scholarly journals Heterologous Expression and Characterization of a High-Efficiency Chitosanase From Bacillus mojavensis SY1 Suitable for Production of Chitosan Oligosaccharides

2021 ◽  
Vol 12 ◽  
Author(s):  
Jianrong Wang ◽  
Xiaoming Li ◽  
Hao Chen ◽  
Bilian Lin ◽  
Liangzhong Zhao
Keyword(s):  
Recombinant Strain ◽  
High Efficiency ◽  
Batch Cultivation ◽  
Maximum Activity ◽  
Gene Encoding ◽  
Enzymatic Production ◽  
Bacillus Mojavensis ◽  
Chitosan Oligosaccharides ◽  
High Level

Chitosanase plays an important role in enzymatic production of chitosan oligosaccharides (COSs). The present study describes the gene cloning and high-level expression of a high-efficiency chitosanase from Bacillus mojavensis SY1 (CsnBm). The gene encoding CsnBm was obtained by homologous cloning, ligated to pPICZαA, and transformed into Pichia pastoris X33. A recombinant strain designated X33-C3 with the highest activity was isolated from 120 recombinant colonies. The maximum activity and total protein concentration of recombinant strain X33-C3 were 6,052 U/ml and 3.75 g/l, respectively, which were obtained in fed-batch cultivation in a 50-l bioreactor. The optimal temperature and pH of purified CsnBm were 55°C and 5.5, respectively. Meanwhile, CsnBm was stable from pH 4.0 to 9.0 and 40 to 55°C. The purified CsnBm exhibited high activity toward colloidal chitosan with degrees of deacetylation from 85 to 95%. Furthermore, CsnBm exhibited high efficiency to hydrolyze different concentration of colloidal chitosan to produce COSs. The result of this study not only identifies a high-efficiency chitosanase for preparation of COSs, but also casts some insight into the high-level production of chitosanase in heterologous systems.

Gene cloning and characterization of thiourocanate hydratase from Burkholderia sp. HME13

2019 ◽  
Vol 167 (3) ◽  
pp. 333-341
Author(s):  
Hisashi Muramatsu ◽  
Haruna Miyaoku ◽  
Syuya Kurita ◽  
Hidenori Matsuo ◽  
Takehiro Kashiwagi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Crude Extract ◽  
Maximum Activity ◽  
Luria Bertani ◽  
Sole Nitrogen Source ◽  
Gene Encoding ◽  
Gene Cloning And Characterization

Abstract A novel enzyme, thiourocanate hydratase, which catalyses the conversion of thiourocanic acid to 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid, was isolated from the ergothioneine-utilizing strain, Burkholderia sp. HME13. When the HME13 cells were cultured in medium containing ergothioneine as the sole nitrogen source, thiourocanate-metabolizing activity was detected in the crude extract from the cells. However, activity was not detected in the crude extract from HME13 cells that were cultured in Luria-Bertani medium. The gene encoding thiourocanate hydratase was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. The enzyme showed maximum activity at pH 7.5 and 55°C and was stable between pH 5.0 and 10.5, and at temperatures up to 45°C. The Km and Vmax values of thiourocanate hydratase towards thiourocanic acid were 30 μM and 7.1 μmol/min/mg, respectively. The enzyme was strongly inhibited by CuCl2 and HgCl2. The amino acid sequence of the enzyme showed 46% identity to urocanase from Pseudomonas putida, but thiourocanate hydratase had no urocanase activity.

Characterization of L-fucose isomerase from Paenibacillus rhizosphaerae to produce L-fuculose from hydrolyzed fucoidan and commercial fucose

2019 ◽  
Author(s):  
Muhammad Waheed Iqbal ◽  
Tahreem Riaz ◽  
Shahid Mahmood ◽  
Yingying Zhu ◽  
Dawei Ni ◽  
...  
Keyword(s):  
Industrial Applications ◽  
Maximum Activity ◽  
Lysosomal Disease ◽  
Simple Method ◽  
Enzymatic Production ◽  
Native Molecular Mass ◽  
Anti Cancer ◽  
Viral Hepatitis B ◽  
High Production

