Tadayuki Imanaka

A PERSPECTIVE ON THE APPLICATION OF GENETIC ENGINEERING: STABILITY OF RECOMBINANT PLASMID

DNA cloning with vector plasmid and/or phage is stimulating not only in the interests of biological science but also in applications such as production of a new medicine, enhancement of yield in a particular metabolite in the fermentation industry, development of a new plant in the agricultural field, etc. Despite the substantial “species barrier” described schematically in FIGURE 1, not a few examples of cloning foreign DNA molecules in host cells have been made available.’.’ For instance, once a specific gene is cloned, metabolites originating from the specific gene might be produced in large quantities due to the gene dosage effect.3 To exaggerate, the contribution of genetic engineering to the biological world is deemed to be revolutionary rather than evolutionary. Although the aspects and/or regions to which genetic engineering is closely related are comprehensive in this context, this paper will pay attention only to a limited area, i.e., the subject of expression and stability of a cloned gene or genes in the cultivation of host cells. Unless cloned genes are kept in situ in vector plasmid during replication in coordination with the growth of host cells, it is not possible to employ the recombinant plasmid as an agent to enhance production of specific materials in industry. Before proceeding to clarify the significance of genetic engineering in this work, a few examples on “stability” and “instability” in plasmid manipulation will be presented.

Two Replication Determinants of an Antibiotic-resistance Plasmid, pTB19, from a Thermophilic Bacillus

Two different replication determinants were found on an antibiotic resistance plasmid, pTB19, from a thermophilic bacillus. One replication determinant (designated RepA) was functional only in Bacillus subtilis, whereas the other (designated RepB) functioned in both B. subtilis and Bacillus stearothermophilus. A deletion plasmid, pTB90, carrying the RepB derived from pTB19 coincidentally contained the specific 1.0 MDal EcoRI fragment of a cryptic plasmid pBSO2 from B. stearothermophilus. The presence of this 1.0 MDal EcoRI fragment in various deletion plasmids from pTB90 increased transformation frequencies for B. stearothermophilus 10(3) to 10(4) times and lowered plasmid copy numbers in the host strain to about one-tenth of those found for plasmids lacking this fragment.

Continuous production of panose by immobilized neopullulanase

Abstract Panose is a mildly sweet trisaccharide composed of three glucose units which has the nature of an anti-cariogenic sugar in foods. Continuous production of panose from pullulan was investigated by using a column of immobilized neopullulanase. Although enzyme immobilization by ionic bond is one of the most general methods for the production of non-ionic materials from non-ionic substrates, the activity of the neopullulanase immobilized on DEAE-cellulose significantly decreased. When the neopullulanase was immobilized on a carrier with spacer arms by covalent bond through the formation of the schiff base, the activity was fully expressed. Optimum temperature and pH for the reaction of the immobilized neopullulanase were 55–60°C and 6.0, which were almost the same as those of the free enzyme. The immobilized neopullulanase column was employed for the continuous production of panose from pullulan. More than 92% of pullulan was converted by this system into the final products; panose, maltose, and glucose. The ratio of panose in the mixture of products was 84%, which was significantly higher than that (around 70%) of a batch system using the free enzyme. The immobilized neopullulanase was very stable, and more than 90% of the initial activity was retained after 150-h continuous reaction at 55°C.

Isolation of streptomycin-nonproducing mutants deficient in biosynthesis of the streptidine moiety or linkage between streptidine 6-phosphate and dihydrostreptose.

Eight streptidine idiotrophic mutants (SD20, SD81, SD141, SD189, SD245, SD261, SD263, and SD274) which required streptidine to produce streptomycin were derived from Streptomyces griseus ATCC 10137 by UV mutagenesis. By both the characterization of intermediates accumulated by the idiotrophs and the assay of enzymes involved in streptidine biosynthesis, the biochemical lesions of the mutants were deduced as follows: SD20 and SD263, transamination; SD81, SD261, and SD274, phosphorylation; SD141, transamidination; SD189, dehydrogenation; SD245, linkage between streptidine 6-phosphate and dihydrostreptose. An accumulation of streptidine 6-phosphate was found in SD245 to impair its aminotransferase activity. This finding suggests that aminotransferase activity might have been negatively controlled by the end product, streptidine 6-phosphate, of the streptidine biosynthetic pathway. Images

Purification and characterization of a new metal protease which hydrolyzes the cyclic decapeptide, gramicidin S

Abstract We screened a strain which can produce a new protease. The strain, Lactobacillus sp. no. 1, was isolated from a natural environment as an organism which could utilize gramicidin S as a sole nitrogen source. This strain was proved to produce much protease because it formed a large halo on a plate containing casein, and the protease was purified using ion exchange column chromatography. The amino-terminal amino acid sequence of the hydrolyzed products by the cleavage of gramicidin S was determined by a protein sequencer, and sizes of those products were analyzed by a mass spectrometer. The protease could cleave two peptide bonds between l -Orn- l -Leu in gramicidin S. These cleavage sites were different from other reported cleavage sites of gramicidin S by protease. The molecular weight of the protease was 42,000 by SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature for the enzyme activity were 5.5 and 45°C, respectively. The enzyme activity was inhibited by EDTA, but not by phenylmethylsulfonyl fluoride (PMSF). Because the reported protease that can hydrolyze gramicidin S was a serine protease and the cleavage site was different from that of this protease from Lactobacillus sp. no. 1, we concluded that this enzyme was a new type of metal protease which can cleave both linear and cyclic peptide substrates with a unique substrate specificity.

