• Journal of Microbiology and Biotechnology
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• 제25권7호
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• pp.1093-1100
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• 2015

Acetate and lactate in growth media are detrimental to the production of Thermus maltogenic amylase (ThMA), a heterologous protein, as well as to the growth of recombinant Escherichia coli. Only 50 mM of acetate or 10 mM of lactate reduced 90% of specific ThMA activity. In this study, mutant E. coli strains blocked in the ackA-pta or ackA-pta and ldh pathways were created, characterized, and assessed for their culture performace in 300 L-scale fermentation. The ackApta and ldh double-mutant strain formed significantly less lactate and acetate, and produced a concomitant increase in the excretion of pyruvate (17.8 mM) under anaerobic conditions. The ackA-pta mutant strain accumulated significant acetate but had an approximately 2-fold increase in the formation of lactate. The ackA-pta and ldh double-mutant strain had superior overall performance in large-scale culture under suboptimal conditions, giving 67% higher cell density and 66% higher ThMA activity compared with those of the control strain. The doublemutant strain also achieved a 179% improvement in volumetric ThMA production.

• Journal of Animal Science and Technology
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• 제66권2호
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• pp.295-309
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• 2024

To investigate the effect of dietary supplementation with a fermented mixture of bean dregs and wheat bran (FBW) on sow performance. FBW was given to sows during late gestation and lactation; in total, 24 sows were randomly assigned to 4 groups (control diet; 3% FBW diet; 6% FBW diet; 9% FBW diet, n = 6). The weight ratio of bean dregs (wet) to wheat bran was 4:6. Sows were fed different diets from 85 d of gestation until weaning. The results showed that supplementation with FBW increased average daily feed intake (ADFI) during lactation (p < 0.05). FBW supplementation also increased litter weight and milk yield (p < 0.05). The contents of Escherichia coli in the feces of the treatment groups were significantly reduced by FBW supplementation (p < 0.01). FBW supplementation significantly improved the fecal morphology (p < 0.05), alleviating sows' constipation. In conclusion, FBW could increase the ADFI, improve lactation and piglet litter weight in sows and reduce the pathogenic bacterial content in sow feces and constipation.

• Asian-Australasian Journal of Animal Sciences
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• 제21권2호
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• pp.252-261
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• 2008

Two experiments were conducted to compare the effects of feeding organic acids and antibiotic growth promoters in weaned pigs. In Exp. 1, 96 nursery pigs (Large White$\times$Landrace; initial weight $7.80{\pm}0.07kg$) were randomly allotted into one of four dietary treatments. Pigs in treatment 1 were fed a complex starter diet. Treatments 2 to 4 were the same as treatment 1 but supplemented with antibiotics (200 ppm chlortetracycline plus 60 ppm Lincospectin), 0.5% potassium diformate or 0.5% dry organic acid blend ACTIVATE Starter DA (ASD). During the 4-week post-weaning period, pigs fed ASD or antibiotics had better gain (p = 0.03) and feed efficiency (p = 0.04) than pigs fed the control diet. On d 14 post-weaning, pigs fed the control diet had the lowest fecal lactobacilli count among all dietary treatments (p = 0.02), whereas pigs fed ASD or antibiotics had a trend for lower fecal E. coli count compared to the control pigs (p = 0.08). Serum insulin-like growth factor-1 (IGF-1) of pigs fed ASD did not differ from pigs fed the control diet (p>0.05) at d 14 after weaning. In Exp. 2, 24 weaned pigs (Large White$\times$Long White; initial weight $5.94{\pm}0.33kg$) were allotted into four groups and housed individually. Pigs were fed a control diet or diets supplemented with antibiotics (100 ppm colistin sulfate, 50 ppm Kitasamycin plus 60 ppm Olaquindox), 0.5% or 1% ASD. All pigs were orally challenged with E. coli $K88^+$ on d 5. During d 5 to 14 after challenge, pigs fed antibiotics, 0.5% or 1% ASD had better gain (p = 0.01) and feed efficiency (p = 0.03) than pigs fed the control diet. On d 14, compared to the control pigs, pigs fed 0.5% ASD had higher lactobacilli in the duodenum and pigs fed 1% ASD and antibiotics had a trend for higher lactobacilli in the ileum (p = 0.08). Pigs fed antibiotics, 0.5% or 1% ASD diets tended to have decreased ileal E. coli count compared to those fed the control diet (p = 0.08). Serum interleukin-6 and cortisol and digesta pH values were not affected by treatment or time. These results indicate that feeding ASD can improve the growth performance of weaning pigs, mainly via modulating intestinal microflora populations without affecting gastrointestinal pH or immune indices.