Abstract Background L-fuculose is an expensive and rare sugar used against different kinds of diseases such as HIV, anti-cancer, anti-viral, Hepatitis-B, human lysosomal disease (fucosidosis), and cardio-protective drugs. The enzymatic way of converting L-fucose into L-fuculose would be an effective method with great industrial applications. The purpose of this research is to introduce a high production of L-fuculose from cheap and natural sources (fucoidan) and commercially source (Sigma-Aldrich) by a recombinant enzyme L-fucose isomerase from Paenibacillus rhizosphaerae (Pa-LFI).Results Fucose containing polysaccharide (FPs) called fucoidan was extracted, hydrolyzed and characterized by U. pinnatifida for enzymatic production of L-fuculose. The FPs provide 35.9% of fucose along with few other monosaccharides. Pa-LFI was characterized and purified with a single band at 65 kDa. It showed an activity of 104.5 U mg -1 and exhibited as a hexamer with native molecular mass 396 kDa. The maximum activity for recombinant Pa-LFI was detected at pH 6.5 and 50 °C in 1 mM of Mn 2+ . The melting temperature observed 75 °C and half-life at 50 °C was 12.6 h. The isomerizing activity of Pa-LFI with aldose substrate (L-fucose) was higher exposing K m , k cat and k cat / K m 86.2 mM, 32831 min -1 and 335 min -1 mM -1 respectively. The conversion ratio of L-fuculose from 100 g L -1 of FPs and commercial fucose after the equilibrium state was about 6% (5.6 g L -1 ) and 30% (30.2 g L -1 ) respectively.Conclusion Pa-LFI catalyzed the reaction to convert L-fucose into L-fuculose. The enzyme will be helpful in the production of L-fuculose with an efficient and simple method without producing any by-product.

Characterization of a Rhizobium larrymoorei FJ Exhibiting High Level Cr(VI) Reduction Potential

2011 ◽  
Vol 356-360 ◽  
pp. 1009-1014 ◽  
Author(s):  
Jun Hui Zhang
Keyword(s):  
High Efficiency ◽  
Anaerobic Respiration ◽  
Waste Recycling ◽  
Soluble Form ◽  
Rrna Gene ◽  
Resting Cells ◽  
Surface Binding ◽  
Batch Cultures ◽  
High Level

The investigation was conducted to evaluate mechanism of Cr(VI) resistance and reduction by a bacterial strain named FJ under different conditions. This strain, identified as a member of Rhizobium larrymoorei by analysis of its 16S rRNA gene sequence was previously isolated from a paddy soil contaminated by e-waste recycling. Good Cr(VI) reduction ability catalyzed by growing cells of R. larrymoorei FJ was observed in batch cultures conducted at different initial Cr(VI) concentrations. Up to 83.23% reduction was shown in LB medium supplemented with 2.50 mM Cr(VI). Cr(VI) was transformed to some soluble form of Cr(III) due to anaerobic respiration. Biosorption was also observed in the process of bioreduction. But only loosely cell-surface binding Cr(VI) was detected in cells grown in medium supplied with different concentrations of Cr(VI). Present of yeast or citrate could enhance Cr(VI) reduction of resting cells. However, Cr(VI) reduction by resting cells was only observed at Cr(VI) concentration lower than 0.25 mM. R. larrymoorei FJ exhibited a high efficiency of Cr(VI) reduction at temperatures from 28°C to 37°C and pH values from 6.0 to 7.0.

Characterization of amoA and hao Genes Responsible for Ammonia Oxidation Reaction in CANON System

2011 ◽  
Vol 183-185 ◽  
pp. 1014-1019
Author(s):  
Hai Yan Zou ◽  
Jun Li Huang ◽  
Fang Fang ◽  
Jin Song Guo
Keyword(s):  
Nitrogen Removal ◽  
Oxidation Reaction ◽  
High Efficiency ◽  
Ammonia Oxidation ◽  
Residual Activity ◽  
Maximum Activity ◽  
Ammonia Oxidizing Bacteria ◽  
Hydroxylamine Oxidoreductase ◽  
Optimum Ph

