Solid state fermentation of straw withNeurospora crassa for CMCase and β-glucosidase production

Summary CMCase and -glucosidase were produced by the mutantNeurospora crassa 40b cultivated on untreated wheat straw in a solid state fermentation. Best enzyme activities were observed when the growth medium was composed of wheat straw mixed with certain mineral solutions at a ratio 1:2 (w/v). A partially purified enzyme preparation showed optimum enzyme activities of CMCase and -glucosidase at pH 4.0 and 5.0 and temperature 50 and 60°C respectively. The apparent Km values for the same enzymes were 16.8 g/l and 1.03x10^–4 M respectively. At optimum growth and enzyme assay conditions yields as high as 586.2 U CMCase and 58.4 U -glucosidase per gram of straw were obtained.     

Comparison of different strains ofCellulomonas for production of cellulolytic and xylanolytic enzymes from biomass produced on saline lands

A comparison of the rate of carboxymethyl cellulase (CMCase), avicelase, xylanase, -glucosidase and -xylosidase production and rates of growth by 4 different strains ofCellulomonas revealed a wide range of behaviour; with some strains producing more CMCase, avicelase, xylanase, -glucosidase and -xylosidase from complex lignocellulosic (LC) biomass (from saline land) and CMC while some others producing small amounts of these enzymes. One strain,C. biazotea, was better with respect to enzyme production potential and growth behaviour than most of the other strains and has been chosen as a starting strain for genetic improvement for producing enzymes of the cellulase complex.     

Somatic hybrids between Solanum brevidens and Solanum tuberosum: Expression of a late blight resistance gene and potato leaf roll resistance

Hexaploid somatic hybrids resulting from mesophyll protoplast fusions between Solanum brevidens Phil., PI 218228, and Solanum tuberosum L., PI 203900 were tested for late blight resistance using two races of Phytophthora infestans Monte., de Bary. The S. tuberosum parent was a late blight differential possessing the R4 gene which confers resistance to race 0. The S. brevidens parent is resistant to potato leaf roll virus. Inoculations with both compatible (race 1.3.4.5) and incompatible (race 0) races of P. infestans clearly demonstrated the expression of the late blight resistance gene in all of the hybrid progeny tested. Most of the hybrids tested were also resistant to potato leaf roll virus (PLRV), indicating that the S. brevidens genes for PLRV resistance were present and expressed.     

Interspecific somatic hybrids ofRudbeckia hirta andR. Laciniata (Compositae)

Interspecific somatic hybrid plants betweenRudbeckia hirta cv. Marmalade andR.laciniata cv. Irish Eyes were regenerated following the electro-fusion of mesophyll protoplasts ofR.hirta with callus protoplasts ofR.laciniata. A hybrid selection scheme was based on the fact that plant regeneration, from parental protoplasts ofR.hirta, was via shoot regeneration of callus, and only via rhizogenesis forR.laciniata. The other half of the selection strategy was based on the presence of anthocyanin-pigmented roots; a characteristic of theR.hirta parent only. Somatic hybrids were regenerated, via rhizogenesis, alongside normalR.laciniata but were distinguished by the presence of pigmented roots (a feature ofR.hirta). Hybrid plants had a floral morphology that was intermediate as compared to that of the two parents, with an expected somatic chromosome number of 2n=(2x+4x)=74. Pollen viability though was low. Esterase and peroxidase isozyme profiles confirmed the hybrid nature of the regenerated plants with pigmented roots, whilst chloroplast DNA restriction analysis showed that these hybrids had aR.laciniata chloroplast DNA. This demonstration of somatic hybridisation not only opens up the possibility of incorporating novel traits between such ornamentalCompositae species, but provides a selection strategy based on rhizogenesis as the route to plant regeneration coupled with heritable pigmentation production of roots as a confirmatory hybrid marker.ABBREVIATIONS BSA bovine serum albumin - EDTA ethylene diamine tetra acetic acid - FDA fluorescein diacetate - f.wt. fresh weight - IAA indole 3-acetic acid - MS Murashige and Skoog (1962) medium - TEMED N,N,N,N-Tetra methyl ethylene diamine - TES (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid)     

