Synthesis and regulation of D-xylanase from Cellulomonas flavigena wild type and a mutant

The xylanolytic system from Cellulomonas flavigena was enhanced by adding cellulose to the growth medium. The Solka floc:xylan (60:40 w/w) mixture induced xylanase synthesis by more than 3-fold over that induced by growing C. flavigena, wild type and its mutant PN-120 on pure xylan. The hydrolysis pattern of sugar cane bagasse and xylan indicated the presence of debranching endo-;-xylanase activity.     

Molecular genetic evidence for the transfer of Oerskovia species into the genus Cellulomonas

A close genetic relationship among strains of Oerskovia turbata, O. xanthineolytica and various coryneforms is indicated by DNA-DNA reassociation studies. O. xanthineolytica shares high homology values (over 60%) with Cellulomonas cartae, Nocardia cellulans, Brevibacterium fermentans and Corynebacterium manihot. O. turbata and other cellulomonads show lower DNA homology values (20–25%) which are still high enough, however, to indicate a relationship at the genus level. Based on these data and supported by comparative analysis of the ribosomal 16 S RNA and the similarity of peptidoglycan types, the transfer of Oerskovia species into the genus Cellulomonas is justified.This paper is respectively dedicated to our teacher and mentor, Professor Dr. O. Kandler, on the occasion of his 60th birthday.     

Double Mutants of Cellulomonas biazotea for Production of Cellulases and Hemicellulases following Growth on Straw of a Perennial Grass

Summary Deoxyglucose-resistant mutants of Cellulomonas biazotea secreted elevated levels of cellulases and xylanases. The production of β-glucosidase in the constitutive mutant was increased 5-fold over its parent strain. This mutant showed an approximately 1.6-fold enhanced productivity of extracellular endo-glucanase following growth on Leptochloa fusca over the mutant parent. Extracellular production of xylanase, filter-paper cellulase (FPase) and endo-glucanase (CMCase) were also altered in the mutant. Maximum volumetric productivities for xylanase, β-xylosidase, FPase, β-glucosidase and endo-glucosidase were 451, 98, 80, 95, and 143 IU l⁻¹ h⁻¹ which were significantly more than their respective values from the parental strains. The enzyme preparation of the mutants exhibited improved saccharification of kallar grass straw.     

Helicoidal orientation of cellulose microfibrils in Nitella opaca internode cells: ultrastructure and computed theoretical effects of strain reorientation during wall growth

The ultrastructure of the mature internode cell wall of Nitella opaca is described. It is interpreted in terms of a helicoidal array of cellulose microfibrils set in a matrix. A helicoid is a multiple plywood made up of layers of parallel microfibrils. There is a progressive change in direction from ply to ply, giving rise to characteristic arced patterns in oblique sections. A critical tilting test, using an electron microscope fitted with a goniometric stage, showed the expected reversal of direction of the arced pattern. Nitella cell wall is thus more regularly structured than previous studies have shown. From a survey of the cell-wall literature, we show that such arced patterns are common. This indicates that the helicoidal structure may be more widespread than is generally realised, although numerous other cell walls show no signs of it. Nevertheless, there are examples in most major plant taxa, and in several types of cells, including wood tracheids. Most of the examples, however, need confirmation by tilting evidence. There are possible implications for wall morphogenesis. Helicoidal cell walls might arise by selfassembly via a liquid crystalline phase, since it is known that the cholesteric state is itself helicoidal. A computer graphics programme has been developed to plot the expected effects of growth strain on the patterns in oblique sections of helicoids with various original angles between consecutive layers. Herringbone patterns typical of crossed polylamellate texture can be generated in this way, indicating a possible mode of their formation.     

Kinetic of improved production and carboxymethyl cellulose hydrolysis by an endo-glucanase from a derepressed mutant of Cellulomonas biazotea

The maximum product yield of endo-glucanase (650 IU g(-1) substrate) from Cellulomonas biazotea mutant 51 Sm(r) was 1.5- to 2.5-fold more than was produced by the wild type cells and was twice that reported by previous researchers. Mutation substantially improved the enthalpy (DeltaH (*)) and entropy of activation (DeltaS (*)) for product formation, turnover number, specificity constant activation energy, free energies for transition state formation and substrate binding for CMC hydrolysis respectively.     

