Molecular screening for alkane hydroxylase genes in Gram-negative and Gram-positive strains

We have developed highly degenerate oligonucleotides for polymerase chain reaction (PCR) amplification of genes related to the Pseudomonas oleovorans GPo1 and Acinetobacter sp. ADP1 alkane hydroxylases, based on a number of highly conserved sequence motifs. In all Gram-negative and in two out of three Gram-positive strains able to grow on medium- (C₆–C₁₁) or long-chain n -alkanes (C₁₂–C₁₆), PCR products of the expected size were obtained. The PCR fragments were cloned and sequenced and found to encode peptides with 43.2–93.8% sequence identity to the corresponding fragment of the P. oleovorans GPo1 alkane hydroxylase. Strains that were unable to grow on n -alkanes did not yield PCR products with homology to alkane hydroxylase genes. The alkane hydroxylase genes of Acinetobacter calcoaceticus EB104 and Pseudomonas putida P1 were cloned using the PCR products as probes. The two genes allow an alkane hydroxylase-negative mutant of Acinetobacter sp. ADP1 and an Escherichia coli recombinant containing all P. oleovorans alk genes except alkB , respectively, to grow on n -alkanes, showing that the cloned genes do indeed encode alkane hydroxylases.     

Characterization of two 3-ketothiolases possessing differing substrate specificities in the polyhydroxyalkanoate synthesizing organism Alcaligenes eutrophus

Abstract Two constitutive acetyl-CoA acetyltransferases (3-ketothiolases A and B) were purified from Alcaligenes eutrophus . Enzyme A was active with only acetoacetyl-CoA and 3-ketopentanoyl-CoA, whereas enzyme B was active with all the 3-ketoacyl-CoAs (C₄−C₁₀) tested. Enzyme A appeared to be a tetramer ( M _r 70 000) with identical subunits ( M _r 44 000) and enzyme B had a similar M _r of 168 000 (containing M _r 46 000 subunits). Enzymes A and B had isoelectric points of 5.0 and 6.4, respectively. The stoichiometry of the reactions catalysed by each enzyme was confirmed. K _m values of 44 μM and 394 μM for acetoacetyl-CoA, and 16 μM and 93 μM for CoA, were determined with enzymes A and B, respectively. Enzymes A and B gave K _m values of 1.1 mM and 230 μM, respectively, for acetyl-CoA. The condensation reaction was potently inhibited by CoA in both cases.     

Uptake and utilization of n-octacosane and n-nonacosane by Arthrobacter nicotianae KCC B35

Arthrobacter nicotianae KCC B35 isolated from blue-green mats densely covering oil sediments along the Arabian Gulf coast grew well on C₁₀ to C₄₀ n -alkanes as sole sources of carbon and energy. Growth on C₂₀ to C₄₀ alkanes was even better than on C₁₀ to C₁₈ alkanes. Biomass samples incubated for 6 h with n -octacosane (C₂₈) or n -nonacosane (C₂₉) accumulated these compounds as the predominant constituent alkanes of the cell hydrocarbon fractions. The even chain hexadecane C₁₆ and the odd chain pentadecane C₁₅ were the second dominant constituent alkanes in C₂₈ and C₂₉ incubated cells, respectively. n -Hexadecane-incubated cells accumulated in their lipids higher proportions of C₁₆-fatty acids than control cells not incubated with hydrocarbons. On the other hand, C₂₈ and C₂₉-incubated cells did not contain any fatty acids with the equivalent chain lengths, but the fatty acid patterns of the cell lipids suggest that there should have been mid-chain oxidation of these very long chain alkanes. This activity qualifies A. nicotianae KCC B35 to be used in cocktails for bioremediating environments polluted with heavy oil sediments.     

