Optimisation of growth medium for the production of cyclodextrin glucanotransferase from Bacillus stearothermophilus HR1 using response surface methodology

Cyclodextrin glucanotransferase (CGTase) activity was observed when the bacterium was grown in the medium at various initial pH values, containing carbon, nitrogen, phosphorus and mineral salt sources at 50 °C for 24 h in the shake flasks. The optimisation of this growth medium was carried out using response surface methodology. The design contains a total of 32 experimental trials involving 10 star points and 6 replicates at the centre points. The design was employed by selecting sago starch, peptone from casein, K₂HPO₄, CaCl₂ and initial pH as five independent variables in this study. The optimal calculated values of tested variables for maximal production of CGTase were found to be comprised of: sago starch, 16.02 g/l; peptone from casein, 20 g/l; K₂HPO₄, 1.4 g/l; CaCl₂, 0.2 g/l and initial pH, 7.54 with a predicted CGTase activity of 14.20 U/ml. These predicted optimal parameters were tested in the laboratory and the final CGTase activity obtained was very close to the predicted value at 14.80 U/ml.     

Production of raw cassava starch-degrading enzyme by Penicillium and its use in conversion of raw cassava flour to ethanol

A newly isolated strain Penicillium sp. GXU20 produced a raw starch-degrading enzyme which showed optimum activity towards raw cassava starch at pH 4.5 and 50 °C. Maximum raw cassava starch-degrading enzyme (RCSDE) activity of 20 U/ml was achieved when GXU20 was cultivated under optimized conditions using wheat bran (3.0% w/v) and soybean meal (2.5% w/v) as carbon and nitrogen sources at pH 5.0 and 28 °C. This represented about a sixfold increment as compared with the activity obtained under basal conditions. Starch hydrolysis degree of 95% of raw cassava flour (150 g/l) was achieved after 72 h of digestion by crude RCSDE (30 U/g flour). Ethanol yield reached 53.3 g/l with fermentation efficiency of 92% after 48 h of simultaneous saccharification and fermentation of raw cassava flour at 150 g/l using the RCSDE (30 U/g flour), carried out at pH 4.0 and 40 °C. This strain and its RCSDE have potential applications in processing of raw cassava starch to ethanol.     

Direct fermentation of gelatinized sago starch to acetone–butanol–ethanol by Clostridium acetobutylicum

Direct fermentation of gelatinized sago starch into solvent (acetone–butanol–ethanol) by Clostridium acetobutylicum P262 was studied using a 250 ml Schott bottle anaerobic fermentation system. Total solvent production from fermentation using 30 g sago starch/l (11.03g/l) was comparable to fermentation using corn starch and about 2-fold higher than fermentation using potato or tapioca starch. At the range of sago starch concentration investigated (10–80 g/l), the highest total solvent production (18.82 g/l) was obtained at 50 g/l. The use of a mixture of organic and inorganic nitrogen source (yeast extract + NH₄NO₃) enhanced growth of C. acetobutylicum, starch hydrolysis and solvent production (24.47 g/l) compared to the use of yeast extract alone. This gave the yield based on sugar consumed of 0.45 g/g. Result from this study also showed that the individual concentrations of nitrogen and carbon influenced solvent production to a greater extent than did carbon to nitrogen (C/N) ratio.     

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.     

Optimization of cyclodextrin production from sago starch

Cyclodextrin (CD) is synthesized by bacterial cyclodextrin glycosyltransferase (CGTase) and is widely used in food, pharmaceutical, cosmetic, and agricultural industries. In this study, Bacillus circulans CGTase was partially purified by ammonium sulfate precipitation at 50-70% saturation. The optimum pH and temperature for CD production from sago starch were found to be in the ranges of 4.5-5.0 and 55-60 degrees C, respectively. beta-CD was the predominant product, constituting 65% of all CD products. The beta-CD produced using partially purified and crude CGTase were compared and found to have no significant difference in yield and productivity. The appropriate proportion of CGTase to sago starch for beta-CD production was determined by response surface methodology. The most appropriate enzyme:substrate ratio was 50 U g sago starch(-1) CGTase and 60 g l(-1) sago starch.     

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.     

Culture conditions for the production of amylolytic enzymes by a thermophilic Clostridium sp.

