Directed evolution for increased chitinase activity

Directed evolution through DNA shuffling and screening was used to enhance the catalytic ability of a fungal, Beauveria bassiana, chitinase, Bbchit1. The Bbchit gene was first linked to various prokaryotic signal sequences and expressed in Escherichia coli. The signal peptide, PelB, from Erwinia carotovora resulted in greatest chitinase secretion into broth. The nucleotide sequence expressing PelB signal peptide was then incorporated into an E. coli vector to express Bbchit1 variants generated by three rounds of DNA shuffling. A Bbchit1 library with 150,000 variants was constructed with a nucleotide point mutation frequency of 0.6% and screened for chitinolytic activity. Two Bbchit1 variants (SHU-1 and SHU-2) were selected that showed increased chitinolytic activity compared to the wild type. Sequence analysis of these variants revealed mutations in amino acid residues that would not normally be considered for rational design of improved chitinase activity. The amino acid substitutions occurred outside of the two putative substrate-binding sites and the catalytic region.     

Enhanced fungal resistance in Arabidopsis expressing wild rice PR-3 (OgChitIVa) encoding chitinase class IV

Oryza grandiglumis Chitinase IVa (OgChitIVa) cDNA encoding a class IV chitinase was cloned from wild rice (Oryza grandiglumis). OgChitIVa cDNA contains an open reading frame of 867 nucleotides encoding 288 amino acid residues with a predicted molecular weight of 30.4 kDa and isoelectric point of 8.48. Deduced amino acid sequences of OgChitIVa include the signal peptide and chitin-binding domain in the N-terminal domain and conserved catalytic domain. OgChitIVa showed significant similarity at the amino acid level with related monocotyledonous rice and maize chitinase, but low similarity with dicotyledoneous chitinase. Southern blot analysis showed that OgChitIVa genes are present as two copies in the wild rice genome. It was shown that RNA expression of OgChitIVa was induced by defense/stress signaling chemicals, such as jasmonic acid, salicylic acid, and ethephon or cantharidin and endothall or wounding, and yeast extract. It was demonstrated that overexpression of OgChitIVa in Arabidopsis resulted in mild resistance against the fungal pathogen, Botrytis cinerea, by lowering disease rate and necrosis size. RT-PCR analysis showed that PR-1 and PR-2 RNA expression was induced in the transgenic lines. Here, we suggest that a novel OgChitIVa gene may play a role in signal transduction process in defense response against B. cinerea in plants. J.-H. Pak and E.-S. Chung contributed equally to this work.     

A stress responsive alternative splicing mechanism in Citrus clementina leaves

Chitinases are often considered pathogenesis-related proteins since their activity can be induced by viral infections, fungal and bacterial cell wall components, and also by more general sources of stress such as wounding, salicylic acid, ethylene, auxins and cytokinins. In the present study, comparative proteomic analysis showed the defense-related acidic chitinase II to be specifically induced in Citrus clementina leaves infested by the two-spotted spider mite Tetranychus urticae or treated with MeJA. In parallel, changes in the mRNA profiles of two partially homologous chitinase forms were shown by RT-PCR. In particular, the appearance of an additional cDNA chitinase fragment in T. urticae-infested and MeJA-treated leaves was observed. This finding may indicate a specific regulatory mechanism of chitinase expression. We report evidence for alternative splicing in T. urticae-infested C. clementina, where a premature stop codon after the first 135 amino acids was introduced. We observed inducible chitinase activity after MeJA treatment, indicative of a rapid plant response to infestation. This work provides the first evidence of chitinase alternative splicing in C. clementina. In addition, the presence of the dual-band pattern for chitinase cDNA by RT-PCR may represent a suitable predictive marker for early diagnosis of plant biotic stress.     

