• Journal of the Semiconductor & Display Technology
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• v.22 no.3
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• pp.36-40
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• 2023

Recently, in the problem of multi-order processing in logistics warehouses, multi-pickup systems are changing from the form in which workers walk around the warehouse to the form in which goods come to workers. These changes are shortening the time to process multiple orders and increasing production. This study considered the sequence problem of which warehouse the items to be loaded on each truck come first and which items to be loaded first when loading multiple pallet-unit goods on multiple trucks in an industrial smart logistics automation warehouse. To solve this problem efficiently, we use the mutant algorithm, which combines the GA algorithm and ACO algorithm, and compare with original system.

• Microbiology and Biotechnology Letters
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• v.40 no.1
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• pp.10-16
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• 2012

The Streptomyces peucetius OIM (Overproducing Industrial Mutant) strain is a recursively-mutated and optimally-screened strain used for the industrial production of polyketide antibiotics, such as doxorubicin (DXR). Using the S. peucetius OIM mutant strain as a surrogate host, a model minimal polyketide pathway for aloesaponarin II, an actinorhodin shunt product, was cloned in a high-copy conjugative plasmid, followed by functional pathway expression and quantitative metabolite analysis. The level of aloesaponarin II production was noted as being significantly higher in the OIM strain than in the wild-type S. peucetius, as well as in the regulatory network-stimulated S. coelicolor mutant strain. Moreover, the aloesaponarin II production level was seen to be even higher in a down-regulator $wblA_{spe}$-deleted S. peucetius OIM strain, implying that the rationally-engineered S. peucetius OIM mutant strain could be used as an efficient surrogate host for the high expression of foreign polyketide pathways.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.442-449
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• 2004

Improving the light utilization efficiency of photosynthetic cells in photobioreactors (PBRs) is a major topic in algal biotechnology. Accordingly, in the current study we investigated the effect and suitability of photosynthetic pigment reduction for improving light utilization efficiency. The light-harvesting complex II (LH-II) genes of Rhodobacter sphaeroides were removed to construct a mutant strain with less pigment content. The mutant strain exhibited a slower growth rate than the wild-type under a low light intensity, while the mutant grew faster under a high light intensity. In addition, the specific absorption coefficient was lower in the mutant due to its reduced pigment content, thus it seemed that light penetrated deeper into its culture broth. However, the distance (light penetration depth) from the surface of the PBR to the compensation point did not increase, due to an increase in the compensation irradiance of the mutant strain. Experimental data showed that a reduced photosynthetic pigment content, which lessened the photoinhibition under high-intensity light, helped the volumetric productivity of photosynthetic microorganisms.

• BMB Reports
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• v.34 no.1
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• pp.51-58
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• 2001

Attempts using in vitro and in vivo selection procedures have been made to search for hammerhead ribozymes that have higher activities than the wild-type ribozyme and also to determine whether other sequences might be possible in the catalytic core of the hammerhead ribozyme. Active sequences selected in the past conformed broadly to the consensus core sequence except at A9, and no sequences were associated with higher activity than that of the hammerhead with the consensus core, an indication that the consensus sequence derived from viruses and virusoids is probably the optimal sequence [Vaish et al. (1997) Biochemistry 36, 6495-6501]. Recently, during construction of ribozyme expression vectors, we isolated a mutant hammerhead ribozyme, with an insertion of G between A9 and G10.1, that appeared to show significant activity [Kawasaki et al. (1996) Nucleic Acids Res. 24, 3010-3016; Kawasaki et al. (1998) Nature 393, 284-289]. We, therefore, characterized kinetic properties of the G-inserted mutant ribozymes in terms of the NUX rule. We demonstrate that the NUX rule is basically applicable to the G-inserted ribozymes and, more importantly, one type of G-inserted ribozyme was very active with $k_{cat}$, value of $6.4\;min^{-1}$ in 50 mM Tris-HCl (pH 8.0) and 10 mM $MgCl_2$ at $37^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• v.29 no.8
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• pp.1288-1298
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• 2019

