Glycine and Triton X-100 enhanced secretion of recombinant α-CGTase mediated by OmpA signal peptide in Escherichia coli

Li, Bin; Wang, Lei; Su, Lingqia; Chen, Sheng; Li, Zhaofeng; Chen, Jian; Wu, Jang‐Yen

doi:10.1007/s12257-011-0601-x

Cited by 26 publications

(21 citation statements)

References 11 publications

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“…Although 20°C might be more suitable for the production of soluble CSN, the significantly lowered cell density limited its total protein yield. Therefore, E. coli was more conducive to the secretion expression in lower temperature at 25°C, which was in agreement with the previous studies [28,29].…”

Section: Effects Of Temperature On the Secretion Of Csnsupporting

confidence: 92%

Improved extracellular expression and high-cell-density fed-batch fermentation of chitosanase from Aspergillus Fumigatus in Escherichia coli

Huang

Wang

Jiang

et al. 2016

Bioprocess Biosyst Eng

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Chitosanase (CSN) from Aspergillus fumigatus has good thermal stability, wide pH range duration, and effective hydrolysis for chitosan. Inhere, CSN was successfully expressed in Escherichia coli followed by extracellular secretion under the guidance of an N-terminal signal peptide PelB, which effectively prompted its secretion out of E. coli cells. To facilitate its later purification, N-terminal or C-terminal 6xHis epitope tag was added to the PelB-CSN protein complex. Our results indicated that PelB-CSN without 6xHis-tag (PelB-CSN) or with N-terminal 6xHis-tag (PelB-CSN-N) can both be effectively secreted into the medium, while CSN with 6xHis-tag anchored at C-terminus was expressed as inclusion bodies. Process optimization strategies were further developed to improve the secretion efficiency of recombinant PelB-CSN-N in E. coli. Under the induction of 10 g/L lactose in shake-flask culture, the extracellular activity of CSN reached 6015 U/mL at 25 °C in TB medium containing 1 % glycine. Moreover, a fed-batch fermentation strategy for high-cell-density cultivation was applied in a 5-L fermenter, increasing the extracellular CSN activity to 14,000 U/mL in 2-day fermentation with the optimal addition of lactose and glycine.

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Section: Effects Of Temperature On the Secretion Of Csnsupporting

confidence: 92%

Improved extracellular expression and high-cell-density fed-batch fermentation of chitosanase from Aspergillus Fumigatus in Escherichia coli

Huang

Wang

Jiang

et al. 2016

Bioprocess Biosyst Eng

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“…However, the extracellular secretion ratio was still very low because most of the pullulanase accumulated in the periplasmic space. Although the recovery of enzymes secreted into the periplasmic space can be achieved by various approaches (16,17), its application on an industrial scale has been limited by the unavailability of efficient large-scale methods for the selective release of periplasmic proteins from the cell (18). Therefore, a method that allows the secretion of recombinant pullulanase directly into the culture medium is highly desirable.…”

mentioning

confidence: 99%

Enhancing the Secretion Efficiency and Thermostability of a Bacillusderamificans Pullulanase Mutant (D437H/D503Y) by N-Terminal Domain Truncation