Degradation of crude oil by Acinetobacter calcoaceticus and Alcaligenes odorans

Two types of Indian crude oil (Bombay High and Gujarat) were tested for their biodegradability by Acinetobacter calcoaceticus and Alcaligenes odorans. Acinetobacter calcoaceticus S30 and Alc. odorans P20 degraded Bombay High crude oil by 50% and 45%, while only 29% and 37% of Gujarat crude oil (heavy crude oil) was degraded by these isolates, respectively. Acinetobacter calcoaceticus and Alc. odorans in combination deraded 58% and 40% of Bombay High and Gujarat crude oils, respectively, which were significantly higher than that of by individual cultures. Acinetobacter calcoaceticus S30 degraded more of the alkanes fraction than the aromatics fraction of both crude oils. GC fingerprinting of alkane fraction showed major degradation of heptadecane (C₁₇), octadecane (C₁₈), nonadecane (C₁₉), eicosane (C₂₀), docosane (C₂₂), tricosane (C₂₃) and tetracosane (C₂₄) of crude oil, while the Alc. odorans P20 degraded alkanes and aromatics equally. The asphaltenic component increased in both types of crude oil after biodegradation. The two strains grew very well on n -alkane up to C₃₃ as well as on pristane (branched-chain alkane) but could not grow on cycloalkanes. Acinetobacter calcoaceticus S30 could not grow on pure polycyclic aromatic hydrocarbon (PAH) compounds except naphthalene but Alc. odorans P20 could grow on anthracene, phenanthrene, dibenzothiophene, fluorene, fluoranthene, pyrene and chrysene.     

Fermentation of cassava peels for the production of cellulolytic enzymes

Cassava peels were used as a substrate for the production of cellulolytic enzymes. Under solid substrate fermentation conditions and a Rhizopus sp., thermostable cellulolytic enzymes were produced. Optimal production temperature and pH were 45°C and 5.6 respectively. Kinetic studies of the enzymes showed that the cellulase C₁ activity was optimal at pH 5.0 and 50°C, whereas that of cellulase C_x was optimal at pH 7.0 and 60°C. The enzymes degraded ca 44% of sorghum grains in 6 h, thus suggesting a possible use in saccharification processes. The results also showed the possibility of re-cycling cassava peels as a cheap substrate for the enzyme industry. and accepted 6 June 1989     

Enhanced degradation of petrol (Slovene diesel) in an aqueous system by immobilized Pseudomonas fluorescens

The rate of degradation of n -alkanes C₁₂-C₁₈, in petrol (Slovene diesel) in an aqueous system, by free and immobilized Pseudomonas fluorescens in shaking flasks was investigated. Cells were immobilized to a biosupport, Biofix, and a biosorbant, Drizit. Analysis of cellular growth of the free and immobilized bacteria over 8 d of incubation with diesel as the sole carbon source, showed a reduction in the lag phase in the immobilized cultures in comparison to the free system. The free system degraded 52·3% of C₁₂ and 11·6% of C₁₃, but C₁₄-C₁₈ were not degraded. In comparison to the free system and diesel which had not been exposed to experimental conditions (unexposed), the immobilized systems degraded significantly more of C₁₃-C₁₈. Biofix-immobilized cells degraded 14·8% of C₁₂ and an average of 53·5% of C₁₃-C₁₈. Drizit-immobilized cells degraded 24·5% of C₁₂, 52·4% of C₁₃ and an average of 91·2% of C₁₄-C₁₈. This study shows the successful use of immobilized bacteria technology to enhance the degradation of diesel in an aqueous system.     

Alcohol oxidase of Aspergillus flavipes grown on hexadecanol

Abstract: The activity of alcohol oxidase in Aspergillus flavipes was induced by growth on hexadecanol, though highest activities were obtained using a mixture of hexadecanol and olive oil. The enzyme showed a wide range of substrate specificity towards aliphatic primary alcohols from C₈ to C₁₈. The preferred substrate was decanol. The enzyme had an optimum pH of 9.5. It also used cis -unsaturated alcohols better than the trans -isomers. ω-Hydroxy fatty acids and α,ω-diols were not attacked.     

Anaerobic utilization of alkylbenzenes and n-alkanes from crude oil in an enrichment culture of denitrifying bacteria affiliating with the beta-subclass of Proteobacteria

Denitrifying bacteria were enriched from freshwater sediment with added nitrate as electron acceptor and crude oil as the only source of organic substrates. The enrichment cultures were used as laboratory model systems for studying the degradative potential of denitrifying bacteria with respect to crude oil constituents, and the phylogenetic affiliation of denitrifiers that are selectively enriched with crude oil. The enrichment culture exhibited two distinct growth phases. During the first phase, bacteria grew homogeneously in the aqueous phase, while various C₁–C₃ alkylbenzenes, but no alkanes, were utilized from the crude oil. During the second phase, bacteria also grew that formed aggregates, adhered to the crude oil layer and emulsified the oil, while utilization of n -alkanes (C₅ to C₁₂) from the crude oil was observed. During growth, several alkylbenzoates accumulated in the aqueous phase, which were presumably formed from alkylbenzenes. Application of a newly designed, fluorescently labelled 16S rRNA-targeted oligonucleotide probe specific for the Azoarcus / Thauera group within the β-subclass of Proteobacteria revealed that the majority of the enriched denitrifiers affiliated with this phylogenetic group.     

