• Journal of Microbiology and Biotechnology
• /
• v.34 no.8
• /
• pp.1660-1670
• /
• 2024

The aim of this study was to modify phytase YiAPPA via protein surficial residue mutation to obtain phytase mutants with improved thermostability and activity, enhancing its application potential in the food industry. First, homology modeling of YiAPPA was performed. By adopting the strategy of protein surficial residue mutation, the lysine (Lys) and glycine (Gly) residues on the protein surface were selected for site-directed mutagenesis to construct single-site mutants. Thermostability screening was performed to obtain mutants (K189R and K216R) with significantly elevated thermostability. The combined mutant K189R/K216R was constructed via beneficial mutation site stacking and characterized. Compared with those of YiAPPA, the half-life of K189R/K216R at 80℃ was extended from 14.81 min to 23.35 min, half-inactivation temperature (T₅₀³⁰) was increased from 55.12℃ to 62.44℃, and T_m value was increased from 48.36℃ to 53.18℃. Meanwhile, the specific activity of K189R/K216R at 37℃ and pH 4.5 increased from 3960.81 to 4469.13 U/mg. Molecular structure modeling analysis and molecular dynamics simulation showed that new hydrogen bonds were introduced into K189R/K216R, improving the stability of certain structural units of the phytase and its thermostability. The enhanced activity was primarily attributed to reduced enzyme-substrate binding energy and shorter nucleophilic attack distance between the catalytic residue His28 and the phytate substrate. Additionally, the K189R/K216R mutant increased the hydrolysis efficiency of phytate in food ingredients by 1.73-2.36 times. This study established an effective method for the molecular modification of phytase thermostability and activity, providing the food industry with an efficient phytase for hydrolyzing phytate in food ingredients.

• International Journal of Industrial Entomology and Biomaterials
• /
• v.15 no.2
• /
• pp.157-160
• /
• 2007

Nymph of the emma field cricket, Teleogryllus emma, were reared on several types of artificial diets. The development period of nymphs were 55.4 days when only a single food, wheat bran, was provided, and it did not show a significant difference compared to the rearing results of the Danong diet and mixed diet. The supplying period of fish meal as the animal feed, the high emergence rates were obtained at 3rd instar with 90% and 4th instar with 100%. For the added amount test, when more than 40% of the diet was added, it confirmed that the insect weight increased. The characteristics of development according to each added amount of the vegetable food (dry bean-curd residue and corn powder) were investigated to minimize the dangers of the degeneration of diet when rearing with a single feed during the $1st{\sim}3rd$ instar period. First, as the added amount of bean-curd residue increased, nymphal development period became longer and the emergence rate became low. With corn powder as the single diet, all died before becoming adult. However, when corn powder was added up to 30%, no difference existed in the breeding results.

• The Korean Journal of Pesticide Science
• /
• v.19 no.4
• /
• pp.345-353
• /
• 2015

The aim of this study is to develop residue analysis method for fenoxanil, a MBI (melanin biosynthesis inhibitor) propionamide fungicide, had mainly been used to control rice blast, and disease of other crops, fruits, and vegetables by using GLC/NPD and GC/MS. Extraction with acetone and partition with n-hexane/dichloromethane (80/20, v/v) were performed from hulled rice, soybean, Kimchi cabbage, green pepper, and apple, then column clean-up with florisil was applied. Mean recoveries were 82.2%-109.1% with less than 7.2% of coefficients of variation and limit of quantitation was set at the concentration of 0.04 mg/kg from the five agricultural products through the determination by GLC/NPD equipped with DB-5 capillary column and single laboratory validation. As a confirmatory method, GC/MS selected ion monitoring (SIM) was set from m/z 125.0, 188.9, and 293.0. Developed method is expected to apply the single residue analysis of fenoxanil in agricultural products.

• Journal of the Korea Academia-Industrial cooperation Society
• /
• v.19 no.6
• /
• pp.375-381
• /
• 2018

This study was performed to investigate the residual characteristics of the insecticide dinotefuran in asparagus under greenhouse conditions from July to August and consequentially to obtain basic data for guidelines on the safe use of this pesticide in asparagus. Residues of dinotefuran in young stem of asparagus before and after growing mother stem were analyzed from samples collected at 0, 1, 3, 5, and 7 days after single application of a commercial formulation of dinotefuran (wettable powder, 10%) at the recommended dose (2,000 times dilution). The residue of dinotefuran in young stem of asparagus was analyzed by HPLC-UVD, and recovery of dinotefuran in young stem was tested at 0.5 and 1.0 mg/kg concentrations. As a result, the limit of quantitation (LOQ) of dinotefuran was 0.01 mg/kg, and the recovery of dinotefuran was in the range of 83.3-94.0% with a coefficient of variation less than 10%. Residues of dinotefuran in young stem of asparagus before and after growing the mother stem were lower than the tentative limit (0.05 mg/kg) from 5 and 3 days after single application, respectively. Based on these results, single application of dinotefuran (wettable powder, 10%) at the recommended dose at 7 days before harvest would have no deleterious effects on safety issues concerning pesticide residue. This result might provide basic information to construct guidelines for the safe use of dinotefuran in asparagus.

