A novel Method for the selective recovery and purification of γ‐polyglutamic acid from <i>Bacillus licheniformis</i> fermentation broth

Manocha, Bhavik; Margaritis, Argyrios

doi:10.1002/btpr.370

Cited by 39 publications

(30 citation statements)

References 35 publications

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“…The copper sulfate precipitation method was used to obtain only poly-␣-glutamic acid. As a preliminary test, the copper sulfate precipitation was used for only commercially available poly-␣-glutamic acid (Sigma), as described by Margaritis et al (20). Three types of poly-␣-glutamic acids, with molecular weights of 750 to 5,000, 3,000 to 15,000, and 15,000 to 50,000, were examined, but the precipitate was observed only when the poly-␣-glutamic acid with a molecular weight of 15,000 to 50,000 was used.…”

Section: Discussionmentioning

confidence: 99%

Poly-α-Glutamic Acid Synthesis Using a Novel Catalytic Activity of RimK from Escherichia coli K-12

Kino

Arai

Arimura

2011

Appl Environ Microbiol

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Poly-L-␣-amino acids have various applications because of their biodegradable properties and biocompatibility. Microorganisms contain several enzymes that catalyze the polymerization of L-amino acids in an ATP-dependent manner, but the products from these reactions contain amide linkages at the side residues of amino acids: e.g., poly-␥-glutamic acid, poly--lysine, and cyanophycin. In this study, we found a novel catalytic activity of RimK, a ribosomal protein S6-modifying enzyme derived from Escherichia coli K-12. This enzyme catalyzed poly-␣-glutamic acid synthesis from unprotected L-glutamic acid (Glu) by hydrolyzing ATP to ADP and phosphate. RimK synthesized poly-␣-glutamic acid of various lengths; matrix-assisted laser desorption ionization-time of flight-mass spectrometry showed that a 46-mer of Glu (maximum length) was synthesized at pH 9. Interestingly, the lengths of polymers changed with changing pH. RimK also exhibited 86% activity after incubation at 55°C for 15 min, thus showing thermal stability. Furthermore, peptide elongation seemed to be catalyzed at the C terminus in a stepwise manner. Although RimK showed strict substrate specificity toward Glu, it also used, to a small extent, other amino acids as C-terminal substrates and synthesized heteropeptides. In addition, RimK-catalyzed modification of ribosomal protein S6 was confirmed. The number of Glu residues added to the protein varied with pH and was largest at pH 9.5.Poly-L-amino acids possess biodegradable properties and are therefore useful in various fields, including food science, medicine, and cosmetics. Polyaspartic acid is used as a biodegradable substitute for synthetic polyacrylate (25), and poly-␣-glutamic acid finds application in a wide variety of surgical and pharmaceutical products (e.g., for enhancement of solubility and control of half-life of drugs) (27). Furthermore, L-arginine (Arg)-rich peptides, which can permeate cell membranes, are used for intracellular delivery of macromolecules (7). In addition, unique polyamino acids produced by various microorganisms, including poly-␥-glutamic acid, poly-ε-lysine, and cyanophycin (multi-L-arginyl-poly[L-aspartic acid]), have been well studied and widely applied (6,21,26). These acids have a ␥-, ε-, or ␤-amide linkage. Both poly-␥-glutamic acid and poly-ε-lysine have characteristic abilities of high water absorbency and antimicrobial activity, respectively, and are therefore used on a commercial scale. Previous studies have revealed the biosynthetic mechanisms of these polyamino acids and have achieved their mass production (5, 33). In contrast, poly-L-amino acids with only an ␣-amide linkage have not been found in microorganisms, probably because poly-L-␣-amino acids might be hydrolyzed by proteases and peptidases contained in the microorganisms. Therefore, poly-L-␣-amino acids may not be detectable even in organisms containing an enzyme possessing poly-L-␣-amino acid-synthesizing activity.To create a supply of useful poly-␣-amino acids, their potential production by chemical and en...

