Cloning, High-Level Expression, Purification, and Properties of a Novel Endo-β-1,4-Mannanase from Bacillus subtilis G1 in Pichia pastoris

Vu, Thi Thu Hang; Quyen, Dinh Thi; Dao, Thi Tuyet; Nguyen, Sy Le Thanh

doi:10.4014/jmb.1106.06052

Cited by 34 publications

(18 citation statements)

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“…The optimal pH, temperature and the stability of mannanases reported were varying depending on the sources. Some mannanases screened by Katrolia P. [18] and Songsiriritthigul C. [2] had similar enzyme characteristics with mannanase Man23 and mannanase screened by Vu TT [19] had similar optimum pH with mannanase Man23 but the latter had broader pH activity range and higher thermo-stability. The activity and kinetic parameters of the mannanases from different sources varied greatly mainly because there are lots of differences with the structure of these mannanases.…”

Section: Discussionmentioning

confidence: 99%

Comparison of expression systems for the extracellular production of mannanase Man23 originated from Bacillus subtilis B23

Zhou

Yang

et al. 2013

Microb Cell Fact

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BackgroundMannanase is an enzyme that can catalyze random hydrolysis of beta-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans which are the key polymers in hemicellulose. It has been used in a number of different industrial applications including food, feed, pharmaceutical, pulp/paper industries, and second generation biofuel. To optimize the expression system of mannanase Man23 gene, two kinds of vectors and host bacteria were determined and compared.ResultsRecombinants pHY-p43-man23 and pBPS-man23 were constructed and transferred into Bacillus subtilis WB600 and Brevibacillus brevis respectively. For mannanase Man23 gene, recombinant pHY-p43-man23 expressed in Brevibacillus brevis had higher production and activity. Compared to the wild-type Bacillus subtilis B23, the production of recombinant pHY-p43-man23 in B. brevis increased by 10 times and activity increased by 21.3%. pHY-p43-man23 in B. brevis had activity at the range of 20 ~ 70°C but its optimum temperature was 50°C and had activity from pH 4 ~ 10 but its optimum pH was around 7. This demonstrated the recombinant had improved stability as well.ConclusionsMannanase is an important industrial enzyme and combination of vector pHY-p43 and host Brevibacillus brevis is a novel expression system for a mannanase decoding gene. This work aims at exploring a better expression system of mannanase Man23 decoding gene for industrial application.

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Section: Discussionmentioning

confidence: 99%

Comparison of expression systems for the extracellular production of mannanase Man23 originated from Bacillus subtilis B23

Zhou

Yang

et al. 2013

Microb Cell Fact

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“…Progressive increase in the expression of proteins such as glucoamylase, lipase, green fluorescence protein, influenza hemagglutinin and human serum albumin was achieved with up to seven gene copies [30]. Proteins such as cattle tick vaccine [31], endo-b-1,4-mannase [32] and hepatitis B surface antigen [33][34] have been successfully expressed in P. pastoris.…”

Section: Introductionmentioning

confidence: 99%

Cloning, expression and characterization of Brugia malayi abundant larval protein transcript-2 (BmALT-2) expressed in Pichia pastoris

Paul

Saha

Selvaraja

et al. 2016

Biotechnology & Biotechnological Equipment

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Besides mass drug administration, successful elimination of human lymphatic filariasis, a mosquitoborne tropical parasitic disease, requires developing other suitable prophylactic agents such as vaccines. The Brugia malayi abundant larval transcript-2 (BmALT-2) protein has been identified as one possible candidate. So far, it (E-ALT-2) has been expressed as a 24-kDa non-glycosylated protein in Escherichia coli. Easy and low-cost downstream purification of secreted BmALT-2 in Pichia pastoris may be a vital option to inexpensive large-scale production. This study focused on expression and molecular characterization of BmALT-2 (P-ALT-2) in P. pastoris. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that some P. pastoris colonies produced 27 and 24 kDa bands and few colonies produced a 24-kDa band. Preliminary studies confirmed glycosylation of 27 kDa P-ALT-2. The ratio of glycosylated and non-glycosylated P-ALT-2 was 20:80-70:30 in various colonies. The maximum yield of glycosylated and non-glycosylated P-ALT-2 was measured as 7.99 § 1.12 and 9.18 § 1.35 mg L ¡1 compared to 6.5 § 1.2 mg L ¡1 of E-ALT-2 in shake flasks. Their overall expression was about 25% and 35% higher than that in E. coli. The glycosylated P-ALT-2 exhibited about 15% higher immunoreactivity with human endemic normal sera. The enhanced secreted production by P. pastoris may lead to cost-effective large-scale production of BmALT-2.

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“…The rSK was identified by MALDI-TOF mass spectrometry as previously described [18]. The predicted protein band on SDS-PAGE was cut out and the target protein was digested by trypsin treatment into small peptide fragments.…”

Section: Methodsmentioning

confidence: 99%

Highly Effective Renaturation of a Streptokinase fromStreptococcus pyogenesDT7 as Inclusion Bodies Overexpressed inEscherichia coli

Nguyen

Quyen

2014

BioMed Research International

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The streptokinase (SK) is emerging as an important thrombolytic therapy agent in the treatment of patients suffering from cardiovascular diseases. We reported highly effective renaturation of a SK from S. pyogeness DT7 overexpressed in E. coli, purification, and biochemical characterization. A gene coding for the SK was cloned from S. pyogeness DT7. Because accumulation of active SK is toxic to the host cells, we have expressed it in the form of inclusion bodies. The mature protein was overexpressed in E. coli BL21 DE3/pESK under the control of the strong promoter tac induced by IPTG with a level of 60% of the total cell proteins. The activity of the rSK, renatured in phosphate buffer supplemented with Triton X-100 and glycerol, was covered with up to 41 folds of its initial activity. The purified of protein was identified with MALDI-TOF mass spectrometry through four peptide fragments, which showed 100% identification to the corresponding peptides of the putative SK from GenBank. Due to overexpression and highly effective renaturation of large amounts of inclusion bodies, the recombinant E. coli BL21 DE3/pESK system could be potentially applied for large-scale production of SK used in the therapy of acute myocardial infarction.

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Cloning, High-Level Expression, Purification, and Properties of a Novel Endo-β-1,4-Mannanase from Bacillus subtilis G1 in Pichia pastoris

Cited by 34 publications

References 0 publications

Comparison of expression systems for the extracellular production of mannanase Man23 originated from Bacillus subtilis B23

Comparison of expression systems for the extracellular production of mannanase Man23 originated from Bacillus subtilis B23

Cloning, expression and characterization of Brugia malayi abundant larval protein transcript-2 (BmALT-2) expressed in Pichia pastoris

Highly Effective Renaturation of a Streptokinase fromStreptococcus pyogenesDT7 as Inclusion Bodies Overexpressed inEscherichia coli

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