Some properties ofSaccharomyces kluyveri

Four isolates of aSaccharomyces species which differed fromS. kluyveri by their ability to use cellobiose were analyzed genetically in relation to the latter species. Isolated single spores had low viability. Spore tetrads segregated mating types 2 2, with sexual agglutination occurring between complementary mating types. All single-spore isolates assimilated cellobiose indicating that these isolates were not naturally occurring hybrids betweenS. kluyveri and a cellobiose assimilatingSaccharomyces species.Two cell types were exhibited by single-spore cultures ofS. kluyveri, one granulated (G-type) and one vacuolated (g-type). G-type cultures formed fertile hybrids with complementary mating types of both G- and g-type cultures. Hybrids between two g-type cultures were sterile. They would, however, give fertile hybrids when mixed with G-type cultures.Sporulating hybrids betweenSaccharomyces sp. andS. kluyveri were produced. However the percentage spore germination was low. Single-spore cultures examined had cell types atypical of either parent. The ability to assimilate cellobiose was dominant and appeared to segregate with mating type and cell type.Weak mating reactions occurred when the (+) and (-) mating types ofSaccharomyces sp. were mixed with (a) and () mating types ofS. cerevisiae, respectively.The species ofSaccharomyces isolated from the Pacific Coast are designated as strains ofS. kluyveri.     

Molecular cloning ofClostridium thermocellum genes involved in β-glucan degradation in bacteriophage lambda

A genomic library ofClostridium thermocellum DSM 1237 was constructed in a bacteriophage lambda vector and screened for hydrolysis of carboxymethyl cellulose (CMC), lichenan and methylumbelliferyl--glucoside. Recombinant clones expressing four -glucanases and two distinct -glucosidases were obtained. The -glucanase activities could be classified with respect to their substrate specificities as -1, 4-endoglucanase (CMCase), -1, 3-endoglucanase (laminarinase), and -1, 3-1, 4-endoglucanases (lichenanase). The -glucosidases were identified as cellobiases hydrolyzing both aryl--glucosides and cellobiose.     

Comparison of optimal conditions for mass production of carboxymethylcellulase by <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109/A-68 with other recombinants in pilot-scale bioreactor

The optimal conditions for mass production of carboxymethylcellulase (CMCase) by E. coli JM109/A-68 were investigated and compared with other E. coli JM109 recombinants producing CMCase. The optimal agitation speed and aeration rate for cell growth of E. coli JM109/A- 68 were 500 rpm and 0.50 vvm in a 7 L bioreactor, whereas those for production of CMCase were 416 rpm and 0.95 vvm. The optimal vessel pressures for cell growth as well as production of CMCase in a 100 L bioreactor were 0.04 MPa. The maximal production of CMCase by E. coli JM109/A-68 under the optimized conditions in a 100 L bioreactor was 11.0 times higher than its wild type, B. velezensis A-68. Optimal conditions for mass production of CMCase by recombinants were different from those for wild strains. The higher production of CMCase by E. coli JM109/A-68 and other recombinant of E. coli seemed to result from its higher cell growth under the optimal conditions for dissolved oxygen and its mixed-growth associated production pattern compared to the growthassociated production of B. velezensis A-68.     

Improved sensitivity of genetic complementation of nitrate reductase deficient mutants via protoplast fusion

An improved rapid assay for complementation testing of mutants of Nicotiana tabacum deficient in nitrate reductase is described. The test is based on measurement of in vivo nitrate reductase activity in 7 to 10 day old cultures derived from fusion-treated protoplast mixtures of the respective mutants as a criterion for complementation. It allows to detect complementing hybrids induced by the conventional droplet fusion technique in small numbers of protoplasts per assay (8×10⁴).Abbreviations NR nitrate reductase - 2,4-D dichlorophenoxy-acetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - PEG polyethylene glycol     

Construction of a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109/A-68 for production of carboxymethylcellulase and comparison of its production with its wild type, <Emphasis Type="Italic">Bacillus velezensis</Emphasis> A-68 in a pilot-scale bioreactor

A gene encoding carboxymethylcellulase (CMCase) of Bacillus velezensis A-68 had been cloned in Escherichia coli JM109. Based on productivity and economic aspect, rice bran and ammonium chloride were chosen to be optimal carbon and nitrogen sources for production of CMCase by E. coli JM109/A-68. The optimal conditions for rice bran, ammonium chloride, and initial pH of medium for production of CMCase were established by the response surface methodology (RSM). The concentrations of four salts in the medium, K₂HPO₄, NaCl, MgSO₄·7H₂O, and (NH₄)₂SO₄, for production of CMCase also were optimized. The optimal temperatures for cell growth and production of CMCase were 37°C. The maximal production of CMCase by E. coli JM109/A-68 was 880.2 U/mL, which was 10.5 time higher than its wild type, B. velezensis A-68. The production of CMCase by E. coli JM109/A-68 was compared with that by B. velezensis A-68 in a 100 L pilot-scale bioreactor under the optimized conditions. The production of CMCase by E. coli JM109/A-68 was found to be the mixed-growth associated unlike the growthassociated production of CMCase by B. velezensis A-68.     

