Purification of two alcohol dehydrogenases from Zymomonas mobilis and their properties

Summary Two alcohol dehydrogenases (ADHI and ADHII, EC 1.1.1.1) were purified to homogeneity from the cell extract of Zymomonas mobilis. The subunit molecular weights of ADHI and ADHII were 40,000 and 38,000, respectively, and both enzymes were homologous dimers. The optimal pHs of ADHI in ethanol oxidation and acetaldehyde reduction reactions were 9.5 and 4.5, and those of ADHII were 9.5 and 6.5, respectively. The optimal temperatures of ADHI and ADHII were 55° C and 45° C, respectively. ADHI was heat-inactivated at 65° C at a 10-fold higher rate than ADHII. ADHI and ADHII were inhibited by 4 M and 1 mM p-chloromercuribenzoate, respectively, and the inhibitions were reversed by the addition of 70 mM 2-mercaptoethanol. ADHII activity was enhanced by 0.02 to 2 mM CoCl₂ and inhibited by 0.4 mM o-phenanthroline; and the activity of inactivated ADHII was restored by addition of 1 mM CoCl₂ or ZnCl₂.ADHI was active on most primary alcohols but not secondary alcohols. ADHII was active on only ethanol, n-propanol, allylalcohol, and furfuryl alcohol.In the anaerobic culture of Z. mobilis, ADHII activity accounted for more than 80% of total alcohol dehydrogenase activity. In aerobic culture, ADHII was the main enzyme but was produced only in the early growth phase.     

Breeding anl-isoleucine producer by protoplast fusion ofCorynebacterium glutamicum

Summary Corynebacterium glutamicum R-18 is a strain forl-isoleucine production. Polyethylene glycol (PEG)-induced protoplast fusion was applied to improve the strain of thisl-isoleucine producer. Strain R-18 was fused with anl-lysine producerC. glutamicum S-37, becausel-isoleucine andl-lysine are synthesized from a common intermediate, aspartate--semialdehyde. Two thousand fusants were checked for their phenotypes. Most of the fusants accumulatedl-lysine, and only 0.9% of the fusants accumulatedl-isoleucine. Two strains, F-28 and F-91, were selected and cultivated in production medium. Fusant F-28 accumulated 12.1 g/l ofl-isoleucine and 4.8 g/l ofl-lysine, and fusant F-91 accumulated 4.8 g/l ofl-isoleucine and 13.0 g/l ofl-lysine, while the parental strains R-18 and S-37 accumulated 9.5 g/l ofl-isoleucine and 26.8 g/l ofl-lysine, respectively. Sugar consumption activity was improved by protoplast fusion, and thel-isoleucine production rate of F-28 was 2.4 times higher than that of R-18.     

Hyper-accumulation of astaxanthin in a green algaHaematococcus pluvialis at elevated temperatures

Summary When a green algaHaematococcus pluvialis was cultivated at 30°C, astaxanthin production was 3-fold more increased than at 20°C. With acetate supplementation to 30°C culture, the alga synthesized over 2-fold more carotenoid than without addition. Tiron, a radical scavenger, however, severely blocked the stimulated carotenogenesis, suggesting that endogenously generated active oxygen was responsible for the highly stimulated carotenogenesis. From these results, possible roles of the elevated cultivation temperatures for hyperaccumulation of astaxanthin were discussed.     

Systematics and distribution of theDiplophos taenia species complex (Gonostomatidae), with a description of a new species

For the systematic study of the gonostomatid fishes of theDiplophos taenia species complex, a total of 698 specimens was obtained from the three oceans. Four valid species were recognized:D. taenia Günther,D. proximus Parr,D. orientalis Matsubara, andD. australis sp. nov. The diagnostic characters are 90–100 total vertebrae (TV) (37–41 abdominal (AV) +52–60 caudal vertebrae (CV)) and 103–115 IC photophores (IC) for D.taenia, 85–90 TV (36–39 AV + 48–52 CV) and 98–104 IC forD. proximus, 83–86 TV (33–35 AV + 49–52 CV) and 92–100 IC for D.orientalis, and 84–91 TV (33–37 AV+ 50–54 CV) and 99–105 IC forD. australis. In addition to the above,D. proximus has larger orbital diameter (the proportion to head length 21–28%) than the other three species (15–23%) beyond 70mm in standard length. Due to wide distribution,D. taenia shows meristic variations: the numbers of TV, IC and anal fin rays (A) are smaller at lower latitudes and larger at higher ones in all oceans, and the number of A is smaller in the Atlantic (56–71) than in the other oceans (59–72) of the same latitude. Because of these variations, identification to species level is possible only area by area. The distribution of each of the four species is also distinct:D. taenia is a cosmopolitan between about 40° N and 30° S exclusive of the eastern tropical Pacific;D. proximus is endemic to the eastern tropical Pacific;D. orientalis is limited to the North Pacific transitional zone between about 30° and 40° N; andD. australis in a transitional zone of the Southern Ocean south of 20° S.     

