Takashi Utagawa

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

ABSTRACT ε-Poly-l-lysine (ε-PL) is produced by Streptomyces albulus NBRC14147 as a secondary metabolite and can be detected only when the fermentation broth has an acidic pH during the stationary growth phase. Since strain NBRC14147 produces ε-PL-degrading enzymes, the original chain length of the ε-PL polymer product synthesized by ε-PL synthetase (Pls) is unclear. Here, we report on the identification of the gene encoding the ε-PL-degrading enzyme (PldII), which plays a central role in ε-PL degradation in this strain. A knockout mutant of the pldII gene was found to produce an ε-PL of unchanged polymer chain length, demonstrating that the length is not determined by ε-PL-degrading enzymes but rather by Pls itself and that the 25 to 35 l-lysine residues of ε-PL represent the original chain length of the polymer product synthesized by Pls in vivo. Transcriptional analysis of pls and a kinetic study of Pls further suggested that the Pls catalytic function is regulated by intracellular ATP, high levels of which are required for full enzymatic activity. Furthermore, it was found that acidic pH conditions during ε-PL fermentation, rather than the inhibition of the ε-PL-degrading enzyme, are necessary for the accumulation of intracellular ATP.

Phosphorylation of Nucleosides by the Mutated Acid Phosphatase from Morganella morganii

ABSTRACT A novel nucleoside phosphorylation process using the food additive pyrophosphate as the phosphate source was investigated. TheMorganella morganii gene encoding a selective nucleoside pyrophosphate phosphotransferase was cloned. It was identical to theM. morganii PhoC acid phosphatase gene. Sequential in vitro random mutagenesis was performed on the gene by error-prone PCR to construct a mutant library. The mutant library was introduced intoEscherichia coli, and the transformants were screened for the production of 5′-IMP. One mutated acid phosphatase with an increased phosphotransferase reaction yield was obtained. With E. coli overproducing the mutated acid phosphatase, 101 g of 5′-IMP per liter (192 mM) was synthesized from inosine in an 88% molar yield. This improvement was achieved with two mutations, Gly to Asp at position 92 and Ile to Thr at position 171. A decreasedKm value for inosine was responsible for the increased productivity.

Production of Arginine by Fermentation

Studies on the production of L-arginine by fermentation using mutants of Corynebacterium (Brevibacterium), Bacillus, and Serratia have been conducted since the 1960s. More recently, the breeding of L-arginine production strains by gene recombination techniques using Escherichia coli has been investigated. To produce L-arginine efficiently by fermentation, it is necessary to breed strains with a strong biosynthetic pathway to L-arginine. Because L-arginine is biosynthesized from the precursor L-glutamic acid through ornithine and citrulline, the use of strains with a high capability for producing L-glutamic acid is desirable. Corynebacterium (Brevibacterium), which is well known in the production of L-glutamic acid, was selected as a starting strain for the breeding of an L-arginine producer and has been used on a commercial scale. Regarding the fermentation conditions, as for other amino acids, L-arginine fermentation is controlled by regulating pH near the neutral point. Due to its high oxygen requirement, L-arginine production is seriously impaired without sufficient oxygen. Advanced purification methods are necessary to obtain highly pure L-arginine from the fermentation broth. After fermentation is complete, bacterial cells and proteins are removed by means of a membrane or centrifugation, and impurities are removed by means of an ion-exchange resin or activated carbon. Highly pure L-arginine crystals can be obtained through concentration at the end of the process.

Production of adenine arabinoside by gel-entrapped cells of Enterobacter aerogenes in water-organic cosolvent system

SummaryGel-entrapped whole cells of Enterobacter aerogenes, which has a transglycosylation activity, were used to produce adenine arabinoside from uracil arabinoside and adenine, in an appropriate water-organic cosolvent system. Cells of E. aerogenes entrapped with a hydrophilic photo-crosslinkable resin prepolymer, ENT-4000, or a urethane prepolymer, PU-6, had a high and stable transglycosylation activity. To improve the poor solubility in water of the substrate (adenine) and product (adenine arabinoside), dimethyl sulfoxide was selected as the cosolvent based on the criteria of operational stability of the immobilized biocatalyst and solubility of both substrate and product. Addition of 40% dimethyl sulfoxide to the reaction mixture permitted use of a high substrate concentration range which gave high productivity under homogeneous reaction conditions. The immobilized cells of E. aerogenes exhibited a markedly improved operational stability, retaining their initial level of activity during repeated use for at least 35 days at 60°C in 40% dimethyl sulfoxide. When the reaction was carried out with 150 mM uracil arabinoside and 50 mM adenine as the substrates, the yield of adenine arabinoside was maintained at 100% based on the molar ratio of adenine, throughout the reaction.

A new method for the synthesis of some 9-β-D-arabinofuranosylpurines by a combination of chemical and enzymatic reactions

Abstract An enzymatic transarabinosylation between 2-chlorohypoxanthine and 1-β- D -arabinofuranosyluracil gave 9-β- D -arabinofuranosyl-2-chlorohypoxanthine which was chemically converted to 9-β- D -arabinofuranosylguanine and its derivatives.

Characterization of the cell surface protein gene of Corynebacterium ammoniagenes.

Three dominant cell surface proteins of Corynebacterium ammoniagenes ATCC 6872 were identified in the cell wall fraction. The cspA gene, which encodes one of the major cell surface proteins, was cloned using the N-terminal amino acid sequence of the protein. Then the cloned chromosomal fragment containing the cspA gene was sequenced and was shown to encode a mature polypeptide of 333 amino acids with a molecular mass of 36654 Da. The amino acid sequence of the cspA gene showed similarity to the amino acid sequence of C. glutamicum CspA, one of the two major secreted proteins of C. glutamicum, although C. ammoniagenes CspA and C. glutamicum CspA differed in size. Northern blot analysis and primer extension analysis respectively revealed a 1.1 kb transcript and a promoter sequence resembling that of the C. ammoniagenes fatty acid synthase B (fasB) gene.

Screening of bacteria at high temperatures to select nucleoside antibiotics producers

Publisher Summary This chapter describes screening of bacteria at high temperatures to select nucleoside antibiotics producers. Many nucleoside analogs have been found occurring naturally or have been synthesized chemically. The most commonly used methods for the chemical synthesis of nucleoside analogs include the sugar-based coupling reaction and modification of naturally occurring nucleosides. For the synthesis of stereo-specific nucleosides, microbial enzymes have been applied as a bio-catalyst in the chemical process. Adenine arabinoside is one of anitiviral agents that have been used as an anti-herpes agent. This compound is synthesized by chemical method and is later found in the culture filtrate of Streptomyces antibioticus. The enzyme substrate uracil arabinoside (ara-U) can be prepared from uridine through reaction with ethylene carbonate followed by the hydrolysis of cyclo-uridine (cyclo-U) by acid. Microorganisms from typeculture and natural resources are incubated at 30°C for 24 hours. The reaction temperature is considered very important and a key to the synthesis of ara-A.