Rice alpha-mannosidase digesting the high mannose glycopeptide of glutelin

α -Mannosidase (EC 3.2.1.24) from rice dry seeds was purified to homogeneity. Optimum pH and K_m for pNP- α -Man hydrolysis were pH 4.3–4.5 and 1.04 m M , respectively. The enzyme digested mannobioses such as Man α -1,2Man, Man α -1,6Man, Man α -1,3Man but Man α -1,4Man. Zn²⁺ ion was required for the activity, whereas EDTA and swainsonine inhibited the activity by 80 and 96%, respectively. The rice storage protein, glutelin was prepared and its basic subunits were shown to have high mannose-type sugar chains by two-dimensional mapping using NH₂-P and C₁₈ silica columns. They were Man₉GlcNAc₂, Man₈GlcNAc₂, Man₇GlcNAc₂, Man₆GlcNAc₂ and Man₅GlcNAc₂. All these oligosaccharides were digested by the purified α -mannosidase, and Man-GlcNAc₂ and mannose were formed. Glycopeptides, having these high mannose-type sugar chains, could also be digested by the α -mannosidase. Subunits were prepared from glutelin basic subunit and the richest subunit among them, subunit 2 (isoform 2), was digested by the α -mannosidase. Isoform 2 was digested by V8 protease only partially and slowly. However, isoform 2, pre-treated with the α -mannosidase, was rapidly and completely digested by V8 protease.     

Leaf blade structure and C4 acid decarboxylation enzymes in xCynochloris spp. (Poaceae), intergeneric hybrids between species of different C4 type

PRENDERGAST, H. D. V., STONE, N. E. & HATTERSLEY, P. W., 1988. Leaf blade structure and C₄ acid decarboxylation enzymes in x Cynochloris spp. (Poaceae), intergeneric hybrids between species of different C₄ type. x Cynochloris macivorii Clifford and Everist and x C. reynoldensis B. K. Simon (Poaceae) are intergeneric C₄ hybrids between Cynodon dactylon (L.) Pers., and NAD-malic enzyme (NAD-ME) species, and two different PEP carboxykinase (PCK) species of Chloris . Parental species of each hybrid species have the 'classical' leaf blade structure of their respective C₄ acid decarboxylation types. The outline of the photosynthetic carbon reduction (PCR or Kranz) bundle sheath and the position of the PCR cell chloroplasts in x Cynochloris are intermediate between those of the parental species, C. macivorii being more like Cynodon dactylon and x C. reynoldensis more like Chloris spp. The PCR chloroplast shape in x C. macivorii and x C. reynoldensis is like that of Cynodon dactylon and Chloris spp., respectively. Differences between the hybrids in their enzyme activities complement these structural differences: x C. macivorii has more NAD—ME and less PCK activity than x C. reynoldensis , although in both species PCK activity is the greater. Both hybrids, however, have a suberized lamella in PCR cell walls as do Chloris spp. The close taxonomic relationship between Cynodon and Chloris make these genera especially suitable for reciprocal crossing experiments aimed at increasing understanding of the genetic relationships between subtypes of the C₄ photosynthetic pathway.     

New butenolides from the photoconductivity screening of Streptomyces antibioticus (Waksman and Woodruff) Waksman and Henrici 1948

Abstract Streptomyces antibioticus strain TÜ 99, from which a wide variety of active compounds had been isolated previously, was reinvestigated using an HPLC photoconductivity screening system. Four new compounds were isolated, characterized and their constitutions determined. All four were α,β-unsaturated γ-lactones; the most abundant compound 3 (C₁₀H₁₆O₄), as well as compound 1 (C₉H₁₄O₄) had a hydroxy group at C(5) of the lactone ring. The four lactones showed antibiotic activity against Pseudomonas aeruginosa and also a weak inhibition of the chitinase from Serratia marcescens .     

