Production of raw cassava starch-digestive glucoamylase by Rhizopus sp. in liquid culture

Among about 200 Rhizopus strains isolated in Thailand, Rhizopus sp. MB46 was selected as a producer of raw cassava starch-digestive glucoamylase. Rice bran was effective for the enzyme production in a solid culture as well as wheat bran. Addition of turpentine oil into the rice bran solid culture increased the productivity. Rhizopus sp. MB46 was found to produce glucoamylase in a liquid culture containing 1% rice bran but not in one consisting of 10% raw cassava starch of 2% glucose. The productivity per 1 g solids in the medium in liquid culture was finally improved 6-times by utilization of n-hexane-treated rice bran, supplement of 0.1% meat extract and addition of gauze as a support. The activity was superior to that in turpentine oil-supplemented solid culture.     

Raw Starch-digestive Glucoamylase Productivity of Protease-less Mutant from Aspergillus awamori var. kawachi

Mutation experiments were performed to decrease the protease productivity of Aspergillus awamori var. kawachi using ultraviolet light and N-methyl-N'-nitro-N-nitrosoguanidine. The selected mutant HF-15 showed reductions in protease productivity of 93%, 84% and 50% in solid wheat bran culture, shaking Medium B and wheat bran cultures, respectively, as compared with the parent. Protease-less mutant HF-15 failed to produce α-mannosidase, and N-acetyl-β-d-glucosaminidase productivity decreased by 35%. Mutant HF-15 specifically produced a high amount of raw starch-adsorbable and raw starch-digestive glucoamylase similar to GA I under all tested cultural conditions. On the contrary, high protease-producing mutant HF-10 produced a glucoamylase with very limited adsorption and digestion capacity on raw corn starch, and lower hydrolysis toward gelatinized potato starch and glycogen that was similar to GA I'.     

Comparative study of amylolytic enzymes production byAspergillus niger in liquid and solid-state cultivation

Summary Amylolytic enzymes produced by a strain ofAspergillus niger cultivated on cassava starch in liquid or solid culture were found to be mainly glucoamylases. For the same initial amount of substrate, the glucoamylase activity increased even after 60 h of culture on solid medium whereas it decreased in liquid culture. Some characteristics of the amylases produced in both culture conditions were compared. The pH optima for enzymes produced in solid and liquid cultures were 4.5 and 5.0 respectively. Glucoamylase synthetized in solid cultures was significantly more thermostable than that from liquid culture and was maximally active at 70°C compared to 50°C for the enzyme from liquid cultures. The Km values expressed as mg soluble starch/100 ml were 0.1% for crude enzyme from solid culture and 0.057% for crude enzyme from liquid culture.     

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%).     

Production of erythritol from glucose by an osmophilic mutant of Candida magnoliae

Candida magnoliae and its mutants were analyzed to produce erythritol from glucose with high yield and productivity. One mutant, M2, showed higher erythritol conversion yield and productivity than the wild strain. The osmophilic mutant produced 25 g erythritol l^–1 after 83 h of a flask culture in a medium containing 10% (w/v) glucose, corresponding to a 25% increase in erythritol and a 30% increase in erythritol productivity compared with the wild type. The fermentation properties were further improved by cultivating the osmophilic mutant in a fermenter containing 20% (w/v) glucose medium with 0.54 g l^–1 h^–1 of erythritol productivity and 43% of erythritol conversion yield based on glucose.     

Characteristics of raw-starch-digesting glucoamylase from thermophilic Rhizomucor pusillus

A newly isolated thermophilic fungus, NH-139, identified as Rhizumucor pusillus (Lindt) Schipper produced only a single form of raw-starch-absorbable, raw-starch-digesting glucoamylase on solid wheat bran medium at 45°C. The electrophoretically homogenous preparation of glucoamylase, molecular weight 68,000, had its optimal temperature on gelatinized starch at 65°C and on raw corn starch at 50°C. However, this raw-starch-digesting glucoamylase, unlike other glucoamylases, could not completely hydrolyze glycogen but hydrolyzed it to the extent of 80% as glucose, and is classified as type B. The subtilisin-modified glucoamylase of this strain, molecular weight 60,000, still belonged to type B in the hydrolysis curve on glycogen and lost the ability to digest and adsorb onto raw starch.     