Cellobiose phosphorylase (EC 2.4.1.20) of Cellulomonas: occurrence,induction, and its role in cellobiose metabolism

The occurrence of cellobiose cleavage by phosphorolysis and by hydrolysis was investigated in Cellulomonas spec., C. uda, C. flavigena, and C. cartalyticum. Cellobiose phosphorylase (EC 2.4.1.20) was shown to be produced by Cellulomonas spec. when cellobiose or cellulose was used as sole source of energy and carbon but not with glycerol or glucose. Using inhibitors of protein synthesis as well as double labelling techniques it was shown that cellobiose phosphorylase is synthesized de novo in Cellulomonas spec. Aryl--D-glucosidase which was shown to be present in crude extracts of this microorganism as well is not involved in cellobiose cleavage.Abbreviations oNPGluc ortho-nitrophenyl--D-glucopyranoside - oNPGlucase ortho-nitrophenyl--D-glucopyranoside hydrolase (aryl--D-glucosidase) - CMC carboxymethyl-cellulose - CMCase carboxymethyl-cellulase - PAGE polyacrylamde disc gel electrophoresis Parts of this work were presented on the Herbsttagung der Gesellschaft für Biologische Chemie (Schimz et al. 1979) and on the 14th FEBS Meeting (Schimz et al. 1981)     

Effects of heavy metals on growth and ultrastructure ofChara vulgaris

Summary The toxicity of some heavy metals to the common macrophytic freshwater algaChara vulgaris was studied under laboratory conditions. For experiments, apical tips of algae containing two internodes were cultivated for fourteen days in the presence of various concentrations of cadmium, mercury or lead (as triethyl lead or lead nitrate). Fifty percent growth inhibition occurred with concentrations of 8.5×10^–8 M (9.5 ppb) cadmium, 7.5×10^–7M (150ppb) mercury, 1.6×10^–6 M (330ppb) organic lead or 4× 10^–5 M (8000 ppb) inorganic lead. Sublethal concentrations of these metals caused alterations in the fine structure of internodal cells which turned out to be at least partly metal-specific or in the case of lead, the effects depended on whether the lead was ionic or organically bound. Cadmium and inorganic lead induced disorders of cell wall microfibrils which resulted in local wall protuberances. Mercury affected the chloroplasts which mostly showed considerably increased grana stacks. In addition, mercury caused a dilation of the endoplasmic reticulum and of the mitochondrial tubuli. Organic lead damaged the membrane system of chloroplasts; sheet- or tubule-like thylakoids were disarranged and showed whorl-like structures. At higher concentrations of organic lead, tubular invaginations of the plasmalemma (charasomes) disappeared. The fine structure of nuclei was not altered by any of the metals.     

Characterization of a novel cellulose synthesis inhibitor

The physiological effects of an experimental herbicide and cellulose synthesis inhibitor, N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine, called AE F150944, are described. In the aminotriazine molecular class, AE F150944 is structurally distinct from other known cellulose synthesis inhibitors. It specifically inhibits crystalline cellulose synthesis in plants without affecting other processes that were tested. The effects of AE F150944 on dicotyledonous plants were tested on cultured mesophyll cells of Zinnia elegans L. cv. Envy, which can be selectively induced to expand via primary wall synthesis or to differentiate into tracheary elements via secondary wall synthesis. The IC₅₀ values during primary and secondary wall synthesis in Z. elegans were 3.91×10^–8 M and 3.67×10^–9 M, respectively. The IC₅₀ in suspension cultures of the monocot Sorghum halapense (L.) Pers., which were dividing and synthesizing primary walls, was 1.67×10^–10 M. At maximally inhibitory concentrations, 18–33% residual crystalline cellulose synthesis activity remained, with the most residual activity observed during primary wall synthesis in Z. elegans. Addition to Z. elegans cells of two other cellulose synthesis inhibitors, 1 M 2,6-dichlorobenzonitrile and isoxaben, along with AE F150944 did not eliminate the residual cellulose synthesis, indicating little synergy between the three inhibitors. In differentiating tracheary elements, AE F150944 inhibited the deposition of detectable cellulose into patterned secondary wall thickenings, which was correlated with delocalization of lignin as described previously for 2, 6-dichlorobenzonitrile. Freeze-fracture electron microscopy showed that the plasma membrane below the patterned thickenings of AE F150944-treated tracheary elements was depleted of cellulose-synthase-containing rosettes, which appeared to be inserted intact into the plasma membrane followed by their rapid disaggregation. AE F150944 also inhibited cellulose-dependent growth in the rosette-containing alga, Spirogyra pratensis, but it did not inhibit cellulose synthesis in Acetobacter xylinum or Dictyostelium discoideum, both of which synthesize cellulose via linear terminal complexes. Therefore, AE F150944 may inhibit crystalline cellulose synthesis by destabilizing plasma membrane rosettes.Abbreviations AE F150944 N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine - CBI cellulose biosynthesis inhibiting - CGA CGA 325615, 1-cyclohexyl-5-(2,3,4,5,6-pentafluorophenoxy)-14,2,4,6-thiatriazin-3-amine - DCB 2,6-dichlorobenzonitrile - TE tracheary element     

