On the mechanism of sucrose utilisation by mycelial felts of Rhizoctonia solani

Summary The utilisation of sucrose and its constituent monosaccharides, as well as of maltose and raffinose by mycelial felts of Rhizoctonia solani was studied with a view to throwing some light on the mechanism of utilisation of sucrose by the fungal mats being tested. The results obtained suggest that sucrose is, most probably, utilised through a process of hydrolysis by a -heterofructosidase enzyme.     

Enhanced hydroxylation of imidacloprid by <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> upon addition of sucrose

Sucrose’s ability to promote the hydroxylation of imidacloprid (IMI) by bacterium Stenotrophomonas maltophilia strain CGMCC 1.1788 was examined. Both growing culture and resting cells could transform IMI into 5-hydroxy IMI. Adding 2% sucrose to the growing culture transformation broth and 5% sucrose to the resting cell transformation broth resulted in biotransformation yields, respectively, 2.5 and 9 times greater than without sucrose. In the growing culture transformation, sucrose increased biomass, which led to enhance hydroxylation of IMI. In the resting cell transformation, sucrose was used not as a carbon source but as an energy source for cofactor regeneration for hydroxylation of IMI. The hydroxylation activity of IMI was promoted eightfold by adding reduced nicotinamide adenine dinucleotide (NADH) to the cell-free extract. The hydroxylation of IMI was significantly inhibited by P450 inhibitor piperonyl butoxide. It seems that the hydroxylation of IMI by S. maltophilia CGMCC 1.1788 might proceed through a system by cooperating with P450 enzyme.     

Phosphorolytic cleavage of sucrose by sucrose-grown ruminal bacterium <Emphasis Type="Italic">Pseudobutyrivibrio ruminis</Emphasis> strain k3

Rumen bacterium Pseudobutyrivibrio ruminis strain k3 utilized over 90 % sucrose added to the growth medium as a sole carbon source. Zymographic studies of the bacterial cell extract revealed the presence of a single enzyme involved in sucrose digestion. Thin layer chromatography showed fructose and glucose-1-phosphate (Glc1P) as end products of the digestion of sucrose by identified enzyme. The activity of the enzyme depended on the presence of inorganic phosphate and was the highest at the concentration of phosphate 56 mmol/L. The enzyme was identified as the sucrose phosphorylase (EC 2.4.1.7) of molar mass ≈54 kDa and maximum activity at pH 6.0 and 45 °C. The calculated Michaelis constant (K _m) for Glc1P formation and release of fructose by partially purified enzyme were 4.4 and 8.56 mmol/L while the maximum velocities of the reaction (v _lim) were 1.19 and 0.64 μmol/L per mg protein per min, respectively.     

Biosynthesis of dextrans with different molecular weights by selecting the concentration of Leuconostoc mesenteroides B-512FMC dextransucrase, the sucrose concentration, and the temperature

Leuconostoc mesenteroides B-512FMC dextransucrase was found to synthesize dextrans of varying molecular weights by selecting the concentrations of dextransucrase and sucrose, as well as the temperature. Four enzyme concentrations (50, 10, 1.0, and 0.1 U/mL), five sucrose concentrations (20, 50, 100, 200 and 1000 mM), and two temperatures (20 °C and 30 °C) were studied. The highest amount of enzyme (50 U/mL), with the lowest concentration of sucrose (20 mM), and the lower temperature of 20 °C gave the lowest number-average molecular weight (MW_n) of 20,630 Da, respectively. As the sucrose concentration was increased, 50 mM, 100 mM, and 200 mM, the MW_n was 49,240 Da, 63,350 Da, and 126,720 Da, respectively. The next enzyme concentration (10 U/mL) gave a similar upward trend, starting at 73,130 Da and ending at 237,870 Da at 20 °C and 130,040 Da and ending at 415,770 Da at 30 °C. The upward trend continued for the 1.0 and 0.1 U/mL enzyme concentrations. An increase in the temperature had the overall effect of increasing the MW_n for each decreasing concentration of enzyme and increasing concentration of sucrose. For 0.1 U/mL and 1000 mM sucrose at 30 °C, the MW_n was 1,645,700 Da. The results of the study show that the molecular weights of the synthesized dextrans were inversely proportional to the concentration of the enzyme and directly proportional to the concentration of sucrose and the temperature.     

