Research letterIncreased chitinase production by a non-pigmented mutant of Serratia marcescens
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Abstract:
Ultraviolet light and nitrosoguanidine were used to mutagenize a red pigmented culture of Serratia marcescens, strain EB415, which produced chitinase. After mutagenesis, a stable, non-pigmented mutant designated BL40 was isolated which produced larger colonies and zones of clearing on solid medium containing colloidal chitin.
In liquid medium with colloidal chitin as the sole carbon source both strains grew similarly but BL40 produced 160 units/ml of chitinase compared with 60 units/ml for EB 415, an increase of 167%. When chitin concentration was increased in the medium, chitinase production also increased. Chitinase appeared to be extracellular, since the supernatant from washed, sonicated cells for both strains showed no detectable amount of chitinolytic activity.Keywords:
Chitinase
Serratia
Carbon source
Ultraviolet light
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Strain (injury)
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After the mutagenesis of Penicillium funiculosum with UV light and N-nitroso-N-methylurea, 83 of 2237 grown colonies were surrounded with increased zones of glucose oxidase diffusion. Analysis of the glucose oxidase activity of selected mutant strains grown in submerged cultures allowed 18 mutant strains to be obtained whose glucose oxidase activity was 5-153% higher (in a medium with glucose) and 4-83% higher (in a medium with sucrose) than that of the parent strain. Two of these mutant strains, UV6.31 and NMU95-132, possessed high glucose oxidase activity when grown in media with glucose or sucrose and produced large amounts of mycelia. The active and morphologically stable mutant P. funiculosum NMU95-132 was chosen for further selection work.
Glucose oxidase
Strain (injury)
Aspergillus niger
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Trichoderma
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Trichoderma harzianum wild-type strains were implanted by N+ to produce mutagenesis.A mutant stain H-13 with higher activity of chitinase was obtained by screening the mutants with transparent zone,and analyzing chitinase activity by DNS method.Single-factor design and orthogonal test design were used for optimizing cultural conditions of chitinase production from mutant strain H-13.Single-factor experimentation showed that the media added 1.0% peptone led to highest chitinase production,and 1.0% colloidal chitin was the best media for inducing chitinase.The mutant strain showed higher enzymatic activity in this media with initial pH 5.5 for 96 h of culture.Orthogonal test showed that chitinase activity of strain H-13 could reach 731.69 U·mL-1 cultured in the optimal medium with 1.0% peptone,0.5% colloidal chitin and initial pH 5.0 for 96 h,which was one fold increase compared to the wild strain enzyme vigor.
Chitinase
Strain (injury)
Trichoderma harzianum
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Bacillus pumilus and Bacillus alvei, among alkaline protease producing strains, were used to examine the changes in alkaline protease gene expression following UV irradiation. Induction of mutation in Bacillus mutant strains was carried out by 0, 5, 10, 15 and 20 min. exposure times of UV irradiation and different distances between the treated bacterial cultures and UV source. Results revealed that alkaline protease activity assayed under submerged culture conditions was more accurate than the relative growth production (C/G) method, because there was no proportional correlation between zone diameter and the ability to produce the enzyme in submerged cultures. No enzyme activity was scored with B. pumilus mutants, while the activity was pronounce, in case of B. alvei mutants. Mutants No.15, 55 and 34 were the most efficient in enzyme production under submerged conditions being 68.8, 81.1 and 99 U/ml, respectively. Their alkaline protease activities were 2.6, 3.02 and 3.7 folds than those of the original strains. The optimal enzyme production was achieved after 48 hr at air: medium ratio of 39: 1. Results also proved that the inoculum size in all tested mutant ranges had no significant effect on the enzyme production .The supplementation with glucose to growth medium gave the highest level of enzyme productivity, while lactose showed the lowest. The addition of arabinose and xylose completely inhibited the enzyme production by both tested strains, while incorporation into the culture medium of sucrose and maltose failed to produce the enzyme in B. alvei, but in mutant No.8, they enhanced high levels of productivity.
