Cell wall invertases from sugar cane

Invertases in sugar cane leaf sheaths are firmly bound to the cell wall. They consist of three different enzymes distinguished on the basis of optimum pH, K_m and the response to inhibitors. This invertase complex differs from the single enzyme described for immature and mature stalk tissues.     

A regulatory invertase from sugar cane leaf-sheaths

A soluble β-fructofuranosidase was isolated from sugar cane leaf-sheaths. The enzyme attacks sucrose with an activation energy of 5700 cal/mol above 30° and 17 000 cal/mol below 30°. The enzyme was inhibited by the reaction products. Glucose is a simple non-competitive inhibitor, but fructose is a competitive inhibitor. Kinetic studies using double reciprocal plots and replots of 1/K_i, slope vs inhibitor concentration showed that fructose binds to two interacting sites of the enzyme. Per cent residual activity plotted against inhibitor concentration, and Hill plots confirmed the regulatory properties of the invertase. n was found to be close to 2, the number of binding sites established with the double reciprocal method. The tissue and cellular levels of sucrose, fructose and glucose were measured. Fructose was found at inhibitory concentrations confirming that the activity of the enzyme is probably modulated by the hexose pool of the leaf-sheaths.     

Cell wall invertases of sugar cane

A study of the cell wall invertases in the different organs of a sugar cane cultivar has been undertaken. The enzymes could not be separated from the cell wall. They are compared on the basis of optimum pH, K_m, the effect of various chemicals and the substrate specificities of the preparations. According to the results each organ appears to possess a set of cell wall invertases with predominance of a different activity in each case.     

β-galactosidase from sugar cane

β-Galactosidase activity occurs in all of the organs of the sugar cane plant, and is also of general occurrence among different cultivars and species. Most of the activity was associated with the cell wall, and only ca 12–16% was an intracellular form. Both activities posess similar optimum pH and K_itm both are activated by Mn²⁺ and ethanol, and inhibited by Hg²⁺, and both attack the same substrates.     

Characterization and inhibition of invertases in sugar cane juice

(NH₄)₂SO₄ fractionation followed by Sephadex G-200 chromatography of sugar cane juice gave an acid invertase with MW of 380 000 and 23.5% carbohydrate and a neutral invertase with MW of 66 000 and 22% carbohydrate. For acid invertase, K_m is 2.8 mM and V_max is 2.7 μmol sucrose hydrolysed/hr/mg protein. For neutral invertase, K_m and V_max are 0.32 mM and 2.8 μmol hydrolysed/hr/mg protein, respectively. Inhibition of both invertases by either lauryl sulfate or metasilicate is not competitive.     

Sugar transformations associated with hexose transport in immature internodal tissue of sugar cane

After a 15 sec incubation in d-glucose-¹⁴C(U), 53–70% of the intracellular radioactivity in immature internodal tissue of sugarcane was in glucose-6-phosphate, and the remainder was in free glucose. Two unmetabolized glucose analogs, 2-deoxy-d-glucosce and 3-O-methyl-d-glucose, were transported at rates comparable to glucose but neither of these analogs was phosphorylated. Doubly-labeled d-glucose-1-¹⁴C-6-phosphate-³²P was dephosphorylated prior to deposition in the inner space, and ¹⁴C was transported into this tissue twice as rapidly as ³²P. It was also shown that ³²P in exogenously supplied glucose-6-³²P was not the source of phosphate for the intracellular synthesis of glucose-6-P. Galactose transport was similar to that of glucose in that the first major product recovered intracellularly was a phosphorylated sugar, i.e. ¹⁴C-galactose-1-P, when the tissue was incubated in d-galactose-¹⁴C(U). Although fructose, glucose, and galactose competed for transport into this tissue, free fructose and glucose predominated in the tissue extract after a 15-sce incubation in d-fructose-¹⁴C(U). This contrasted sharply, with the products of ¹⁴C-glucose transport which were comprised of phosphorylated sugars after 15 sec.     

o-Diphenol: Oxygen oxidoreductase from leaves of sugar cane

A non-particulate o-diphenol: O₂ oxidoreductase (phenolase) has been isolated from leaves of sugar cane. Gel filtration produced two fractions MW 32000 and 130000. The preferred substrate was chlorogenic acid. Other o-diphenols (caffeic acid, catechol, pyrogallol, dihydroxyphenylalanine) all of which were slowly oxidized when tested alone, increased the rates of O₂ consumption obtained with catalytic amounts of chlorogenic acid. Both enzyme fractions were inhibited by thiols; thioglycollate, which acted in a non-competitive manner, was most effective.     

Ammonium heptamolybdate,an inhibitor of plant invertases

Ammonium heptamolybdate was an inhibitor of plant invertases. The inhibition was a linear mixed type and the constants K_i and aK_i were determined. α- and β-glycerophosphate, 2,3-diphosphoglycerate, glucose-1-phosphate, phosphoenolpyruvate, pyruvate and malate suppressed the inhibition. The curves of enzyme recovery against the concentrations of these activators were sigmoid. UV spectrophotometry showed complex formation between inhibitor and each activator, and indicated that sucrose did not form a complex with the inhibitor. Consequently, heptamolybdate is postulated to act by a reversible binding to the enzyme.     

