• Journal of Microbiology and Biotechnology
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• 제29권12호
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• pp.1882-1893
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• 2019

β-Glucosidases and β-xylosidases are two categories of enzymes that could cleave out non-reducing, terminal β-D-glucosyl and β-D-xylosyl residues with release of D-glucose and D-xylose, respectively. In this paper, two functional β-glucosidase Dth3 and β-xylosidase Xln-DT from Dictyoglomus thermophilum were heterologously expressed in E.coli BL21 (DE3). Dth3 and Xln-DT were relatively stable at 75℃ and were tolerant or even stimulated by glucose and xylose. Dth3 was highly tolerant to glucose with a K_i value of approximately 3 M. Meanwhile, it was not affected by xylose in high concentration. The activity of Xln-DT was stimulated 2.13-fold by 1 M glucose and 1.29-fold by 0.3 M xylose, respectively. Furthermore, the βglucosidase Dth3 and β-xylosidase Xln-DT showed excellent selectivity to cleave the outer C-6 and C-3 sugar moieties of ASI, which established an effective and green method to produce the more pharmacologically active CAG, an exclusive telomerase activator. We measured temperature, pH and dosage of enzyme using a single-factor experiment in ASI biotransformation. After optimization, the optimal reaction conditions were as follows: 75℃, pH 5.5, 1 U of Dth3 and 0.2 U of Xln-DT, respectively. Under the optimized conditions, 1 g/l ASI was transformed into 0.63 g/l CAG with a corresponding molar conversion of 94.5% within 3 h. This is the first report to use the purified thermostable and sugar-tolerant enzymes from Dictyoglomus thermophilum to hydrolyze ASI synergistically, which provides a specific, environment-friendly and cost-effective way to produce CAG.

• Journal of Microbiology and Biotechnology
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• 제24권9호
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• pp.1162-1169
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• 2014

Glyphosate is the active component of the top-selling herbicide, the phytotoxicity of which is due to its inhibition of the shikimic acid pathway. 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the shikimic acid pathway. Glyphosate tolerance in plants can be achieved by the expression of a glyphosate-insensitive aroA gene (EPSPS). In this study, we used a PCR-based two-step DNA synthesis method to synthesize a new aroA gene ($aroA_{R.\;leguminosarum}$) from Rhizobium leguminosarum. In vitro glyphosate sensitivity assays showed that $aroA_{R.\;leguminosarum}$ is glyphosate tolerant. The new gene was then expressed in E. coli and key kinetic values of the purified enzyme were determined. Furthermore, we transformed the aroA gene into Arabidopsis thaliana by the floral dip method. Transgenic Arabidopsis with the $aroA_{R.\;leguminosarum}$ gene was obtained to prove its potential use in developing glyphosate-resistant crops.

• BMB Reports
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• 제40권1호
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• pp.44-52
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• 2007

Kinesins, as a kind of microtubule-based motor proteins, have a conserved microtubule-binding site in their motor domain. Here we report that two homologous kinesins in Arabidopsis thaliana, KatB and KatC, contain a second microtubule-binding site in their tail domains. The prokaryotic-expressed N-terminal tail domain of the KatC heavy chain can bind to microtubules in an ATP-insensitive manner. To identify the precise region responsible for the binding, a serious of truncated KatC cDNAs encoding KatC N-terminal regions in different lengths, KatC1-128, KatC1-86, KatC1-73 and KatC1-63, fused to Histidine-tags, were expressed in E. coli and affinity-purified. Microtubule cosedimentation assays show that the site at amino acid residues 74-86 in KatC is important for microtubule-binding. By similarity, we obtained three different lengths of KatB N-terminal regions, KatB1-384, KatB1-77, and KatB1-63, and analyzed their microtubule-binding ability. Cosedimentation assays indicate that the KatB tail domain can also bind to microtubules at the same site as and in a similar manner to KatC. Fluorescence microscopic observations show that the microtubule-binding site at the tail domain of KatB or KatC can induce microtubules bundling only when the stalk domain is present. Through pull-down assays, we show that KatB1-385 and KatC1-394 are able to interact specifically with themselves and with each other in vitro. These findings are significant for identifying a previously uncharacterized microtubule-binding site in the two kinesin proteins, KatB and KatC, and the functional relations between them.

• BMB Reports
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• 제46권7호
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• pp.358-363
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• 2013

In this paper, we firstly reported a C-type lectin cDNA clone of 1029 bps from the larvae of A. Pernyi (Ap-CTL) using PCR and RACE techniques. The full-length cDNA contains an open reading frame encoding 308 amino acid residues which has two different carbohydrate-recognition domains (CRDs) arranged in tandem. To investigate the biological activities in the innate immunity, recombinant Ap-CTL was expressed in E. coli with a 6-histidine at the amino-terminus (Ap-rCTL). Besides acted as a broad-spectrum recognition protein binding to a wide range of PAMPs and microorganisms, Ap-rCTL also had the ability to recognize and trigger the agglutination of bacteria and fungi. In the proPO activation assay, Ap-rCTL specifically restored the PO activity of hemolymph blocked by anti-Ap-rCTL antibody in the presence of different PAMPs or microorganisms. In summary, Ap-rCTL plays an important role in insect innate immunity as an pattern recognition protein.