Probing enzyme–substrate interactions at the catalytic subsite of Leuconostoc mesenteroides sucrose phosphorylase with site-directed mutagenesis: the roles of Asp49 and Arg395

Abstract

Sucrose phosphorylase is a bacterial α-transglucosidase that catalyses glucosyl transfer from sucrose to phosphate, releasing d-fructose and α-d-glucose 1-phosphate as the product of the first (enzyme glucosylation) and second (enzyme deglucosylation) step of the enzymatic reaction, respectively. The transferred glucosyl moiety of sucrose is accommodated at the catalytic subsite of the phosphorylase through a network of charged hydrogen bonds whereby a highly conserved residue pair of Asp and Arg points towards the equatorial hydroxyl at C₄. To examine the role of this ‘hyperpolar’ binding site for the substrate 4-OH, we have mutated Asp⁴⁹ and Arg³⁹⁵ of Leuconostoc mesenteroides sucrose phosphorylase individually to Ala (D49A) and Leu (R395L), respectively, and also prepared an ‘uncharged’ double mutant harbouring both site-directed substitutions. The efficiency for enzyme glucosylation from sucrose was massively decreased in purified preparations of D49A (10⁷-fold) and R395L (10⁵-fold) as compared to wild-type enzyme. The double mutant was not active above the detection limit. Enzyme deglucosylation to phosphate proceeded relatively efficient in D49A as well as R395L, about 500-fold less than in the wild-type phosphorylase. Substrate inhibition by phosphate and a loss in selectivity for reaction with phosphate as compared to water were new features in the two mutants. Asp⁴⁹ and Arg³⁹⁵ are both essential in the catalytic reaction of L. mesenteroides sucrose phosphorylase.     

Extracellular invertase from Aspergillus flavus

An extracellular invertase was induced in cultures of Aspergillus flavus Link during growth in liquid medium that contained sucrose as the sole carbon source. Synthesis of this enzyme was repressed by the addition of glucose or fructose to sucrose-metabolizing cells, and was induced in a glucose or fructose-metabolizing culture by the addition of sucrose. A. flavus invertase had a pH optimum of 6.0 and an apparent Km of approximately 133 mM for sucrose. The enzyme required potassium phosphate for maximum activity, optimum concentration being 250 mM. The enzyme was partially purified by ammonium sulphate precipitation followed by dialysis and separated by molecular exclusion into three components with molecular weights ranging from approximately 40,000 to 55,000.     

Sucrose phosphorylase: a powerful transglucosylation catalyst for synthesis of α-D-glucosides as industrial fine chemicals

Abstract

Sucrose phosphorylase is a bacterial transglucosidase that catalyzes conversion of sucrose and phosphate into α-D-glucose-1-phosphate and D-fructose. The enzyme utilizes a glycoside hydrolase-like double displacement mechanism that involves a catalytically competent β-glucosyl enzyme intermediate. In addition to reaction with phosphate, glucosylated sucrose phosphorylase can undergo hydrolysis to yield α-D-glucose or it can decompose via glucosyl transfer to a hydroxy group in suitable acceptor molecules, giving new α-D-glucosidic products. The glucosyl acceptor specificity of sucrose phosphorylase is reviewed, focusing on applications of the enzyme in glucoside synthesis. Polyhydroxylated compounds such as sugars and sugar alcohols are often glucosylated efficiently. Aryl alcohols and different carboxylic acids also serve as acceptors for enzymatic transglucosylation. The natural osmolyte 2-O-(α-D-glucopyranosyl)-sn-glycerol (GG) was prepared by regioselective glucosylation of glycerol from sucrose using the phosphorylase from Leuconostoc mesenteroides. An industrial process for production of GG as active ingredient of cosmetic formulations has been recently developed. General advantages of sucrose phosphorylase as a transglucosylation catalyst lie in the use of sucrose as a high-energy glucosyl donor and the usually weak hydrolase activity of the enzyme towards substrate and product.     

