Continuous production of high-glucose syrup by chitin-immobilized amylase

A simple method of preparing a chitin-immobilized alpha-amylase and glucoamylase from the protease- and glycosidase-less Mutant HF-15 of Aspergillus awamori var. kawachi was developed and used for the production of high-glucose syrup. The glucoamylase was tightly bound onto chitin without the aid of a crosslinking agent because the enzyme contained a specific binding site for chitin. Continuous production of high glucose concentrate from a highly concentrated alpha-amylasetreated gelatinized starch substrate (about 45% total solids) was undertaken successfully with the use of a column-packed chitin-immobilized amylase. The activity of the column was stable for more than 20 days of continuous operation and the product was found to be only glucose with an average dextrose equivalent value of more than 97%. The final product showed no isomaltose or panose contamination, indicating that the immobilized amylase had no transglucosidation activity. The immobilized amylase was most active in the conversion of gelatinized starch to glucose at 55 degrees C and pH 2.5 to 5.0. Drying the chitin-immobilized amylase resulted in the decrease of activity and shortening of storage life, whereas a storage life of up to 80 days was attained without affecting its original activity when kept under moist condition at 4 degrees C.     

Anaerobic fermentation of gelatinized sago starch-derived sugars to acetone—1-butanol—Ethanol solvent byClostridium acetobutylicum

A study of the kinetics and performance of solvent-yielding batch fermentation of individual sugars and their mixture derived from enzymic hydrolysis of sago starch byClostridium acetobutylicum showed that the use of 30 g/L gelatinized sago starch as the sole carbon source produced 11.2 g/L total solvent,i.e. 1.5–2 times more than with pure maltose or glucose used as carbon sources. Enzymic pretreatment of gelatinized sago starch yielding maltose and glucose hydrolyzates prior to the fermentation did not improve solvent production as compared to direct fermentation of gelatinized sago starch. The solvent yield of direct gelatinized sago starch fermentation depended on the activity and stability of amylolytic enzymes produced during the fermentation. The pH optima for α-amylase and glucoamylase were found to be at 5.3 and 4.0–4.4, respectively. α-Amylase showed a broad pH stability profile, retaining more than 80% of its maximum activity at pH 3.0–8.0 after a 1-d incubation at 37°C. SinceC. acetobutylicum α-amylase has a high activity and stability at low pH, this strain can potentially be employed in a one-step direct solvent-yielding fermentation of sago starch. However, theC. acetobutylicum glucoamylase was only stable at pH 4–5, maintaining more than 90% of its maximum activity after a 1-d incubation at 37°C.     

Raw Starch-digestive Chitin-immobilized Amylase from a Protease—Glycosidase-less Mutant of Aspergillus awamori var. kawachi

The α-amylase and glucoamylase produced by a protease-, glycosidase-less mutant HF-15 of Aspergillus awamori var. kawachi were found to be adsorbable onto chitin. This adsorption was pH-independent, different from the adsorption onto raw corn starch. The binding between amylases and chitin was so tight that a chitin-immobilized amylase was obtained without the aid of a cross linking agent, glutaraldehyde, and it retained more than 90% of the original activity of the free enzyme. The immobilized amylase digested gelatinized potato starch, glycogen and even raw corn starch to the same high extent as glucose similar to the free enzyme, but it was different from the unbound crude enzyme in the lack of transglucosidase activity, and slightly different in pH- and thermo-stabilities. An experiment using the immobilized amylase for alcohol fermentation demonstrated the possibility of recycling the enzyme for raw starch saccharification.     

Immobilization of glucoamylase on naphthyl cotton cloth

Summary Aspergillus niger glucoamylase was adsorbed to -naphthyl cotton cloth by hydrophobic interaction. The adsorbed enzyme was cross-linked with glutaraldehyde. The immobilized glucoamylase exhibited greater pH dependence though the optimal pH did not change. The immobilized glucoamylase in a packed bed column completely hydrolysed 5% soluble starch at a specific velocity of about 4. Used naphthyl cloth could be regenerated by heating in 2 N NaOH at 100°C for 1 hour.     

Production of thermostable and neutral glucoamylase using immobilized <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis>

Thermomucor indicae-seudaticae was immobilized in alginate, κ-carrageenan, agarose, agar, polyacrylamide and loofah (Luffa cylindrica) sponge (as such or coated with alginate/starch/Emerson YpSs agar), and used for the production of glucoamylase in submerged fermentation. The mycelium developed from alginate-immobilized sporangiospores secreted higher glucoamylase titres (22.7 U ml⁻¹) than those immobilized in other gel matrices and the freely growing mycelial pellets (18.5 U ml⁻¹). Loofah network provided a good support for mycelial growth, but the enzyme production was lower than that attained with alginate beads. Glucoamylase production increased with inoculum density and the optimum levels were achieved when 40 calcium alginate beads (∼5 × 10⁶ immobilized spores) were used to inoculate 50 ml production medium. The alginate bead inoculum displayed high storage stability at 4°C and produced comparable enzyme titres up to 120 days. The glucoamylase production by hyphae emerged from the immobilized sporangiospores was almost stable over eight batches of repeated fermentation. Scanning electron micrographs of alginate beads, after batch fermentation, revealed extensive mycelial growth inside and around the beads.     