Effect of cobalt and nickel on growth and carboxymethyl cellulase activity ofCellulomonas spp

ACellulomonas spp isolated from soil produced carboxymethyl cellulase (CMCase) and xylanase enzymes. Cobalt (0.1mm) and nickel (0.1mm) decreased the growth rate ofCellulomonas spp. These metal ions activated CMCase activity but not xylanase activity; cobalt being the greater stimulatory ion than nickel. A predominant long lag phase was observed in adapted cells when compared with the non-adapted cells. However, the growth level of control cells was never obtained by the adapted cells.     

Effects of surfactants on cell growth and pigment production in suspension cultures ofPerilla frutescens

The effects of surfactants, adecanol LG-294 and silicone A, on anthocyanin accumulation and the growth ofPerilla frutescens cells in suspension cultures were studied. Production of the red pigment was remarkably reduced from about 1.9 g/l to 0.4 g/l by adecanol LG-294 at 0.06 ml/l but not by silicone A up to 0.4 ml/l. Several repeated shake-flask cultures also demonstrated no adverse effects of silicone A on the metabolite accumulation by the suspended cells. Furthermore, the addition of silicone A to a culture in a stirred bioreactor produced a three-fold higher growth rate and a seven-fold increase in anthocyanin compared with surfactant-free cultures. The improvement was due to the substantial reduction or prevention of foaming and of cell adhesion to the bioreactor wall.     

Antibiotic inhibition of pectolytic and cellulolytic enzyme activity in twoFusarium species

Among all antibiotics tested, amoxycillin (500 ppm) completely inhibited the polygalacturonase and pectinmethylgalacturonase enzyme activity inF. oxysporum; none of the antibiotics did so inF. moniliforme. No antibiotic completely inhibited the cellulase activity in both test organisms, however, amoxycillin was better than other antibiotics in inhibiting the cellulase activity in both the organisms.     

Effects of heavy metals on pollen tube growth and ultrastructure

Summary The influence of different concentrations of the heavy metals cadmium (Cd²⁺), cobalt (Co²⁺), copper (Cu²⁺), iron (Fe²⁺ and Fe³⁺), mercury (Hg²⁺), manganese (Mn²⁺), and zinc (Zn²⁺), plus aluminium (Al³⁺) (a toxic metal in polluted areas), on pollen germination and tube growth ofLilium longiflorum was investigated using light microscopy. Effects could be observed with 3 M and 100 M of heavy metal, added as chloride salts to the medium. Cd²⁺, Cu²⁺, and Hg²⁺, showed the greatest toxicity, whereas germination and growth rate was less affected by Mn²⁺. Affected tubes showed swelling of the tip region. Tubes treated with Cd²⁺, Co²⁺, Fe²⁺, Fe³⁺, Hg²⁺, and Mn²⁺ were also prepared for ultrastructural studies. In all cases, the main effect was abnormal cell wall organization, mostly at the tip, where round, fibrillar aggregates, the shape and size of secretory Golgi vesicles were formed. They built up a loose network which could be up to 10 m thick compared to untreated tubes where the cell wall was composed of thin layers of long fibrils and about 100 nm thick. Cd²⁺ was the only metal which produced effects at the intracellular level: organelle distribution within the tip region appeared disorganized. A general mechanism of heavy metal action on pollen tube growth is discussed.     