新台糖25号甘蔗愈伤组织诱导试验

以甘蔗新台糖25号心叶为试材,分别在添加2,4-D1．5、2．5、3．5、4．5、5．5、6．5mg/L6 个处理的MS培养基上培养。结果表明,2,4-D对愈伤组织诱导及其继代培养有显著影响,诱导效果以2,4-D2．5mg/L为佳。     

Root dynamics in plant and ratoon crops of sugar cane

The root system of a sugar cane crop on an Ultisol in northeastern Brazil was examined throughout the plant and first ratoon crop cycles, using both coring and minirhizotron methods. Total root masses (living plus dead, 0.9–1.1 kg m^-2) and live root lengths (14.0–17.5 km m^-2) were greater during the ratoon cycle than at the end of the plant cane cycle (0.75 kg m^-2 and 13.8 km m^-2, respectively). Root die-back during the two weeks following ratoon harvest was estimated to be 0.15 kg m^-2, about 17% of the total root mass. Root die-back after the plant cane harvest was lower because fire was not used at this harvest and soil humidity was higher under the accumulated litter. A small amount of fine roots proliferated in the litter layer, amounting to 1% of the total mass and 3% of the total length. Root turnover could not be accurately assessed from minirhizotron observations due to variation in the relationship between coring data and the minirhizotron data with both time and soil depth.     

Environmental parameters affecting xylitol production from sugar cane bagasse hemicellulosic hydrolyzate by Candida guilliermondii

The bioconversion of xylose to xylitol by Candida guilliermondii FTI 20037 cultivated in sugar cane bagasse hemicellulosic hydrolyzate was influenced by cell inoculum level, age of inoculum and hydrolyzate concentration. The maximum xylitol productivity (0.75 g L⁻¹ h⁻¹) occurred in tests carried out with hydrolyzate containing 54.5 g L⁻¹ of xylose, using 3.0 g L⁻¹ of a 24-h-old inoculum. Xylitol productivity and cell concentration decreased with hydrolyzate containing 74.2 g L⁻¹ of xylose. Received 02 February 1996/ Accepted in revised form 15 November 1996     

Simultaneous saccharification and fermentation of pretreated sugar cane leaves to ethanol

The simultaneous saccharification and fermentation (SSF) of pretreated sugar cane leaves to produce ethanol using a cellulolytic enzyme complex from Trichoderma reesei QM 9414 and Saccharomyces cerevisiae NRRL-Y-132 was optimized. Enzymic saccharification parameters were evaluated prior to SSF studies. A 92% conversion of 2·5% substrate (alkaline hydrogen peroxide pretreated) to sugars was achieved at 50°C and pH 4·5, using T. reesei cellulase (40 FPU/g substrate), in 48 h. The pretreated substrate was then subjected to an SSF process using the cellulase complex and S. cerevisiae cells. Optimization of the SSF system is described.     

Technical approaches to inoculate micropropagated sugar cane plants were Acetobacter diazotrophicus

Micropropagated plantlets of sugar cane were inoculated with the N2-fixing bacterium Acetobacter diazotrophicus. Various modifications on the basic plant culture medium MS were made for the plant/bacteria association. The protocol required the inoculation of the bacteria at the end of the rooting period in a medium without hormones or vitamins, and with the concentration of sugar and mineral nutrients reduced by a factor of 10. Individual plants were inoculated with A. diazotrophicus and maintained under the appropriate light and temperature condition used for micropropagation up to 7 days. The system favored the infection and the establishment of the bacteria within the plant tissue. Bacteria colonized the plant tissue and accumulated in inter-cellular cavities and the region of lateral root emergence and also colonizes the xylem vessels. The inoculated plantlets were subsequently transferred to the acclimatization phase and after 30 days it was possible to isolate the bacteria from plant tissue. This protocol permitted studies of infection and comparison among strains.     

Accumulation of recombinant cellobiohydrolase and endoglucanase in the leaves of mature transgenic sugar cane

