Batch production of high-content fructo-oligosaccharides from sucrose by the mixed-enzyme system of β-fructofuranosidase and glucose oxidase

The production of high-content fructo-oligosaccharides from sucrose by the mixed-enzyme system of β-fructofuranosidase and glucose oxidase was investigated. The mixed-enzyme reaction was carried out in a stirred tank reactor containing 0.7 l of sucrose solution with coupled β-fructofuranosidase and glucose oxidase for 25 h. The optimum conditions for the mixed-enzyme reaction were as follows: pH, 5.5; temperature, 40°C; sucrose concentration, 400 g/l; agitation speed, 550 rpm; oxygen flow rate, 0.7 l/min; enzyme dosage, 10 units of β-fructofuranosidase with the combination of 15 units of glucose oxidase per gram sucrose. Under optimum conditions, high-content fructo-oligosaccharides up to 98% were obtained with complete consumption of sucrose and glucose by the mixed-enzyme system. Compared with the fructo-oligosaccharides produced by the β-fructofuranosidase, the high-content fructo-oligosaccharides produced by the mixed-enzyme system showed a significant difference with respect to sugar composition; i.e., a higher content of nystose was accumulated and only a trace amount of fructofuranosyl nystose was detected.     

Production of high fructo-oligosaccharide syrup with two enzyme system of fructosyltransferase and glucose oxidase

Summary A novel two enzyme system of fructosyltransferase and glucose oxidase to enhance the content of the net fructo—oligosaccharide (FOS) fractions in the industrial production of FOS syrup from sucrose was devised. The net FOS content in the commercial FOS syrup has been limited only to 55–60 % due to the accumulation of glucose which acts as a feedback inhibitor of the fructosyltransferase. By supplementing glucose oxidase to the conventional FOS reaction system, we could convert the glucose to gluconic acid readily separable from neutral sugars by simple ion exchange operation in the next step. The simultaneous removal of glucose was proved effective in proceeding the reaction by fructosyltransferase further by relieving the product inhibition caused by glucose. By this way, we could raise the net FOS content as high as 90 %.     

Purification and evaluation of the enzymatic properties of a novel fructosyltransferase from Aspergillus oryzae: a potential biocatalyst for the synthesis of sucrose 6-acetate

A novel fructosyltransferase (AoFT) capable of synthesizing sucrose 6-acetate (S6A) from sucrose and glucose 6-acetate has been purified to homogeneity from Aspergillus oryzae ZZ-01. Its molecular mass was ~50 kDa by SDS-PAGE; optimal activity was at 45 °C and it was stable from pH 4.5 to 7.5 with an optimum pH of 6. Mg²⁺, K⁺ (5 mM), propanol, toluene (50 %, v/v), Tween 20 or Triton X-100 (1 %, w/v) increased the transfructosylation activity by 20, 17, 17, 10, 25 and 20 %, respectively. An overall conversion of 32 % was achieved under optimal conditions over 24 h. This is the first report that the purified and characterized the fructosyltransferase from Aspergillus capable of synthesis of S6A from sucrose and glucose 6-acetate.     

Development of biosensors for the simultaneous determination of sucrose and glucose, lactose and glucose, and starch and glucose

Sensors for the simultaneous determinations of sucrose and glucose, lactose and glucose, and starch and glucose were prepared by a combination of the enzyme system shown below and an oxygen electrode: The mechanism for separating the substrates with the proposed sensors is based on the time lag arising from reaction and diffusion. Invertase, beta-galactosidase, amyloglucosidase, mutarotase, and glucose oxidase were covalently immobilized on triacetyl cellulose membranes containing 1,8-diamino-4-aminomethyloctane. A glucose oxidase membrane, mutarotase membrane, three sheets of triacetyl cellulose membranes, and invertase, or beta-galactosidase or amyloglucosidase membrane were placed in that order on the tip of the oxygen electrode. Calibration curves for sucrose, lactose, and starch were linear up to 40 mM, 60-180 mM, and 10%, respectively. The simultaneous determination of sucrose and glucose, lactose and glucose, and starch and glucose was possible when the amount of glucose coexised was in the range of 2-16% sucrose, 2.8-8.3% lactose, or 0.1-1% starch. The relative errors were +/-4% for sucrose and +/-3% for lactose in 100 assays. The starch sensor was reused only five times. Each enzyme membrane was fairly stable for more than 10 days.     