The role of NADH- and NADPH-linked acetoacetyl-CoA reductases in the poly-3-hydroxybutyrate synthesizing organism Alcaligenes eutrophus

Abstract Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Alcaligenes eutrophus . Incorporation of [1-¹⁴C]-acetyl-CoA into poly-3-hydroxybutyrate (PHB) by systems reconstituted from purified preparations of either 3-ketothiolase, AcAc-CoA reductase and PHB synthase, occurred only when NADPH-AcAc-CoA reductase was present. The NADH reductase was active with all of the d (−)- and l (+)-3-hydroxyacyl-CoA substrates tested (C₄-C₁₀), whereas the NADPH reductase was only active with d (−)-3-hydroxyacyl-CoAs (C₄-C₆). The products of AcAc-CoA reduction by the NADH- and NADPH-linked enzymes were l (+)-3-hydroxybutyryl-CoA and d (−)-3-hydroxybutyryl-CoA, respectively. The NADH-linked enzyme had an M _r of 150,000 (containing identical M _r 30,000 sub-units) and the NADPH-linked enzyme appeared to be a tetramer ( M _r 84,000) with identical sub-units ( M _r 23,000). K _m^app values of 22 μM and 5 μM for AcAc-CoA and 13 μM (NADH) and 19 μM (NADPH) for the coenzymes were determined for the NADH- and NADPH-linked enzymes, respectively.     

Diverse cuticular hydrocarbons from Australian canebeetles (Coleoptera: Scarabaeidae)

Abstract  Cuticular hydrocarbon components in beetles of six Australian melolonthines whose larvae damage sugarcane, Antitrogus parvulus (Britton), A. consanguineus (Blackburn), Lepidiota negatoria (Blackburn), L. picticollis (Lea), L. noxia (Britton) and Dermolepida alborhirtum (Arrow), are identified and compared. These species demonstrate species-specific cuticular hydrocarbon profiles with a number of unprecedented structures. Major components have been identified as polymethylated hydrocarbons, 3-methyl substituted n -alkanes, 9,10-allenes and the corresponding C9 alkenes. The similarity of these compounds shows some correlation with the phylogeny of the beetles, but two polymethylated C₂₂ hydrocarbons are unique to A. parvulus. One C₂₅ allene is shown to have a potential role in mate recognition in A. consanguineus.     

A survey of photosynthetic carbon metabolism in 4 ecotypes of Phragmites australis in northwest China: Leaf anatomy, ultrastructure, and activities of ribulose 1,5-bisphosphate carboxylase, phosphoenolpyruvate carboxylase and glycollate oxidase

Four ecotypes of Phragmites australis from different habitats in northwest China were examined to compare their photosynthetic characteristics. In a swamp ecotype, the Δ ¹³C value of leaf materials was −34.0‰, and bundle sheath cells contained a small amount of organelles and round-shaped chloroplasts, as being similar to typical C₃ plants. In a dune ecotype, the Δ ¹³C value was −20.9‰ and bundle sheath cells contained oval-shaped chloroplasts with poorly-developed grana. In light and heavy salt meadow ecotypes, Δ ¹³C values were −30.6‰ and −35.6‰, respectively. The shape of bundle sheath chloroplasts in the light salt meadow ecotype was intermediate between those of the swamp and dune ecotypes. Abundance of bundle sheath organelles in the heavy salt meadow ecotype was intermediate. The swamp ecotype had photosynthetic enzyme activities typical of C₃ type plants, whereas the dune ecotype had an increased activity of phosphoenolpyruvate carboxylase (PEPC), a key C₄ enzyme, and a decreased ribulose 1,5-bisphosphate carboxylase (Rubisco) activity. The light salt meadow and heavy salt meadow ecotypes had substantial activities of PEPC, which indicates potential for C₄ photosynthesis. These data suggest that this species evolved the C₃-like ecotype in swamp environments and the C₄-like C₃-C₄ intermediate in dune desert environments, and C₃-like C₃-C₄ intermediates in salt environments.     

Chromosome numbers of C3 and C4 variants within the species Alloteropsis sernialata (R.Br.) Hitchc. (Poaceae)

FREAN, M. L. & MARKS, E., 1988. Chromosome numbers of C₃ and C₄ variants within the species Alloteropais semialata (R.Br.) Hitch. (Poaceae). In a study of mid-lamina leaf sections, different variants of A. semialata were found to have C₃ or C₄ anatomy. The C₄ leaf showed a specialized photosynthetic vascular bundle sheath not present in the C₃ form. Chromosome counts made from pollen mother cell squashes showed that the C₃ form of A. semialata is a fertile diploid 2n = 2x = 18 and the C₄ form, a fertile allohexaploid 2n = 6x = 54. The cytological evidence suggests that the two forms should be considered as separate species.     