Characterization of exopolysaccharide (EPS) produced by Weissella hellenica SKkimchi3 isolated from kimchi

Weissella hellenica SKkimchi3 produces the higher exopolysaccharide (EPS) on sucrose than lactose, glucose, and fructose at pH 5 and 20°C. Sucrose was exclusively used to cultivate SKkimchi3 in all experiments base on the EPS production tests. The molecular mass of EPS, as determined by gel permeation chroma-tography, was 203,000. ¹H and ¹³C NMR analysis indicated that the identity of EPS may be a glucan. When EPS, starch, and cellulose was treated with a-amylase, glucoamylase, glucosidase, and cellulase, glucose was produced from starch and cellulose but was not produced from EPS. Based on HPLC analysis, elemental analysis, ¹H and ¹³C NMR analysis, and enzymatic hydrolysis tests, EPS was estimated to be a glucan. EPS suspension was not precipitated even by centrifugation at 10,000×g for 60 min, and EPS made the fermented milk and bacterial culture viscous.     

Enzymatic processes for the purification of trehalose

A dual‐enzyme process aiming at facilitating the purification of trehalose from maltose is reported in this study. Enzymatic conversion of maltose to trehalose usually leads to the presence of significant amount of glucose, by‐product of the reaction, and unreacted maltose. To facilitate the separation of trehalose from glucose and unreacted maltose, sequential conversion of maltose to glucose and glucose to gluconic acid under the catalysis of glucoamylase and glucose oxidase, respectively, is studied. This study focuses on the hydrolysis of maltose with immobilized glucoamylase on Eupergit® C and CM Sepharose. CM Sepharose exhibited a higher protein adsorption capacity, 49.35 ± 1.43 mg/g, and was thus selected as carrier for the immobilization of glucoamylase. The optimal reaction temperature and reaction pH of the immobilized glucoamylase for maltose hydrolysis were identified as 40°C and 4.0, respectively. Under such conditions, the unreacted maltose in the product stream of trehalose synthase‐catalyzed reaction was completely converted to glucose within 35 min, without detectable trehalose degradation. The conversion of maltose to glucose could be maintained at 0.92 even after 80 cycles in repeated‐batch operations. It was also demonstrated that glucose thus generated could be readily oxidized into gluconic acid, which can be easily separated from trehalose. We thus believe the proposed process of maltose hydrolysis with immobilized glucoamylase, in conjunction with trehalose synthase‐catalyzed isomerization and glucose oxidase‐catalyzed oxidation, is promising for the production and purification of trehalose on industrial scales. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Glucoamylase and α-amylase production by immobilized Aspergillus niger

Summary Aspergillus niger mycelia or spores were immobilized in calcium alginate gel beads and employed for production of glucoamylase and -amylase by repeated batch process. The immobilized mycelium produced lower enzyme activities than immobilized spores germinated in a growth medium and subsequently cultured in an enzyme production medium. In repeated batch experiments, free cells could be used for only 4 4-day batches, whereas with immobilized spores at least 11 4-day batches with a gradual increase in enzyme activities in each successive batch were possible. The activity ratio of glucoamylase and -amylase produced was altered by immobilization.     

Starch conversion by soluble and immobilized α-amylase

B. subtilis α-amylase was immobilized on cyanogen bromide activated carboxymethyl cellulose. The conversion of wheat starchwas carried out at 72°C in a stirred tank by soluble and immobilized α-amylase. The initial reaction rate with immobilized α-amylase was lower than with the soluble enzyme, but after 1 hr immobilized α-amylase produced a higher quantity of reducing sugars than the soluble enzyme. The action pattern of immobilized α-amylase was different from that of the soluble enzyme: immobilized α-amylase produced relatively more glucose and maltose, except at the beginning of conversion. Immobilized α- readily hydrolyze G₆. The starch conversion by immobilized α-amylase was not diffusion controlled at a stirring rate of 100-300 rpm.     