• The Korean Journal of Pesticide Science
• /
• v.17 no.2
• /
• pp.107-116
• /
• 2013

Polar pesticides like pymetrozine (log $P_{ow}$: -0.18) are known to be difficult to analyze. The analytical method of pymetrozine using hydromatrix included in the official method of KFDA was uncommon and provided ambiguous evidence to confirm both the identity and the quantity. Therefore, precise single residue analytical method was developed in representative crops for using liquid-liquid extraction (LLE). The pymetrozine residue was extracted with methanol from 11 representative crops which comprised apple, blueberry, broccoli, cabbage, cherry, crown daisy, hulled rice, Korean cabbage, potato, rice and watermelon. The extract was purified serially by liquid-liquid extraction (LLE) and silica solid phase extraction (SPE). For rice and hulled rice samples, n-hexane partition was additionally adopted to remove nonpolar interferences, mainly lipids. The residue levels were analyzed by HPLC with DAD, using $C_8$ column. LOQ (limit of quantitation) of pymetroizinie was 1 ng (S/N > 10) and MQL (method quantitation limit) was 0.01 mg/kg. Mean recoveries from 11 crop samples fortified at three levels (MQL, 10 ${\times}$ MQL and 50 ${\times}$ MQL) in triplicate were in the range of 83.1~98.5% with coefficients of variation (CV) of less than 10%, regardless of sample type, which satisfies the criteria of KFDA. The method established in this study could be applied to most of crops as an official and general method for analysis of pymetrozine residue.

• The Korean Journal of Pesticide Science
• /
• v.21 no.1
• /
• pp.75-83
• /
• 2017

The single residue analytical method was developed for determining fungicide pencycuron residues in various agricultural commodities with high-performance liquid chromatography (HPLC). Pencycuron residue was extracted with acetone from representative crops such as Korean cabbage, apple, brown rice and green pepper. After ethyl acetate/n-hexane partition and subsequent clean-up with silica gel chromatography, pencycuron residue was quantified by reversed phase HPLC with UV detection at 240 nm. The suspected residue of pencycuron was confirmed using selected-ion monitoring (SIM) LC/mass spectrometry (MS). Instrumental limit of quantitation (ILOQ) and method LOQ (MLOQ) were set at 2 ng and 0.02 mg/kg, respectively. Overall recoveries of pencycuron from different crop samples fortified at three levels (MLOQ, 10MLOQ, 100MLOQ) were 72~108%. This proposed method could be useful as official analytical method for quantification of pencycuron residues in agricultural commodities.

• Korean Journal of Environmental Agriculture
• /
• v.30 no.4
• /
• pp.446-452
• /
• 2011

BACKGROUND: To investigate the dissipation patterns of 3 pesticides, azoxystrobin, difenoconazole and iprodione, on green garlic after field treatment pesticides were treated as foliar treatment by single application at recommended and double the recommended rates. METHODS AND RESULTS: Residue samples were harvested at 0, 1, 2, 5, 7 and 10 days post-treatment for azoxystrobin and 0, 1, 2, 5, 7, 10, 15 and 21 days post-treatment for difenoconazole and iprodione. After preparation the fortified samples were extracted and analyzed by gas chromotography-electron capture detector (GC-ECD) to determine the residue levels. Recoveries ranged from 87 to 109% for azoxystrobin, difenoconazole and iprodione at two different levels. The limit of Quantification (LOQ) values were 0.002 mg/kg for azoxystrobin and difenoconazole and 0.01 mg/kg for iprodione. CONCLUSION(S): Half-lives of azoxystrobin, difenoconazole and iprodione in green garlic after treatment were 1.2, 3.8 and 3.2 days at recommended and 1.4, 3.3 and 3.2 at double the recommended rate, respectively. Residue level of azoxystrobin, difenoconazole and iprodione in green garlic were below the maximum residue limits (MRLs) at 0 day, 0 day and 5 days, respectively. Therefore, these pesticide were considered that residues was satisfied to the requirement of domestic trade related to the consumer safety.

