High-level expression of the xylanase from Thermomyces lanuginosus in Escherichia coli

Er-kang, Yin; Le, Yilin; Pei, Jianjun; Shao, Weilan; Yang, Qian

doi:10.1007/s11274-007-9469-5

Cited by 28 publications

(18 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Many enzymes had been expressed successfully with the heat-shock vector without isopropyl-β-D-1-thiogalactopyranoside (Shao et al 2006;Wu et al 2008;Yin et al 2008). By using the temperature sensitive vector pHsh, the target gene was successfully expressed in E. coli JM109.…”

Section: Resultsmentioning

confidence: 99%

Expression and characterization of an α-glucosidase from Thermoanaerobacter ethanolicus JW200 with potential for industrial application

et al. 2009

View full text Add to dashboard Cite

In this study, a new α-glucosidase gene from Thermoanaerobacter ethanolicus JW200 was cloned and expressed in Escherichia coli by a novel heat-shock vector pHsh. The recombinant α-glucosidase exhibited its maximum hydrolytic activity at 70• C and pH 5.0∼5.5. With p-nitrophenyl-α-D-glucoside as a substrate and under the optimal condition (70 • C, pH 5.5), Km and Vmax of the enzyme was 1.72 mM and 39 U/mg, respectively. The purified α-glucosidase could hydrolyze oligosaccharides with both α-1,4 and α-1,6 linkages. The enzyme also had strong transglycosylation activity when maltose was used as sugar donor. The transglucosylation products towards maltose are isomaltose, maltotriose, panose, isomaltotriose and tetrasaccharides. The enzyme could convert 400 g/L maltose to oligosaccharides with a conversion rate of 52%, and 83% of the oligosaccharides formed were prebiotic isomaltooligosaccharides (containing isomaltose, panose and isomaltotriose).

show abstract

Section: Resultsmentioning

confidence: 99%

Expression and characterization of an α-glucosidase from Thermoanaerobacter ethanolicus JW200 with potential for industrial application

et al. 2009

View full text Add to dashboard Cite

show abstract

“…Enhanced recombinant xylanase production may be achieved following manipulation of codon hierarchy within the heterologous gene [45]. The efficacy of the signal sequence peptide has also been documented to influence heterologous expression [46].…”

Section: Discussionmentioning

confidence: 99%

Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris

Gaffney

Carberry

Doyle

et al. 2009

Enzyme and Microbial Technology

View full text Add to dashboard Cite

a b s t r a c tA xylanase produced by Thermomyces lanuginosus 195 by solid state fermentation (SSF) was purified 9.3-fold from a crude koji extract, with a 7.6% final yield. The purified xylanase (with an estimated mass of 22 kDa by SDS-PAGE) retained 18% relative activity when treated for 10 min at 100• C and approximately 90% relative activity when incubated at pH values ranging from 6 to 10. Xylanase activity in the purified preparation was significantly enhanced following treatment with manganese and potassium chlorides (p < 0.05) but significantly reduced by calcium, cobalt and iron (p < 0.05). The purified enzyme was also shown to be exclusively xylanolytic. The gene encoding xylanase activity from T. lanuginosus 195 was functionally expressed by Pichia pastoris. MALDI-ToF mass spectrometry and zymography were employed to confirm functional recombinant expression. Maximum xylanase titres were achieved following 120 h induction of the recombinant culture, yielding 26.8 U/mL. Achieving functional protein expression facilitates future efforts to optimise the cultivation conditions for heterologous xylanase production.

show abstract

“…A xylanase gene from Bacillus that was expressed in E. coli had been reported to be distributed in extracellular, intracellular and periplasmic fractions (Huang et al, 2006). However, recombinant xylanases were commonly found to be in the insoluble fraction of the cytoplasm, hinting that even after optimization, the expression of the xylanase gene produces protein that is insoluble and accumulates in inclusion bodies (Yin et al, 2008). In many cases the expressed protein is insoluble and accumulates in inclusion bodies, especially under conditions of high level expression.…”

Section: Resultsmentioning

confidence: 99%

Characterisation of Klebsiella pneumoniae Xylanase and Increment of Its Activity in Heterologous Expression System

Hasnain¹,

Suhaila²,

Chong³

et al. 2016

BJRST

View full text Add to dashboard Cite

A xylanase DNA sequence with a total length of 642 bp was previously isolated from a xylanolytic Klebsiellapneumoniae. Xylanase gene primers were designed with the addition of BamH1 and EcoR1 restriction enzymesites in order get a full xylanase gene that is in-frame with pSTAG expression vector. The isolated xylanasegene was amplified using the designed primers through PCR, then cloned and expressed in E. coli BL21 (DE3).In-silico characterization showed that the recombinant xylanase has a molecular weight of 23.9 kDa and a pI of9.32. The signal peptide cleavage site for the recombinant xylanase was predicted to be between residues 61and 62. The activity of the crude recombinant xylanase was 2.015 U/mL, which was higher than the crudenative xylanase activity, with maximum at 0.642 U/mL. Staining of the birchwood xylan agar plate with Congored showed a clearing zone around E. coli BL21 (DE3) colonies with recombinant pSTAG plasmid evenwithout being induced with IPTG. This implied leaky expression of the E. coli BL21 (DE3) secretion system,which recognized the signal sequence of the recombinant xylanase, and proceeded to cleave and secreted outthe mature protein into the culture medium. MALDI-TOF analysis of a 20 kDa protein present in the culturemedium confirmed that the recombinant xylanase had been secreted into the culture medium.

show abstract

High-level expression of the xylanase from Thermomyces lanuginosus in Escherichia coli

Cited by 28 publications

References 19 publications

Expression and characterization of an α-glucosidase from Thermoanaerobacter ethanolicus JW200 with potential for industrial application

Expression and characterization of an α-glucosidase from Thermoanaerobacter ethanolicus JW200 with potential for industrial application

Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris

Characterisation of Klebsiella pneumoniae Xylanase and Increment of Its Activity in Heterologous Expression System

Contact Info

Product

Resources

About