In this research the genes (amoA and hao) for ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) responsible for ammonia oxidation reaction in completely autotrophic nitrogen removal over nitrite process were cloned and sequenced, and the recombinant protein of AMO and HAO was expressed and characterized. The optimum temperature for AMO activity was 55 °C and more than 40% of the maximum activity was retained from 15-50 °C. The optimum pH for the enzyme was found to be pH 11.0. The highest activity for HAO was observed at 45 °C. More than 50% of the maximum activity was retained even at 55 °C. The dependence of HAO on pH was strong and only average 15% of residual activity left at pH ranging from 3.0-9.0. Study on the molecular and biochemistry properties of recombinant AMO and HAO will benefit for the manipulation of ammonia-oxidizing bacteria to achieve the goal of high efficiency of nitrogen removal.

scholarly journals New Aspects of the Interplay between Penicillin Binding Proteins, murM, and the Two-Component System CiaRH of Penicillin-Resistant Streptococcus pneumoniae Serotype 19A Isolates from Hungary

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Inga Schweizer ◽  
Sebastian Blättner ◽  
Patrick Maurer ◽  
Katharina Peters ◽  
Daniela Vollmer ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Morphological Changes ◽  
Penicillin Resistance ◽  
Penicillin Binding Protein ◽  
Two Component System ◽  
Gene Encoding ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
High Level

ABSTRACT The Streptococcus pneumoniae clone Hungary19A-6 expresses unusually high levels of β-lactam resistance, which is in part due to mutations in the MurM gene, encoding a transferase involved in the synthesis of branched peptidoglycan. Moreover, it contains the allele ciaH232, encoding the histidine kinase CiaH (M. Müller, P. Marx, R. Hakenbeck, and R. Brückner, Microbiology 157:3104–3112, 2011, https://doi.org/10.1099/mic.0.053157-0 ). High-level penicillin resistance primarily requires the presence of low-affinity (mosaic) penicillin binding protein (PBP) genes, as, for example, in strain Hu17, a closely related member of the Hungary19A-6 lineage. Interestingly, strain Hu15 is β-lactam sensitive due to the absence of mosaic PBPs. This unique situation prompted us to investigate the development of cefotaxime resistance in transformation experiments with genes known to play a role in this phenotype, pbp2x, pbp1a, murM, and ciaH, and penicillin-sensitive recipient strains R6 and Hu15. Characterization of phenotypes, peptidoglycan composition, and CiaR-mediated gene expression revealed several novel aspects of penicillin resistance. The murM gene of strain Hu17 (murM Hu17), which is highly similar to murM of Streptococcus mitis, induced morphological changes which were partly reversed by ciaH232. murM Hu17 conferred cefotaxime resistance only in the presence of the pbp2x of strain Hu17 (pbp2x Hu17). The ciaH232 allele contributed to a remarkable increase in cefotaxime resistance in combination with pbp2x Hu17 and pbp1a of strain Hu17 (pbp1a Hu17), accompanied by higher levels of expression of CiaR-regulated genes, documenting that ciaH232 responds to PBP1aHu17-mediated changes in cell wall synthesis. Most importantly, the proportion of branched peptides relative to the proportion of linear muropeptides increased in cells containing mosaic PBPs, suggesting an altered enzymatic activity of these proteins.

scholarly journals Cloning and Characterization of a Gene Encoding the Major Surface Protein of the Bacterial EndosymbiontWolbachia pipientis

1998 ◽  
Vol 180 (9) ◽  
pp. 2373-2378 ◽  
Author(s):  
Henk R. Braig ◽  
Weiguo Zhou ◽  
Stephen L. Dobson ◽  
Scott L. O’Neill
Keyword(s):  
Molecular Mechanisms ◽  
Cytoplasmic Incompatibility ◽  
Surface Protein ◽  
Gene Encoding ◽  
Developmental Abnormalities ◽  
Intracellular Symbiont ◽  
Different Strains ◽  
Major Surface ◽  
High Level

ABSTRACT The maternally inherited intracellular symbiont Wolbachia pipientis is well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning and characterization of the gene (wsp) that encodes it. The functionality of the wsp promoter region was also successfully tested in Escherichia coli. Comparison of sequences of this gene from different strains of Wolbachiarevealed a high level of variability. This sequence variation correlated with the ability of certain Wolbachia strains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects. As such, this gene will be a very useful tool forWolbachia strain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction ofWolbachia with its host.

