Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B.

In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.     

Relationship between emulsifying activity and carbohydrate backbone structure of emulsan from Acinetobacter calcoaceticus RAG-1

Various emulsan samples with the different degrees of branching of the carbohydrate backbone were obtained from Acinetobacter calcoaceticus under different culture conditions. The emulsifying activity of emulsan had a linear correlation to the branching degrees of the carbohydrate backbone (r²= 0.930) suggesting that the structure of carbohydrate backbone was an important factor influencing emulsifying activity. This revised version was published online in November 2006 with corrections to the Cover Date.     

Effect of soybean oil and glucose on sophorose lipid fermentation by Torulopsis bombicola in continuous culture

The effect of soybean oil and glucose on the growth of Torulopsis bombicola and sophorose lipid production in continuous culture was investigated. As the dilution rate in 100 g/l glucose and 100 g/l soybean oil medium was increased, the dry cell weight and sophorose lipid concentration decreased. Sophorose lipid productivity, however, was maximum at a dilution rate of 0.03 h⁻¹. The cell yield from glucose and the sophorose lipid production from soybean oil were approximately constant regardless of the dilution rate. The specific consumption rate of soybean oil was closely related to the specific production rate of sophorose lipid. These results suggest that soybean oil was used only for sophorose lipid production whereas glucose was used only for cell mass and maintenance. When the soybean oil concentration was varied at fixed dilution rate in 100 g/l glucose medium, a high concentration of soybean oil was found to inhibit sophorose lipid production. Received: 9 January 1997 / Received revision: 5 March 1997 / Accepted: 13 April 1997     

Production of a high viscosity polysaccharide, methylan, in a novel bioreactor

The effect of shear stress on the production of a high viscosity polysaccharide, methylan, from methanol by Methylobacterium organophilum was investigated by using a multidisk mixer. It was observed in the multidisk mixer with defined shear stresses that the specific production rate of methylan increased gradually with increasing shear stress up to 30 Pa, and the production rate was constant beyond 30 Pa. This result suggested that the limited mass transfer from the medium into cells reduced methylan production. A novel bioreactor that provided the large volume of a high shear region was used to increase methylan production. Fed-batch cultures in the novel bioreactor were performed by the dissolved oxygen-stat method of methanol. When 1.13 g/L ammonium ion was added, the concentrations of cells of methylan were 31 and 20.6 g/L, respectively. The productions of cells and methylan in our designed bioreactor were 20 and 50% higher than those obtained in a conventional fermentor. The methylan content reached a maximum of 20.7 g/L in the bioreactor and the viscosity of the fermentation broth was 127 Pa . s, which corresponds to 68 g/L as a xanthan. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 115-121, 1997.     

Plant regeneration from callus cultures ofLithospermum erythrorhizon

We previously studied the production of shikonin derivatives by cell lines ofLithospermum erythrorhizon. As a result, we have obtained a cell line LE 87, which exhibited high cell growth and high shikonin production. In the present study, the effects of auxins (2,4-D, IAA, picloram, and NAA) and cytokinins (BAP and kinetin) on organogenesis and somatic embryogenesis in this shikonin-producing cell line were investigated. The highest organogenic and embryogenic efficiency was obtained on MS medium supplemented with 10 µM NAA and 0.3 µM kinetin. Subcultured calli showed different morphogenic frequencies depending on the NAA and kinetin concentration. Morphologically normal plants have been regenerated via mostly organogenesis. Shoots subsequently produced roots on plant growth regulator-free MS medium and developed into plantlets. In most cases, a few thin roots were formed at the bases of the shoots after four weeks on the rooting medium. More than fifty green plantlets were transplanted to soil in pots and developed into phenotypically normal plants 8 weeks after being transferred to soil. The regenerated plants grew to maturity, flowered, and set seeds by only artificial pollination.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA -naphthalene-acetic acid - MS Murashige and Skoog (1962) medium Communicated by S. Gleddie     

Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds

A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.     

Isolation of a root-specific cDNA encoding a ns-LTP-like protein from the roots of bean (Phaseolus vulgaris L.) seedlings

A root-specific cDNA clone, PVR3, was isolated from a bean (Phaseolus vulgaris L.) root cDNA library by a differential screening procedure. The nucleotide sequence of PVR3 contains an open reading frame coding for an 11.14 kDa polypeptide of 102 amino acid residues; the first 25 amino acids correspond to the sequence characteristic of a signal peptide. Comparison of the deduced PVR3 polypeptide sequence with the polypeptide sequences of previously cloned genes indicates that PVR3 may encode a ns-LTP-like protein. Molecular modelling of the PVR3 protein predicts that it has a three-dimensional structure that is similar to the three-dimensional model determined from the maize ns-LTP. The PVR3 mRNA accumulated mainly in the roots of young seedlings. It can be detected at low levels in flowers, but it is not detected in other organs. Genomic Southern blot analysis indicates that the genomic DNA corresponding to PVR3 cDNA is encoded by a single gene or small gene family in the bean genome.     

Use of electrophoretic enzyme patterns for streptomycete systematics

Abstract The relative electrophoretic mobilities of various enzymes from 24 different streptomycetes were determined on polyacrylamide gels in order to examine the relatedness of species and strains of the genus Streptomyces . Of 11 different enzymes tested in this study, hexokinase, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, malate dehydrogenase and isocitrate dehydrogenase showed a limited number of constant and reproducible polymorphic enzyme patterns, by comparing which the inter-specific relationships could be examined. In contrast, glucose dehydrogenase, alcohol dehydrogenase, 3-hydroxybutyrate dehydrogenase, phosphoglucose isomerase, peroxidase and esterase exhibited either weak non-reproducible or highly heterogeneous band patterns which were suitable for dissecting the strains within a species and a cluster group.