Bacterial ATP synthases drive ATP synthesis by a rotary mechanism, and play a vital role in physiology and cell metabolism. Corynebacterium glutamicum is well known as an industrial workhorse for amino acid production, and its ATP synthase operon contains eight structural genes and two adjacent genes, cg1360 and cg1361. So far, the physiological functions of Cg1360 (GenBank CAF19908) and Cg1361 (GenBank CAF19909) remain unclear. Here, we showed that Cg1360 was a hydrophobic protein with four transmembrane helices (TMHs), while no TMH was found in Cg1361. Deletion of cg1360, but not cg1361, led to significantly reduced cell growth using glucose and acetic acid as carbon sources, reduced F1 portions in the membrane, reduced ATP-driven proton-pumping activity and ATPase activity, suggesting that Cg1360 plays an important role in ATP synthase function. The intracellular ATP concentration in the ${\Delta}cg1360$ mutant was decreased to 72% of the wild type, while the NADH and NADPH levels in the ${\Delta}cg1360$ mutant were increased by 29% and 26%, respectively. However, the ${\Delta}cg1361$ mutant exhibited comparable intracellular ATP, NADH and NADPH levels with the wild-type strain. Moreover, the effect of cg1360 deletion on L-valine production was examined in the L-valine-producing V-10 strain. The final production of L-valine in the $V-10-{\Delta}cg1360$ mutant reached $9.2{\pm}0.3g/l$ in shake flasks, which was 14% higher than that of the V-10 strain. Thus, Cg1360 can be used as an effective engineering target by altering energy metabolism for the enhancement of amino acid production in C. glutamicum.

• Journal of Microbiology and Biotechnology
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• v.23 no.11
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• pp.1519-1528
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• 2013

Bacillus pumilus SG2, a halotolerant strain, expresses two major chitinases designated ChiS and ChiL that were induced by chitin and secreted into the supernatant. The present work aimed to obtain a mutant with higher chitinolytic activity through mutagenesis of Bacillus pumilus SG2 using a combination of UV irradiation and nitrous acid treatment. Following mutagenesis and screening on chitin agar and subsequent formation of halos, the mutated strains were examined for degradation of chitin under different conditions. A mutant designated AV2-9 was selected owing to its higher chitinase activity. To search for possible mutations in the whole operon including ChiS and ChiL, the entire chitinase operon, including the intergenic region, promoter, and two areas corresponding to the ChiS and ChiL ORF, was suquenced. Nucleotide sequence analysis of the complete chitinase operon from the SG2 and AV2-9 strains showed the presence of a mutation in the catalytic domain (GH18) of chitinase (ChiL). The results demonstrated that a single base change had occurred in the ChiL sequence in AV2-9. The wild-type chitinase, ChiL, and the mutant (designated ChiLm) were cloned, expressed, and purified in E. coli. Both enzymes showed similar profiles of activity at different ranges of pH, NaCl concentration, and temperature, but the mutant enzyme showed approximately 30% higher catalytic activity under all the conditions tested. The results obtained in this study showed that the thermal stability of chitinase increased in the mutant strain. Bioinformatics analysis was performed to predict changes in the stability of proteins caused by mutation.

• Journal of Microbiology and Biotechnology
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• v.29 no.3
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• pp.419-428
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• 2019

Phytases are enzymes that can hydrolyze phytate and its salts into inositol and phosphoric acid, and have been utilized to increase the availability of nutrients in animal feed and mitigate environmental pollution. However, the enzymes' low thermostability has limited their application during the feed palletization process. In this study, a combination of B-value calculation and protein surface engineering was applied to rationally evolve the heat stability of Escherichia coli phytase. After systematic alignment and mining for homologs of the original phytase from the histidine acid phosphatase family, the two models 1DKL and 1DKQ were chosen and used to identify the B-values and spatial distribution of key amino acid residues. Consequently, thirteen potential amino acid mutation sites were obtained and categorized into six domains to construct mutant libraries. After five rounds of iterative mutation screening, the thermophilic phytase mutant P56214 was finally yielded. Compared with the wild-type, the residual enzyme activity of the mutant increased from 20% to 75% after incubation at $90^{\circ}C$ for 5 min. Compared with traditional methods, the rational engineering approach used in this study reduces the screening workload and provides a reference for future applications of phytases as green catalysts.