Raw cassava starch-digestive glucoamylase of Aspergillus sp. N-2 isolated from cassava chips

One hundred and eighty strains of black aspergilli isolated from cassava fields and factories in Thailand were screened for the activity of raw cassava starch-digestive glucoamylase. Aspergillus sp. N-2 was selected as the best producer and its extracellular glucoamylase production was investigated. Conditions for the production were optimized for both liquid and solid cultures, and solid culture was found to be approximately three times more efficient than liquid culture. The culture filtrate showed strong glucoamylase activity at low pH (pH 2.0) and high temperature (55°C), and could digest high concentration raw cassava starch. The glucoamylase activity was separated to four fractions (A, B, C and D) by DEAE-Sephacel column chromatography. Fraction C was obtained in a homogeneous state with a molecular weight of 92,000. Each fraction was characterized in terms of the properties of the glucoamylase activity and the efficiency of digestion of cooked and raw cassava starch.     

Production of novel oligosaccharide oxidase by wheat bran solid-state fermentation

A search for oxidases that catalyze the oxidation of oligosaccharides has resulted in the isolation of several soil-derived fungus strains which produced novel oligosaccharide oxidases with different substrate specificity on wheat bran solid culture. One of these oxidases produced by Acremonium strictum T1 strain has been characterized. This enzyme showed high reactivity toward maltose, lactose, cellobiose and maltooligosaccharides composed of up to seven glucose units, and was named as glucooligosaccharide oxidase based on its substrate specificity. Strain T1 was subjected to a strain improvement program, and an enzyme hyper-producing mutant strain T1-38 was selected. This mutant strain produced glucooligosaccharide oxidase 75 times higher than the wild type strain T1. When cultivated in a solid medium comprised of 1 part of wheat bran and 1 part of water (w/w), enzyme activity reached a maximum level of 6 units per g of culture medium after 4 days cultivation. Characteristics of the enzyme including the substrate specificity were compared with two other novel oligosaccharide oxidases isolated in this laboratory. Batch type conversion of lactose to lactobionic acid using crude enzyme was also discussed.     

Amylolytic enzymes from Aspergillus hennebergi (A. niger Group): Purification and characterization of amylases from solid and liquid cultures

Two glucoamylases (I and II) were produced during solid-state culture of Aspergillus hennebergi (A. niger group) on cassava meal, whereas one glucoamylase and one alpha-amylase were synthesized by the mould in liquid culture. These glucoamylases were acidic proteins with thermotolerant activities. Glucoamylase I was not a glycoprotein, but glucoamylase II and the glucoamylase from liquid cultures contained 15% of sugars. The alpha-amylase was significantly less thermotolerant and of smaller molecular weight. The influence of culture conditions on the production of different amylases by the same Aspergillus strain on the same substrate is discussed.     

Optimization of the production of aroma compounds by Kluyveromyces marxianus in solid-state fermentation using factorial design and response surface methodology

Studies were carried out for the production of aroma compounds in solid-state fermentation using factorial design and response surface methodology (RSM) experiments. Five agro-industrial residues were evaluated as substrate for cultivating a strain of Kluyveromyces marxianus. The results proved the feasibility of using cassava bagasse and giant palm bran (Opuntia ficus indica) as substrates to produce fruity aroma compounds by the yeast culture. In order to test the influence of the process parameters on the culture to produce volatile compounds, two statistical experimental designs were performed. The parameters studied were initial substrate pH, addition of glucose, cultivation temperature, initial substrate moisture and inoculum size. Using a 2(5) factorial design, addition of glucose and initial pH of the substrate was found statistically significant for aroma compounds production on palm bran. Although this experimental design showed that addition of glucose did not have a significant role with cassava bagasse, 2(2) factorial design revealed that glucose addition was significant at higher concentrations. Head-space analysis of the culture by gas chromatography showed the production of nine and eleven compounds from palm bran and cassava bagasse, respectively, which included alcohols, esters and aldehyde. In both the cases, two compounds remained unidentified and ethyl acetate, ethanol and acetaldehyde were the major compounds produced. Esters produced were responsible for the fruity aroma in both the cases. With palm bran, ethanol was the compound produced in highest concentration, and with cassava bagasse (both supplemented with 10% glucose), ethyl acetate was produced at highest concentration, accumulating 418 and 1395μmoll(-1) head-spaceg(-1) substrate in 72h, respectively.     

