• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.6
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• pp.482-493
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• 2005

Biomass is originally photosynthesized from inorgainic compounds such as $CO_2$, minerals, water and solar energy. Recent studies have shown that anaerobic bacteria have the ability to convert recalcitrant biomass such as cellullosic or chitinoic materials to useful compounds. The biomass containing agricultural waste, unutilized wood and other garbage is expected to utilize as feed, food and fuel by microbial degradation and other metabolic functions. In this study we isolated several anaerobic, cellulolytic and chitinolytic bacteria from rumen fluid, compost and soil to study their related enzymes and genes. The anaerobic and cellulolytic bacteria, Clostridium thermocellum, Clostridium stercorarium, and Clostridium josui, were isolated from compost and the chitinolytic Clostridium paraputrificum from beach soil and Ruminococcus albus was isolated from cow rumen. After isolation, novel cellulase and xylanase genes from these anaerobes were cloned and expressed in Escherichia coli. The properties of the cloned enzymes showed that some of them were the components of the enzyme (cellulase) complex, i.e., cellulosome, which is known to form complexes by binding cohesin domains on the cellulase integrating protein (Cip: or core protein) and dockerin domains on the enzymes. Several dockerin and cohesin polypeptides were independently produced by E. coli and their binding properties were specified with BIAcore by measuring surface plasmon resonance. Three pairs of cohesin-dockerin with differing binding specificities were selected. Two of their genes encoding their respective cohesin polypeptides were combined to one gene and expressed in E. coli as a chimeric core protein, on which two dockerin-dehydrogenase chimeras, the dockerin-formaldehyde dehydrogenase and the dockerin-NADH dehydrogenase are planning to bind for catalyzing $CO_2$ reduction to formic acid by feeding NADH. This reaction may represent a novel strategy for the reduction of the green house gases. Enzymes from the anaerobes were also expressed in tobacco and rice plants. The activity of a xylanase from C. stercorarium was detected in leaves, stems, and rice grain under the control of CaMV35S promoter. The digestibility of transgenic rice leaves in goat rumen was slightly accelerated. C. paraputrificum was found to solubilize shrimp shells and chitin to generate hydrogen gas. Hydrogen productivity (1.7 mol $H_2/mol$ glucos) of the organism was improved up to 1.8 times by additional expression of the own hydrogenase gene in C. paraputrficum using a modified vector of Clostridiu, perfringens. The hydrygen producing microflora from soil, garbage and dried pelletted garbage, known as refuse derived fuel(RDF), were also found to be effective in converting biomass waste to hydrogen gas.

• The Plant Pathology Journal
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• v.25 no.4
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• pp.344-351
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• 2009

A biocontrol bacterium Bacillus licheniformis N1 grown in nutrient broth showed no chitinolytic activity, while its genome contains a gene which encodes a chitinase. The gene for chitinase from B. licheniformis N1 was amplified by PCR and the deduced amino acid sequence analysis revealed that the chitinase exhibited over 95% identity with chitinases from other B. licheniformis strains. Escherichia coli cells carrying the recombinant plasmid displayed chitinase activity as revealed by the formation of a clear zone on chitin containing media, indicating that the gene could be expressed in E. coli cells. Chitinase gene expression in B. licheniformis N1 was not detected by RT-PCR analysis. The protein was over-expressed in E. coli BL21 (DE3) as a glutathione S-transferase fusion protein. The protein could also be produced in B. subtilis 168 strain carrying the chitinase gene of N1 strain. The crude protein extract from E. coli BL21 carrying GST fusion protein or culture supernatant of B. subtilis carrying the chitinase gene exhibited enzyme activity by hydrolyzing chitin analogs, 4-methylumbelliferyl-$\beta$-D-N,N'-diacetylchitobioside and 4-methylumbelliferyl-$\beta$-D-N,N',N"-triacetylchitotrioside. These results indicated that even though the chitinase gene is not expressed in the N1 strain, the coding region is functional and encodes an active chitinase enzyme. Furthermore, B. subtilis 168 transformants expressing the chitinase gene exhibited antifungal activity against Fulvia fulva by suppressing spore germination. Our results suggest that the proper engineering of the expression of the indigenous chitinase gene, which will lead to its expression in the biocontrol strain B. licheniformis N1, may further enhance its biocontrol activity.