Functional expression of cDNAs for bovine 11 beta-hydroxylase-aldosterone synthases, P450(11 beta)-2 and -3 and their chimeras.

Two molecular species of bovine P450(11β), P450(11β)-2 and P450(11β)-3 have been identified, in which the amino acid differences were found at the 6th, 36th and 82nd positions from the NH₂-termini of the mature proteins. They catalyzed the 11β-, 18- and 19-hydroxylation and aldosterone formation from 11-deoxycorticosterone, and the rate of production of 18-hydroxycorticosterone and aldosterone by P450(11β)-3 was greater than that by P450(11β)-2 [Morohashi et al., J. Biochem. 107 (1990) 635–640].

In this study, chimeric clones were constructed whose 6th, 36th and 82nd amino acid residues were exchanged with each other. Two original clones and six chimeric clones were expressed in COS-7 cells, and their steroidogenic activities studied. The ratio of aldosterone or 18-hydroxycorticosterone production to corticosterone production by one clone was compared with that of the other. The ratios for the four clones having Gly36 [P450(11β)-3 type] were 0.08–0.22, whereas those for the clones having Ser36 [P450(11β)-2 type] were 0.03–0.05, suggesting that the Gly36 structure is important for aldosterone production.     

Profiiles of Proteins in Guard-cell and Mesophyll Protoplasts from Vicia faba L. Fractionated by Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis

Polypeptides of low molecular weight unique to protoplasts ofVicia guard cells were found by sodium dodecylsulfate-polyacrylamidegel electrophoresis. The polypeptides were 16 kDa (2 species),15 kDa and 12.5 kDa, and were concentrated in membrane-richfractions. The large subunit and holoenzyme of ribulose bisphosphatecarboxylase were identified in guard-cell protoplasts by immunoblotting. ³Present address: Biological Laboratory, College of GeneralEducation, Kyushu University, Ropponmatsu, Fukuoka, 810 Japan. (Received January 25, 1989; Accepted April 24, 1989)     

Partial contig map of the smallest chromosome inCoprinus cinereus

We have constructed a chromosome-specific cosmid library from electrophoretically separated chromosomes of the basidiomyceteCoprinus cinereus and performed contig mapping and analysis of chromosome length polymorphisms (CLPs) for the smallest chromosome of the 5302 strain. A contig map of about 300 kb indicated that the novel size chromosomes in the F₁ progeny were apparently recombinants containing physical markers derived from both ends and central regions in this map. This may be the first case in which the formation of CLPs in the F₁ generation has been explained using the contig map. The results obtained were consistent with the hypothesis that novel CLPs were produced by meiotic recombination between the parental homologous chromosomes of unequal sizes.     

Characterization of cold-responsive extracellular chitinase in bromegrass cell cultures and its relationship to antifreeze activity

A cold-responsive chitinase gene, BiCHT1, was isolated from bromegrass (Bromus inermis) 'Manchar' suspension cells. BiCHT1 messenger RNA was detected at low levels in nonstressed bromegrass cells, whereas its accumulation was induced by incubation at 10 degrees C and 4 degrees C as detected by northern- and western-blot analyses. BiCHT1 was highly homologous to rye CHT9, known to encode an antifreeze protein. BiCHT1 was overexpressed in Escherichia coli and bromegrass cells using genetic transformation procedures. BiCHT1 products expressed in both systems had chitinase activity, but the expressed proteins did not affect the growth of ice crystals in any conditions tested. Besides cold stress, the expression of the BiCHT1 gene was up-regulated by exposure to 35 degrees C, but not by salt or osmotic stress, abscisic acid, or ethephon. BiCHT1 messenger RNA did not accumulate in response to methyl jasmonate and salicylic acid, but was slightly increased by prolonged culture at 25 degrees C and only transiently by chitin. Antifreeze activity detected in the culture medium was induced at 4 degrees C but only slightly at 10 degrees C. It was also induced by ethephon treatment, but not by abscisic acid, chitin, or prolonged incubation at 25 degrees C. The results of transgenics and expression analyses suggest that the BiCHT1 product is a major protein with chitinase activity secreted in the medium of cold-treated cells and is unlikely to be responsible for the antifreeze activity detected in the culture medium.