Biohydrogen production from cellulosic hydrolysate produced via temperature-shift-enhanced bacterial cellulose hydrolysis

A “temperature-shift” strategy was developed to improve reducing sugar production from bacterial hydrolysis of cellulosic materials. In this strategy, production of cellulolytic enzymes with Cellulomonas uda E3-01 was promoted at a preferable temperature (35 °C), while more efficient enzymatic cellulose hydrolysis was achieved under an elevated culture temperature (45 °C), at which cell growth was inhibited to avoid consumption of reducing sugar. This temperature-shift strategy was shown to markedly increase the reducing sugar (especially, monosaccharide and disaccharide) concentration in the hydrolysate while hydrolyzing pure (carboxymethyl-cellulose, xylan, avicel and cellobiose) and natural (rice husk, rice straw, bagasse and Napier-grass) cellulosic materials. The cellulosic hydrolysates from CMC and xylan were successfully converted to H₂ via dark fermentation with Clostridium butyricum CGS5, attaining a maximum hydrogen yield of 4.79 mmol H₂/g reducing sugar.     

Cellulase production and activity in a species ofCladosporium

ACladosporium species produced large amounts of cellulase enzyme components when grown in shake-culture with medium containing carboxymethylcellulose. There was significantly less activity when Avicel, filter paper or cotton were used as substrates. KNO₃ was better than NH₄Cl or urea for the production of cellulase. Tween 80 at 0.1% (w/v) increased the production of cellulase by 1.5 to 4.5-fold. All the cellulase components were optimally active in the assay at pH 5.0 and 60°C.     

Effect of <Emphasis Type="Italic">poxB</Emphasis> gene knockout on metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis> based on growth characteristics and enzyme activities

The effect of poxB gene knockout on metabolism in Escherichia coli was investigated in the present paper based on the growth characteristics and the activities of the enzymes involved in the central metabolic pathways. The absence of pyruvate oxidase reduced the glucose uptake rate and cell growth rate, and increased O₂ consumption and CO₂ evolution. The enzyme assay results showed that although glucokinase activity increased, the flux through glycolysis was reduced due to the down-regulation of the other glycolytic enzymes such as 6-phosphofructosekinase and fructose bisphosphate aldolase in the poxB mutant. TCA cycle enzymes such as citrate synthase and malate dehydrogenase were repressed in the poxB mutant when the cells were cultivated in LB medium. The pyruvate oxidase mutation also resulted in the activation of glucose-6-phosphate dehydrogenase and acetyl-CoA synthetase. All these results suggest that pyruvate oxidase is not only a stationary-phase enzyme as previously known, and that the removal of the poxB gene affects the central metabolism at the enzyme level in E. coli.     

Congo red absorption and cellulose synthesis by Rhizobiaceae

Congo red uptake by Rhizobium colonies from yeast extract-mannitol-mineral salts-Congo red-agar plates was related with the cellulose content in the cell capsule of the bacteria.     

Saccharification of native and degraded cotton cellulose and commercial microcrystalline cellulose by Trichoderma viride cellobiohydrolase I

The degree of polymerization of samples of acid degraded cotton cellulose has no appreciable influence on the saccharification by cellobiohydrolase I from Trichoderma viride. The increase in the number of cellulose molecule ends, achieved by a 30-fold decrease in molecular weight, does not produce the effect which could be expected for a pure end-wise mode of action of this exoglucanase. Microcrystalline celluloses saccharified by the same enzyme yield considerably more reducing sugars than cotton cellulose, either with a similar degree of polymerization or one of about 7000. It appears, therefore, that the difference in the susceptibility of the commercial substrates is not a consequence of their low degree of polymerization.     