Bacillus pumilus
Bacillaceae
Bacillus (shape)
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A wild-type strain of the fungus Aphanocladium album was mutagenized by UV irradiation in order to obtain chitinase-overproducing mutants. Mutants were screened on agar medium containing colloidal chitin and selected for their ability to produce large clearing zones around the colonies. Two mutant strains, designated E3 and E12, showed respectively a 26- and a 2·5-fold increase in maximal extracellular chitinase activity, determined in liquid medium with crystalline chitin as sole carbon source, compared to the wild-type strain. This is believed to be the first report on the induction of stable chitinase-overproducing mutants in a filamentous fungus. Regulation of enzyme activity was investigated in mutant E3 and the wild-type strain.
Chitinase
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Wild type
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Purpose: In order to increase the ability to produce isoamylase, the isoamylolytic wild strain of Rhizopus oryzae PR 7, MTCC 9642 was subjected to chemical mutagenesis by N-methyl-N'-nitro-nitroso guanidine (MNNG) . Methods: Among the surviving strains, those strains showing improved amylolytic activity both by in situ assay on iodine stained starch agar plate and in vitro assay by reducing sugar measurement were selected and subcultured subsequently for three generations. Results: Strain named K1 was found to be the best amylase synthesizing mutant strain. Glycogen (0.5%) was found to be the best inducer followed by pure and indigenous starch types. The selected mutant strain showed maximum production on 96 th hour of cultivation and a persistent enzyme production till 120 th hour. At this hour glycogen induced production by K1 strain showed a 5.59 times increase in enzyme production than that of the wild strain. Preliminary characterization showed an achievement of increased pH and thermo stability by the enzyme of the mutant strain. Conclusion: Hence the mutant strain K1 could be used for commercial production of isoamylase using cheap or waste sources of starchy materials.
Rhizopus oryzae
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Isoamylase
Rhizopus
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Five mutant strains of Monascus ruber were obtained by gamma and e-beam processing and were characterized in terms of enzyme production, growth rate and pigmentogenesis. These strains were cultivated onto glucose-yeast extract agar slants for maintenance and on rice medium for pigments biosynthesis. It was demonstrated that the parental strain does not produce any amylolytic, lypolytic, proteolytic or cellulolytic enzymes, while mutant strains have enzymatic activity. The biosynthesis of natural pigments was achieved in solid state fermentation on sterilized rice. The biopigments were extracted in ethanol and hexane and the absorption spectra were recorded for the five mutant strains. M5 and M2 mutants produced the highest amount of red and yellow pigments.
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Solid-State Fermentation
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Genetic modification of Serratia marcescens QMB1466 was undertaken to isolated mutants which produce increased levels of chitinolytic activity. After mutagenesis with ultraviolet light, ethyl methane sulfonate or N-methyl-N'-nitro-N-nitrosoguanidine, 19,940 colonies were screened for production of enlarged zones of clearing (indicative of chitinase activity) on chitin-containing agar plates. Forty-four chitinase high producers were tested further in shake flask cultures. Mutant IMR-1E1 was isolated which, depending on medium composition, produced two to three times more than the wild type of the other components of the chitinolytic enzyme system--a factor involved in the hydrolysis of crystalline chitin and chitobiase. After induction by chitin, endochitinase and chitobiase activity appeared at similar times for both IMR-1E1 and QMB1466, suggesting possible coordinate control of these enzymes. The results are consistent with IMR-1E1 containing a regulatory mutation which increased production of the components of the chitinolytic enzyme system and/or with IMR-1E1 containing a tandem duplication of the chitinase genes. The high rate of reversion of IMR-1E1 to decreased levels of chitinase production suggests that the overproduction of chitinase by IMR-1E1 is due to a tandem gene duplication.
Chitinase
Serratia
Ultraviolet light
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