https://pubs.rsc.org/en/content/articlepdf/2017/gc/c6gc02328b

Transaminase biocatalysis: optimization and application

Methods for the fermentative production of four carbon alcohols is provided. Specifically, butanol, preferably isobutanol is produced by the fermentative growth of a recombinant bacterium expressing an isobutanol biosynthetic pathway.

Fermentive Production of Four Carbon Alcohols

Bioinformatic analysis of a PLP-dependent enzyme superfamily suitable for biocatalytic applications

Chiral amines in enantiopure forms are important chemical building blocks, which are most well recognized in the pharmaceutical industries for imparting desirable biological activity to chemical entities. A number of synthetic strategies to produce chiral amines via biocatalytic as well as chemical transformation have been developed. Recently, ω-transaminase (ω-TA) has attracted growing attention as a promising catalyst which provides an environment-friendly access to production of chiral amines with exquisite stereoselectivity and excellent catalytic turnover. To obtain enantiopure amines using ω-TAs, either kinetic resolution of racemic amines or asymmetric amination of achiral ketones is employed. The latter is usually preferred because of twofold higher yield and no requirement of conversion of a ketone product back to racemic amine. However, the choice of a production process depends on several factors such as reaction equilibrium, substrate reactivity, enzyme inhibition, and commercial availability of substrates. This review summarizes the biochemical features of ω-TA, including reaction chemistry, substrate specificity, and active site structure, and then introduces recent advances in expanding the scope of ω-TA reaction by protein engineering and public database searching. We also address crucial factors to be considered for the development of efficient ω-TA processes.

Features and technical applications of ω-transaminases.

Four (R)-ω-transaminases originating from Hyphomonas neptunium (HN-ωTA), Aspergillus terreus (AT-ωTA) and Arthrobacter sp. (ArR-ωTA), as well as an evolved transaminase (ArRmut11-ωTA) were successfully employed for the amination of prochiral ketones leading to optically pure (R)-amines. The first three transaminases displayed perfect stereoselectivity for the amination of all substrates tested (ee >99%). Furthermore, the transaminase AT-ωTA led in most cases to better conversion than ArR-ωTA and HN-ωTA using D-alanine as amine donor. α-Tetralone, which was the only substrate not accepted by HN-ωTA, ArR-ωTA, and AT-ωTA, was successfully transformed with perfect enantioselectivity (ee >99%) into the corresponding optically pure amine employing the variant ArRmut11-ωTA.

Stereoselectivity of Four (R)-Selective Transaminases for the Asymmetric Amination of Ketones

A transaminase from Vibrio fluvialis JS17 showing activity toward chiral amines was purified to homogeneity and its enzymatic properties were characterized. The transaminase showed an apparent molecular mass of 100 kDa as determined by gel filtration chromatography and a subunit mass of 50 kDa by MALDI-TOF mass spectrometry, suggesting a dimeric structure. The enzyme had an isoelectric point of 5.4 and its absorption spectrum exhibited maxima at 320 and 405 nm. The optimal pH and temperature for enzyme activity were 9.2 and 37°C, respectively. Pyruvate and pyridoxal 5′-phosphate increased enzyme stability whereas (S)-α-methylbenzylamine reversibly inactivated the enzyme. The transaminase gene was cloned from a V. fluvialis JS17 genomic library. The deduced amino acid sequence (453 residues) showed significant homology with ω-amino acid:pyruvate transaminases (ω-APT) from various bacterial strains (80 identical residues with four ω-APTs). However, of 159 conserved residues in the four ω-APTs, 79 were not conserved in the transaminase from V. fluvialis JS17. Taken together with the sequence homology results, and the lack of activity toward β-alanine (a typical amino donor for the ω-APT), the results suggest that the transaminase is a novel amine:pyruvate transaminase that has not been reported to date.

Purification, characterization, and molecular cloning of a novel amine:pyruvate transaminase from Vibrio fluvialis JS17

Development and characterization of membrane surface display system using molecular chaperon, prsA, of Bacillus subtilis.