TiO2膜吸附固定糖化酶特性的研究

分别以醋酸纤维素TiO2膜(AC.TiO2膜)、羧甲基纤维TiO2膜(CMC.TiO2膜)和聚丙烯TiO2膜(PP.TiO2膜)为载体吸附固定糖化酶,并与醋酸纤维素、羧甲基纤维素和聚丙烯固定糖化酶的性能进行了比较,得出以AC.TiO2膜和PP.TiO2膜对糖化酶的吸附性能及稳定性能均较好,PP.TiO2膜固定的糖化酶使用8次后其剩余酶活仍能保持在72%.     

Sago starch as a low-cost carbon source for exopolysaccharide production by Lactobacillus kefiranofaciens

Low-cost sago starch was used as a carbon source for production of the exopolysaccharide kefiran by Lactobacillus kefiranofaciens. A simultaneous saccharification and fermentation process of sago starch for kefiran production was evaluated. Factors affecting the process such as an initial pH, temperature, starch concentration, including a mixture of α-amylase and glucoamylase were determined. The highest kefiran concentration of 0.85 g/l was obtained at the initial pH of 5.5, temperature of 30 °C, starch concentration of 4% and mixed-enzymes with activity of 100 U/g-starch. The use of a mixture of α-amylase and glucoamylase could enhance the productivity compared to the use of α-amylase alone. The optimal ratio of α-amylase to glucoamylase of 60:40 gave the highest kefiran production rate of 11.83 mg/l/h. This study showed that sago starch could serve as a low-cost substrate for kefiran production.     

Kojic acid production byAspergillus flavus using gelatinized and hydrolyzed sago starch as carbon sources

Direct conversion of gelatinized sago starch into kojic acid byAspergillus flavus strain having amylolytic enzymes was carried out at two different scales of submerged batch fermentation in a 250-mL shake flask and in a 50-L stirred-tank fermentor. For comparison, fermentations were also carried out using glucose and glucose hydrolyzate from enzymic hydrolysis of sago starch as carbon sources. During kojic acid fermentation of starch, starch was first hydrolyzed to glucose by the action of α-amylase and glucoamylase during active growth phase. The glucose remaining during the production phase (non-growing phase) was then converted to kojic acid. Kojic acid production (23.5g/L) using 100 g/L sago starch in a shake flask was comparable to fermentation of glucose (31.5 g/L) and glucose hydrolyzate (27.9 g/L) but in the 50-L fermentor was greatly reduced due to non-optimal aeration conditions. Kojic acid production using glucose was higher in the 50-L fermentor than in the shake flask.     

Mixture design of starchy substrates hydrolysis by an immobilized glucoamylase from Aspergillus brasiliensis

Starch has great importance in human diet, since it is a heteropolymer of plants, mainly found in roots, as potato, cassava and arrowroots. This carbohydrate is composed by a highly-branched chain: amylopectin; and a linear chain: amylose. The proportion between the chains varies according to the botanical source. Starch hydrolysis is catalyzed by enzymes of the amilolytic system, named amylases. Among the various enzymes of this system, the glucoamylases (EC 3.2.1.3 glucan 1,4-alpha-glucosidases) are the majority because they hydrolyze the glycosidic linkages at the end of starch chains releasing glucose monomers. In this work, a glucoamylase secreted in the culture medium, by the ascomycete Aspergillus brasiliensis, was immobilized in Dietilaminoetil Sepharose-Polyethylene Glycol (DEAE-PEG), since immobilized biocatalysts are more stable in long periods of hydrolysis, and can be recovered from the final product and reused for several cycles. Glucoamylase immobilization has shown great thermal stability improvement over the soluble enzyme, reaching 66% more activity after 6?h at 60?°C, and 68% of the activity after 10 hydrolysis cycles. A simplex centroid experimental mixture design was applied as a tool to characterize the affinity of the immobilized enzyme for different starchy substrates. In assays containing several proportions of amylose, amylopectin and starch, the glucoamylase from A. brasiliensis mainly hydrolyzed the amylopectin chains, showing to have preference by branched substrates.     

Isoglucose production from raw starchy materials based on a two-stage enzymatic system

A new low-cost glucoamylase preparation for liquefaction and saccharification of starchy raw materials in a one-stage system was developed and characterized. A non-purified biocatalyst with a glucoamylase activity of 3.11 U/mg, an alpha-amylase activity of 0.12 WU/mg and a protein content of 0.04 mg protein/mg was obtained from a shaken-flask culture of the strain Aspergillus niger C-IV-4. Factors influencing the enzymatic hydrolysis of starchy materials such as reaction time, temperature and enzyme and substrate concentration were standardized to maximize the yield of glucose syrup. Thus, a 90% conversion of 5% starch, a 67.5% conversion of 5% potato flour and a 55% conversion of 5% wheat flour to sweet syrups containing up to 87% glucose was reached in 3 h using 1.24 glucoamylase U/mg hydrolyzed substrate. The application of such glucoamylase preparation and a commercially immobilized glucose isomerase for the production of glucose-fructose syrup in a two-stage system resulted in high production of stable glucose/fructose blends with a fructose content of 50%. A high concentration of fructose in obtained sweet syrups was achieved when isomerization was performed both in a batch and repeated batch process.     