Characterization of Antifungal Chitinase from Marine Streptomyces sp. DA11 Associated with South China Sea Sponge Craniella Australiensis

The gene cloning, purification, properties, kinetics, and antifungal activity of chitinase from marine Streptomyces sp. DA11 associated with South China sponge Craniella australiensis were investigated. Alignment analysis of the amino acid sequence deduced from the cloned conserved 451 bp DNA sequence shows the chitinase belongs to ChiC type with 80% similarity to chitinase C precursor from Streptomyces peucetius. Through purification by 80% ammonium sulfate, affinity binding to chitin and diethylaminoethyl-cellulose anion-exchange chromatography, 6.15-fold total purification with a specific activity of 2.95 Umg⁻¹ was achieved. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a molecular weight of approximately 34 kDa and antifungal activities were observed against Aspergillus niger and Candida albicans. The optimal pH, temperature, and salinity for chitinase activity were 8.0, 50°C, and 45 g‰ psu, respectively, which may contribute to special application of this marine microbe-derived chitinase compared with terrestrial chitinases. The chitinase activity was increased by Mn²⁺, Cu²⁺, and Mg²⁺, while strongly inhibited by Fe²⁺ and Ba²⁺. Meanwhile, SDS, ethyleneglycoltetraacetic acid, urea, and ethylenediaminetetraacetic acid were found to have significantly inhibitory effect on chitinase activity. With colloidal chitin as substrates instead of powder chitin, higher V _max (0.82 mg product/min·mg protein) and lower K _m (0.019 mg/ml) values were achieved. The sponge’s microbial symbiont with chitinase activity may contribute to chitin degradation and antifungal defense. To our knowledge, it was the first time to study sponge-associated microbial chitinase.     

Cloning, expression, and characterization of a highly thermostable family 18 chitinase from Rhodothermus marinus

A family 18 chitinase gene chiA from the thermophile Rhodothermus marinus was cloned and expressed in Escherichia coli. The gene consisted of an open reading frame of 1,131 nucleotides encoding a protein of 377 amino acids with a calculated molecular weight of 42,341 Da. The deduced ChiA was a non-modular enzyme with one unique glycoside hydrolase family 18 catalytic domain. The catalytic domain exhibited 43% amino acid identity with Bacillus circulans chitinase C. Due to poor expression of ChiA, a signal peptide-lacking mutant, chiAsp, was designed and used subsequently. The optimal temperature and pH for chitinase activity of both ChiA and ChiAsp were 70°C and 4.5–5, respectively. The enzyme maintained 100% activity after 16 h incubation at 70°C, with half-lives of 3 h at 90°C and 45 min at 95°C. Results of activity measurements with chromogenic substrates, thin-layer chromatography, and viscosity measurements demonstrated that the chitinase is an endoacting enzyme releasing chitobiose as a major end product, although it acted as an exochitobiohydrolase with chitin oligomers shorter than five residues. The enzyme was fully inhibited by 5 mM HgCl₂, but excess ethylenediamine tetraacetic acid relieved completely the inhibition. The enzyme hydrolyzed 73% deacetylated chitosan, offering an attractive alternative for enzymatic production of chitooligosaccharides at high temperature and low pH. Our results show that the R. marinus chitinase is the most thermostable family 18 chitinase isolated from Bacteria so far.     

Cloning, expression, characterization and crystallization of BRP39, a signalling glycoprotein expressed during mammary gland apoptosis

Breast regression protein (BRP39) is a glycoprotein, which is expressed during mammary gland involution in mouse. The physiological function of BRP39 is not known. High levels of expression of BRP39 have also been associated with breast cancer development. In the present investigation a cDNA encoding rBRP39 (recombinant BRP39) was cloned by PCR techniques. It consists of 1,143 nucleotides and encodes an open reading frame of 381 amino acid residues including a signal sequence of 21 amino acids. Recombinant BRP39 was produced in E. coli in a soluble form at low temperature (15 °C). Expression and purification of rBRP39 was confirmed by western blot analysis. Purified rBRP39 showed high chitin-binding activity but no chitinase activity. The lack of chitinase activity may be attributed to the mutation of critical active site residue Glu120 to Leu120 and Asp118 to Ala118 in BRP39. However, a mutant in which the residue was reverted back to Glu, by site directed mutagenesis, displayed no chitinase activity. Purified recombinant BRP39 was crystallized and the crystals diffracted X-rays to 2.8 Å resolution. The crystals belonged to the space group C2 with unit cell parameters a = 130.4 Å, b = 81.3 Å, c = 229.2 Å, β = 105.9°. The structure refinement is in progress.     

