Expression of Bacillus subtilis MA139 β-mannanase in Pichia pastoris and the Enzyme Characterization

Qiao, Jie; Rao, Zhenghua; Dong, Bing; Cao, Yunhe

doi:10.1007/s12010-009-8688-7

Cited by 33 publications

(18 citation statements)

References 25 publications

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“…These observations confirm that foreign T4 lysozyme protein can be expressed in H. polymorpha under the control of the P. pastoris pGAP promoter. Our data are in accordance with previous reports which show that pGAP is useful for large-scale constitutive expression of many heterologous proteins in P. pastoris [18][19][20].…”

Section: Production and Characterization Of Recombinant T4 Lysozymesupporting

confidence: 93%

Expression, characterization, and antimicrobial ability of T4 lysozyme from methylotrophic yeast Hansenula polymorpha A16

Wang

Zhang

et al. 2011

Sci. China Life Sci.

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Lysozyme is an enzyme that is essential for protection against bacterial infections. In this study, a T4 lysozyme gene was cloned into the yeast expression vector pPIC9K under the control of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP). A Hansenula polymorpha-derived ribosomal DNA (rDNA)-targeting element was inserted into the expression vector and was critical for stable DNA integration into the H. polymorpha chromosome. Recombinant T4 lysozyme was successfully expressed in the yeast H. polymorpha A16; 0.49 g L −1 secreted recombinant T4 lysozyme was obtained 72 h after incubation in culture broth that had an initial pH of 6.0. Recombinant T4 lysozyme showed lytic activity against the cell walls of the gram positive bacteria, Micrococcus lysodeikticus, and the gram negative bacteria Xanthomonas campestris pv. malvacearum and Xanthomonas oryzae pv. oryzae. The zone of inhibition assay was used to evaluate antimicrobial activity. Mass spectrometry showed the N-terminal sequence of recombinant T4 lysozyme was identical to that of the native enzyme. SDS-PAGE indicated that the molecular mass of recombinant T4 lysozyme was 18.7 kD which corresponds to a monomer of the native enzyme. SDS-PAGE without 0.2 mol L −1 dithiothreitol treatment detected two bands (15 and 31 kD) suggesting that some recombinant T4 lysozyme formed inter-and intra-molecular disulfide bonds which resulted in loss of enzyme activity.

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Section: Production and Characterization Of Recombinant T4 Lysozymesupporting

confidence: 93%

Expression, characterization, and antimicrobial ability of T4 lysozyme from methylotrophic yeast Hansenula polymorpha A16

Wang

Zhang

et al. 2011

Sci. China Life Sci.

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“…Strain BE-91 fermented for 9 h exhibited the highest activity, up to 273.7 IU/mL. Wild-type B. subtilis MA139 yielded a maximum β -mannanase activity of 170 IU/mL after 3 days of fermentation, and the maximum enzyme activity of B. subtilis TJ-102 was 205.3 IU/mL at 38 h [25, 26]. Notably, BE-91 secreted β -mannanase with higher activity in shorter time.…”

Section: Resultsmentioning

confidence: 99%

Purification and Characterization of a Thermostableβ-Mannanase fromBacillus subtilisBE-91: Potential Application in Inflammatory Diseases

Cheng

Duan

Feng

et al. 2016

BioMed Research International

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β-mannanase has shown compelling biological functions because of its regulatory roles in metabolism, inflammation, and oxidation. This study separated and purified the β-mannanase from Bacillus subtilis BE-91, which is a powerful hemicellulose-degrading bacterium using a “two-step” method comprising ultrafiltration and gel chromatography. The purified β-mannanase (about 28.2 kDa) showed high specific activity (79, 859.2 IU/mg). The optimum temperature and pH were 65°C and 6.0, respectively. Moreover, the enzyme was highly stable at temperatures up to 70°C and pH 4.5–7.0. The β-mannanase activity was significantly enhanced in the presence of Mn2+, Cu2+, Zn2+, Ca2+, Mg2+, and Al3+ and strongly inhibited by Ba2+ and Pb2+. K m and V max values for locust bean gum were 7.14 mg/mL and 107.5 μmol/min/mL versus 1.749 mg/mL and 33.45 µmol/min/mL for Konjac glucomannan, respectively. Therefore, β-mannanase purified by this work shows stability at high temperatures and in weakly acidic or neutral environments. Based on such data, the β-mannanase will have potential applications as a dietary supplement in treatment of inflammatory processes.