Two Forms of Glucoamylase from Mucor rouxianus

Both of the two forms of glucoamylase (glucoamylases I and II) from the wheat bran culture of Mucor rouxianus hydrolyzed amylopectin, amylose, glycogen, soluble starch, maltotriose, and maltose, but did not act on isomaltose and isomaltotriose. Phenyl α-maltoside was hydrolyzed into glucose and phenyl α-glucoside by both glucoamylases. Maltose was hydrolyzed about one-fifth as rapidly as amylopectin. Both enzymes produced glucose from amylopectin, amylose, glycogen, soluble starch in the yields of almost complete hydrolysis. They hydrolyzed amylose with the inversion of configuration, producing the β-anomer of glucose. Glucoamylase II hydrolyzed raw starch at 3-fold higher rate than glucoamylase I. The former hydrolyzed rice starch almost completely into glucose, whereas the latter hydrolyzed it incompletely (nearly 50%).     

Effect of C/N ratio and starch concentration on ethanol production from sago starch using recombinant yeast

Recombinant Saccharomyces cerevisiae YKU 131 (capable of expressing glucoamylase) was used to produce ethanol from sago starch. The optimum C/N ratio for ethanol production by the recombinant yeast was 7.9, where 4.7 and 10.1 g/l ethanol was produced from 20 and 40 g/l sago starch, respectively. At sago starch concentration higher than 40 g/l and C/N ratio higher than 10.4, glucoamylase production and rate of starch hydrolysis were reduced, which in turn, reduced ethanol production significantly. The theoretical yield of ethanol based on sago starch consumed in fermentation using 40 g/l was 72.6%. This yield was slightly lower than those obtained in fermentation using soluble starch such as potato and corn starch, which ranged from 80–90% as reported in the literature. However, S. cerevisiae YKU 131 could only utilize 62% of the total amount of starch added to a medium.     

Hydrolysis of cellulose in a cellulase-bead fluidized bed reactor

Cellulase was immobilized in a collagen fibril matrix, and no leakage of cellulase from the collagen fibril matrix was observed. The immobilized cellulase was more stable than the native cellulase. The substrate cellulose was hydrolyzed quantitatively with immobilized cellulase. The final reaction product was identified as glucose. Immobilized cellulase was used in a fluidized bed reactor where the pressure drop of the fluidized bed reactor was low and constant. Cellulose was hydrolyzed to glucose by the cellulase-bead fluidized bed reactor. The minimum flow velocity (U_mf) was 0.5 cm/sec and the optimum flow velocity of the cellulose hydrolysis was 1 cm/sec.     

Culture condition for production of glucoamylase from rice bran byAspergillus terreus

Summary Optimal conditions for the production of glucoamylase from rice bran usingAspergillus terreus in stationary culture were a medium containing 20 g rice bran/l, 0.3% (w/v) (NH₄)₂SO₄ and 0.2% (w/v) peptone at 30°C with an initial pH of 3.0. Enzymatic activity was maximal after 4 d. Glucose was the major reducing sugar produced by hydrolysis of starch. Carbohydrates favouring induction of glucoamylase were, in order: maltose, starch, cellobiose, lactose, glucose, fructose and galactose. Amino acids, in particular glycine, lysine, isoleucine and histidine, were vital for glucoamylase synthesis. Tween 80 and Triton X-100 enhanced the growth but suppressed glucoamylase synthesis.

---

Conditions de culture pour la production de glucoamylase à partir de son de riz parAspergillus terreus

Résumé Les conditions optimales pour la production de glucoamylase à partir de son de riz en utilisantAspergillus terreus en culture en état stationnaire, consistent en un milieu contenant 20 g de son de riz par litre, 0.3 % (poids/vol.) de (NH₄)₂ SO₄ et 0.2 % (poids/vol.) de peptone, à 30 °C avec un pH initial de 3.0. L'activité enzymatique est maximum après 4 jours. Le glucose est le principal sucre réducteur produit par hydrolyse de l'amidon. Les hydrates de carbone qui favorisent l'induction de la glucoamylase, sont, dans l'ordre: le maltose, l'amidon, la cellobiose, le lactose, le glucose, le fructose et le galactose. Les acides aminés, en particulier la glycine, la lysine, l'isoleucine et l'histidine sont vitales pour la synthèse de glucoamylase. Le tween 80 et le triton X-100 augmentent la croissance mais suppriment la synthèse de glucoamylase.