Purification and anti-fungal activity of chitinase against Pyricularia grisea in finger millet

Pseudomonas fluorescens isolate 1 (Pfl) protected finger millet plants treated with the ragi blast fungus, Pyricularia grisea, by upto 27% depending on the cultivar. Induction of pathogenesis-related proteins, viz., chitinase by Pfl isolate, was studied against Py. grisea. The activity of chitinase from plants treated with Pfl was significantly higher than the control plant after pathogen inoculation in all cultivars tested. Chitinase in the cultivars, with and without challenge by Py. grisea, revealed changes in the isoform pattern by western blot analysis. Chitinase was purified by affinity chromatography from the Pfl-treated leaves. It showed a single band at 57 kDa after SDS-PAGE. Western blot analysis using barley chitinase antiserum confirmed a 57 kDa chitinase. The chitinase had anti-fungal activity against Py. grisea in vitro. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cloning,characterization and expression of the chitinase gene of <Emphasis Type="Italic">Enterobacter</Emphasis> sp. NRG4

A chitinase producing bacterium Enterobacter sp. NRG4, previously isolated in our laboratory, has been reported to have a wide range of applications such as anti-fungal activity, generation of fungal protoplasts and production of chitobiose and N-acetyl D-glucosamine from swollen chitin. In this paper, the gene coding for Enterobacter chitinase has been cloned and expressed in Escherichia coli BL21(DE3). The structural portion of the chitinase gene comprised of 1686 bp. The deduced amino acid sequence of chitinase has high degree of homology (99.0%) with chitinase from Serratia marcescens. The recombinant chitinase was purified to near homogeneity using His-Tag affinity chromatography. The purified recombinant chitinase had a specific activity of 2041.6 U mg⁻¹. It exhibited similar properties pH and temperature optima of 5.5 and 45°C respectively as that of native chitinase. Using swollen chitin as a substrate, the K_m, k_cat and catalytic efficiency (k_cat/K_m) values of recombinant chitinase were found to be 1.27 mg ml⁻¹, 0.69 s⁻¹ and 0.54 s⁻¹M⁻¹ respectively. Like native chitinase, the recombinant chitinase produced medicinally important N-acetyl D-glucosamine and chitobiose from swollen chitin and also inhibited the growth of many fungi.     

Functional expression in Beauveria bassiana of a chitinase gene from Ophiocordyceps unilateralis,an ant-pathogenic fungus

A gene encoding chitinase was cloned from Ophiocordyceps unilateralis, a Formamidae-specific fungus, collected from Sirindhorn Peat Swamp Forest, Thailand. The O. unilateralis chitinase (OuChi) full-length gene (1311 bp) encodes 436 amino acids with the first 20 amino acids as a putative signal peptide. The gene showed highest identity (78%) to Isaria farinose endochitinase. To investigate if cross-species chitinase expression also enhances fungal toxicity, the mature OuChi gene was subcloned into an Agrobacterium binary vector pPZP-bar and then transformed into Beauveria bassiana strain BCC2659. Chitinase activity was detected using 4-methylumbelliferyl-β-D-N,N′-diacetylchitobioside. The fungal transformant expressing O. unilateralis chitinase showed higher toxicity against Spodoptera exigua. These results support the hypothesis that chitinolytic enzymes are one of several ‘virulence’ factors produced by entomopathogenic fungi during host encounter.     