The occurrence and intracellular localization of sucrase activity in rat liver

The presence of an enzyme in rat liver which hydroluzes sucrose is demonstrated in this report. The hydrolysis of sucrose was studied in vivo after injecting [¹⁴C]sucrose into rats, and a method was developed for the extraction and analysis of the radioactive sugars stored within subcellular particles. The results show that, besides sucrose, glucose and fructose are also found in the lysosomal fraction of the liver homogenate. In vitro studies reveal the presence of a sucrase, although the activity of the enzyme is very low. Intracellular distribution studies indicate that sucrase is present in the lysosomes as well as in the microsomes, the microsomal enzyme having a pH optimum different from that of the lysosomal enzyme.     

Characterization of a novel low molecular weight sucrase from filamentous fungus Termitomyces clypeatus

An extracellular sucrase from the culture filtrate of filamentous basidiomycota Termitomyces clypeatus grown on high sucrose (5%, w/v) was purified by gel filtration chromatography, ion exchange chromatography and HPGPLC. The biochemical properties, molecular weight and conformation of sucrase produced were significantly different from the sucrase earlier purified from sucrose (1%, w/v) medium in the fungus. Purified sucrase was characterized as a low molecular weight protein of 13.5 kDa as approximated by SDS-PAGE and HPGPLC and exhibited predominantly random coil conformation in far-UV CD spectra. The enzyme was optimally active at 47 °C and pH 5.0. K_m and catalytic activity of the enzyme for sucrose were found to be 3.5 mM and 1.06 U/mg/mM, respectively. The enzyme was maximally active towards sucrose than to raffinose and sucrase activity was significantly inhibited by bivalent metal ions and reducing group agents. The results indicated that due to changes in aggregation pattern, molecular organization of purified sucrase, produced in high sucrose medium, was altered and was different from the previously reported enzyme. This is the first report of a sucrase of such low size showing activity.     

Purification and properties of sucrose synthetase from morning-glory callus cells

Sucrose synthetase was purified about 130-fold from morning-glory (Pharbitis nil Choisy cv. Murasaki) callus cells, and the properties of sucrose synthesis and cleavage activities of the enzyme were compared. The enzyme preparation gave a single band by disc electrophoresis. The molecular mass of the enzyme was estimated to be 4.2 × 10⁵ by gel filtration. The enzyme preparation gave two bands by SDS disc electrophoresis, suggesting the molecular mass of about 3.8 ×10⁴ and 7.0 × 10⁴. The pH optima of sucrose synthesis and cleavage activities of the enzyme were different from each other, giving pH 9.0 and pH 6.5 respectively. MgCl², MnCl² and CaCl² activated the sucrose synthesis activity about two times the normal rate and conversely inhibited the sucrose cleavage activity. F-6-P was not replaced by fructose. UDP was the only valuable substrate as a nucleotide diphosphate. The enzyme showed the negative ecoperativity effect of UDPG suggesting to be an allosteric enzyme. The Km values of sucrose and fructose were calculated to be 167 mM and 5 mM, respectively. UDP suggested substrate inhibition. The apparent equilibrium constant varied between 1 to 3. Based on these results, the role of the enzyme in the sucrose metabolism of morning-glory callus cells will be discussed.     

Hydrolysis of fish oil by hyperactivated rhizomucor miehei lipase immobilized by multipoint anion exchange

Rhizomucor miehei lipase (RML) is greatly hyperactivated (around 20‐ to 25‐fold toward small substrates) in the presence of sucrose laurate. Hyperactivation appears to be an intramolecular process because it is very similar for soluble enzymes and covalently immobilized derivatives. The hyperactivated enzyme was immobilized (in the presence of sucrose laurate) on cyanogen bromide‐activated Sepharose (very mild covalent immobilization through the amino terminal residue), on glyoxyl Sepharose (intense multipoint covalent immobilization through the region with the highest amount of Lys residues), and on different anion exchangers (by multipoint anionic exchange through the region with the highest density of negative charges). Covalent immobilization does not promote the fixation of the hyperactivated enzyme, but immobilization on Sepharose Q retains the hyperactivated enzyme even in the absence of a detergent. The hydrolysis of fish oils by these hyperactivated enzyme derivatives was sevenfold faster than by covalently immobilized derivatives and three and a half times faster than by the enzyme hyperactivated on octyl‐Sepharose. The open structure of the hyperactivated lipase is fairly exposed to the medium, and no steric hindrance should interfere with the hydrolysis of large substrates. These new hyperactivated derivatives seem to be more suitable for hydrolysis of oils by RML immobilized inside porous supports. In addition, the hyperactivated derivatives are fairly stable against heat and organic cosolvents. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Process control of an ethanol fermentation with an enzyme thermistor as a sucrose sensor

