality or weight of F1 male pupae between treatments, but F1 female pupae were significantly smaller and prevalence of mortality was higher for the Serratia treatment. Serratia marcescens was not isolated from any of the control F1 pupae, but 6% of pupal cadavers for the Serratia treatment were positive for the bacterium. No S. marcescens was recovered from the meconia of any of the F1 adults (n = 2,600) regardless of treatment, and there were no differences in adult weights between treatments. Although sublethal effects of S. marcescens were detected, the impact and prevalence of the bacterium were tremendously reduced over the F1 generation in the absence of all but the most basic management strategies.     

Chitinases A,B, and C1 of Serratia marcescens 2170 produced by recombinant Escherichia coli: enzymatic properties and synergism on chitin degradation

To discover the individual roles of the chitinases from Serratia marcescens 2170, chitinases A, B, and C1 (ChiA, ChiB, and ChiC1) were produced by Escherichia coli and their enzymatic properties as well as synergistic effect on chitin degradation were studied. All three chitinases showed a broad pH optimum and maintained significant chitinolytic activity between pH 4 and 10. ChiA was the most active enzyme toward insoluble chitins, but ChiC1 was the most active toward soluble chitin derivatives among the three chitinases. Although all three chitinases released (GlcNAc)2 almost exclusively from colloidal chitin, ChiB and ChiC1 split (GlcNAc)6 to (GlcNAc)3, while ChiA exclusively generated (GlcNAc)2 and (GlcNAc)4. Clear synergism on the hydrolysis of powdered chitin was observed in the combination between ChiA and either ChiB or ChiC, and the sites attacked by ChiA on the substrate are suggested to be different from those by either ChiB or ChiC1.     

Detection of chitinase activity after polyacrylamide gel electrophoresis

Commercial Streptomyces griseus and Serratia marcescens chitinases and purified wheat germ W1A and hen egg white lysozymes were subjected to polyacrylamide gel electrophoresis under native conditions at pH 4.3. After electrophoresis, an overlay gel containing 0.01% (W/V) glycol chitin as substrate was incubated in contact with the separation gel. Lytic zones were revealed by uv illumination with a transilluminator after staining for 5 min with 0.01% (W/V) Calcofluor white M2R. As low as 500 ng of purified hen egg lysozyme could be detected after 1 h incubation at 37 degrees C. One band was observed with W1A lysozyme and several bands with the commercial microbial chitinases. The same system was also used with native polyacrylamide gel electrophoresis at pH 8.9. Several bands were detected with the microbial chitinases. The same enzymes were also subjected to denaturing polyacrylamide gel electrophoresis in gradient gels containing 0.01% (W/V) glycol chitin. After electrophoresis, enzymes were renatured in buffered 1% (V/V) purified Triton X-100. Lytic zones were revealed by uv after staining with Calcofluor white M2R as for native gels. The molecular weights of chitinolytic enzymes could thus be directly estimated. In denaturing gels, as low as 10 ng of purified hen egg white lysozyme could be detected after 2 h incubation at 37 degrees C. Estimated molecular weights of St. griseus and Se. marcescens were between 24,000 and 72,000 and between 40,500 and 73,000, respectively. Some microbial chitinases were only resistant to denaturation with sodium dodecyl sulfate while others were resistant to sodium dodecyl sulfate and beta-mercaptoethanol.     

Mutation of a conserved tryptophan in the chitin-binding cleft of Serratia marcescens chitinase A enhances transglycosylation

Family 18 chitinases have the signature peptide DGXDXDXE forming the fourth beta-strand in the (beta/alpha)8-barrel of their catalytic domain. The carboxyl-end glutamic acid, E315 in Serratia marcescens chitinase A, serves as the acid/base during chitin hydrolysis, and the side-chain of the preceding aspartic acid, D313, helps to position correctly the N-acetyl moiety of the glycosyl sugar undergoing hydrolysis. Chitin substrates are bound within a long cleft across the top of the barrel, whose floor consists of aromatic residues that hydrophobically stack with every other GlcNAc. Alanine substitution of the conserved Trp167 at the -3 subsite in Serratia marcescens chitinase A enhanced transglycosylation. Higher oligosaccharides were formed from both chitin tetra- and pentasaccharide, and the only hydrolytic product from chitin trisaccharide was the disaccharide. Greater retention of the glycosyl fragment at the active site of the -3 mutant of Serratia marcescens chitinase A might favor transglycosylation due to a stabilized conformation of its D313.     