On the polarity of cellulose in the cell wall of Valonia

The orientation of the triclinic phase of cellulose in the cell wall of Valonia ventricosa J. Agardh was investigated by X-ray- and electron-diffraction analysis. In addition to the well-documented uniplanar-axial organization of the cell wall which requires that the a ^* axis should be always perpendicular to the wall surface, the direction of this axis was also found to be pointing outward from the plasma membrane side of the wall. This unidirectionality was persistent throughout the various layers that constitute the cell wall and also for the three microfibrillar orientations that occur in Valonia cell walls. The unidirectionality of the a ^* axis indicates, in particular, that the Valonia cellulose microfibrils are not twisted along their axis. These observations are consistent with a cellulose biosynthetic scheme where a close association exists between terminal-complex orientations and those of the cellulose microfibrils. In this context, the unidirectionality of the a ^* axis of cellulose seems to be related to the restricted mobility of the terminal complexes which are able to slide in the plasma membrane but not to rotate along their long axis.Abbreviations TC terminal complex This work was initiated during a visit of J.F.R at Grenoble in the framework of a France-Québec exchange program. J.S. was recipient of a CNRS fellowship. The diagram in Fig. 8 was kindly drawn for us by Miss Yukie Saito from the Department of Forest Products, the University of Tokyo.     

Effects of acidity on the growth of two Euglena species

Our study separates the effects of elevated protons (at pH <3) and elevated metals (Al, Cd, Cu, Fe, Ni, Zn) on the growth of E. mutabilis Schmitz, a pioneering phototroph in acid mine drainage (AMD) and E. gracilis Klebs, a closely-related species rarely found in severely AMD-impacted sites. Both species were acid tolerant, growing optimally at pH 2.5–7. At pH values typical of AMD (pH 2.5–4) in the absence of elevated metals, E. gracilis outcompeted E. mutabilis (growth rates of 1.0 and 0.8 div d^–1, respectively). Relative metal toxicities were evaluated based on the Effective Exposure causing 50% growth reduction (= EE50). With total metal additions similar to AMD levels, E. mutabilis demonstrated significantly greater tolerance to all metals, except Cu. E. gracilis showed two-fold higher tolerance to Cu²⁺ than E. mutabilis (EE50 of 91.6 vs. 45.7 pmol cell^–1). The EE50 for Zn²⁺ was similar for both species (368 pmol cell^–1 for E. gracilis and 423 pmol cell^–1 for E. mutabilis). With Cd and Ni, E. mutabilis tolerated an order of magnitude higher exposure than E. gracilis(EE50 of 1.6 vs. 0.2 pmol Cd²⁺ cell^–1; EE50 of 942 vs. 87 pmol Ni²⁺ cell^–1). Al and Fe were tolerated at high total metal concentrations (up to 100 mM) by E. mutabilis, but toxicity was evident with E. gracilisat much lower levels. E. mutabilis grew at double the Al³⁺ exposure tolerated by E. gracilis (EE50 of 398 vs. 188 pmol Al³⁺ cell^–1). There was an 18-fold difference in Fe tolerance levels between E. mutabilis and E. gracilis with EE50s of 8773 and 502 pmol Fe²⁺ cell^–1, respectively. We conclude that differential metal tolerance, particularly to Fe²⁺, accounts for the mutually exclusive distribution of E. gracilis and E. mutabilis in AMD-impacted habitats.     

一株玉米秸秆纤维素分解菌株的分离鉴定及酶学性质

【目的】筛选分离可以分解玉米秸秆纤维素的菌株。【方法】采用平板稀释法从土样中分离纯化得到能够分解玉米秸秆纤维素的菌株,对分离得到的菌株进行生理生化及分子鉴定,同时以菌株Penicillium spp.(CICC40361)作为对照,比较菌株纤维素酶和木质素酶的活性,研究不同因素对菌株纤维素酶活力的影响,确定菌株纤维素酶动力学常数Km值。【结果】分离得到的菌株命名为PL2#,经生理生化及分子鉴定后,确定菌株PL2#为羊毛状青霉(Penicillium lanosum)。菌株PL2#的纤维素酶和木质素酶活力高于对照菌株Penicillium spp.;菌株PL2#的最优酶活测定条件为:1%CMC底物浓度,pH 4.8,50°C,酶反应时间60 min以及2 m L DNS添加量。【结论】羊毛状青霉(Penicillium lanosum)菌株PL2#比对照菌株Penicillium spp.具有更好地降解玉米秸秆纤维素的性能。