The growth of a thermophilic Clostridium sp. and the production of α-glucosidase, α-amylase and pullulanase were studied under anaerobic conditions using different carbon and nitrogen sources and varying pH values and temperatures. Growth and enzyme activities were highest with soybean meal as the nitrogen source. The optimum concentration was 2.5% [w/v] for the production of α-amylase as well as pullulanase and 2% [w/v] for α-glucosidase. The best carbon source proved to be soluble starch for α-amylase, and pullulanase and maltose for α-glucosidase. Growth and enzyme production reached their optimum at pH 6.5 to 7.0 and 70°C. Under these conditions, the enzyme activities followed exponential growth with maximum yields of α-glucosidase, α-amylase and pullulanase at 28, 36, and 44 h.     

Optimization of fermentation conditions for the production of ethanol from sago starch using response surface methodology

The quantitative effects of temperature, pH and time of fermentation were investigated on simultaneous saccharification and fermentation (SSF) of ethanol from sago starch with glucoamylase (AMG) and Zymomonas mobilis ZM4 using a Box–Wilson central composite design protocol. The SSF process was studied using free enzyme and free cells and it was found that with sago starch, maximum ethanol concentration of 70.68 g/l was obtained using a starch concentration of 140 g/l, which represents an ethanol yield of 97.08%. The optimum conditions for the above yield were found to be a temperature of 36.74 °C, pH of 5.02 and time of fermentation of 17 h. Thus by using the central composite design, it is possible to determine the accurate values of the fermentation parameters where maximum production of ethanol occurs.     

The fermentative production of acetone-butanol by Clostridium acetobutylicum.

Fourteen different media were used in the fermentative production of acetone-butanol. The highest total yields were achieved in medium I. Potato starch and soluble starch were suitable as carbon sources. The best concentrations of potato starch and soluble starch were 500.0 and 10.0 g/l, respectively. Peptone was the most favourable nitrogen source. The best concentration of peptone was 4.0 g/l. Calcium carbonate in 3.6 g/l acted as buffering agent in the fermentation process. The best initial pH value of the fermentation medium was 6.0. The optimum temperature was 32--33degreesC. The fermentation process required 120 h to obtain maximum yields of acetone-butanol.     

Preparation of Triglyceride Hydroperoxides

Corticium rolfsii AHU 9627, which we isolated from a tomato stem, is one of the most promising producers of a raw starch saccharifying enzyme. The effects of the cultural conditions and medium components on the enzyme production were investigated. The enzyme production was improved by increasing both the concentrations of carbon sources and organic nutrients in the medium. Under the optimum cultural conditions, the enzyme activity of the culture supernatant against raw starch reached a maximum after 8-days incubation at 27°C and the activity reached 80 units per ml (when determined at 40°C and pH 4.0). The optimal pH and temperature for the enzyme reaction were 4.0 and 65°C, respectively. The saccharifying reaction was scarcely inhibited even with a high substrate concentration, and raw starch was rapidly hydrolyzed into glucose.     

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.     

Itaconic acid production using sago starch hydrolysate by Aspergillus terreus TN484-M1

Sago starch was hydrolyzed using either chemical agents, or enzymes at various pH and concentrations. Hydrolysis using 5000 AUN/ml (0.5%, w/v) glucoamylase exhibited the highest itaconic acid yield up to 0.36 g/g sago starch, whereas hydrolysis using nitric acid at pH 2.0 yielded 0.35 g/g sago starch. The medium was optimized and the composition was (g/l) 140 sago starch, 1.8 corn steep liquor, 1.2 MgSO(4).7H(2)O and 2.9 NH(4)NO(3). When the optimal conditions of hydrolysis and medium composition were applied to itaconic acid production in a 3-l jar fermentor, the itaconic acid production was 48.2 g/l with a yield of 0.34 g/g sago starch. This was filtered from the cultured broth and 37.1g of itaconic acid was recovered with a purity of 97.2%. This result showed that sago starch could be converted to a value-added product with only a simple pretreatment.     

Production and properties of fibrinolytic enzyme fromStreptomyces sp. NRC 411

Of 16Streptomyces spp. investigated for the production of extracellular fibrinolytic enzyme, one species was chosen as the most promising producer. Using shaken cultures grown for 7 days, optimal conditions for enzyme production were pH 6.0, 5% (w/v) starch as carbon source, (NH₄)₂SO₄ and soybean flour as nitrogen sources and KH₂PO₄ at 1.2 g/l. Maximal activity of the crude enzyme was at pH 6.0 and 45°C. Holding the enzyme at 37°C for 2 h decreased the activity by only 10%.     