• Journal of Ginseng Research
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• 제44권5호
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• pp.690-696
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• 2020

Background: As the main metabolites of ginsenosides, 20(S, R)-protopanaxadiol [PPD(S, R)] and 20(S, R)-protopanaxatriol [PPT(S, R)] are the structural basis response to a series of pharmacological effects of their parent components. Although the estrogenicity of several ginsenosides has been confirmed, however, the underlying mechanisms of their estrogenic effects are still largely unclear. In this work, PPD(S, R) and PPT(S, R) were assessed for their ability to bind and activate human estrogen receptor α (hERα) by a combination of in vitro and in silico analysis. Methods: The recombinant hERα ligand-binding domain (hERα-LBD) was expressed in E. coli strain. The direct binding interactions of ginsenosides with hERα-LBD and their ERα agonistic potency were investigated by fluorescence polarization and reporter gene assays, respectively. Then, molecular dynamics simulations were carried out to simulate the binding modes between ginsenosides and hERα-LBD to reveal the structural basis for their agonist activities toward receptor. Results: Fluorescence polarization assay revealed that PPD(S, R) and PPT(S, R) could bind to hERα-LBD with moderate affinities. In the dual luciferase reporter assay using transiently transfected MCF-7 cells, PPD(S, R) and PPT(S, R) acted as agonists of hERα. Molecular docking results showed that these ginsenosides adopted an agonist conformation in the flexible hydrophobic ligand-binding pocket. The stereostructure of C-20 hydroxyl group and the presence of C-6 hydroxyl group exerted significant influence on the hydrogen bond network and steric hindrance, respectively. Conclusion: This work may provide insight into the chemical and pharmacological screening of novel therapeutic agents from ginsenosides.

• The Korean Journal of Physiology and Pharmacology
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• 제22권3호
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• pp.269-275
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• 2018

$Na^+/H^+$ exchangers (NHEs) have been shown to be involved in regulating cell volume and maintaining fluid and electrolyte homeostasis. Pooled evidences have suggested that loss of $Na^+/H^+$ exchanger isoform 8 (NHE8) impairs intestinal mucosa. Whether NHE8 participates in the pathology of infectious colitis is still unknown. Our previous study demonstrated that somatostatin (SST) could stimulate the expression of intestinal NHE8 so as to facilitate $Na^+$ absorption under normal condition. This study further explored whether NHE8 participates in the pathological processes of infectious colitis and the effects of SST on intestinal NHE8 expression in the setting of infectious colitis. Our data showed that NHE8 expression was reduced in Citrobacter rodentium (CR) infected mice. Up-regulation of NHE8 improved diarrhea symptom and mucosal damage induced by CR. In vitro, a similar observation was also seen in Enteropathogenic E. coli (EPEC) infected Caco-2 cells. Seglitide, a SST receptor (SSTR) 2 agonist, partly reversed the inhibiting action of EPEC on NHE8 expression, but SSTR5 agonist (L-817,818) had no effect on the expression of NHE8. Moreover, SST blocked the phosphorylation of p38 in EPEC-infected Caco-2 cells. Taken together, these results suggest that enhancement of intestinal NHE8 expression by SST could ameliorate the symptoms of mice with infectious colitis.

• International Journal of Industrial Entomology and Biomaterials
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• 제22권2호
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• pp.59-64
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• 2011

Protein kinase C (PKC) is involved in many cellular signaling pathways, it participates in many physiological processes, such as cell cycle, growth, proliferation, differentiation and apoptosis. To investigate the effect of PKC on the silkworm midgut tissue infection of Bombyx mori parvo-like virus (BmPLV), a B. mori atypical protein kinase C (BmaPKC) gene was cloned from larval midgut tissue, expressed in E. coli and purified. Additionally, the BmPLV susceptible silkworm strain and resistant silkworm strain were used to test the effect of the B. mori infection on BmPLV. The result showed that BmaPKC encodes a predicted 586 amino acid protein, which contains a C-terminal kinase domain and an N-terminal regulatory domain. The maximum expression amount of the soluble (His)6-tagged fusion protein was detected after 0.8 mmol/L IPTG was added and cultured at $21^{\circ}C$. The (His) 6-tagged fusion protein revealed about 73 kDa molecular weight which confirmed by western blot and mass spectrography. Furthermore BmaPKC protein were detected at 0-72 h post-infection in BmPLVinfected larval midgut tissue, western blot showed that as time went on, the expression of BmaPKC increased gradually in susceptible strain, the expression quantity on 72 h is 5 times of 0 h. However, in resistant strain, the expression quantity is slightly lower than susceptible strain. But no significant change in resistant strain was observed as time went on. The available data suggest that BmaPKC may involve in the regulation of BmPLV proliferation.