Duan

2015

Appl Environ Microbiol

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Pullulanase (EC 3.2.1.41), an important enzyme in the production of starch syrup, catalyzes the hydrolysis of ␣-1,6 glycosidic bonds in complex carbohydrates. A double mutant (DM; D437H/D503Y) form of Bacillus deramificans pullulanase was recently constructed to enhance the thermostability and catalytic efficiency of the enzyme (X. Duan, J. Chen, and J. Wu, Appl Environ Microbiol 79:4072-4077, 2013, http://dx.doi.org/10.1128/AEM.00457-13). In the present study, three N-terminally truncated variants of this DM that lack the CBM41 domain (DM-T1), the CBM41 and X25 domains (DM-T2), or the CBM41, X25, and X45 domains (DM-T3) were constructed. Upon expression, DM-T3 existed as inclusion bodies, while 72.8 and 74.8% of the total pullulanase activities of DM-T1 and DM-T2, respectively, were secreted into the medium. These activities are 2.8-and 2.9-fold that of the DM enzyme, respectively. The specific activities of DM-T1 and DM-T2 were 380.0 ؋ 10 8 and 449.3 ؋ 10 8 U · mol ؊1, respectively, which are 0.94-and 1.11-fold that of the DM enzyme. DM-T1 and DM-T2 retained 50% of their activity after incubation at 60°C for 203 and 160 h, respectively, which are 1.7-and 1.3-fold that of the DM enzyme. Kinetic studies showed that the K m values of DM-T1 and DM-T2 were 1.5-and 2.7-fold higher and the K cat /K m values were 11 and 50% lower, respectively, than those of the DM enzyme. Furthermore, DM-T1 and DM-T2 produced D-glucose contents of 95.0 and 94.1%, respectively, in a starch saccharification reaction, which are essentially identical to that produced by the DM enzyme (95%). The enhanced secretion and improved thermostability of the truncation mutant enzymes make them more suitable than the DM enzyme for industrial processes. P ullulanase (EC 3.2.1.41), which catalyzes the hydrolysis of ␣-1,6-glucosidic linkages in pullulan, amylopectin, and the dextrins of amylopectin, is a well-known starch-debranching enzyme (1). It belongs to the ␣-amylase family, which is identified as glycoside hydrolase family 13 in the CAZy database. When combined with ␣-amylase, ␤-amylase, glucoamylase, or cyclodextrin glucosyltransferase, pullulanase can be used in the production of glucose, fructose, maltose, cyclodextrins, and amylose (2, 3). The addition of pullulanase allows the reaction time to be reduced, the substrate concentration to be increased, and the conversion rate to be improved (4, 5). Recently, pullulanase has been used as a biocatalyst for the production of fuel ethanol, low-calorie beers, resistant starch, maltotriose, and other important products (6, 7). With this plethora of potential industrial applications, pullulanase production has attracted a great deal of attention in recent years (8,9).A large number of microorganisms, including mesophiles, thermophiles, and hyperthermophiles, have been shown to produce pullulanase (10, 11). Although numerous pullulanases have been identified and heterologously expressed (12)(13)(14), reports have shown that the secretion ratio of recombinant pullulanase is low. Low extracellular prod...

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“…Fu et al reports that the chemical triton X-100 could release recombinant protein to the extracellular medium by permeabilization of the cells [20]. Triton X-100 is reported to inhibit synthesis of membrane phospholipids, which would promote extracellular translocation of recombinant proteins by affecting integrity of the cell membrane [12].…”

Section: Discussionmentioning

confidence: 99%

“…Aside from their individual ability to permeate the E. coli membrane, Bin Lee et al reveals a potential mechanism in the interaction effect of glycine and triton X-100 [12]. Research has shown that combination of glycine and triton X-100 could result in drastically increased permeability of both inner and outer membranes [12].…”

Section: Discussionmentioning

confidence: 99%

“…Some medium supplements such as lysozyme, magnesium, calcium, EDTA, glycine, and triton X-100 could increase membrane permeability and subsequent protein secretion to the culture medium [9][10][11]. The existence certain values of triton X-100 and glycine in the culture medium had a strong effect on extracellular production and could greatly increase both inner and outer cell membrane permeability [12]. Divalent cations, such as Ca +2 and Mg +2 in medium rich with glycine can effectively protect cells from lysing [13].…”

Section: Introductionmentioning

confidence: 99%

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Optimization of culture media for extracellular expression of streptokinase in Escherichia coli using response surface methodology in combination with Plackett-Burman Design

Aghaeepoor¹,

Kobarfard²,

Eidgahi³

et al. 2018

Trop. J. Pharm Res

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Glycine and Triton X-100 enhanced secretion of recombinant α-CGTase mediated by OmpA signal peptide in Escherichia coli

Cited by 26 publications

References 11 publications

Improved extracellular expression and high-cell-density fed-batch fermentation of chitosanase from Aspergillus Fumigatus in Escherichia coli

Improved extracellular expression and high-cell-density fed-batch fermentation of chitosanase from Aspergillus Fumigatus in Escherichia coli

Enhancing the Secretion Efficiency and Thermostability of a Bacillusderamificans Pullulanase Mutant (D437H/D503Y) by N-Terminal Domain Truncation

Optimization of culture media for extracellular expression of streptokinase in Escherichia coli using response surface methodology in combination with Plackett-Burman Design

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