Mechanism of ε-Poly-
            <scp>l</scp>
            -Lysine Production and Accumulation Revealed by Identification and Analysis of an ε-Poly-
            <scp>l</scp>
            -Lysine-Degrading Enzyme

Yamanaka, Kazuya; Kito, Naoko; Imokawa, Yuuki; Maruyama, Chitose; Utagawa, Takashi; Hamano, Yoshimitsu

doi:10.1128/aem.00853-10

Cited by 104 publications

(96 citation statements)

References 21 publications

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“…In fact, we recently reported that a mutant with a knockout of the pld II gene, which encodes an ε-PL-degrading enzyme that plays a central role in ε-PL degradation in S. albulus NBRC14147, was found to produce an ε-PL of unchanged polymer chain length (25-to 35-mer) (18). This finding revealed that in vivo, the length is determined not by ε-PLdegrading enzymes but rather by Pls itself and that the 25-to 35-mer is an original-chain-length polymer product that Pls synthesizes.…”

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confidence: 99%

ε-Poly- l -Lysine Peptide Chain Length Regulated by the Linkers Connecting the Transmembrane Domains of ε-Poly- l -Lysine Synthetase

Hamano

Kito

Kita

et al. 2014

Appl Environ Microbiol

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b-Poly-L-lysine (-PL), consisting of 25 to 35 L-lysine residues with linkages between the ␣-carboxyl groups and -amino groups, is produced by Streptomyces albulus NBRC14147. -PL synthetase (Pls) is a membrane protein with six transmembrane domains (TM1 to TM6) as well as both an adenylation domain and a thiolation domain, characteristic of the nonribosomal peptide synthetases. Pls directly generates -PL chain length diversity (25-to 35-mer), but the processes that control the chain length of -PL during the polymerization reaction are still not fully understood. Here, we report on the identification of Pls amino acid residues involved in the regulation of the -PL chain length. From approximately 12,000 variants generated by random mutagenesis, we found 8 Pls variants that produced shorter chains of -PL. These variants have one or more mutations in two linker regions connecting the TM1 and TM2 domains and the TM3 and TM4 domains. In the Pls catalytic mechanism, the growing chain of -PL is not tethered to the enzyme, implying that the enzyme must hold the growing chain until the polymerization reaction is complete. Our findings reveal that the linker regions are important contributors to grasp the growing chain of -PL.

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confidence: 99%

ε-Poly- l -Lysine Peptide Chain Length Regulated by the Linkers Connecting the Transmembrane Domains of ε-Poly- l -Lysine Synthetase

Hamano

Kito

Kita

et al. 2014

Appl Environ Microbiol

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“…In the medium, S. albulus CR1 (wild type strain) was cultivated for 3 days at 30 C in the same manner as that described previously. 7 …”

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confidence: 99%

“…5 Also, the synthetic enzyme, εPL-synthetase, was identified, and the biosynthetic mechanism has been elucidated. 6,7 Thus, screening the synthetic enzyme to produce higher-or lower-molecular-weight εPL species as well as a more efficient one has become an important subject for further applications.…”

Section: Introductionmentioning

confidence: 99%

Separation and Purification of ε-Poly-L-lysine from the Culture Broth Based on Precipitation with the Tetraphenylborate Anion

et al. 2012

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“…5,6 Thus, the applied study is being developed. Recently, the biosynthetic mechanism has been elucidated, 7,8 and the screening of the synthetic enzyme to produce higher-or lower-molecular-weight εPL species as well as the more efficient one has become an important subject for further applications.…”

Section: Introductionmentioning

confidence: 99%

Separation and Purification of ε-Poly-l-lysine with Its Colorimetric Determination Using Dipicrylamine

et al. 2015

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A separation method for a biopolymer, ε-poly-L-lysine (εPL), with its colorimetric determination using a yellow anionic dye, dipicrylamine anion (DPA -), is presented. The εPL-producing culture broth was mixed with a NaDPA solution to precipitate the εPL in polycationic form with the DPA -anion. The precipitate was dissolved into acetonitrile (AN). The AN solution was yellowish, and gave an absorption maximum at around 420 nm. Thus, εPL in the culture broth can be assayed colorimetrically a with multiwell microtiter plate. By the addition of bis(triphenylphosphoranylidene)ammonium chloride, the polycationic εPL was precipitated from the AN solution as the hydrochloride salt. On the other hand, the DPA -anion remained in the AN solution as the quaternary ammonium salt. Thus, εPL larger than tetramer can be separated and purified after the high-throughput screening of the synthetic enzyme.Keywords Separation, colorimetry, ε-poly-L-lysine, dipicrylamine, high-throughput screening

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Mechanism of ε-Poly- l -Lysine Production and Accumulation Revealed by Identification and Analysis of an ε-Poly- l -Lysine-Degrading Enzyme

Cited by 104 publications

References 21 publications

ε-Poly- l -Lysine Peptide Chain Length Regulated by the Linkers Connecting the Transmembrane Domains of ε-Poly- l -Lysine Synthetase

ε-Poly- l -Lysine Peptide Chain Length Regulated by the Linkers Connecting the Transmembrane Domains of ε-Poly- l -Lysine Synthetase

Separation and Purification of ε-Poly-L-lysine from the Culture Broth Based on Precipitation with the Tetraphenylborate Anion

Separation and Purification of ε-Poly-l-lysine with Its Colorimetric Determination Using Dipicrylamine

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