Characterization of gastrointestinal chitinase in the lizard Sceloporus undulatus garmani (Reptilia: Phrynosomatidae)

Most studies on chitinase activity in lizards have been concerned with Palaearctic (European) and Laurasian (Middle Eastern and Asian) taxa. Several genera of Old World lizards, Anguis, Uromastix, Chamaeleo and Lacerta, have been shown to possess chitinolytic activity. To date, only one New World lizard, Anolis carolinensis, has been reported to exhibit chitinolytic activity. In the present study, chitinase activity was characterized in a second New World taxon, Sceloporus undulatus garmani, a New World, phrynosomatid lizard. Chitinolytic activity was measured by incubating tissue extracts with a radioactive chitin substrate, acetyl-[H³]chitin and determining acid soluble radioactivity as an estimate for chitin hydrolysis. Chitinolytic activity was present in stomach, small intestine and pancreas extracts, with the stomach and pancreas having the highest specific activities. Chitinolytic activity was higher at pH 4.5 than at pH 7.5. The stomach chitinase is immunologically similar to the gastric chitinase previously described for rainbow trout. Western blot analysis showed anti-chitinase cross-reactivity in the extracts of the stomach, but no cross-reactivity in the pancreatic or intestinal extracts, suggesting different isoforms of chitinase. There was no detected lysozyme activity (less than 0.01 mg/ml lysozyme) present in the extracts of the stomach, small intestine and pancreas. The localization of chitinolytic activity in S. u. garmani is in agreement with earlier reptilian reports on the distribution of chitinase.     

Enhanced expression of chitinase during the autolysis of mushroom in <Emphasis Type="Italic">Coprinellus congregatus</Emphasis>

Fungal cell walls consist of various glucans and chitin. An inky cap, Coprinellus congregates, produced mushrooms at 25°C in a regime of 15 h light/9 h dark, and then the mushroom was autolyzed rapidly to generate black liquid droplets where no cell wall was detected by microscopy. A chitinase cDNA from the matured mushroom cells of C. congregates that consisted of 1,541 nucleotides was successfully cloned using the rapid amplification of cDNA ends (RACE)-PCR technique. Its deduced 441 amino acid sequence had the conserved catalytic domain as in other fungal chitinase family 18. Chitinase activity was higher at the matured mushroom stage than primordial and young mushroom stage. When the expression of the cloned chitinase was examined by real-time PCR using the chitinase-specific primers, it was increased more than twice to 20 times during the autolytic process of mushroom than young mushroom or primordial stages, respectively.     

Characterization of β-N-acetylglucosaminidase from a marine Pseudoalteromonas sp. for application in N-acetyl-glucosamine production

The psychrotolerant Pseudoalteromonas issachenkonii PAMC 22718 was isolated for its high exo-acting chitinase activity in the Kara Sea, Arctic. An exo-acting chitinase (W-Chi22718) was homogeneously purified from the culture supernatant of PAMC 22718, the molecular weight of which was estimated to be approximately 112?kDa. Due to its β-N-acetylglucosaminidase activity, W-Chi22718 was able to produce N-acetyl-D-glucosamine (GlcNAc) monomers from chitin oligosaccharide substrates. W-Chi22718 displayed chitinase activity from 0 to 37°C (optimal temperature of 30°C) and maintained activity from pH 6.0 to 9.0 (optimal pH of 7.6). W-Chi22718 exhibited a relative activity of 13 and 35% of maximal activity at 0 and 10°C, respectively, which is comparable to the activities of previously characterized, cold-adapted bacterial chitinases. W-Chi22718 activity was enhanced by K⁺, Ca²⁺, and Fe²⁺, but completely inhibited by Cu²⁺ and SDS. We found that W-Chi22718 can produce much more (GlcNAcs) from colloidal chitin, working together with previously characterized cold-active endochitinase W-Chi21702. Genome sequencing revealed that the corresponding gene (chi22718_IV) was 2,856?bp encoding a 951?amino acid protein with a calculated molecular weight of approximately 102?kDa.     