Unusual glycoconjugates in the oesophagus of a tilapine polyhybrid

The aim of this work is to elucidate the glycoconjugate composition of the secretory products of the oesophageal mucous cells in a tilapine polyhybrid. Lectin histochemistry gave evidence of the presence of β-galactose, α-N-acetylgalactosamine and sialic acid residues in the terminal position. The majority of sialic acid belongs to short side chains; a few sialic acid residues are acetylated at the C₇ and/or C₈ and/or C₉ level. The heterogeneity of the carbohydrate chains may mask potential receptor sites for micro-organisms and hamper the formation of multiple bonds.     

Carbon isotope ratios of plants and occurrences of C4 species under different soil moisture regimes in arid region of Northwest China

Carbon isotope ratio of leaf dry matter, δ ¹³C, was measured on species occurring within Baiyin desert community, consisting of valley, slope and ridge microhabitats, and within Shandan desert community, consisting of Gobi desert and seasonal flooded creek microhabitats, in Northwest China. δ ¹³C of C₃ species increased with a decrease in soil water availability, suggesting that water-use efficiency (WUE) increased with decreasing soil moisture, whereas for all C₄ species, δ ¹³C tended to decrease with decreasing soil water availability, suggesting that WUE also increased with decreasing soil moisture. Above results indicated that water-use pattern was conservative under drought for C₄ and C₃ plants. In this present study, C₄ species' occurrences within different microhabitats were investigated and C₄ plants were observed to be absent and/or scarce within relatively lower soil moisture microhabitats, whereas they occurred and/or even had a high abundance within relatively higher soil moisture microhabitats, suggesting limited moisture available was a key factor of limiting C₄ distribution in arid region of Northwest China.     

Inactivation of the streptokinase gene prevents Streptococcus equisimilis H46A from acquiring cell-associated plasmin activity in the presence of plasminogen

Abstract The effects of some physico-chemical parameters on production of extracellular α-L-arabinofuranosidase by Aspergillus nidulans were examined. Highest levels of α-L-arabinofuranosidase were generated with cultures grown on 1% (w/v) purified beet pulp arabinan at 30°C and at an initial pH of 7.0. The enzyme was shown to be very sensitive to the action of proteases. Zymogram overlay of a protein profile obtained by SDS-PAGE revealed the occurrence of a band ( M _r 36 000) exhibiting α-L-arabinofuranosidase activity. The isoelectric pH of the enzyme lay near 4.3. Temperature and pH optima for the activity of crude α-L-arabinofuranosidase preparations were 55°C and 5.5, respectively. Enzyme activity was greatly reduced by thiol reagents such as Hg²⁺ and p -hydroxymercuribenzoate and showed a K _m value of 2.7 mM on p -nitrophenyl α-L-arabinofuranoside as substrate.     

Polygalacturonase biosynthesis by Lactobacillus plantarum: effect of cultural conditions on enzyme production

Lactobacillus plantarum was found to produce extracellular polygalacturonase (EC 3.2.1.15.). Maximum enzyme production was obtained in a medium containing 0.5% glucose and 1.5% low methyl-pectin as inducer at 27°C at an initial pH of 6.8. Enzyme production was strongly inhibited by 5 μmol/l NiCl₂, 5 μmol/l CoCl₂, 5 μmol/l CuSO₄, and 10 μmol/l ZnCl₂. MnSO₄ and MgSO₄ at 200 μmol/l and 50 μmol/l respectively seemed to enhance enzyme biosynthesis. The optimal pH and temperature for enzyme activity were 4.5 and 30°C respectively. Enzyme production in batch culture accompanied growth.     

CMC-liquefying enzyme, a low molecular mass initial cellulose-decomposing cellulase responsible for fragmentation from Streptomyces sp. LX

Streptomyces sp. LX, newly isolated from soil, was shown to secrete a carboxylmethylcellulose (CMC)-liquefying enzyme that cleaves the CMC chains, releasing negligible reducing terminals. The new enzyme, named component C₂, was purified to homogeneity by dialysation. It has a molecular mass of 9·8 kDa. The pH optimum of the enzyme activity is 6·4 and its temperature optimum is 50°C. It retains full activity at pH 4–6·4 upon incubation at 50°C for 30 min. The enzyme has significant fragmentation activity on filter paper despite the absence of weight loss, release of reducing sugars and depolymerization during incubation with filter paper. The one-electron oxidative reaction is shown not to participate in the fragmentation of filter paper by enzyme C₂.