Purification and characterization of a fructosyltransferase from onion bulbs and its key role in the synthesis of fructo-oligosaccharides in vivo

A fructosyltransferase that transfers the terminal (2 --> 1)-beta-linked D-fructosyl group of fructo-oligosaccharides (1(F)(1-beta-D-fructofuranosyl)(n) sucrose, n >/= 1) to HO-6 of the glucosyl residue and HO-1 of the fructosyl residue of similar saccharides (1(F)(1-beta-D-fructofuranosyl)(m) sucrose, m >/= 0) has been purified from an extract of the bulbs of onion (Allium cepa). Successive column chromatography using DEAE-Sepharose CL-6B, Toyopearl HW65, Toyopearl HW55, DEAE-Sepharose CL-6B (2nd time), Sephadex G-100, Concanavalin A Sepharose, and Toyopearl HW-65 (2nd time) were applied for protein purification. The general properties of the enzyme, were as follows: molecular masses of 66 kDa (gel filtration chromatography), and of 52 kDa and 25 kDa (SDS-PAGE); optimum pH of c. 5.68, stable at 20-40 degrees C for 15 min; stable in a range of pH 5.30-6.31 at 30 degrees C for 30 min, inhibited by Hg(2+), Ag(+), p-chloromercuribenzoic acid (p-CMB) and sodium dodecyl sulfate (SDS), activated by sodium deoxycholate, Triton X-100 and Tween-80. The amino acid sequence of the N-terminus moiety of the 52-kDa polypeptide was ADNEFPWTNDMLAWQRCGFHFRTVRNYMNDPSGPMYYKGWYHLFYQHNKDFAYXG and the amino acid sequence from the N-terminus of the 25-kDa polypeptide was ADVGYXCSTSGGAATRGTLGPFGLL VLANQDLTENTATYFYVSKGTDGALRTHFCQDET. The enzyme tentatively classified as fructan: fructan 6(G)-fructosyltransferase (6G-FFT). The enzyme is proposed to play an important role in the synthesis of inulin and inulinneo-series fructo-oligosaccharides in onion bulbs.     

Sucrose:sucrose fructosyltransferase and fructan:fructan fructosyltransferase from allium cepa

Sucrose: sucrose fructosyltransferase and fructan:fructan fructosyltransferase were isolated from the inner leaf bases of bulbing onion plants (Allium cepa) and separated by gel filtration on Bio-Gel P-150. Sucrose:sucrose fructosyltransferase produced only one trisaccharide, 1^F-fructosylsucrose, from sucrose. Fructan:fructan fructosyltransferase produced tetrasaccharide and higher polymers from trisaccharide. The trisaccharide found in the greatest concentration in onion, 6^G-fructosylsucrose, was produced from 1^F-fructosylsucrose by fructan:fructan fructosyltransferase and was not a product of sucrose:sucrose fructosyltransferase.     

Fructosyltransferase activity of commercial enzyme preparations used in fruit juice processing

Summary Of twenty-two commercial fungal enzyme preparations used in fruit juice processing examined, Pectinex Ultra SP-L, was found to possess the highest activity of fructosyltransferase (44.8 units/ml). The enzyme preparation converted sucrose into a high fructooliogosaccharide syrup containing 42.3 % kestose, 17.2% nystose, 10.6% sucrose, 27.8% glucose, and 2.1% fructose. The efficiency was 69% based the amount of sucrose consumed.     

Enhancement of operational stability of an enzyme biosensor for glucose and sucrose using protein based stabilizing agents

With the incorporation of lysozyme during the immobilization step, considerable enhancement of the operational stability of a biosensor has been demonstrated in the case of an immobilized single enzyme (glucose oxidase) system for glucose and multienzyme (invertase, mutarotase and glucose oxidase) system for sucrose. Thus an increased number of repeated analyses of 750 samples during 230 days for glucose and 400 samples during 40 days of operation for sucrose have been achieved. The increased operational stability of immobilized single and multienzyme system, will improve the operating cost effectiveness of the biosensor.     