Highly branched oligosaccharides produced by the transglycosylation reaction of neopullulanase

Abstract We previously reported a new way of producing isomalto-oligosaccharide syrup from starch by using the transglycosylation reaction of neopullulanase (Kuriki, T., Yanase, M., Takata, H., Takesada, Y., Imanaka, T., and Okada, S., Appl. Environ. Microbiol., 59, 953–959, 1993). The syrup contained isomaltose, a mixture of isopanose and panose, and 62-O-α-maltosyl-maltose as isomalto-oligosaccharides with degrees of polymerization (DP) of 2, 3, and 4, respectively. Isomalto-oligosaccharides with DP ≧ 5 were simultaneously obtained by the system, and the structures were analyzed. The results indicated that most of the isomalto-oligosaccharides with DP ≧ 5 contained two or three α-(1→6)-glucosidic linkages with α-(1→4)-glucosidic linkages. It was also proved that these highly branched oligosaccharides were not limit dextrins from the degradation of starch, but were synthesized by the transglycosylation reaction of neopullulanase.

Crystallization and Preliminary X-Ray Diffraction Studies of Bacterial Alginate Lyase

Alginate lyase (EC 4.2.2.3) from a soil bacterium (Flavobacterium sp.), which was overexpressed in Bacillus subtilis, has been crystallized by the vapor-diffusion method. Tetragonal crystals were obtained using ammonium sulfate as a precipitant. The crystal was resistant to X-ray radiation damage and diffracted to about 2.2 A resolution. The diffraction pattern indicated that the crystal belongs to the orthorhombic system, space group P212121 with unit-cell dimensions of a=82.7, b=142.1 and c=80.9 A. It is supposed that the asymmetric unit consists of two alginate lyase molecules.

A simple method for determination of substrate specificity of alginate lyases

Abstract When two types of alginate lyases [poly-mannuronate (poly M)-specific and poly-guluronate (poly G)-specific lyases] were incubated on agar plates containing alginate and the surfaces of the plates covered with CaCl 2 solution, poly M-specific and poly G-specific lyases yielded white-halos and white-rings, respectively. The substrate specificity of alginate lyase was easily determined by discriminating between the types of gellation ( i.e. halo or ring formation) caused by the interaction between calcium ions and depolymerized alginates.

Protein Engineering of Penicillinase as Affinity Ligands for Bioprocessing

Abstract The active site and the substrate binding site of penicillinase (β-lactamase) from Bacillus licheniformis were altered in this study so that the enzyme retains the specific binding capability to the β-lactam antibiotics, but fails to hydrolyze them. When Lys47 in the enzyme molecule was replaced by Ala47, the mutant protein PenP(KA) lost not only its catalytic activity but also the substrate binding ability. In contrast, when Ser44 was replaced by Ala, the mutant protein PenP(SA) lost its catalytic activity but still kept the substrate binding ability. It was found that PenP(SA) exhibited the characteristic association and dissociation with penicillin G, but the dissociation constant was much larger than expected. Possible use of this mutant protein as an affinity ligand is also discussed.

Biological threshold values of procaryotic gene expression which is controlled by the DNA inverted repeat sequence and the mRNA secondary structure

Abstract Procaryotic gene expression is generally controlled at three levels, i.e., transcription, translation and degradation of mRNA. The effects of the DNA inverted repeat sequence and the mRNA secondary structure on each step in the gene expression were analyzed by the compilation of quantitative data published in the literature. When the ΔG of the mRNA secondary structure in an SD sequence and the flanking region is larger (i.e., more negative) than −5∼−6 kcal/mol, the efficiency of translation initiation drops drastically to 30% or less of the full expression. When the ΔG values of mRNA secondary structures in an open reading frame, the 3′-terminus region and the inverted repeat sequence of the terminator are larger than −20 kcal/mol, reduction of translation efficiency, mRNA stability, and transcription termination are maximized, respectively. In other words, the biological threshold values of the mRNA secondary structure for the regulation of translation initiation and other steps (termination of transcription, degradation of mRNA, and elongation of peptide chain) are −5∼−6 kcal/mol and −20 kcal/mol in ΔG value, respectively. Reasons for the difference are discussed.