• Journal of Mushroom
• /
• v.18 no.4
• /
• pp.380-386
• /
• 2020

The study was conducted for the safety evaluation of 320 pesticide residues in 768 Lentinula edodes fruit body samples and 143 L. edodes media samples, which are distributed nationwide in South Korea. The monitoring method was the second of the multi-residue methods in the Korean Food Code. GC-ECD, GC-NPD, and GC-MSD were used as evaluation equipment for analysis. Single-analysis of the target pesticides was performed for mepiquat chloride. Through the analysis of collected L. edodes samples, pesticide residues were detected in total seven cases, including four L. edodes fruit body samples and three L. edodes media samples. The detected pesticide residues were carbendazim, diflubenzuron, fluopyram, and dinotefuran. In this study, carbendazim was detected in three L. edodes fruit body samples and one L. edodes media sample. The detected amount of carbendazim was 0.056, 0.17, 0.043, and 0.09 mg/kg, respectively. The amount of carbendazim in the collected L. edodes samples was detected below the MRLs (maximum residue level). The detected amounts of fluopyram and dinotefuran were 0.068 mg/kg and 0.06 mg/kg, respectively. Two pesticide residues were detected in the medium in one case. Mepiquat chloride was not detected in this study. These results suggested that residual pesticides were detected in a small number of collected L. edodes. However, the PLS for unregistered pesticides MRL was 0.01 ppm; therefore, we have to conduct research on preparing safety standards for mushrooms, including L. edodes.

• Korean Journal of Environmental Agriculture
• /
• v.32 no.2
• /
• pp.128-135
• /
• 2013

BACKGROUND: This study was performed to develop a single residue analytical method for new herbicide metazosulfuron in crops. METHODS AND RESULTS: Brown rice, apple, mandarin, Kimchi cabbage and soybean were selected as representative crops, and clean-up system, partition solvent and extraction solvent were optimized. Instrumental limit of quantitation (ILOQ), linearity of calibration curve and method limit of quantitation (MLOQ) were determined based on the chromatography and whole procedures. For recovery tests, brown rice, apple, mandarin, Kimchi cabbage and soybean samples were macerated and fortified with metazosulfuron standard solution at three levels (MLOQ, 10 MLOQ and 100 MLOQ). And then those were extracted with acetonitrile, concentrated, and partitioned with ethyl acetate. Then the extracts were concentrated again and cleaned-up through $NH_2$ (aminopropyl) SPE cartridge with acetone : dichloromethane (1% acetic acid) (20 : 80, v/v) before concentration and analysis with HPLC. CONCLUSION(S): ILOQ of metazosulfuron was 2 ng (S/N${\geq}$10) and good linearity was achieved between 0.05 and 12.5 mg/Kg of metazosulfuron standard solutions, with coefficients of determination of 0.9999. MLOQ was 0.02 mg/Kg. Good recoveries from 74.1 to 116.9% with coefficients of variation (C.V.) of less than 10% were obtained, regardless of sample type, which satisfies the criteria of Korea Food and Drug Administration (KFDA). Those results were reconfirmed with LC-MS (SIM). The method established in this study is simple, economic and efficient to be applied to most of crops as an official and general method for residue analysis of metazosulfuron.

• Journal of Microbiology and Biotechnology
• /
• v.16 no.6
• /
• pp.988-992
• /
• 2006

The ptsHI operon from Leuconostoc mesenteroides ssp. mesenteroides SY1 (L. mesenteroides SY1), a strain isolated from kimchi, was cloned and characterized. The ptsH open reading frame (ORF) was 273 bp in size, which can encode a protein of 90 amino acid residues with a molecular weight of 9,212 Da. The pfsI ORF was 1,719 bp in size, which was capable of encoding a protein of 572 amino acids with a molecular mass of 62,549 Da. ptsH and pfsI genes were transcribed as a single transcript of 2.0 kb in size regardless of carbon sources, supporting the operon structure. Although the deduced amino acid sequences of the HPr and EI were highly homologous with those of other Gram-positive bacteria, an additional amino acid (glutamine at the $3^{rd}$ amino acid) was present in HPr from L. mesenteroides SY1. Phosphorylation sites of HPr included the histidine residue ($16^{th}$) and serine residue ($47^{th}$). Mutant HPrs, in which each phosphorylation site was mutated into alanine, were obtained, and phosphorylation with HPr and mutated HPrs showed that HPr was phosphorylated at the serine residue ($47^{th}$) by HPr kinaseiphosphorylase (HPr K/P).