• Asian Pacific Journal of Cancer Prevention
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• 제15권12호
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• pp.5055-5061
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• 2014

In this study, an anti-oxidant and anti-tumor protein Latcripin-3 of Lentinula edodes C91-3 was expressed in Escherichia coli. for the first time. According to the cDNA library, the full-length gene of Latcripin-3 was cloned by the methods of 3'-full rapid amplification of cDNA Ends (RACE) and 5'-full RACE. The structural domain gene of Latcripin-3 was inserted into the pET32 a(+). The functional protein of Latcripin-3 was expressed in Rosetta-gami (DE3) E. coli, evaluated by Western blotting and mass spectrometry. DPPH testing showed that the protein Latcripin-3 can scavenge free radicals remarkably well. The activity of functional protein Latcripin-3 on A549 cells was studied with flow cytometry and the MTT method. The MTT assay results showed that there was a decreases in cell viability in a dose-dependent and time-dependent manner in protein Latcripin-3 treated groups. Flow cytometry demonstrated that Latcripin-3 can induce apoptosis and block S phase dramatically in human A549 lung cancer cells as compared to the control group. At the same time, the cell ultrastructure observed by transmission electron microscopy supported the results of flow cytometry. This research offers new insights and advantages for identifying anti-oxidant and anti-tumor proteins.

• Asian-Australasian Journal of Animal Sciences
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• 제27권2호
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• pp.237-246
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• 2014

Two experiments were conducted to evaluate the efficacy of feeding an Escherichia coli (E. coli) derived phytase to pigs fed P deficient, corn-soybean meal diets. In Exp. 1, one hundred and twenty crossbred piglets ($9.53{\pm}0.84$ kg) were allocated to one of five treatments which consisted of four low P diets (0.61% Ca, 0.46% total P and 0.24% non-phytate P) supplemented with 0, 500, 1,000, or 20,000 FTU/kg E. coli phytase as well as a positive control formulated to be adequate in all nutrients (0.77% Ca, 0.62% total P and 0.42% non-phytate P). The treatments were applied to six pens with four pigs per pen for 28 days. In Exp. 2, ten crossbred pigs ($19.66{\pm}1.16$ kg) fitted with ileal T-cannula were used in a nutrient balance study. The pigs were assigned to treatments similar to those used in Exp. 1 in a doubly replicated $5{\pm}4$ incomplete Latin square design (5 diets with 4 periods). Each period consisted of a 5-d adjustment period followed by a 3-d total collection of feces and urine and then a 2-d collection of ileal digesta. Supplementation with phytase linearly increased (p<0.05) weight gain, feed intake, feed efficiency, bone breaking strength and fat-free dry and ash bone weight. There were linear increases (p<0.01) in the apparent ileal digestibility (AID) of DM, GE, CP, Ca, total P, inositol hexaphosphate ($IP_6$) and some AA with increasing dose of E. coli phytase. Pigs fed 20,000 FTU/kg had a greater (p<0.05) AID of IP6 (80% vs 59% or 64%, respectively) than pigs fed diets with 500 or 1,000 FTU/kg phytase. There were linear increases (p<0.05) in the total tract digestibility of Ca, total P, Na, K, Mg, and Zn as well as in the retention of Mg and Zn with increased phytase dose. The retention and utilization of Cu, and the total tract digestibility of CP and Cu quadratic increased (p<0.05) with increased phytase dose. In conclusion, supplementation of 500 FTU of phytase/kg and above effectively hydrolyzed phytate in low-P corn-soybean diets for pigs. In addition, a super dose of phytase (20,000 FTU/kg) hydrolyzed most of the IP6 and consequently further improved mineral use, protein utilization and performance.