Isolation and Characterization of the Genes Encoding Basic and Acidic Chitinase in Arabidopsis thaliana

Plants synthesize a number of antimicrobial proteins in response to pathogen invasion and environmental stresses. These proteins include two classes of chitinases that have either basic or acidic isoelectric points and that are capable of degrading fungal cell wall chitin. We have cloned and determined the nucleotide sequence of the genes encoding the acidic and basic chitinases from Arabidopsis thaliana (L.) Heynh. Columbia wild type. Both chitinases are encoded by single copy genes that contain introns, a novel feature in chitinase genes. The basic chitinase has 73% amino acid sequence similarity to the basic chitinase from tobacco, and the acidic chitinase has 60% amino acid sequence similarity to the acidic chitinase from cucumber. Expression of the basic chitinase is organ-specific and age-dependent in Arabidopsis. A high constitutive level of expression was observed in roots with lower levels in leaves and flowering shoots. Exposure of plants to ethylene induced high levels of systemic expression of basic chitinase with expression increasing with plant age. Constitutive expression of basic chitinase was observed in roots of the ethylene insensitive mutant (etr) of Arabidopsis, demonstrating that root-specific expression is ethylene independent. Expression of the acidic chitinase gene was not observed in normal, untreated Arabidopsis plants or in plants treated with ethylene or salicylate. However, a transient expression assay indicated that the acidic chitinase promoter is active in Arabidopsis leaf tissue.     

Molecular cloning and characterization of a pea chitinase gene expressed in response to wounding,fungal infection and the elicitor chitosan

The fungicidal class I endochitinases (E.C.3.3.1.14, chitinase) are associated with the biochemical defense of plants against potential pathogens. We isolated and sequenced a genomic clone, DAH53, corresponding to a class I basic endochitinase gene in pea, Chil. The predicted amino acid sequence of this chitinase contains a hydrophobic C-terminal domain similar to the vacuole targeting sequences of class I chitinases isolated from other plants. The pea genome contains one gene corresponding to the chitinase DAH53 probe. Chitinase RNA accumulation was observed in pea pods within 2 to 4 h after inoculation with the incompatible fungal strain Fusarium solani f. sp. phaseoli, the compatible strain F. solani f.sp. pisi, or the elicitor chitosan. The RNA accumulation was high in the basal region (lower stem and root) of both fungus challenged and wounded pea seedlings. The sustained high levels of chitinase mRNA expression may contribute to later stages of pea's non-host resistance.     

Site-directed Mutagenesis of Chitinase from Alteromonas sp. Strain O–7

Chitinase (Chi85) from Alteromonas sp. strain 0–7 contains the two conserved regions common to microbial and plant chitinases. We did site-directed mutagenesis of Chi85 to investigate the effects of the conserved amino acid residues on chitinase activity. We suggest that Asp-290 and Glu-292 of Chi85 may be the essential amino acid residues for the cleavage of β-glycosidic linkage of chitin.     

Identification and characterization of chitinolytic bacteria isolated from a freshwater lake

To develop a novel type of biocontrol agent, we focus on bacteria that are characterized by both chitinase activity and biofilm development. Chitinolytic bacteria were isolated from sediments and chitin flakes immersed in the water of a sand dune lake, Sakata, in Niigata, Japan. Thirty-one isolates from more than 5100 isolated strains were examined chitinase activity and biofilm formation. Phylogenetic analysis of these isolates based on the 16S rRNA gene sequences revealed that most isolates belonged to the family Aeromonadaceae, followed by Paenibacillaceae, Enterobacteriaceae, and Neisseriaceae. The specific activity of chitinase of four selected strains was higher than that of a reference strain. The molecular size of one chitinase produced by Andreprevotia was greater than that of typical bacterial chitinases. The dialyzed culture supernatant containing chitinases of the four strains suppressed hyphal growth of Trichoderma reesei. These results indicate that these four strains are good candidates for biocontrol agents.     