Characterization of two β-glucosidase genes fromClostridium thermocellum

Summary Two -glucosidase genes, designatedbglA andbglB, were isolated from a gene bank ofClostridium thermocellum DSM 1237. The coding sequences forbglA andbglB were located on non-homologous DNA fragments of 3.2– and 3.4-kb, respectively. Both genes direct inEscherichia coli the synthesis of cytoplasmic -glucosidases, which differ with respect to substrate specificity and temperature profile. The properties of thebglA-encoded -glucosidase A closely resemble that of a -glucosidase previously isolated fromC. thermocellum cultures.     

Conjugal transfer of Streptococcal antibiotic resistance plasmids intoClostridium acetobutylicum

Summary Streptococcal plasmids pAM1, pVA797, pVA797Tn917 and pAM610 were transferred or mobilized toClostridium acetobútylicum fromStreptococcus sanguis,S. faecalis andS. lactis donors.Clostridum transconjugants were able to retransfer pAM1 and pVA797 to a plasmid-freeS. lactis recipient.     

Intraspecific gametosomatic hybridisation in Petunia hybrida

Following PEG and high pH induced fusion of haploid tetrad protoplasts of a normal purple flowered variety of P. hybrida with cell suspension protoplasts of a nuclear albino mutant of the variety Blue Lace, triploid gametosomatic hybrid plants were recovered. These hybrids possessed an intermediate floral morphology and the expected chromosome number of 2n=3x=21. Selection was based on the fact that pollen tetrad protoplasts failed to divide in culture and that, following complementation to chlorophyll proficiency in the gametosomatic hybrid, the hybrid cells were visualised against a background of albino cells of the variety Blue Lace. The production of such gametosomatic hybrid plants in Petunia has shown that the concept of gametosomatic hybridisation can be extended to genera other than Nicotiana and that alternative selection strategies are available.Abbreviations BAP 6-benzylaminopurine - IAA 3-indole acetic acid - NAA naphthalene acetic acid - Z zeatin - ABN bromonaphthalene - MS Murashige and Skoog (1962) - MW molecular weight - PEG polyethylene glycol     

Fates and expression ofBacillus subtilis endo-β-1, 4-glucanase gene andAlcaligenes faecalis β-glucosidase gene introduced inEscherichia coli andBacillus cells

Summary Two kinds of cellulase genes coding for endo--1, 4-glucanase and -glucosidase, isolated fromBacillus subtilis andAlcaligenes faecalis respectively, were separately or combinedly put on a newly constructedEscherichia coli-Bacillus shuttle vector plasmid. When the recombinant plasmids having cellulase gene(s) were introduced intoE. coli orBacillus cells, drastic differences in fates and expression of the two genes were observed.     

Production and characterization of intergeneric somatic hybrids of Trachystoma ballii and Brassica juncea

Intergeneric somatic hybrids, Trachystoma ballii (2n=16)+B. juncea (2n=38), were obtained by fusing mesophyll protoplasts of T. ballii and hypocotyl protoplasts of B. juncea using polyethylene glycol. The heterokaryotic fusion frequency was around 23%. Plants were regenerated from 10 out of 2×10⁴ calli of which four were hybrids. Hybrids were intermediate between the parents in general morphology. However, in some characters one of the parents dominated. All the plants were symmetric in their chromosomal constitution as revealed by the formation of 26 bivalents at metaphase-I of meiosis. Two trivalents and 2 univalents were also observed in some pollen mother cells. Hybrid nature was also confirmed by Southern hybridization of DNA of one regenerated plant restricted with Hind III and probed with the nick translated plasmid pTA71 carrying a wheat nuclear r-DNA sequence. Hybrid plant RT 1 showed bands characteristic of both parents. All the plants were absolutely pollen sterile. However, on backcrosses to B. juncea seeds were obtained.     