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1176-1183
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• 2009

Lactobacillus crispatus (L. crispatus) ZJ001 is highly adhesive to epithelial cells and expresses S-layer proteins. In this study, S-S-layer layer genes were sequenced and expressed in E. coli to characterize the function of proteins with this particular strain. L. crispatus ZJ001 harbored two S-layer genes slpA and slpB, and only slpA gene was expressed in the bacterium, as revealed by RT-PCR and immunoassays. The mature SlpA showed 47% amino acid sequence identity to SlpB. The SlpA and SlpB of L. crispatus ZJ001 were highly homologous at the C-terminal region to other Lactobacillus S-layer proteins, but were substantially variable at N-terminal and middle regions. Electron microscopic analysis indicated that His-slpA expressed in E. coli was able to form a sheet-like structure similar to the natural S-layer, but His-slpB formed as disc-like structures. In the cell binding experiments, HeLa cells were able to bind to both recombinant His-slpA and His-slpB proteins to the extent similar to the natural S-layer. The cell binding domains remain mostly in the N-terminal regions in SlpA and SlpB, as shown by high binding of truncated peptides SlpA2-228 and SlpB2-249. Our results indicated that SlpA was active and high binding to HeLa cells, and that the slpA gene could be targeted to display foreign proteins on the bacterial surface of ZJ001 as a potential mucosal vaccine vector.

• Journal of Microbiology and Biotechnology
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• 제28권8호
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• pp.1293-1298
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• 2018

Phosphomannomutase (ManB) converts mannose-6-phosphate (M-6-P) to mannose-1-phosphate (M-1-P), which is a key metabolic precursor for the production of GDP-D-mannose used for production of glycoconjugates and post-translational modification of proteins. The aim of this study was to express the manB gene from Escherichia coli in Lactococcus lactis subsp. cremoris NZ9000 and to characterize the encoded enzyme. The manB gene from E. coli K12, of 1,371 bp and encoding 457 amino acids (52 kDa), was cloned and overexpressed in L. lactis NZ9000 using the nisin-controlled expression system. The enzyme was purified by Ni-NTA column chromatography and exhibited a specific activity of 5.34 units/mg, significantly higher than that of other previously reported ManB enzymes. The pH and temperature optima were 8.0 and $50^{\circ}C$, respectively. Interestingly, the ManB used in this study had two substrate specificity for both mannose-1-phosphate and glucose-1-phosphate, and the specific activity for glucose-1-phosphate was 3.76 units/mg showing 70% relative activity to that of mannose-1-phosphate. This is the first study on heterologous expression and characterization of ManB in lactic acid bacteria. The ManB expression system constructed in this study canbe used to synthesize rare sugars or glycoconjugates.

• KSBB Journal
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• 제10권5호
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• pp.575-581
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• 1995