Production and Characteristics of Raw-Starch-Digesting alpha-Amylase from a Protease-Negative Aspergillus ficum Mutant

Mutational experiments were carried out to decrease the protease productivity of Aspergillus ficum IFO 4320 by using N-methyl-N'-nitro-N-nitrosoguanidine. A protease-negative mutant, M-33, exhibited higher alpha-amylaseactivity than the parent strain under submerged culture at 30 degrees C for 24 h. About 70% of the total alpha-amylase activity in the M-33 culture filtrate was adsorbed onto starch granules. The electrophoretically homogeneous preparation of raw-starch-adsorbable alpha-amylase (molecular weight, 88,000), acid stable at pH 2, showed intensive raw-starch-digesting activity, dissolving corn starch granules completely. The preparation also exhibited a high synergistic effect with glucoamylase I. A mutant, M-72, with higher protease activity produced a raw cornstarch-unadsorbable alpha-amylase. The purified enzyme (molecular weight, 54,000), acid unstable, showed no digesting activity on raw corn starch and a lower synergistic effect with glucoamylase I in the hydrolysis of raw corn starch. The fungal alpha-amylase was therefore divided into two types, a novel type of raw-starch-digesting enzyme and a conventional type of raw-starch-nondigesting enzyme.     

Enhanced Cellulase Production by a Mutant of Sclerotium rolfsii

A mutant of Sclerotium rolfsii CPC 142 that secretes about two times more filter paper-degrading activity in NM-2 growth medium in submerged cultures than the parent strain was obtained by ultraviolet mutagenesis of crushed sclerotia. The production of endo-β-glucanase in the mutant was affected to a lesser extent. With the parent strain, the addition of 3% rice bran to NM-2 medium was essential for optimal formation of cellulase, including filter paper-degrading activity. However, with the mutant the addition of rice bran to NM-2 medium increased the formation of endo-β-glucanase but not filter paper-degrading or cellobiase activity. An altered control mechanism for the production of filter paper-degrading enzymes is suggested. The genome(s) controlling the cellulase complex of enzymes in the UV-8 mutant is not under coordinate control.     

Fermentation of Maize Bran, Oat Bran, and Wheat Bran by Bacteroides ovatus V975

Bacteroides ovatus is a Gram-negative obligate anaerobe that was isolated from the human colon and is capable of utilizing xylan. The objective of this study was to evaluate the ability of B. ovatus V975 to digest maize bran, oat bran, and wheat bran as well as the isolated cell walls from each bran source. Strain V975 was incubated in basal medium that contained either 0.1 or 0.3 g of each bran or each bran cell wall for 0, 24, 48, and 72 h. Acetate and succinate were the main products detected from each fermentation; however, less of each end product was produced from the isolated cell walls than from each bran. More of the oat bran was digested (in vitro dry matter disappearance = 74.8%) during the 72 h incubation than any other bran source. While each bran contained arabinose and xylose, more glucose, galactose, and mannose were utilized by strain V975 during the 72-h incubation than either pentose sugar. Compared with each bran, the bran cell walls had lower concentrations of most sugars, and more glucose than any other sugar was utilized by strain V975. These results suggest that strain V975 preferentially utilizes glucose, galactose, and mannose in each bran, while glucose is the main sugar fermented in bran cell walls. Received: 19 June 1997 / Accepted: 31 July 1997     