Genotypic effects on the amino acid relationships in maize (Zea mays L.) pollen and style

Summary The free amino acid content of the pollen grains and the style from three single cross hybrids (Wf9 x H55, Ky49 x Ky27, K64 x K55) and two inbred lines (Oh43, H55) was determined. No tyrosine was detected in the pollen grains of any genotype. Significant differences between pollen genotypes were found for aspartic acid, threonine, serine, lysine and histidine with no effect resulting from the vigor of the pollen source. No proline was found in the style of any genotype. Significant differences between style genotypes were obtained for threonine, serine, glutamic acid, leucine, tyrosine, ethanolanine, aminobutyric acid and histidine with a relationship between vigor of the style genotype present for tyrosine, ethanolanine and aminobutyric acid. The relationship between the pollen and style level for each amino acid as influenced by genotype was analyzed. A significant negative correlation was found only for threonine. Apparently, a complementary relationship between the pollen and style exists for some amino acids. Proline and tyrosine are available only from the pollen and style respectively. Threonine levels are balanced with varying contributions from both pollen and style depending on the genotype.     

Secondary product formation by cultures of Beta vulgaris and Nicotiana rustica transformed with Agrobacterium rhizogenes

Hairy root cultures of Beta vulgaris and Nicotiana rustica were established after roots were induced on plants following infection with Agrobacterium rhizogenes. The transformed cultures of B. vulgaris and N. rustica synthesised their characteristic secondary products, the betalain pigments and nicotine alkaloids respectively, at levels comparable with those of in vivo roots from the same variety. Betalains were entirely retained inside the root tissue. In contrast, a proportion of the nicotine alkaloids was secreted into the medium. The potential of this type of in vitro plant tissue culture for the production of valuable plant secondary products is identified and confirmed.     

Activity and thermostability of carboxymethylcellulase from Aspergillus niger is strongly influenced by noncovalently attached polysaccharides

Removal of non-covalently attached polysaccharides from carboxymethylcellulase (CMCase) of Aspergillus niger improved its activity but decreased its thermostability and protease resistance. The activation energy profile of the hydrolysis of carboxymethylcellulose (CMC) was triphasic with increasing values of 17,-55 and-562 kJ/mol for polysaccharide-free and 19, -21 and -207 kJ/mol for polysaccharide-complexed CMCase. The specificity constant (V_max/K_m) of polysaccharide-free CMCase was 1.41 compared to polysaccharide-complexed CMCase which was only 0.68. The polysaccharide free CMCase had lower thermostability (melting point = 82°C) and higher protease susceptibility compared to polysaccharide-complexed CMCase (melting point>100°C).The authors are with the National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Jhang Road, Faisalabad, Pakistan;     

Isolation,culture and callus regeneration of protoplasts of wild and cultivated Helianthus species

Protoplasts were isolated from seedling roots, hypocotyls, and cotyledons of four cultivars of Helianthus annuus and from leaves of axenic shoot cultures of the wild species H. praecox, H. scaberimus and H. rigidus. Optimal culture conditions were established for the respective protoplast systems, using the agarose bead method of culture. Protoplast division was induced for all the species examined. In the case of the cultivars of H. annuus, hypocotyl and cotyledon protoplast division was sustained leading to callus formation, which in turn, could be induced to produce roots and organised meristematic regions in the presence of NAA and 6-BAP.Abbreviations 6-BAP 6-benzylaminopurine - NAA -naphthalene acetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog     

Towards a classification ofE. coli ribosomal proteins: A hypothetical ‘small ribosome’ as a primitive protein-synthesizing apparatus

Homologies were searched among the published primary sequences of 51E. coli ribosomal proteins, partly by eye and partly by computer-assisted methods. By employing Moore and Goodman's alignment statistics for evaluating homology levels, 33 out of these 51 ribosomal proteins has been classified into 9 homology groups, some of which being yet tentative and remaining to be further analyzed. Taking it into consideration that most ribosomal protein genes are clustered atstr-stc region,rif region and several other regions, these results strongly suggest that most or all of the contemporary ribosomal proteins must have evolved by repeated gene duplications ofvery few (oronly one) primitive ancestral ribosomal protein gene(s). Thus it is most reasonable to propose that a small ribosome consisting of very few (or only one) ribosomal protein(s) must have existed as a primitive protein-synthesizing apparatus.     

Characterization of the purified multifunctional cellulase component of Penicillium funiculosum

Summary The endoglucanase component (CMCase I) ofPenicillium funiculosum cellulase was purified to apparent homogeneity by ultrafiltration and gel chromatography. It consists of a single polypeptide chain with a molecular weight of 56000 and is a glycoprotein. Viscometric and end-product analysis revealed the randomness of enzyme action. Multifunctional characteristic of CMCase I was studied with various carbohydrate substrates.NCL Communication No.: 4307