The industrial-scale production of phenylalanine ammonia-lyase (PAL) mainly uses strains of Rhodotorula. However, the PAL gene from Rhodotorula has not been cloned. Here, the full-length gene of PAL from Rhodotorula glutinis was isolated. It was 2,121 bp, encoding a polypeptide with 706 amino acids and a calculated MW of 75.5 kDa. Though R. glutinis is an anamorph of Rhodosporium toruloides, the amino acid sequences of PALs them are not the same (about 74 % identity). PAL was expressed in E. coli and characterized. Its specific activity was 4.2 U mg(-1) and the k cat/K m was 1.9 × 10(4) mM(-1) s(-1), exhibiting the highest catalytic ability among the reported PALs. The genetic and biochemical information reported here should facilitate future application in industry.

BackgroundActivators of Nitrile hydratase (NHase) are essential for functional NHase biosynthesis. However, the activator P14K in P. putida is difficult to heterogeneously express, which retards the clarification of the mechanism of P14K involved in the maturation of NHase. Although a strep tag containing P14K (strep-P14K) was over-expressed, its low expression level and low stability affect the further analysis.ResultsWe successfully expressed P14K through genetic modifications according to N-end rule and analyzed the mechanism for its difficult expression. We found that mutation of the second N-terminal amino-acid of the protein from lysine to alanine or truncating the N-terminal 16 amino-acid sequence resulted in successful expression of P14K. Moreover, fusion of a pelB leader and strep tag together (pelB-strep-P14K) at the N-terminus increased P14K expression. In addition, the pelB-strep-P14K was more stable than the strep-P14K.ConclusionsOur results are not only useful for clarification of the role of P14K involved in the NHase maturation, but also helpful for heterologous expression of other difficult expression proteins.

We have elucidated the significance of three key amino acid residues of L-aspartate α-decarboxylase that act remotely from its cleavage site for its functional self-cleavage as well as for its catalytic activity. These results provide useful fundamental information for engineering L-aspartate α-decarboxylase. L-Aspartate α-decarboxylase (ADC) from Corynebacterium glutamicum, and encoded by panD, was cloned and expressed in Escherichia coli and then purified. Three amino acid residues were found to be related to ADC self-cleavage. Mutating R3 to either A, Q, N, L, D, or E produced only the unprocessed pro-enzyme. Although mutating R54 and Y58 into A or K and A or T, respectively, partly influenced ADC self-cleavage, the specific activity of each of the four ßmutants decreased to 3.5, 4, 2.4, and 2.6 U mg(-1), respectively, compared with a specific activity of 690 U mg(-1) for the wild-type enzyme. Thus, R3 triggers ADC self-cleavage and completes the modification of the active site with assistance by R54 and Y58. These results will help to engineer ADC for improved industrial applications.

Two TGases derived from the same zymogen from S. hygroscopicus were discovered. These two active forms of TGase may be due to different activation processes: one of them is catalysed by its own active TGase, while the other is activated by an exogenous protease.

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