• Journal of Microbiology and Biotechnology
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• 제27권1호
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• pp.92-100
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• 2017

Acetoin (AC) is a volatile platform compound with various potential industrial applications. AC contains two stereoisomeric forms: (3S)-AC and (3R)-AC. Optically pure AC is an important potential intermediate and widely used as a precursor to synthesize novel optically active materials. In this study, chiral (3R)-AC production from meso-2,3-butanediol (meso-2,3-BD) was obtained using recombinant Escherichia coli cells co-expressing meso-2,3-butanediol dehydrogenase (meso-2,3-BDH), NADH oxidase (NOX), and hemoglobin protein (VHB) from Serratia sp. T241, Lactobacillus brevis, and Vitreoscilla, respectively. The new biocatalyst of E. coli/pET-mbdh-nox-vgb was developed and the bioconversion conditions were optimized. Under the optimal conditions, 86.74 g/l of (3R)-AC with the productivity of 3.61 g/l/h and the stereoisomeric purity of 97.89% was achieved from 93.73 g/l meso-2,3-BD using the whole-cell biocatalyst. The yield and productivity were new records for (3R)-AC production. The results exhibit the industrial potential for (3R)-AC production via whole-cell biocatalysis.

• Asian-Australasian Journal of Animal Sciences
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• 제26권8호
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• pp.1181-1188
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• 2013

Two experiments were conducted to evaluate the effect of supplementing a corn-soybean meal-based diet with an enzyme complex containing amylase, protease and xylanase on the performance, intestinal health, apparent ileal digestibility of amino acids and nutrient digestibility of weaned pigs. In Exp. 1, 108 piglets weaned at 28 d of age were fed one of three diets containing 0 (control), 100, or 150 ppm enzyme complex for 4 wks, based on a two-phase feeding program namely 1 to 7 d (phase 1) and 8 to 28 d (phase 2). At the end of the experiment, six pigs from the control group and the group supplemented with 150 ppm enzyme complex were chosen to collect digesta samples from intestine to measure viscosity and pH in the stomach, ileum, and cecum, as well as volatile fatty acid concentrations and composition of the microflora in the cecum and colon. There were linear increases (p<0.01) in weight gain, gain: feed ratio and digestibility of gross energy with the increasing dose rate of enzyme supplementation during the whole experiment. Supplementation with enzyme complex increased the digesta viscosity in the stomach (p<0.05) and significantly increased (p<0.01) the concentrations of acetic, propionic and butyric acid in the cecum and colon. Enzyme supplementation also significantly increased the population of Lactobacilli (p<0.01) in the cecum and decreased the population of E. coli (p<0.05) in the colon. In Exp. 2, six crossbred barrows (initial body weight: $18.26{\pm}1.21$ kg), fitted with a simple T-cannula at the distal ileum, were assigned to three dietary treatments according to a replicated $3{\times}3$ Latin Square design. The experimental diets were the same as the diets used in phase 2 in Exp. 1. Apparent ileal digestibility of isoleucine (p<0.01), valine (p<0.05) and aspartic acid (p<0.05) linearly increased with the increasing dose rate of enzyme supplementation. In conclusion, supplementation of the diet with an enzyme complex containing amylase, protease and xylanase improved piglet performance. This is likely a result of improvement in nutrient digestibility, volatile fatty acid concentrations and bacteria ratio in the large intestine.

• 한국축산식품학회지
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• 제27권3호
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• pp.363-370
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• 2007

김치의 L. plantarum의 생균제적 특성을 조사하기 위하여 김치에서 산생산과 성장능력 등이 우수한 25종의 Lactobacillus sp.를 분리하였고, API kit 분석에 의하여 L. plantarum을 동정하고 재차 16S rDNA 염기서열(99.9% 상동성)을 비교한 후 L. plantarum JK-01로 표기하였다. L. plantarum JK-01은 MRS broth에서 배양시 18시간 후 $2.9{\times}10^{10}CFU/ml$로 최대로 증식하는 빠른 성장특성을 나타냈으며 pH도 4.5로 조속히 하강하였다. 효소활성은 xylanase, amylase, protease, phytase의 순으로 높은 활성을 포함하고 있는 것으로 예측되었다. L. plantarum JK-01은 pH 2에서도 $1.36{\times}10^5CFU/mL$가 생존하였고, 1%의 담즙산에서도 약 $10^6CFU/mL$이상이 생존하여 내산성과 내담즙산성이 강한 것으로 나타났다. 또, $60^{\circ}C$에서도 $3.3{\times}10^3$ CFU/mL 정도로 생존하는 내열성이 있었으며, 대장균과 함께 배양시 18시간 후 대장균을 사멸시키는 것으로 나타났다. 이상의 결과로부터 분리한 L. plantarum JK-01은 생균제로서 충분히 이용가치가 있는 것으로 사료되었다.