Characterization of d-succinylase from Cupriavidus sp. P4-10-C and its application in d-amino acid synthesis.

d-Amino acids are important building blocks for various compounds, such as pharmaceuticals and agrochemicals. A more cost-effective enzymatic method for d-amino acid production is needed in the industry. We improved a one-pot enzymatic method for d-amino acid production by the dynamic kinetic resolution of N-succinyl amino acids using two enzymes: d-succinylase (DSA) from Cupriavidus sp. P4-10-C, which hydrolyzes N-succinyl-d-amino acids enantioselectively to their corresponding d-amino acid, and N-succinyl amino acid racemase (NSAR, EC.4.2.1.113) from Geobacillus stearothermophilus NCA1503. In this study, DSA and NSAR were purified and their properties were investigated. The optimum temperature of DSA was 50°C and it was stable up to 55°C. The optimum pH of DSA and NSAR was around 7.5. In d-phenylalanine production, the optical purity of product was improved to 91.6% ee from the examination about enzyme concentration. Moreover, 100 mM N-succinyl-dl-tryptophan was converted to d-tryptophan at 81.8% yield with 94.7% ee. This enzymatic method could be useful for the industrial production of various d-amino acids.

Expression analyses of casein kinase 2alpha and casein kinase 2beta in the silkmoth, Bombyx mori.

A period-timeless (per-tim) based feedback loop is considered to be essential in generating circadian rhythms in Drosophila melanogaster. In addition to transcriptional regulation, the post-transcriptional modification is essential to the circadian oscillation of core clock proteins in the circadian system. Here we present expression profiles of the catalytic subunit of casein kinase 2alpha (ck2alpha) and casein kinase 2beta (ck2beta) in Bombyx mori. Southern blot analyses showed that ck2alpha and ck2beta of B. mori were single copy genes. Northern blot analyses demonstrated that both subunits were expressed in eggs, larval heads, adult heads, testes and ovaries. In situ hybridization analyses indicated that subunits were expressed in brain neurons expressing PER-like protein. Surprisingly, antisense RNAs of ck2alpha and ck2beta were also detected in the putative clock neurons. Temporal expressions of ck2alpha and ck2beta mRNAs were constant in adult heads under LD12:12. The core clock genes per and tim showed daily fluctuations of mRNA abundance in the embryonic stage that is photoperiod sensitive period to determine egg diapause in the next generation whereas the expression of ck2alpha and ck2beta was constant. No evidence supports that ck2alpha and ck2beta of B. mori were transcriptionally regulated by circadian oscillation, but histological data show a close association of ck2alpha and ck2beta with circadian system in B. mori.

Cloning and characterization of insect arylalkylamine N-acetyltransferase from Bombyx mori.

Arylalkylamine N-acetyltransferase (AANAT) catalyzes N-acetylation of arylarkylamines. A cDNA of Bombyx mori insect AANAT (Bm-iAANAT) was found by searching an expressed-sequence tag (EST) database of B. mori (SilkBase). The cDNA encoded a 261 amino acid protein. The mRNA of Bm-iAANAT was expressed in eggs, larvae, adults and various tissues. Recombinant Bm-iAANAT protein was expressed in Sf9 cells by a baculovirus expression system. The AANAT activity of Bm-iAANAT was inhibited by high concentrations (over 0.01 mM) of tryptamine used as a substrate. The Bm-iAANAT acetylated tryptamine, serotonin, dopamine, octopamine, tyramine and norepinephrine. This is the first report of a cloned AANAT that acetylated norepinephrine. These results suggest that Bm-iAANAT is a novel member of insect AANAT family with unique kinetic properties and a broad substrate range.

Expression analysis of two types of transcripts from circadian output gene lark in Bombyx mori.

We analyzed expression patterns of lark (Bmlark) in Bombyx mori. Southern blot analysis demonstrated that Bmlark was a single copy gene. Northern blot analyses revealed two types of Bmlark transcripts, one being of 1.38 kb (Bmlark-PA) and the other of 0.85 kb (Bmlark-PB). Both transcripts were detected in the eggs, larval and adult heads, testes, ovaries and flight muscles. Both types of transcripts are constitutively expressed with no clear rhythmicity in the adult heads under light:dark (LD) cycles but the amount of the Bmlark-PA transcript was twice as much as that of the Bmlark-PB transcript in the adult heads throughout a day. Real-Time PCR assays also indicated constant expressions of the two types of Bmlark in the pupal brains under LD12:12 and LD16:8.