In this paper, the possibility of using a mutation of ftsZ as a pseudo-spore mutant is investigated. ftsZ, which is essential for cell division and sporulation of Bacillus subtilis, was placed under the spac promoter, which is inducible with isopropyl thiogalactose (IPTG). Cell growth of the ftsZ mutant and its β-galactosidase activity under the aprE promoter were compared with the wild type. In the presence of 1 mM IPTG, cell growth of the ftsZ mutant was almost the same as that of the wild type and its sporulation frequency was slightly lower than that of the wild type. However, under uninduced conditions, cell growth of ftsZ mutant was severely impaired. When induced with 0.2 mM IPTG, the ftsZ mutant showed about 13 times higher β-galactosidase activity than the wild type. When the ftsZ mutant was used for secretory production of subtilisin, only three times higher extracellular subtilisin activity was measured, compared with the wild type. By real-time PCR investigation, it was revealed that the ftsZ mutant intracellular mRNA level for subtilisin was more than 16 times higher, compared with the wild type. However, it appears that the secretion pathway is somewhat damaged in the ftsZ mutant. These results suggest that the cell division mutant can also be used like a sporulation mutant to produce recombinant proteins, with a precise control of cell growth and induction.

The effects of ftsZ mutation on the production of recombinant protein in Bacillus subtilis.

The genome of Bacillus subtilis contains three different genes encoding RNase H homologs: RNases HI, HII and HIII. RNase HIII from B. subtilis degrades RNA in RNA-DNA hybrids in an Mg(2+)-dependent manner like Escherichia coli RNase HI. However, they belong to different classes; the former belongs to the 'class II' or 'large' RNase H family, while the latter belongs to the 'class I' or 'small' RNase H family. RNase HIII of B. subtilis has been overexpressed in E. coli and crystallized at 296 K using sodium formate as a precipitant. The native X-ray diffraction data have been collected to 2.8 A resolution using synchrotron radiation. The crystals are hexagonal, belonging to the space group P6(1), with unit-cell parameters a = b = 86.89, c = 214.49 A, alpha = beta = 90.0, gamma = 120.0 degrees. A self-rotation function calculation indicated the presence of two monomers of the recombinant RNase HIII in the crystallographic asymmetric unit, giving a V(M) of 3.43 A(3) Da(-1) and a solvent content of 64.2%.

Crystallization and preliminary X-ray crystallographic analysis of RNase HIII from Bacillus subtilis.

A Gram-stain-negative, facultatively anaerobic, flagellated and coccoid, ovoid or rod-shaped bacterial strain, NIFS-20-8T, was isolated from the intestine of an olive flounder (Paralichthys olivaceus) from the East Sea, Republic of Korea. The neighbour-joining phylogenetic tree of 16S rRNA gene sequences showed that strain NIFS-20-8T fell within the clade comprising the type strains of Enterovibrio species. Strain NIFS-20-8T exhibited 16S rRNA gene sequence similarities of 97.2 and 97.1 % to the type strains of Enterovibrio nigricans and Enterovibrio norvegicus, respectively, and of 96.6-97.0 % to the type strains of the other Enterovibrio species. The average nucleotide identity and digital DNA-DNA hybridization values between the genomic sequence of strain NIFS-20-8T and those of the type strains of four Enterovibrio species were 73.8-75.0 and 19.8-21.1 %, respectively. The DNA G+C content of strain NIFS-20-8T from genomic sequence data was 50.55 mol%. Strain NIFS-20-8T contained Q-8 as the predominant ubiquinone and summed feature 3 (C16 : 1  ω7c and/or C16 : 1  ω6c), C16 : 0 and C18 : 1  ω7c as the major fatty acids. The major polar lipids detected in stain NIFS-20-8T were phosphatidylethanolamine and phosphatidylglycerol. Distinguishing phenotypic properties, together with phylogenetic and genetic distinctiveness, revealed that strain NIFS-20-8T is separated from recognized Enterovibrio species. On the basis of the data presented here, strain NIFS-20-8T is considered to represent a novel species of the genus Enterovibrio, for which the name Enterovibrio paralichthyis sp. nov. is proposed. The type strain is NIFS-20-8T (= KCTC 82873T=NBRC 115237T).

Enterovibrio paralichthyis sp. nov., isolated from the gut of an olive flounder Paralichthys olivaceus.

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