Enzymic synthesis of levan and fructo-oligosaccharides by <Emphasis Type="Italic">Bacillus circulans</Emphasis> and improvement of levansucrase stability by carbohydrate coupling

Bacillus circulans was able to produce extracellular levansucrase using sucrose as carbon source optimally at 35°C. The enzymic synthesis of levan and fructo-oligosaccharides was studied using a 50% ethanol fraction of crude extract. The molecular weight of the synthesized levan was markedly affected by sucrose concentration, the molecular weight of levan decreased with increased sucrose concentration up to 32% whereby fructo-oligosaccharides were isolated. Temperature and the reaction time clearly affected the conversion of fructose to levan with molecular weight values ranging from 10 to 38 kDa. Identification of levan indicated that fructose was the building unit of the levan obtained. Thermal and pH stabilities of B. circulans levansucrase could be improved by enzyme glycosylation using sodium metaperiodate treatment. Chemical modification provides additional points of attachment of the enzyme to the support which offered the modified enzyme greater stabilization than did the free enzyme. The modified enzyme exhibited thermal tolerance up to 50°C, where it retained 88.25% of its activity, while the free enzyme only retained 64.55% of its original activity. The half-life significantly increased from 130 min for the free enzyme to 347 min for the modified enzyme at 50°C, however, it increased from 103 min for the free enzyme to 210 min for the modified enzyme at 60°C. Other properties i.e., the response to some metal ions as well as the ability to convert higher substrate levels and tolerance to an extension of the reaction periods were also improved upon modification. Obviously, the results obtained outlined the conditions leading to the formation of important high or low molecular weight or levan and fructo-oligosaccharides suitable for different industrial applications.     

蔗糖-葡萄糖双功能酶传感器的研究

结合蔗糖转化酶(INV)酶管与葡萄糖氧化酶(GOD)-葡萄糖变旋酶(MUT)双酶电极构成一种新的蔗糖传感器。该传感器可以分别用于蔗糖及葡萄糖的测定。蔗糖经酶管作用产生α-D-葡萄糖,再用COD-MUT双酶电极定糖。若是样品中蔗糖和葡萄糖共存,比较样品流经不同路径(Ws和Wg)时传感器的响应值,可以排除葡萄糖对蔗糖测定的干扰。传感器的最适pH和温度范围分别为:5.0—6.5和30—40℃。在稳态法实验中,传感器的线性范围为:2.5×10~(-4)—5×10~(-3)mol/L。传感器的重复性很好,CV<1％。该传感器在用于测定发酵培养基(含葡萄糖)的蔗糖含量,平均回收率为97.9％。传感器与糖度计法测定的相关系数为0.997。传感器至少可以稳定使用8天以上。     

The effects of potassium and chloride ions on the ethanolic fermentation of sucrose by Zymomonas mobilis 2716

Summary The inclusion of specific salts in Zymomonas mobilis batch sucrose fermentations can limit by-product formation. Sorbitol and fructo-oligosaccharide formation can be reduced and ethanol production enhanced by manipulating mineral salt concentrations. Chloride salts reduced the production of biomass and sorbitol in favour of fructo-oligosaccharide formation at concentrations lower than 10 g NaCl/l or MgCl₂. Higher concentrations led to the accumulation of glucose and fructose. Low concentrations of KH₂PO₄ (<20 g/l) enhanced biomass formation, and the concomitant reduction in sorbitol and fructo-oligosaccharides favoured enhanced ethanol formation. At concentrations above 20 g/l, its effects were similar to those obtained with the chloride salts. Invertase addition at the start of fermentation increased sorbitol formation, whereas addition after the completion of sucrose hydrolysis resulted in the conversion of fructo-oligosaccharides formed into fructose or ethanol. Fermentation with 250 g/l of sugar-cane syrup ( = 130 g sucrose/l) in the presence of 8 g KH₂PO₄/l, with 0.05 g invertase/l added on the completion of sucrose hydrolysis, resulted in a conversion efficiency of 94% with complete carbon accountability, and only 7 g sorbitol/l. Offprint requests to: H. W. Doelle     

Study of the production of fructose and ethanol from sucrose media by Saccharomyces cerevisiae