Degradation of raw starch granules by alpha-amylase purified from culture of Aspergillus awamori KT-11

Raw-starch-digesting alpha-amylase (Amyl III) was purified to an electrophoretically pure state from the extract of a koji culture of Aspergillus awamori KT-11 using wheat bran in the medium. The purified Amyl III digested not only soluble starch but also raw corn starch. The major products from the raw starch using Amyl III were maltotriose and maltose, although a small amount of glucose was produced. Amyl III acted on all raw starch granules that it has been tested on. However, it was considered that the action mode of the Amyl III on starch granules was different from that of glucoamylase judging from the observation of granules under a scanning electron microscope before and after enzyme reaction, and also from the reaction products. Glucoamylase (GA I) was also isolated and it was purified to an electrophoretically pure state from the extract. It was found that the electron micrographic features of the granules after treatment with the enzymes were quite different. A synergistic effect of Amyl III and GA I was observed for the digestion of raw starch granules.     

Alcoholic fermentation of raw cassava starch by Rhizopus koji without cooking

Using only wheat bran koji from the Rhizopus strain, raw cassava starch and cassava pellets converted reasonably well to alcohol (ethanol) without cooking at 35 degrees C and pH 4.5-5.0. When the initial broth contained 30 g raw cassava starch, 10 g Rhizopus sp. koji, and 100 mL tap water, 12.1 g of alcohol was recovered by final distillation from fermented broth. In this case, 12.1 g alcohol corresponds to an 85.5% conversion rate based on the theoretical values of the starch content. When the initial broth contained 40 g cassava starch, 14.1 g of alcohol was recovered, where 14.1 g corresponds to a 74.5% conversion rate. The alcoholic fermentation process described in the present work is considered more effective and reasonable than the process using raw starch without cooking reported until now, since the new process makes it unnecessary to add yeast cells and glucoamylase preparation.     

Production of acidic lipase by a mutant of Aspergillus niger NCIM 1207 in submerged fermentation

Mutants of Aspergillus niger NCIM 1207, isolated by subjecting conidia to UV-irradiation, were tested for the production of lipase (glycerol ester hydrolase EC 3.1.1.3). Mutants UV-10 and ANCR-1 showed seven fold and five fold enhanced productivity of enzyme, respectively, over the wild strain in shake flask culture when grown in SOB medium containing 1% olive oil. Maximum lipase activity (41 IU/ml) was obtained in the culture broth when UV-10 was grown in medium supplemented with 0.5% Triton X-100. A higher concentration of oil in the medium did not help lipase production in the case of mutant UV-10. Similarly no increase in enzyme levels was observed when mutant UV-10 was grown in medium supplemented with glucose. However, the addition of glucose in the medium resulted in increased levels of lipase production by wild strain, Aspergillus niger NCIM 1207.     

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.

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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.

     

Selection and study of a Candida molischiana mutant derepressed for β-glucosidase production

Abstract A mutant strain of Candida molischiana was selected. Analysis of the exocellular activity of Candida molischiana 35M5N grown on different carbon sources revealed that the biosynthesis of β-glucosidase is derepressed in this yeast strain. The strain is not a hyper-producer mutant. There were no observed differences in the endocellular and parietal activities of the wild and mutant strains. However, the mutant strain produced 35-fold more enzyme than the wild-type in the culture medium with glucose as carbon source. When glucose was used as carbon source, the mutant strain produced 90% more exocellular enzyme than when cellobiose was used as the carbon source.     

木聚糖酶高产菌株的诱变*

出发菌株Aspergillus niger M1经过紫外线诱变得到一株木聚糖酶活力提高30％的突变株A．niger J506。木聚糖酶谱带检测发现,突变株成熟发酵液中有3种类型的木聚糖酶,而出发菌株中只有两种。经过正交试验得出突变株产酶的最佳发酵条件为：主碳源浓度4％、麸皮与玉米芯的比例为5：5、葡萄糖浓度0．1％、草酸铵浓度2．0％,培养基初始pH为5．0,250mL三角瓶的装液量为100mL。