Crystal structure of imaginal disc growth factor-2. A member of a new family of growth-promoting glycoproteins from Drosophila melanogaster

Imaginal disc growth factor-2 (IDGF-2) is a member of a recently described family of Drosophila melanogaster-soluble polypeptide growth factors that promote cell proliferation in imaginal discs. Although their precise mode of action has not been established, IDGFs cooperate with insulin in stimulating the growth of imaginal disc cells. We report the crystal structure of IDGF-2 at 1.3-A resolution. The structure shows the classical (betaalpha)(8) barrel-fold of family 18 glycosyl hydrolases, with an insertion of an alpha + beta domain similar to that of Serratia marcescens chitinases A and B. However, amino acid substitutions in the consensus catalytic sequence of chitinases give IDGF-2 a less negatively charged environment in its putative ligand-binding site and preclude the nucleophilic attack mechanism of chitin hydrolysis. Particularly important is the replacement of Glu by Gln at position 132, which has been shown to abolish enzymatic activity in chitinases. Nevertheless, a modest conservation of residues that participate in oligosaccharide recognition suggests that IDGF-2 could bind carbohydrates, assuming several conformational changes to open the partially occluded binding site. Thus, IDGFs may have evolved from chitinases to acquire new functions as growth factors, interacting with cell surface glycoproteins implicated in growth-promoting processes, such as the Drosophila insulin receptor.     

Cloning, sequence, and expression of a chitinase gene from a marine bacterium, Altermonas sp. strain O-7.

The gene encoding an extracellular chitinase from marine Alteromonas sp. strain O-7 was cloned in Escherichia coli JM109 by using pUC18. The chitinase produced was not secreted into the growth medium but accumulated in the periplasmic space. A chitinase-positive clone of E. coli produced two chitinases with different molecular weights from a single chitinase gene. These proteins showed almost the same enzymatic properties as the native chitinase of Alteromonas sp. strain O-7. The N-terminal sequences of the two enzymes were identical. The nucleotide sequence of the 3,394-bp SphI-HindIII fragment that included the chitinase gene was determined. A single open reading frame was found to encode a protein consisting of 820 amino acids with a molecular weight of 87,341. A putative ribosome-binding site, promoter, and signal sequence were identified. The deduced amino acid sequence of the cloned chitinase showed sequence homology with chitinases A (33.4%) and B (15.3%) from Serratia marcescens. Regardless of origin, the enzymes of the two bacteria isolated from marine and terrestrial environments had high homology, suggesting that these organisms evolved from a common ancestor.     

Purification and properties of two chitinolytic enzymes of Serratia plymuthica HRO-C48

The chitinolytic rhizobacterium Serratia plymuthica HRO-C48 was previously selected as a biocontrol agent of phytopathogenic fungi. One endochitinase (E.C. 3.2.1.14), CHIT60, and one N-acetyl-beta-1,4- D-hexosaminidase (E.C. 3.2.1.52), CHIT100, were purified and characterized. The endochitinase CHIT60, with an apparent molecular mass of 60.5 kDa, had a N-terminal amino acid sequence highly similar to that of chitinases A from Serratia liquefaciens and Serratia marcescens. The enzyme activity had its peak at 55 degrees C and pH 5.4, and increased by more than 20% in the presence of 10 mM Ca(2+), Co(2+) or Mn(2+). Activity was inhibited by 80% in the presence of 10 mM Cu(2+). CHIT100 appeared to be a monomeric enzyme with a molecular mass of 95.6 kDa and a pI of 6.8. Optimal activity was obtained at 43 degrees C and pH 6.6, and decreased by more than 90 % in the presence of 10 mM Co(2+) or Cu(2+). CHIT100 (100 microg ml(-1)) inhibited spore germination and germ tube elongation of the phytopathogenic fungus Botrytis cinerea by 28 % and 31.6 %, respectively. With CHIT60 (100 microg ml(-1)), the effect was more pronounced: 78 % inhibition of of germination and 63.9 % inhibition of germ tube elongation.     