• Journal of Sericultural and Entomological Science
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• v.39 no.2
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• pp.161-168
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• 1997

To clarify the effect of anti-juvenile hormone analogue (AJH) on the larval ecdysis by feeding at early stage of the 4th instar, the total amount of protein and activity of chitinolytic enzymes in the integument of Bombyx mori were analyzed, PAGE pattern of the protein was observed and the morphological changes of integument during molting period were also observed and the morphological changes of integument during molting period were also observed by means of TEM. The total amount of protein was greatly increased in premolting, then reached maximum level just before ecdysis, and rapidly decreased after the larval ecdysis in the control, while in the AJH treatment, increased 12 hr later than the control and its maximum was only 82.6% of the control. Two specific proteins, which were presumed as the protein originated from endocuticle, also appeared 12 hr later than the control and were maintained to 132 hr after AJH treatment from the aspects of the Native- and SDS-PAGE patterns, although those of the control disappeared instantly after ecdysis. Chitinase and $\beta$-N-acetylglucosaminidase activities were also suppressed and delayed by AJH treatment. Furthermore, it was observed that the apolysis took place 12 hr later than the control but new epicuticle was not formed at least until 132 hr after AJH treatment. From these results, it is suggested that the larval molting process of silkworm develops 12 hr later than the control but new epicuticle was not formed at least until 132 hr after AJH treatment. From these results, it is suggested that the larval molting process of silkworm develops 12 hr later than the control by AJH treatment but no further processing takes place just after apolysis.

• Journal of Marine Life Science
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• v.8 no.1
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• pp.56-67
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• 2023

Marine macroalgae are important in coastal ecosystems and interact with marine microorganisms. In this study, we isolated fungi from seven types of marine macroalgae including Cladophora sp., Gloiopeltis furcate, Gracilariopsis chorda, Hydroclathrus clathratus, Prionitis crispata, Sargassum micracanthum, and Ulva lactuca collected in Korea. Morphological and phylogenetic analyses identified the isolates as four Aspergillus spp. (A. fumigatus, A. sydowii, A. tamarii, and A. terreus), three Penicillium spp. (P. crustosum, P. jejuense, and P. rubens), and Cladosporium tenuissimum. Among them, A. fumigatus TOP-U2, A. tamarii SH-Sw5, and A. terreus GJ-Gf2 strains showed the activities of all enzymes examined (amylase, chitinase, lipase, and protease). Based on the enzymatic index (EI) values in solid media, A. terreus GJ-Gf2 and C. tenuissimum UL-Pr1 exhibited the highest amylase and lipase activities, respectively. Chitinolytic activity was only observed in A. terreus GJ-Gf2, A. tamarii SH-Sw5, and A. fumigatus TOP-U2. Penicillium crustosum UL-Cl2 and C. tenuissimum UL-Pr1 showed the highest protease activities. To the best of our knowledge, this is the first report of lipolytic and proteolytic activities in a marine-derived C. tenuissimum strain. Overall, the fungal strains isolated from the marine macroalgae in this study actively produced industrially important enzymes.

• Korean Journal of Soil Science and Fertilizer
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• v.43 no.4
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• pp.490-497
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• 2010

A chitinolytic bacterium having a strong antagonistic activity against various pathogens including Phytophtora capsici was isolated from rhizosphere soil, and identified as Lysobacter enzymogenes (named as LE429) based on 16S rRNA gene sequence analysis. This strain produced a number of substances such as chitinase, ${\beta}-1$, 3-glucanase, lipase, protease, gelatinase and an antibiotic compound. This antibiotic compound was purified by diaion HP-20, sephadex LH-20 column chromatography and HPLC. The purified compound was identified as phenylacetic acid by gas chromatography-electron ionization (GC-EI) and gas chromatography-chemical ionization (GC-CI) mass spectrometry. In field experiment, pepper plants were treated by the strain LE429 culture (CB), neem oil solution (NO), combination (CB+NO) or control (CON). Plant height and number of branches, flowers and pods of pepper plant in CB treatment were generally highest, and followed by CB+NO, CON and NO. The fungal pathogens were strongly inhibited, while several insect pests were discovered in CB treatment. Any insect pests were not found, while all fungal pathogens tested were not suppressed in NO treatment. However, in CB+NO treatment, non incidence of fungal pathogens and insect pests were found. The strain LE429 producing secondary metabolites with neem oil should be a potential agent to control fungal diseases and insect pests.