• KSBB Journal
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• v.25 no.5
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• pp.429-436
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• 2010

Keratinase is widely used in certain industrial applications. The present study sought to improve the culture conditions of Bacillus subtilis SMMJ-2 to facilitate mass production of keratinase. Strain SMMJ-2 was irradiated by ultraviolet light and the resulting isolates were tested for keratinase activity. Isolates displaying elevated keratinase activity were selected and used to determine the optimum temperature (24, 30, 37, 45, $55^{\circ}C$) for bacterial keratinase production during a 4 day incubation period. The highest enzyme activity (55 units/mL/min), from a Bacillus subtilis SMMJ-2 mutant (mutant No. 2) was demonstrated following incubation at $30^{\circ}C$. The effects of carbon and nitrogen sources on keratinase production were confirmed by measuring the enzyme activity from the culture broth of the mutant strain cultured in various media containing different carbon source and nitrogen sources during a 4 day period. The optimal medium composition for producing keratinase consisted of 1% glucose, 0.7% $K_2HPO_4$, 0.2% $K_2HPO_4$, and 1.2% soybean meal. Optimal initial pH and temperature for producing keratinase were 7.0 and $30^{\circ}C$, respectively. Keratinases produced by B. subtilis SMMJ-2 and the mutant No. 2 were purified from the culture broth which used soybean meal as a nitrogen source. Membrane ultrafiltration, DEAE-sephacel ion exchange and Sephadex G-100 gel chromatography were used to purify the enzymes. The purified keratinases from both B. subtilis SMMJ-2 and the mutant No. 2 showed single bands and their molecular weights were estimated as 28 kDa and 42 kDa, respectively on SDS-polyacrylamide gel electrophoresis.

• BMB Reports
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• v.30 no.3
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• pp.167-172
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• 1997

The yeast Saccharomyces cerevisiae, mutant CH117, shows a drug-hypersensitivity (dhs) to cycloheximide, bleomycin, actinomycin D, 5-fluorouracil. nystatin, nigericin and several other antibiotics. CH 117 was also temperature-sensitive (ts). being unable to grow at $37^{\circ}C$ and secreted more invertase and acid phosphatase into the medium than the parent yeast. CH117 grows very slowly and the cell shape is somewhat larger and more sensitive to zymolyase than the wild type cells. Light microscopic and electron microscopic observation also revealed abnormality of the mutant cell wall. These characteristics indicate that CH117 has a defect in an essential component of the cell surface and that the cell wall which performs barrier functions has become leaky in the mutant. We screened a genomic library of wild type yeast for clones that can complement the mutation of CH117. A plasmid, pCHX1, with an insert of 3.6 kilobases (kbs) could complement the dhs and ts of CH117. Deletion and subcloning of the 3.6 kb insert showed that a gene for the complementation of mutant phenotypes was located in 1.9 kbs Puvll-Hindlll fragment.

• International Journal of Industrial Entomology and Biomaterials
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• v.19 no.1
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• pp.199-204
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• 2009

The newly cloned Bacillus thuringiensis cry1-5 gene showed high activity to both Plutella xylostella and Spodoptera exigua, while cry1Ac only showed high activity against P. xylostella but low to S. exigua. Through the alignment of amino acid sequences between Cry1Ac and Cry1-5, we found 12 different residues in domain I (6 residues) and domain II (6 residues). In this study, the modified cry1Ac gene, which is constructed according to a crop-preferring codon usage, was used as a template to construct mutant B. thuringiensis cry1Ac genes based on cry1-5 gene through multi site-directed mutagenesis. Total 63 various mutant cry genes were obtained at 12 positions randomly. Among them, ten mutant cry genes, whose domain I was totally converted and domain II was randomly, were selected to express in baculovirus expression system as a polyhedrin fusion form. The recombinant proteins were 95 kDa in size and were stably activated as 65 kDa by trypsin. The expressed mutant Cry proteins were applied to bioassays against P. xylostella and S. exigua. All mutants showed high insecticidal activity both to P. xylostella and S. exigua similar to cry1-5. These results suggest that these mutant cry genes might be expected of desirable cry genes for introduction to transgenic crops.