Cloning and sequencing of Serratia protease gene.

The gene encoding an extracellular metalloproteinase from Serratia sp. E-15 has been cloned, and its complete nucleotide sequence determined. The amino acid sequence deduced from the nucleotide sequence reveals that the mature protein of the Serratia protease consists of 470 amino acids with a molecular weight of 50,632. The G+C content of the coding region for the mature protein is 58%; this high G+C content is due to a marked preference for G+C bases at the third position of the codons. The gene codes for a short pro-peptide preceding the mature protein. The Serratia protease gene was expressed in Escherichia coli and Serratia marcescens; the former produced the Serratia protease in the cells and the latter in the culture medium. Three zinc ligands and an active site of the Serratia protease were predicted by comparing the structure of the enzyme with those of thermolysin and Bacillus subtilis neutral protease.     

Optimal dissolved oxygen concentration for the production of chitinases by Serratia marcescens

Chitinase production by Serratia marcescens 990E was maximal (34 U/ml) for a dissolved oxygen concentration ranging from 20 to 50% saturation. Chitin addition (6 g/l) at the end of fermentation reactivated the growth but did not bring about further chitinase accumulation. Four chitinases with different isoelectric points were separated.     

Effects of nitric oxide and nitrogen dioxide on bacterial growth

The effects of low concentrations of nitric oxide (NO) and nitrogen dioxide (NO2) on actively dividing cultures of Staphylococcus aureus, Micrococcus luteus, Micrococcus roseus, Serratia marcescens, Bacillus subtilis, Bacillus circulans, Bacillus megaterium, and Bacillus cereus were studied. Fresh cultures of each organism were incubated for 24 h at 25 degrees C on both nutrient agar and mineral salts glucose agar plates under atmospheres containing various low concentrations of NO in air (0 to 1.9 ppm [0 to 2.0 micrograms/g of air]), NO2 in air (0 to 5.5 ppm [0 to 8.8 micrograms/g of air]), or NO and NO2 in air. Bacteria grown under air only were used as controls. After incubation, the colonies that developed on the plates were counted. None of the bacteria tested was affected by NO or NO2 at the indicated concentrations while growing on nutrient agar. Serratia marcescens, B. circulans, B. subtilis, B. megaterium, and B. cereus grown on mineral salts glucose agar were not significantly affected by NO or NO2. Low concentrations (0 to 1.9 ppm) of NO were bacteriostatic to log-phase cultures of M. roseus, M. luteus, and Staphylococcus aureus grown on mineral salts glucose agar. Bacteriostatic activity over a 24-h interval was maximal at an initial NO concentration of 1 ppm. Appreciable amounts of NO2 were produced in 24 h at initial NO concentrations greater than 1 ppm. These results suggest that NO2 may reduce the bacteriostatic activity of NO. Low concentrations (0 to 5.5 ppm) of NO2 in air did not affect any of the bacteria tested. At these low concentrations, NO affected bacterial growth, although NO2, NO2-, and NO3- did not. In addition, it was determined that the bacteriostatic activity observed in this study was not due to an increase in the acidity of the medium.     

Effects of nitric oxide and nitrogen dioxide on bacterial growth.