The production of ethanol and enriched fructose syrups from a synthetic medium with various sucrose concentrations using the mutant Saccharomyces cerevisiae ATCC 36858 was investigated. In batch tests, fructose yields were above 90% of theoretical values for the sucrose concentrations between 35 g/l and 257 g/l. The specific growth rates and biomass yields were from 0.218 to 0.128 h(-1) and from 0.160 to 0.075 g biomass/g of glucose and fructose consumed, respectively. Ethanol yields were in the range of 72 to 85% of theoretical value when sucrose concentrations were above 81 g/l. The volumetric ethanol productivity was 2.23 g ethanol/(l h) in a medium containing 216 g/l sucrose. Fructo-oligosaccharides and glycerol were also produced in the process. A maximum fructo-oligosaccharides concentration (up to 9 g/l) was attained in the 257 g/l sucrose medium in the first 7 h of the fermentation. These sugars started to be consumed when the concentrations of sucrose in the media were less than 30% of its initial values. The fructo-oligosaccharides mixture was composed of 6-kestose (61.5%), neokestose (29.7%) and 1-kestose (8.8%). The concentration of glycerol produced in the process was less than 9 g/l. These results will be useful in the production of enriched fructose syrups and ethanol using sucrose-based raw materials.     

Production of fructosylxyloside by the new fructosyltransferase from Bacillus macerans EG-6

The new fructosyltransferase (FTase) from Bacillus maceransEG-6 showed a broad acceptor specificity, and resulted in the formation of fructosylxyloside (FX) with d-xylose being the most effective acceptor. The optimal FTase concentration for FX production was 0.6 unit per g sucrose, which gave the highest transfer ratio, 83%, of fructosyl moiety from sucrose to d-xylose. Maximum yield of FX was 114 g l^–1with 200 g sucrose l^–1and 300 g d-xylose l^–1.     

Selective production of GF4-fructooligosaccharide from sucrose by a new transfructosylating enzyme

A new fructosyltransferase produced by a new isolate of Bacillus macerans EG-6 showed mainly hydrolyzing activity at 10 g sucrose/l, while both hydrolyzing and transfructosylating activity were observed at 100 g sucrose/l. In contrast, the new enzyme catalyzed an almost exclusively fructosyl transfer reaction with 500 g sucrose/l, producing selectively 42% of fructooligosaccharide (GF4) without forming other fructooligosaccharides. The reaction was optimal at pH 6.0 and 37°C. © Rapid Science Ltd. 1998     

Production of fructosyltransferase by <Emphasis Type="Italic">Aureobasidium</Emphasis> sp. ATCC 20524 in batch and two-step batch cultures

A comparison of fructosyltransferase (EC 2.4.1.9) production by Aureobasidium sp. ATCC 20524 in batch and two step batch cultures was investigated in a 1-l stirred tank reactor using a sucrose supply of 200 g/l. Results showed that the innovative cultivation in two step of Aureobasidium sp. produced more fructosyltransferase (FFase) than the single batch culture at the same sucrose concentration with a maximal enzyme production of 523 U/ml, which was 80.5% higher than the one obtained in the batch culture. The production of fructooligosaccharides (FOSs) was also analyzed; their concentration reached a maximum value of 160 g/l the first day in the two-step culture and 127 g/l in the single-batch mode. The use of the two-step batch culture with Aureobasidium sp. ATCC 20524 in allowing the microorganism to grow up prior to the induction of sucrose (second step), proved to be a powerful method for producing fructosyltransferase and FOSs.     

Production of fructooligosaccharides in high yield using a mixed enzyme system of β-fructofuranosidase and glucose oxidase

A mixed enzyme system, with -fructofuranosidase (obtained from Aspergillus japonicus) and commercial glucose oxidase (Gluzyme, Novo Nordisk), produced fructooligosaccharides (FOS) in high yield from sucrose. The reaction was performed in an aerated stirred tank reactor controlled at pH 5.5 by a slurry of CaCO₃. Glucose, an inhibitor of -fructofuranosidase, produced in the reaction was converted by glucose oxidase to gluconic acid, which was then precipitated to calcium gluconate in solution. The system produced more than 90% (w/w) FOS on a dry weight basis, the remainder was glucose, sucrose and a small amount of calcium gluconate. Most of the FOS and sucrose was hydrolyzed to fructose in the mixed enzyme system with glucose oxidase and -fructofuranosidase from Asp. niger.     