Isolation and Identification of Streptomyces fradiae SU-1 from Thailand and Protoplast Transformation with the Chitinase B Gene from Nocardiopsis prasina OPC-131

Thirty-two strains of actinomycetes obtained from soil samples of Thailand were selected. Actinomycete strain SU-1 is the most effective in terms of antagonism of Fusarium moniliforme. It produces antifungal substances on agar medium against F. moniliforme. On the basis of microscopical observations of its morphology and biochemical tests as well as analysis of cell wall and fatty acid pattern, this strain was identified as Streptomyces fradiae. The chitinase gene B (chiB337) from Nocardiopsis prasina OPC-131 was inserted into an integrating plasmid pFIS318, an Escherichia coli–Streptomyces shuttle vector. The new plasmid pFIS319-1 carrying the chitinase gene was used to transform protoplasts of S. fradiae strain SU-1. The obtained recombinant strain SU-1 pFIS319-1 exhibited higher chitinase activity than the wild-type in chitinase induction medium. Chitinase activity after renaturing protein from SDS-PAGE was detected rapidly by using 4-methylumbelliferyl β-D-N,N′’-diacetylchitobioside as the substrate. S. fradiae SU-1 secreted two chitinases with estimated molecular masses of 26 kDa and 43 kDa whereas the recombinant strain secreted three chitinases of about 26 kDa, 31.5 kDa (ChiB), and 43 kDa. The supernatant of the recombinant strain grown in chitinase induction medium inhibited the hyphal extension of F. moniliforme.     

Isolation of an osmotic stress- and abscisic acid-induced gene encoding an acidic endochitinase from Lycopersicon chilense

We have identified one osmotic stress- and abscisic acid-responsive member of the endochitinase (EC 3.2.1.14) gene family from leaves of drought-stressed Lycopersicon chilense plants, a natural inhabitant of extremely arid regions in South America. The 966-bp full-length cDNA (designated pcht28) encodes an acidic chitinase precursor with an amino-terminal signal peptide. The mature protein is predicted to have 229 amino acid residues with a relative molecular mass of 24 943 and pI value of 6.2. Sequence analysis revealed that pcht28 has a high degree of homology with class II chitinases (EC 3.2.1.14) from tomato and tobacco. Expression of the pcht28 protein in Escherichia coli verified that it is indeed a chitinase. Northern blot analysis indicated that this gene has evolved a different pattern of expression from that of other family members reported thus far. It is highly induced by both osmotic stress and the plant hormone abscisic acid. Southern blot analysis of genomic DNA suggested that the pcht28-related genes may form a small multigene family in this species. The efficiency of induction of the gene by drought stress, in leaves and stems, is significantly higher in L. chilense than in the cultivated tomato. It is speculated that, besides its general defensive function, the pcht28-encoded chitinase may play a particular role in plant development or in protecting plants from pathogen attack during water stress.     

A new class of tobacco chitinases homologous to bacterial exo-chitinases displays antifungal activity

A novel chitinase gene of tobacco was isolated and characterized by DNA sequence analysis of a genomic clone and a cDNA clone. Comparative sequence analysis of both clones showed an identity of 94%. The proteins encoded by these sequences do not correspond to any of the previously characterized plant chitinases of classes I–IV and are designated as class V chitinases. Comparison of the chitinase class V peptide sequence with sequences in the Swiss Protein databank revealed significant sequence similarity with bacterial exo-chitinases from Bacillus circulans, Serratia marcescens and Streptomyces plicatus. It was demonstrated that class V chitinase gene expression is induced after treatment of tobacco with different forms of stress, like TMV-infection, ethylene treatment, wounding or ultraviolet irradiation. Two related chitinase class V proteins of 41 and 43 kDa were purified from Samsun NN tobacco leaves inoculated with tobacco mosaic virus. The proteins were purified by Chelating Superose chromatography and gel filtration. In vitro assays demonstrated that class V chitinases have endo-chitinase activity and exhibit antifungal activity toward Trichoderma viride and Alternaria radicina. In addition, it was shown that class V chitinase acts synergistically with tobacco class I β-1,3-glucanase against Fusarium solani germlings.