• Korean Journal of Microbiology
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• v.48 no.4
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• pp.293-297
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• 2012

Of the 169 strains of microorganisms stored in Polar and Alpine Microbial Collection of Korea Polar Research Institute, 27 strains were selected for their chitinase activity on ZoBell plates supplemented with 0.4% colloidal chitin. Among them, PAMC 21693 strain have shown the highest chitinolytic enzyme activity toward pNP-$(GlcNAc)_1$ at low temperature and the highest growth rate at $4^{\circ}C$. We cloned a full-length chitinase gene of 2,857 bp which contains an open reading frame of 2,169 bp encoding 872-amino acid polypeptide. Recombinant chitinase protein was expressed in E. coli and its molecular weight was confirmed 96 kDa. In this paper, we suggest the potential use of cold-active chitinase from polar microorganisms in the field of biotechnology.

• Korean Journal of Soil Science and Fertilizer
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• v.39 no.1
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• pp.15-20
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• 2006

Biological control by chitinolytic microorganisms is being evaluated as management options for soilborne diseases. Forty kilograms of chitin compost (CTC) and control compost (CC) were amended on tomato plots ($15m{\times}0.5m$) 7 d before transplanting to evaluate enzymatic activities and the control of Fusarium wilt. Samples were taken on day 1, 3, 5, and 7, the day 1 corresponded to the 66 d after transplanting, the day on which the initial wilting symptoms occurred in plants of CC treated plots. The chitinase activity in soil of CTC was always higher compared to the control. Pathogenesis related (PR) protein (chitinase, ${\beta}$-1, 3-glucanase and peroxidase) activities in tomato roots in CC increased every day and showed marked differences compared to CTC. Wilting symptoms (96 d after transplanting) were reduced by 25% in CTC compared to the control. Protection of tomato plant may be correlated with the high levels of soil enzyme activities resulting from the chitin compost.

• Korean Journal of Soil Science and Fertilizer
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• v.39 no.4
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• pp.185-194
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• 2006

A fungal strain of Trichoderma having strong chitinolytic activity was isolated from field soil enriched with crabshell for several years. Based on 5.8S rRNA, partial 18S, 28S rRNA genes, ITS1, ITS2 sequence analysis and morphological characteristics, the fungus was identified as Trichoderma asperellum and named as Trichoderma asperellum T-5 (TaT-5). The fungus released lytic enzymes such as chitinase and ${\beta}$-1, 3-glucanse, and produced six antifungal substances in chitin broth medium. To demonstrate the protective effect of TaT-5 against damping-off in cucumber plant caused by Rhizoctonia solani, TaT-5 culture broth (TA), chitin medium (CM) and distilled water (DW) were applied to each pot at 10 days after sowing, respectively. Then, the homogenized hyphae of R. solani were infected to each pot at 1 week after TaT-5 inoculation. During experimental period, fresh weight of shoot and root in cucumber plant more increased at TA treatment compared to other treatments. PR-proteins (${\beta}$-1, 3-glucanase and chitinase) activities in cucumber leaves markedly increased at CM and DW treatments, but the activity slightly increased and then decreased at TA treatment at 3 days after infection of R. solani. The activity of PR-proteins activities in cucumber roots at all treatments decreased with time where the degree of decrement was more alleviated at TA treatment than CM and DW. These results suggest that the lytic enzymes (chitinase and ${\beta}$-1, 3-glucanse) and antifungal substances produced by TaT-5 can reduce the pathogenic attack by R. solani in cucumber plants.

• Korean Journal of Soil Science and Fertilizer
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• v.44 no.6
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• pp.1150-1157
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• 2011

Streptomyces sampsonii KK1024 having strong chitinolytic activity was isolated from crab-shell rich soil at Muan, Jeolanamdo. The KK1024 produced chitinase, protease, gelatinase and lipase. When 50% of KK1024 culture broth was treated to juveniles and eggs of root-knot nematode, juvenile mortality at 3 days was 81.67% and egg hatch rate at 5 days was 2.00%. When $183.7{\mu}g\;mL^{-1}$ of crude enzyme produced by KK1024 was treated, juvenile mortality at 3 days was 96.00% and egg hatch rate at 5 days was 5.33%. At 1% of butanol extract from KK1024, juvenile mortality was highest with 90.00% and egg hatch rate was lowest with 0%. The comparison of the effect of KK1024 culture broth with only medium, synthetic fertilizer, and commercial nematicide on tomato growth and nematode infection was examined in pot trials. KK1024 culture broth showed lower number of egg mass and gall in plant, and population of juveniles in soil compared with only medium and synthetic fertilizer treatment, but not in commercial nematicide. However, the highest shoot weight and length was discovered in KK1024 culture broth. These results suggest that Streptomyces sampsonii KK1024 producing lytic enzymes and nematicidal compounds can be one of candidates for biocontrol agents against root-knot nematodes.