Kinetic studies and mathematical model for sucrose conversion by <Emphasis Type="Italic">Aspergillus niger</Emphasis> fructosyl-transferase under high hydrostatic pressure

The effect of pressure on the kinetically controlled synthesis reaction catalyzed from sucrose by Aspergillus niger fructosyl-transferase was investigated at pH 5.5 and 40 degrees C. The overall reaction was split up into five main reactions that were studied under pressure in initial rate conditions with various substrate concentrations in the absence or in the presence of glucose 50 g/l. A global reaction model was worked out according to the mathematical expression of the initial rates as the products of a polynomial rational function of substrate concentration and a corrective term introducing pressure. Experimental data from sugar concentrations were correctly described by the model during the course of the reaction under pressure. Raising the pressure induced a decrease in fructo-oligosaccharides yield by inhibiting the main transfer reaction without affecting sucrose hydrolysis.     

Chromatographic separation and kinetic properties of fructosyltransferase from Aureobasidium pullulans

Efficient chromatographic separation of fructosyltransferase from Aureobasidium pullulans was achieved on a preparative scale using a weak anion-exchanger Sepabeads FP-DA. The recovery yield was about 70% and the purification factor reached a value of 28. The molecular weight of the enzyme determined by size-exclusion chromatography was 570,000. The enzyme exhibited both hydrolytic and transferase activity when the latter was higher in the whole concentration range and completely dominating at higher sucrose concentrations. It was found that sucrose was the only donor of fructosyl moiety used in the transfer reaction. The initial rate method was used for the investigation of the kinetics of the action of fructosyltransferase on sucrose in the concentration range 30–2430 mM. The initial transfructosylation rate was well fitted with a linear function of the sucrose activity where the activity coefficient was an exponentially decreasing function of the sucrose concentration.     

蔗糖:蔗糖-1-果糖基转移酶的表面展示及酶学性质分析

【目的】蔗糖:蔗糖-1-果糖基转移酶催化1分子蔗糖上的果糖基转移到另一个蔗糖分子上,形成1-蔗果三糖和葡萄糖。在低聚果糖中,1-蔗果三糖益生素活性最高。本研究将该酶展示在酵母菌细胞表面上,并用于1-蔗果三糖的制备。【方法】将来自莴苣的蔗糖:蔗糖-1-果糖基转移酶基因克隆到用于酵母细胞表面展示的表达载体上,并在解脂亚罗酵母菌中进行异源表达,表达的酶展示在该细胞表面上,然后以蔗糖为底物,研究表面展示的蔗糖:蔗糖-1-果糖基转移酶的性质。【结果】免疫荧光实验结果表明蔗糖:蔗糖-1-果糖基转移酶已展示在酵母菌的细胞表面上,高效液相色谱结果表明酵母表面展示的该酶具有转移酶的催化活性。该酶的最适作用温度、最适作用p H分别为45°C和7.5;该酶的催化活性受Zn2+和Cu2+的抑制,受Ca2+激活;该酶重复使用7次后,酶活下降50%。表面展示的蔗糖:蔗糖-1-果糖基转移酶和3%蔗糖混合后在40°C条件下孵育30 min后,所产1-蔗果三糖含量最高为20.8 mmol/L。【结论】蔗糖:蔗糖-1-果糖基转移酶在解脂亚罗酵母菌中得到成功表达,并展示在其细胞表面上,生化研究表明该重组蛋白具有果糖基转移酶活性,且催化蔗果三糖的生成。表面展示的蔗糖:蔗糖-1-果糖基转移酶作为一种全细胞催化剂能够用于1-蔗果三糖的制备。     

Purification and characterisation of 6G-fructosyltransferase from the roots of asparagus (Asparagus officinalis L.)

A fructosyltransferase that catalyses the transfer of the terminal (2 → 1)-β-linked d-fructosyl group of fructo-oligosaccharides [1^F(1-β-d-fructofuranosyl)_msucrose, m > 0] to HO-6 of the glucosyl group of similar saccharides [1^F(1-β-d-fructofuranosyl)_nsucrose, n > 0] has been purified (760-fold) from an extract of the roots of asparagus (Asparagus officinalis L.) by successive fractionation with ammonium sulfate, treatment with calcium phosphate gel, and then chromatography on octyl-Sepharose, DEAE-cellulose, Sephadex G-200, and raffinose-coupled Sepharose 6B. The enzyme, tentatively termed 6^G-fructosyltransferase, was homogeneous in disc electrophoresis, had a mol. wt. of ~69,000 and an optimum pH of ~5.5, was stable at pH 5.0–6.0 on heating for 20 mins at 45° and for 10 min at 20–37°, and was inhibited by Hg²⁺, p-chloromercuribenzoate, and Ag⁺.