scholarly journals High-Level Heterologous Expression of Endo-1,4-β-Xylanase from Penicillium citrinum in Pichia pastoris X-33 Directed through Codon Optimization and Optimized Expression

Molecules ◽  
2019 ◽  
Vol 24 (19) ◽  
pp. 3515 ◽  
Author(s):  
Chanika Ouephanit ◽  
Nassapat Boonvitthya ◽  
Sophie Bozonnet ◽  
Warawut Chulalaksananukul
Keyword(s):  
Pichia Pastoris ◽  
Xylanase Activity ◽  
Alcohol Oxidase ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Gene Encoding ◽  
Original Activity ◽  
Native Strain ◽  
Wide Range ◽  
High Level

Most common industrial xylanases are produced from filamentous fungi. In this study, the codon-optimized xynA gene encoding xylanase A from the fungus Penicilium citrinum was successfully synthesized and expressed in the yeast Pichia pastoris. The levels of secreted enzyme activity under the control of glyceraldehyde-3-phosphate dehydrogenase (PGAP) and alcohol oxidase 1 (PAOX1) promoters were compared. The Pc Xyn11A was produced as a soluble protein and the total xylanase activity under the control of PGAP and PAOX1 was 34- and 193-fold, respectively, higher than that produced by the native strain of P. citrinum. The Pc Xyn11A produced under the control of the PAOX1 reached a maximum activity of 676 U/mL when induced with 1% (v/v) methanol every 24 h for 5 days. The xylanase was purified by ion exchange chromatography and then characterized. The enzyme was optimally active at 55 °C and pH 5.0 but stable over a broad pH range (3.0–9.0), retaining more than 80% of the original activity after 24 h or after pre-incubation at 40 °C for 1 h. With birchwood xylan as a substrate, Pc Xyn11A showed a Km(app) of 2.8 mg/mL, and a kcat of 243 s−1. The high level of secretion of Pc Xyn11A and its stability over a wide range of pH and moderate temperatures could make it useful for a variety of biotechnological applications.

scholarly journals Molecular characterization of the gene encoding high-level mupirocin resistance in Staphylococcus aureus J2870.

1994 ◽  
Vol 38 (5) ◽  
pp. 1205-1208 ◽  
Author(s):  
J E Hodgson ◽  
S P Curnock ◽  
K G Dyke ◽  
R Morris ◽  
D R Sylvester ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Molecular Characterization ◽  
Gene Encoding ◽  
Mupirocin Resistance ◽  
High Level

scholarly journals Identification and Characterization of an O-Succinyl-L-Homoserine Sulfhydrylase From Thioalkalivibrio sulfidiphilus

2021 ◽  
Vol 9 ◽  
Author(s):  
Wen-Yuan Zhu ◽  
Kun Niu ◽  
Peng Liu ◽  
Yu-Hang Fan ◽  
Zhi-Qiang Liu ◽  
...  
Keyword(s):  
Large Scale ◽  
Total Yield ◽  
Maximum Activity ◽  
Potential Candidate ◽  
Natural Amino Acid ◽  
Gene Encoding ◽  
Broad Application ◽  
Identification And Characterization ◽  
Application Prospects

L-methionine is an important natural amino acid with broad application prospects. A novel gene encoding the enzyme with the ability to catalyze O-succinyl-L-homoserine (OSH) to L-methionine was screened and characterized. The recombinant O-succinyl-L-homoserine sulfhydrylase from Thioalkalivibrio sulfidiphilus (tsOSHS) exhibited maximum activity at 35°C and pH 6.5. OSHS displayed an excellent thermostability with a half-life of 21.72 h at 30°C. Furthermore, the activity of OSHS increased 115% after Fe2+ added. L-methionine was obtained with a total yield reaching 42.63 g/L under the concentration of O-succinyl-L-homoserine 400 mM (87.6 g/L). These results indicated that OSHS is a potential candidate for applying in the large-scale bioproduction of L-methionine.

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