Casein kinases I of the silkworm, Bombyx mori: their possible roles in circadian timing and developmental determination.

Doubletime (DBT), a homolog of casein kinase Iepsilon (CKIepsilon), is an essential circadian clock component and developmental regulator in Drosophila melanogaster. The authors cloned a dbt homolog from the silkworm, Bombyx mori(Bmdbt), and examined its spatial and temporal expression in comparison to a CKI[alpha] homolog (BmCKIalpha). Four Bmdbt splice variants and 2 BmCKIalpha splice variants were detected, and their expression patterns varied in different tissues. The level of Bmdbt transcript in the brain was constant under LD 12:12 while those of BmCKIalpha transcripts fluctuated with a decrease at ZT12. In situ hybridization showed presumably identical distribution of dbt, CKIalpha, and per transcripts in the putative clock neurons of the head ganglia, as well as in the retina, where CKI-and PER-like immunoreactivities were colocalized, suggesting a possible involvement of both CKIs in the B. mori circadian system. Signals were detected at 4 Ia(1) neurons in each dorsolateral protocerebrum, 6 to 8 cells in the pars intercerebralis, about 6 cells in the suboesophageal ganglion, 2 neurons in the frontal ganglion, and most of the photoreceptors. All these cells contained dbt, CKIalpha, and per antisense transcripts. The Northern analysis of dbtand CKIalpha transcripts at different developmental stages showed that both genes were expressed at relatively high levels during early embryogenesis and in the ovary. The levels of CKIalpha transcripts were also high in the late larval stages until the mid-fifth instar and then suddenly disappeared before larval-pupal ecdysis. In contrast, the transcriptional activity of both genes was low in diapausing eggs.

MbIDGF, a novel member of the imaginal disc growth factor family in Mamestra brassicae, stimulates cell proliferation in two lepidopteran cell lines without insulin.

Imaginal disc growth factor (IDGF) is a soluble polypeptide growth factor that was first identified from the conditioned medium of Drosophilia imaginal disc C1.8+ cells. Working with insulin, IDGF stimulated the growth of cultured imaginal disk cells, which suggested that IDGF might function as a cofactor of Drosophila insulin or insulin like peptide. Here we report a new member of the IDGF family, named MbIDGF, from the cabbage armyworm, Mamestra brassicae. Using a cloned cDNA of MbIDGF, recombinant MbIDGF protein was expressed in baculovirus-infected Sf9 cells and purified. Without insulin, the recombinant MbIDGF protein stimulated cell growth of SES-MaBr-4 and NIAS-MaBr-93 cell lines that were derived from the fat bodies and hemocytes of M. brassicae, in a dose-dependent manner. The saturation of growth stimulation by MbIDGF was attained for the two types of cells at 80 ng/ml (0.8 nM) and 300 ng/ml (6 nM), respectively. The results suggest that MbIDGF may stimulate the growth of lepidopteran cells by a new mechanism without associating with the insulin pathway.

Structure and expressions of two circadian clock genes, period and timeless in the commercial silkmoth, Bombyx mori.

We cloned two circadian clock genes period (Bmper) and timeless (Bmtim) from the commercial silkmoth, Bombyx mori. Sequence analysis revealed a high degree of conservation among insects for both genes. BmPER predicted from the DNA sequence is a polypeptide of 1, 113 amino acids with functional domains such as PAS, PAC, nuclear localization signal (NLS) and cytoplasmic localization domain (CLD). Deduced BmTIM consists of 997 amino acids with PER interaction site (PIS) as well as NLS and CLD. Southern blot analyses revealed that Bmper and Bmtim are single copy genes. Northern blot analysis demonstrated that Bmper and Bmtim are expressed both in the head and peripheral tissues. We also examined temporal profiles of Bmper and Bmtim expressions in the head, flight muscle, testis and antenna of adult males under LD12:12 and LD16:8 by Real-Time PCR assays. Our data show that photoperiod differentially affects the temporal expression patterns of Bmper and Bmtim. The mRNA expression of Bmper and Bmtim in the head had a phase lead under LD12:12 compared to that under LD16:8, whereas photoperiod did not affect expression patterns in peripheral tissues relative to light-on. Photoperiod affected not only the phase relationship but also the expression level. In the testis and antenna, the level of transcription of Bmtim was low in LD12:12 but high in LD16:8. The daily differences in amplitudes of the Bmper and Bmtim expression rhythms were 2-fold in the head and 1.5-2.5 folds in the peripheral tissues examined.