Summary An enzyme thermistor was used to monitor and control the sucrose concentration in a conversion of sucrose to ethanol with immobilized yeast. A continuous stirred tank reactor containing calcium alginate to entrap Saccharomyces cerevisiae was used. The enzyme thermistor was continuously measuring the sucrose concentration in the fermenter with an on-line arrangement giving stable and reproducible heat signals. The control of the sucrose concentration level was performed with an analogue PI-controller.     

Production, purification and characterization of an extracellular inulinase from Kluyveromyces marxianus var. bulgaricus

The yeast Kluyveromyces marxianus var. bulgaricus produced large amounts of extracellular inulinase activity when grown on inulin, sucrose, fructose and glucose as carbon source. This protein has been purified to homogeneity by using successive DEAE-Trisacryl Plus and Superose 6HR 10/30 columns. The purified enzyme showed a relative molecular weight of 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 77 kDa by gel filtration in Superose 6 HR 10/30. Analysis by SDS-PAGE showed a unique polypeptide band with Coomassie Blue stain and nondenaturing PAGE of the purified enzyme obtained from media with different carbon sources showed the band, too, when stained for glucose oxidase activity. The optimal hydrolysis temperature for sucrose, raffinose and inulin was 55°C and the optimal pH for sucrose was 4.75. The apparent K _m values for sucrose, raffinose and inulin are 4.58, 7.41 and 86.9 mg/ml, respectively. Thin layer chromatography showed that inulinase from K. marxianus var. bulgaricus was capable of hydrolyzing different substrates (sucrose, raffinose and inulin), releasing monosaccharides and oligosaccharides. The results obtained suggest the hypothesis that enzyme production was constitutive. Journal of Industrial Microbiology & Biotechnology (2000) 25, 63–69. Received 17 November 1999/ Accepted in revised form 30 May 2000     

Production of high-content fructo-oligosaccharides by an enzymatic system from Penicillium rugulosum

Summary The production of high-content fructo-oligosaccharides from sucrose by a crude FTF from a new strain of Penicillium isolated in our Laboratory was investigated. The optimum conditions for the production of the enzyme and for the enzymic reaction have been determined. It has been demonstrated that the crude enzyme acts as a mixed enzyme system of fructosyl transferase (FTF; Class 2 of Enzyme Nomenclature) and glycosidases (Class 3 of Enyme Nomenclature). Under optimum conditions: pH 5.5, temperature 55°C, sucrose concentration 750 g/l, enzyme concentration 5 FTF units/g sucrose, conversion yield up to 80% were obtained and high concentration of nystose (412 g/l) and fructofuranosyl-nystose (176 g/l) were accumulated.     

Thermal Destabilization of Stem Bromelain by Trehalose

Trehalose, a naturally occurring osmolyte, is considered as a universal protein stabilizer. We investigated the effect of the disaccharides, trehalose and sucrose, on the thermal stability and conformation of bromelain. To our surprise, bromelain in the presence of 1 M trehalose/sucrose was destabilized under thermal stress. The average Tm values as determined by UV spectroscopy and CD spectropolarimetry decreased by 5° and 7°C for bromelain in 1 M sucrose or trehalose solutions, respectively. The enzyme was also found to inactivate faster at 60°C in the presence of these osmolytes. The tertiary and secondary structure of bromelain undergoes small changes in the presence of sucrose/trehalose. Studies on the binding of these osmolytes with the native and the heat denatured enzyme revealed that sucrose/trehalose lead to preferential hydration of the denatured bromelain as compared to the native one, hence stabilizing more the denatured conformation. This is perhaps the first report on the destabilization of a protein by trehalose.     