The effects of low concentrations of nitric oxide (NO) and nitrogen dioxide (NO2) on actively dividing cultures of Staphylococcus aureus, Micrococcus luteus, Micrococcus roseus, Serratia marcescens, Bacillus subtilis, Bacillus circulans, Bacillus megaterium, and Bacillus cereus were studied. Fresh cultures of each organism were incubated for 24 h at 25 degrees C on both nutrient agar and mineral salts glucose agar plates under atmospheres containing various low concentrations of NO in air (0 to 1.9 ppm [0 to 2.0 micrograms/g of air]), NO2 in air (0 to 5.5 ppm [0 to 8.8 micrograms/g of air]), or NO and NO2 in air. Bacteria grown under air only were used as controls. After incubation, the colonies that developed on the plates were counted. None of the bacteria tested was affected by NO or NO2 at the indicated concentrations while growing on nutrient agar. Serratia marcescens, B. circulans, B. subtilis, B. megaterium, and B. cereus grown on mineral salts glucose agar were not significantly affected by NO or NO2. Low concentrations (0 to 1.9 ppm) of NO were bacteriostatic to log-phase cultures of M. roseus, M. luteus, and Staphylococcus aureus grown on mineral salts glucose agar. Bacteriostatic activity over a 24-h interval was maximal at an initial NO concentration of 1 ppm. Appreciable amounts of NO2 were produced in 24 h at initial NO concentrations greater than 1 ppm. These results suggest that NO2 may reduce the bacteriostatic activity of NO. Low concentrations (0 to 5.5 ppm) of NO2 in air did not affect any of the bacteria tested. At these low concentrations, NO affected bacterial growth, although NO2, NO2-, and NO3- did not. In addition, it was determined that the bacteriostatic activity observed in this study was not due to an increase in the acidity of the medium.     

Expression of chitinase A (chiA) gene from a local isolate of Serratia marcescens in Coleoptera-specific Bacillus thuringiensis

Aims:  The present study focused on cloning and expression of chiA gene from a highly chitinolytic local isolate of Serratia marcescens in an anti-Coleopteran Bacillus thuringiensis and comparison of the characteristics of the native and recombinant ChiAs.
Methods and Results:  chiA gene from Ser . marcescens was cloned, sequenced and compared with the previously cloned chiA genes. chiA gene was PCR cloned and expressed in anti-Coleopteran B. thuringiensis strain 3023 as verified by Western blot analysis. Specific ChiA activity of the recombinant B. thuringiensis (strain 3023-SCHI) reached its highest level at 21st hour of growth (16·93 U mg⁻¹), which was 5·2- and 1·3-fold higher than that of its parental strain and Ser . marcescens , respectively. Temperature and pH effects on native and recombinant ChiAs were next determined. The recombinant plasmid was quite stable over 240 generations.
Conclusions:  Serratia marcescens ChiA was heterologously expressed in an anti-Coleopteran B. thuringiensis at levels even higher than that produced by the source organism.
Significance and Impact of the Study:  Bacillus thuringiensis 3023-SCHI co-expressing anti-Coleopteran Cry3Aa protein and Ser . marcescens chitinase offers a viable alternative to the use of chitinolytic microbes/enzymes in combination with entamopathogenic bacteria for an increased potency because of synergistic interaction between them.     

Estimating disease resistance in insects: phenoloxidase and lysozyme-like activity and disease resistance in the cricket Gryllus texensis

An animal's ability to resist disease is usually estimated by measuring one or more components of the immune system. There is an assumption that these assays of immunity measure an animal's ability to mount an effective immune response. This paper tests this assumption by examining the relationship between two common estimates of insect immunocompetence, phenoloxidase and lysozyme-like enzyme activity, and resistance to three common insect bacterial pathogens: Serratia marcescens, Serratia liquefaciens, and Bacillus cereus. There was a correlation (Spearman's rs=0.33, p<0.001, n=190 pairs) between total phenoloxidase and baseline lysozyme-like activity within individuals. However, total phenoloxidase and baseline lysozyme-like activity levels did not predict which male crickets would survive any of the three bacterial challenges. Lysozyme-like activity increased after an immune challenge (Friedman, 33.72, p<0.001), and the greater the increase, the greater the chance that the cricket would survive S. marcescens (slope=0.15, chi 2=8.2, p=0.005) or B. cereus (slope=0.8, chi 2=6.4, p=0.01). The crickets with a greater total hemolymph protein concentration were also more likely to survive a challenge with any of the three bacterial pathogens than the crickets with lower total hemolymph protein concentrations (S. liquefaciens: slope=0.02, chi 2=9.2, p=0.002; B. cereus: slope=0.02, chi 2=6.5, p=0.01; S. marcescens: slope=0.03, chi 2=7.8, p=0.005). Because of the complexity of the immune system, empirical tests of the relationship between assays of immunity and resistance to a range of actual pathogens are important for correctly interpreting these measures.