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“…S. cerevisiae is used to consume the oxygen inside the fermentation bag to create an anaerobic condition for S. thermophilus and B. subtilis MA139. B. subtilis MA139 was successfully isolated in our previous study (Guo et al, 2006) and is capable of secreting several active components such as β-mannanase and β-glucanase (Qiao et al, 2009; 2010). B. subtilis MA139 is also able to synthesize anti-microbial substances and prevent the growth of enterobacteriaceae (Ying et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Energy and Ileal Digestible Amino Acid Concentrations for Growing Pigs and Performance of Weanling Pigs Fed Fermented or Conventional Soybean Meal

Wang¹,

Lu²,

Li³

et al. 2014

Asian Australas. J. Anim. Sci

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A new strategy of co-inoculating Bacillus subtilis MA139 with Streptococcus thermophilus and Saccharomyces cerevisiae was used to produce fermented soybean meal (FSBM). Three experiments were conducted to determine the concentration of digestible energy (DE) and metabolizable energy (ME) (Exp. 1), apparent ileal digestibility (AID) and standardized ileal digestibility (SID) of amino acids (AA) (Exp. 2), and feeding value (Exp. 3) of FSBM produced by this new strategy (NFSB) compared with soybean meal (SBM) and conventionally available FSBM (Suprotein). In Exp. 1, twenty-four barrows (initial body weight [BW] of 32.2 ±1.7 kg) were randomly allotted to 1 of 4 diets with 6 replicates per diet. A corn basal diet and 3 diets based on a mixture of corn and 1 of 3 soybean products listed above were formulated and the DE and ME contents were determined by the difference method. The results showed that there were no differences in DE and ME between SBM and either FSBM product (p>0.05). In Exp. 2, eight barrows (initial BW of 26.8±1.5 kg) were fitted with ileal T-cannulaes and used in a replicated 4×4 Latin square design. Three corn-starch-based diets were formulated using each of the 3 soybean products as the sole source of AA. A nitrogen-free diet was also formulated to measure endogenous losses of AA. The results showed that the SID of all AA except arginine and histidine was similar for NFSB and SBM (p>0.05), but Suprotein had greater (p<0.05) SID of most AA except lysine, aspartate, glycine and proline than NFSB. In Exp. 3, a total of 144 piglets (initial BW of 8.8±1.2 kg) were blocked by weight and fed 1 of 4 diets including a control diet with 24% SBM as well as diets containing 6% and 12% NFSB or 12% Suprotein added at the expense of SBM. During d 15 to 28, replacing SBM with 6% NFSB significantly improved average daily gain (ADG) and average daily feed intake (ADFI) (p<0.05) for nursery piglets. During the overall experiment, ADG of piglets fed diets containing 6% NFSB was significantly greater (p<0.05) than that of piglets fed SBM. In conclusion, fermentation with the new strategy did not affect the energy content or the AID and the SID of AA in SBM. However, inclusion of 6% NFSB in diets fed to nursery piglets improved performance after weaning likely as a result of better nutritional status and reduced immunological challenge.

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Expression of Bacillus subtilis MA139 β-mannanase in Pichia pastoris and the Enzyme Characterization

Cited by 33 publications

References 25 publications

Expression, characterization, and antimicrobial ability of T4 lysozyme from methylotrophic yeast Hansenula polymorpha A16

Expression, characterization, and antimicrobial ability of T4 lysozyme from methylotrophic yeast Hansenula polymorpha A16

Purification and Characterization of a Thermostableβ-Mannanase fromBacillus subtilisBE-91: Potential Application in Inflammatory Diseases

Energy and Ileal Digestible Amino Acid Concentrations for Growing Pigs and Performance of Weanling Pigs Fed Fermented or Conventional Soybean Meal

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