Absence of turnover and futile cycling of sucrose in leaves of<Emphasis Type="Italic"> Lolium temulentum</Emphasis> L.: implications for metabolic compartmentation

To study the interdependence of sucrose accumulation and its hydrolyzing enzyme, soluble acid invertase (AI; EC 3.2.1.26), in fructan-accumulating temperate grasses and cereals, experiments were performed in which sucrose synthesis was abolished in leaves of Lolium temulentum by four independent inhibitory factors, each having a distinct mechanism of action. Trials in the light with mannose or vanadate and in the dark with anoxia or cyanide showed that previously accumulated sucrose was stable in the tissue over a 5- to 6-h period. Conversely, putatively vacuolar AI activity in tissue homogenates was sufficient to completely convert endogenous sucrose to monosaccharide within the same period. Continuous invertase-mediated breakdown of sucrose was thus not a feature of this tissue. It is concluded that AI and sucrose were not in metabolic contact in vivo, implying differential compartmentation. In darkness, in uninhibited leaves, sucrose concentrations fell linearly with respect to time at a rate of –0.6 mg g^–1 FW h^–1, over a 5- to 6-h period. This value is equivalent to rates of dark respiration measured by gas exchange. Dark-utilisation of sucrose was not accompanied by monosaccharide accumulation in the tissue. The rate of sucrose loss was 3-fold lower than rates of extractable AI activity. Hence, if AI was involved in dark-utilisation, then this implies at least a partial differential localisation of enzyme and substrate. However, the dark-consumption of sucrose was completely abolished by anoxia and by cyanide. It follows that dark-mobilisation (unlike invertase hydrolysis per se) was respiration-dependent and did not result from a simple co-localisation of sucrose and invertase. Taken together, the results show that sucrose and invertase do not share the same metabolic compartment in grass leaves. It is possible that invertase has no role in the mobilisation of stored sucrose in leaves of the fructan-accumulating grasses.Abbreviations AI Acid invertase - PAR Photosynthetically active radiation - TLC Thin-layer chromatography     

苹果中磷酸蔗糖合酶家族基因的表达特性及其与蔗糖含量的关系

磷酸蔗糖合酶(sucrose phosphate synthase,SPS)是植物中蔗糖合成的主要限速酶,影响植物的生长发育和果实中蔗糖的含量。为探明苹果中SPS基因家族特性及其在蔗糖合成中的作用,该研究从苹果基因组中分离了MdSPS家族基因,分析了它们的进化关系以及mRNA表达特性与酶活性和蔗糖含量的关系。结果显示:(1)在苹果基因组中有8个SPS家族基因表达,它们分别属于双子叶植物的3个SPS亚家族。(2)荧光定量PCR分析显示,苹果C类的MdSPS6基因和A类的MdSPS1a/b基因是苹果中表达丰度最高的SPS基因成员,其中MdSPS6在苹果成熟果中表达丰度最高,其次是成熟叶片,而MdSPS1a/b在不积累蔗糖的幼果中表达丰度最高。(3)在果实发育过程中,除MdSPS1a/b之外,其它5个苹果MdSPS家族基因均随果实的生长表达丰度增加,与SPS活性和蔗糖含量明显呈正相关关系。研究表明,C类家族MdSPS6是苹果果实发育后期和叶片中蔗糖合成的主要SPS基因。     

Aromatic interactions at the catalytic subsite of sucrose phosphorylase: Their roles in enzymatic glucosyl transfer probed with Phe → Ala and Phe → Asn mutants

Mutants of Leuconostoc mesenteroides sucrose phosphorylase having active-site Phe⁵² replaced by Ala (F52A) or Asn (F52N) were characterized by free energy profile analysis for catalytic glucosyl transfer from sucrose to phosphate. Despite large destabilization (?3.5 kcal/mol) of the transition states for enzyme glucosylation and deglucosylation in both mutants as compared to wild-type, the relative stability of the glucosyl enzyme intermediate was weakly affected by substitution of Phe⁵². In reverse reaction where fructose becomes glucocylated, “error hydrolysis” was the preponderant path of breakdown of the covalent intermediate of F52A and F52N. It is proposed, therefore, that Phe⁵² facilitates reaction of the phosphorylase through (1) positioning of the transferred glucosyl moiety at the catalytic subsite and (2) strong cation-π stabilization of the oxocarbenium ion-like transition states flanking the covalent enzyme intermediate.