A major strategic goal in making ethanol from lignocellulosic biomass a cost-competitive liquid transport fuel is to reduce the cost of production of cellulolytic enzymes that hydrolyse lignocellulosic substrates to fermentable sugars. Current production systems for these enzymes, namely microbes, are not economic. One way to substantially reduce production costs is to express cellulolytic enzymes in plants at levels that are high enough to hydrolyse lignocellulosic biomass. Sugar cane fibre (bagasse) is the most promising lignocellulosic feedstock for conversion to ethanol in the tropics and subtropics. Cellulolytic enzyme production in sugar cane will have a substantial impact on the economics of lignocellulosic ethanol production from bagasse. We therefore generated transgenic sugar cane accumulating three cellulolytic enzymes, fungal cellobiohydrolase I (CBH I), CBH II and bacterial endoglucanase (EG), in leaves using the maize PepC promoter as an alternative to maize Ubi1 for controlling transgene expression. Different subcellular targeting signals were shown to have a substantial impact on the accumulation of these enzymes; the CBHs and EG accumulated to higher levels when fused to a vacuolar-sorting determinant than to an endoplasmic reticulum-retention signal, while EG was produced in the largest amounts when fused to a chloroplast-targeting signal. These results are the first demonstration of the expression and accumulation of recombinant CBH I, CBH II and EG in sugar cane and represent a significant first step towards the optimization of cellulolytic enzyme expression in sugar cane for the economic production of lignocellulosic ethanol.     

Enzymatic saccharification of sugar cane bagasse by continuous xylanase and cellulase production from cellulomonas flavigena PR‐22

Cellulase (CMCase) and xylanase enzyme production and saccharification of sugar cane bagasse were coupled into two stages and named enzyme production and sugar cane bagasse saccharification. The performance of Cellulomonas flavigena (Cf) PR‐22 cultured in a bubble column reactor (BCR) was compared to that in a stirred tank reactor (STR). Cells cultured in the BCR presented higher yields and productivity of both CMCase and xylanase activities than those grown in the STR configuration. A continuous culture with Cf PR‐22 was run in the BCR using 1% alkali‐pretreated sugar cane bagasse and mineral media, at dilution rates ranging from 0.04 to 0.22 1/h. The highest enzymatic productivity values were found at 0.08 1/h with 1846.4 ± 126.4 and 101.6 ± 5.6 U/L·h for xylanase and CMCase, respectively. Effluent from the BCR in steady state was transferred to an enzymatic reactor operated in fed‐batch mode with an initial load of 75 g of pretreated sugar cane bagasse; saccharification was then performed in an STR at 55°C and 300 rpm for 90 h. The constant addition of fresh enzyme as well as the increase in time of contact with the substrate increased the total soluble sugar concentration 83% compared to the value obtained in a batch enzymatic reactor. This advantageous strategy may be used for industrial enzyme pretreatment and saccharification of lignocellulosic wastes to be used in bioethanol and chemicals production from lignocellulose. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:321–326, 2016     

Hydrocarbons in leaf waxes of Saccharum and related genera

The composition of the n-alkanes in the leaf waxes of over 80 clones of Saccharum officinarum, S. edule, S. robustum, S. spontaneum and from a number of related species have been compared by GLC. The waxes contain predominantly odd alkanes, C₂₇–C₃₅, the major components being C₂₉ and C₃₁. In a number of clones, particularly of S. edule, a homologous series of alkenes was also present. No chemotaxonomic relationship could be derived from the compositions as the intraspecific variation was greater than the interspecific variations.     

Production of cellulases and hemicellulases by Penicillium echinulatum grown on pretreated sugar cane bagasse and wheat bran in solid-state fermentation

AIM: To evaluate the solid-state fermentation (SSF) production of cellulase and hemicellulases (xylanases), by Penicillium echinulatum 9A02S1, in experiments carried out with different concentrations of the pretreated sugar cane bagasse (PSCB) and wheat bran (WB). METHODS AND RESULTS: This study reports the production of xylanolytic and cellulolytic enzymes by P. echinulatum 9A02S1 using a cheap medium containing PSCB and WB under SSF. The highest amounts of filter paper activity (FPA) could be measured on mixtures of PSCB and WB (32.89 +/- 1.90 U gdm(-1)). The highest beta-glucosidase activity was 58.95 +/- 2.58 U gdm(-1) on the fourth day. The highest activity for endoglucanases was 282.36 +/- 1.23 U gdm(-1) on the fourth day, and for xylanases the activity was around 10 U gdm(-1) from the second to the fourth day. CONCLUSIONS: The present work has established the potential of P. echinulatum for FPA, endoglucanase, beta-glucosidase and xylanase productions in SSF, indicating that WB may be partially substituted by PSCB. SIGNIFICANCE AND IMPACT OF THE STUDY: The incorporation of cheap sources, such as sugar cane bagasse, into media for the production of lignocellulose enzymes should help decrease the production costs of enzymatic complexes that can hydrolyse lignocellulose residues for the formation of fermented syrups, thus contributing to the economic production of bioethanol.     

Sugar transport: Occurrence of trehalase activity in sugar cane

Summary Trehalase activity was detected in extracts of roots, leaves, and stalk tissue from sugar cane. The enzyme was not bound to cell particulates, and had a pH optimum of 6.2 and a Michaelis constant for trehalose of 1×10^-4 M. The level of enzyme detected in mature stalk tissue was too low to account for glucose transport into tissue slices. The enzyme level was high in immature stalk tissue in which the vacuolar sugar pool turns over rapidly. Trehalose synthesis and breakdown may be part of a system for transport of hexose out from the vacuole.