Plasmid pTTY2에 의한 competent cell(P90c/$\phi$ 434)의 형질전환에 의하여 lipase를 생성하는 재조합 대장균(P90c/따434/pTTY2)을 합성하였다. Li pase 활성 조사를 위한 LAT plate, Rhodamine B plate를 이용한 실험에서 P90c/$\phi$434의 경우 halo 형성이 없었고, P90c/$\phi$434/pTTY2의 경우 용해시 켜 얻은 상등액과 용해되지 않은 미생물을 물리적으 로 깨 서 얻은 상등액 모두 halo를 형성하였다. P90c/$\phi$434/pTTY2에 대한 IPTG 유도시점, 유도 시간이 $\phi$434에 의한 미생물 용해에 미치는 영향을 살펴 봄으로써 다음과 같은 결론을 얻었다. 1. 재조합 대장균의 효과적인 용해는 ODGOO 0.5 ~ 2 2.5인 초기 exponential growth phase에서 IPTG 로 유도한 후, mitomycin C 첨가나 uv조사에 의 해 이루어진다. 2. 재조합 대장균의 용해는 IPTG 유도시간이 1 시간일 때 가장 효과적이었으며, 이후 유도시간이 증가할수록 감소하여 유도시간이 4시간일 때는 세포 용해가 이루어지지 않는다. 3. 실험한 범위에서 uv조사보다 mitomycin C 첨가가 세포 용해에 더 효과적이다. 본 연구결과가 대규모의 생물공학 생산물의 정제 에 응용되기 위해서는 고농도 세포에서의 세포용해 에 대한 연구가 펼요한 것으로 판단된다.

• Journal of Microbiology and Biotechnology
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• 제23권11호
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• pp.1500-1508
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• 2013

Ebosin, a novel exopolysaccharide produced by Streptomyces sp. 139, has obvious antirheumatic arthritis activity in vivo, and its biosynthesis gene cluster (ste), consisting of 27 open reading frames, has been identified. This paper reports our study of the gene functionality of ste8, the predicted protein product of which is homologous to some bacterial chain length determinant Wzz proteins. For characterization of Ste8, ste8 was cloned and expressed in the mutant strain E. coli 086:H2 (${\Delta}wzz$). The functional complementation of wzz by ste8 was demonstrated by the restoration of wild-type lipopolysaccharide biosynthesis and increased levels of serum resistance of E. coli 086:H2 (${\Delta}wzz$) (pET30a-ste8). To examine the function of ste8 in ebosin biosynthesis, the gene was knocked out with a double crossover via homologous recombination. The molecular weight of the ebosin derivative EPS-8m produced by the mutant Streptomyces sp. 139 ($ste8^-$) was much lower than that of ebosin, and the binding activity of EPS-8m for IL-1R decreased significantly compared with ebosin. These results demonstrate that ste8 encodes a chain length determinant (Wzz) that functions in ebosin biosynthesis.

• Journal of Microbiology and Biotechnology
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• 제31권11호
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• pp.1583-1590
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• 2021

Studies have demonstrated that PE_PGRS45 is constitutively expressed under various environmental conditions (such as nutrient depletion, hypoxia, and low pH) of the in vitro growth conditions examined, indicating that PE_PGRS45 protein is critical to the basic functions of Mycobacterium tuberculosis. However, there are few reports about the biochemical function and pathogenic mechanism of PE_PGRS45 protein. The fact that this M. tuberculosis gene is not easily expressed in E. coli may be mainly due to the high content of G+C and the use of unique codons. Fusion tags are indispensable tools used to improve the soluble expression of recombinant proteins and accelerate the characterization of protein structure and function. In the present study, His6, Trx, and His6-MBP were used as fusion tags, but only MBP-PE_PGRS45 was expressed solubly. The purification using His6-MBP tag-specific binding to the Ni column was easy to separate after the tag cleavage. We used the purified PE_PGRS45 to immunize New Zealand rabbits and obtained anti-PE_PGRS45 serum. We found that the titer of polyclonal antibodies against PE_PGR45 was higher than 1:256000. The result shows that purified PE_PGRS45 can induce New Zealand rabbits to produce high-titer antibodies. In conclusion, the recombinant protein PE_PGRS45 was successfully expressed in E. coli and specific antiserum was prepared, which will be followed by further evaluation of these specific antigens to develop highly sensitive and specific diagnostic tests for tuberculosis.