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Section: Discussionmentioning

confidence: 99%

Poly-α-Glutamic Acid Synthesis Using a Novel Catalytic Activity of RimK from Escherichia coli K-12

Kino

Arai

Arimura

2011

Appl Environ Microbiol

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“…Bacillus licheniformis is a saprophytic bacterium that is widespread in Nature. It has been applied widely to produce proteases [ 1 ], amylases [ 2 ], lactamase [ 3 ], antibiotics [ 4 ], surfactin [ 5 ] and special chemicals [ 6 ] in the fermentation industry with low risk of adverse effects to human health or the environment. Bacitracin, the first peptide antibiotic derived from cultures of B. licheniformis , has been applied widely in the medical and veterinary area, and several other peptide antibiotics from different B. licheniformis strains have been studied [ 7 ].…”

Section: Introductionmentioning

confidence: 99%

Isolation and Partial Characterization of an Antifungal Protein Produced by Bacillus licheniformis BS-3

2012

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An antifungal protein produced by Bacillus licheniformis strain BS-3 was purified to homogeneity by ammonium sulfate precipitation, DEAE-52 column chromatography and Sephadex G-75 column chromatography. The purified protein was designated as F2 protein, inhibited the growth of Aspergillus niger , Magnaporthe oryzae and Rhizoctonia solani . F2 protein was a monomer with approximately molecular weight of 31 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gave a single peak on High Performance Liquid Chromatography (HPLC). Using Rhizoctonia solani as the indicator strain, the EC 50 of F2 protein was 35.82 µg/mL, displaying a higher antifungal activity in a range of pH 6.0 to pH 10.0, and at a temperature below 70 °C for 30 min. F2 protein was moderately resistant to hydrolysis by trypsin, proteinase K, after which its relative activities were 41.7% and 59.5%, respectively. F2 protein was assayed using various substrates to determine the enzymatic activities, the results showed the hydrolyzing activity on casein, however, no enzymatic activities on colloidal chitin, CM-cellulose, xylan, M. lysodeikticus , and p -nitrophenyl- N -acetylglucosaminide.

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“…Cultures were incubated at 30 ºC, 250 rpm, for 24 h. B. subtilis strains were also grown in similar conditions, except without the addition of IPTG and antibiotic. The PGA produced in the supernatant was either measured directly or purified by a modified version of the copper precipitation method described by (Manocha and Margaritis, 2010;Yuan et al, 2019). In brief, 0.5 M CuSO 4 was added to the supernatant and the solution was mixed by inversion and let stand for 1 h at room temperature.…”

Section: Pga Productionmentioning

confidence: 99%

Characterization of a membrane enzymatic complex for heterologous production of poly-γ-glutamate inE. coli

Nascimento

Nair

2020

Preprint

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Poly-γ-glutamic acid (PGA) produced by many Bacillus species is a polymer with many distinct and desirable characteristics. However, the multi-subunit enzymatic complex responsible for its synthesis, PGA Synthetase (PGS), has not been well characterized yet, in native nor in recombinant contexts. Elucidating structural and functional properties are crucial for future engineering efforts aimed at altering the catalytic properties of this enzyme. This study focuses on expressing the enzyme heterologously in the Escherichia coli membrane and characterizing localization, orientation, and activity of this heterooligomeric enzyme complex. In E. coli, we were able to produce high molecular weight PGA polymers with minimal degradation at titers of approximately 13 mg/L in deep-well microtiter batch cultures. Using fusion proteins, we observed, for the first time, the association and orientation of the different subunits with the inner cell membrane. These results elucidate provide fundamental structural information on this poorly studied enzyme complex and will aid future fundamental studies and engineering efforts.

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A novel Method for the selective recovery and purification of γ‐polyglutamic acid from Bacillus licheniformis fermentation broth

Cited by 39 publications

References 35 publications

Poly-α-Glutamic Acid Synthesis Using a Novel Catalytic Activity of RimK from Escherichia coli K-12

Poly-α-Glutamic Acid Synthesis Using a Novel Catalytic Activity of RimK from Escherichia coli K-12

Isolation and Partial Characterization of an Antifungal Protein Produced by Bacillus licheniformis BS-3

Characterization of a membrane enzymatic complex for heterologous production of poly-γ-glutamate inE. coli

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