Research on Expression Conditions of Endoglucanase in the Recombinant E. coli Strain
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In order to improve the expression level of endoglucanase gene in E.coli,the expression conditions of the recombinant were studied in this experiment.The results showed that: the recombinant E.coli was incubated at 43 ℃ for 5 h,and when induced by 0.1 mmol/L IPTG or 0.1% lactose,its yield of endoglucanase can come to 141.22 U/mL which is about 2 times higher than that of the control.Keywords:
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Maltose-binding protein
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Using two different expression vectors pET-30a-C and pET-28a-C the endoglucanase of Bacillus. sp. bs-1 have been expressed in E. Coli,and their optimal condition for the soluble over expression has been investigated. The expression of the endoglucanase using expression vector pET-30a-C has achieved its best result at the condition of 25℃,induction with 1mM of IPTG and expression for 3 hours. The expression using the expression vector pET-28a-C shows the best soluble over expression at the condition of 25℃,induction with 0. 8mM of IPTG and expression for 5 hours. The activities of the expressed endoglucanases have been determined,and the optimal reaction conditions of the enzyme have been investigated. The expressed endoglucanases were purified by Ni-NTA column,and the specific activities of purified endoglucanase have been determined. The enzyme expressed by pET-30a-C shows the best volume activity of 1489U / ml with a specific activity of 723U / mg. The endoglucanase expressed by pET-28a-C shows a volume activity of 683U / ml. and shows a specific activity of 808U / mg. This result provides a theoretical basis and suggestion for the industrial applications of the endoglucanase.
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Alpha-amylase are of considerable commercial value. It can be produced by a wide variety of microorganisma. The alpha-amylase gene (amyE) from Bacillus licheniformis, which is widely used for the industrial hydrolysis of starch, was mutated (amyEM), then amplified by PCR and inserted into pBV220 and pPIC9k to obtain the recombinant vector pBV220-amyEM and pPIC9k-amyEM. These recombinant vectors were transformed into corresponding competent cell E. coli DH5alpha and P. pastoris GS115 respectively. The resulting recombinant strains, DH5alpha/pBV220-amyEM and GS115/ pPIC9k-amyEM, were then screened by measuring the enzymatic activity and SDS-PAGE. DH5alpha/pBV220-amyEM was induced by temperature and GS115/pPIC9k-amyEM by methanol. In contrast to the parent cells, the a-amylases were expressed in both the recombinant strains. In E. coli the molecular weight was approximately 55kDa; optimal temperature and pH of the recombinant a-amylase were 80 degrees C - 90 degrees C and 6.0 respectively. The recombinant amylase had high activity in pH 5.0 - 5.5 compared to wild type. In Pichia pastoris, the recombinant amylase was secreted to the medium; molecular weight was 60kDa for the putative post-translational modifications; optimal pH shifted to 5.5. The specific activities of alpha-amylase produced by E. coli and P. pastoris were 8.1U/mg and 102U/mg respectively. This result indicated that the alpha-amylase were secreted into the culture medium with high efficiency in the recombinant P. pastoris High activity in high temperature and lower pH properties impart the recombinant amylase potential applications in industry.
Alpha-amylase
Bacillus licheniformis
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β-galactosidase can be used to hydrolyze lactose of milk into glucose and galactose. Athermostable β-galactosidase gene bgaB from Bacillus stearothermophilus was cloned and successfully expressed in E. coli T7 expression system. The optimum induction conditions, i.e. induction time, inducer concentration and induction length were studied on SOB media by IPTG and Lactose as inducers. The specific enzyme activity reached 11.5 and 7.7 U/mg protein, 90 and 60 times higher than that of original strain, respectively.
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Galactosidases
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The rhIFN α2a is expressed as a fusion protein containing thioredoxine and polyhistidine sites at its N terminal. Our previous research has obtained recombinant human IFN α2a (rhIFN α2a) protein that expressed predominantly as a soluble form in E. coli BL21(DE3). Through systematic approach of various culture conditions, the aim of current this research is to acquire the best condition and its stability of recombinant rhIFN α2a fusion protein in a culture under study. Expression optimization performed by using three parameters, i.e.: temperature, induction time and inducer concentration. Various IPTG concentrations are 0.25 mM, 0.5 mM, 0.75 mM and 1.0 mM. The incubation time of bacterial cell culture carried out in 3, 4, and 5 hours at temperature 28, 30, and 37°C. The best condition was used to analyze the stability of rhIFN α2a protein expression up to ten generation. The expressed protein was analyzed using SDS PAGE and CBB staining. The optimal culture condition was found to be 37°C temperature with 4 hours time of induction and 1 mM IPTG concentration. Stability analysis revealed that the rhFN α2a protein expression remained stable until the tenth generation with molecular weight, approximately, 36 kDa.
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The possibility of using lactose as an inducer to substitute IPTG in the fermentation of rhCu/Zn-SOD in E.coli BL21(DE3) was investigated. The growth of E.coli and the expression of the recombinant protein were compared after induction by lactose or IPTG. The influences of concentrations of lactose and metal ions and induction temperatures on the expression of the recombinant protein were examined. Finally the recombinant E.coli was cultured in a 5 L fermentor. The maximum enzyme activity of rhCu/Zn-SOD reached 1810 U per ml fermentation broth. The results suggested that rhCu/Zn-SOD could be expressed in E.coli with lactose as a substitute inducer and the maximum enzyme activity was about 89% of that with IPTG.
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Liquid medium
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Heterologous expression
Yeast extract
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Amidase
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We induced the acquired recombinant Pichia pastoris strains with the gene encoding A/D antigenic domain of hog cholera virus E2 protein and optimized expression condition by studying the relations beween expression yield and growth conditions with different inductions time,recombinant strain,strain density,pH value and methanol dose of medium,respectively.The optimal conditions of the recombinant protein expression were:28-30 ℃,225 r/min,in BMMY medium of pH 6.0 which was 4 times volume of BMGY with OD_(600) being 3.0-3.5,was incubated 72 h and methanol was added to keep its concentration up to 0.5% between 24 h.After induction under optimal conditions,the yield of recombinant protein could be added up to 275.38 μg/mL.After comparing the effects of different convervation temperature on lysis of recombinant protein,it was found that the supernatant should be conserved in -20 ℃ and these jobs had provided a good basis for large-scaled ferment and application of recombinant protein.
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Pichia
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