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.     

Tylosin production by Streptomyces fradiae using raw cornmeal in airlift bioreactor

Using a 50-l airlift bioreactor, for the effective production of tylosin from Streptomyces fradiae TM-224 using raw cornmeal as the energy source, various environmental factors were studied in flask cultures. The maximum tylosin concentration was obtained at 32 degrees C and pH between 7.0 and 7.5. When seed was inoculated after 24 h of culture, the maximum tylosin concentration, 5.7 g/l, was obtained after 4 days of culture. Various concentrations of raw cornmeal were tested to investigate the optimum initial concentration for the tylosin production. An initial raw cornmeal concentration of 80 g/l gave the highest tylosin concentration, 5.8 g/l, after 5 days of culture. Of the various nitrogen sources, soybean meal and fish meal were found to be the most effective for the production of tylosin. In particular, with the optimal mixing ratio, 12 g/l of soybean meal to 14 g/l of fish meal, 7.2 g/l of tylosin was obtained after 5 days of culture. To compare raw cornmeal and glucose for the production oftylosin in the 50-1 airlift bioreactor for 10 days, fed-batch cultures were carried out under the optimum culture conditions. When raw corn meal was used as the energy source, the tylosin production increased with increasing culture time. The maximum tylosin concentration after 10 days of culture was 13.5 g/l, with a product yield from raw cornmeal of 0.123 g/g of consumed carbon source, which was about 7.2 times higher than that obtained when glucose was used as the carbon source.     

Production and characterization of thermostable alpha-amylase by thermophilic Geobacillus stearothermophilus

Studies on the alpha-amylase-producing thermophilic bacterium isolated and identified from a hot spring in Jordan and designated as Geobacillus stearothermophilus JT2 were carried out. The optimum conditions for growth and enzyme production were pH 7 and 55 degrees C. The study of the kinetics of cellular growth indicated a mu(max) of 0.22/h, a K(s) of 1.2 g/L, a tau(d) of 3.15 h and a Y(x/s) of 0.43 g cell/g starch. In addition, the activation energy for growth and death were estimated and found to be 30.5 and 210 J/mol, respectively. The effect of different carbon and nitrogen sources on the cellular growth was tested.     

Production and partial characterization of high molecular weight extracellular alpha-amylase from Thermoactinomyces vulgaris isolated from Egyptian soil

Optimizing production of alpha-amylase production by Thermoactinomyces vulgaris isolated from Egyptian soil was studied. The optimum incubation period, temperature and initial pH of medium for organism growth and enzyme yield were around 24 h, 55 degrees C and 7.0, respectively. Maximum alpha-amylase activity was observed in a medium containing starch as carbon source. The other tested carbohydrates (cellulose, glucose, galactose, xylose, arabinose, lactose and maltose) inhibited the enzyme production. Adding tryptone as a nitrogen source exhibited a maximum activity of alpha-amylase. Bactopeptone and yeast extract gave also high activity comparing to the other nitrogen sources (NH4CI, NH4NO3, NaNO3, KNO3, CH3CO2NH4). Electrophoresis profile of the produced two alpha-amylase isozymes indicated that the same pattern at about 135-145 kDa under different conditions. The optimum pH and temperature of the enzyme activity were 8.0 and 60 degrees C, respectively and enzyme was stable at 50 degrees C over 6 hours. The enzyme was significantly inhibited by the addition of metal ions (Na+, Co2+ and Ca2+) whereas CI- seemed to act as activator. The enzyme was not affected by 0.1 mM EDTA while higher concentration (10 mM EDTA) totally inactivated the enzyme.     

Influence of growth conditions on the production of xylanolytic enzymes by Trametes trogii

Various nitrogen and carbon sources were examined as inducers of the production of endoxylanase and -xylosidase by Trametes trogii. T. trogii grown on xylan plus crystalline cellulose provided supernatants with the highest enzymatic activities. Organic nitrogen sources (especially asparagine and casamino acids) were the best for enzyme production. The increase in xylan concentration stimulated endoxylanase production whereas significant differences were not attained in -xylosidase production with more than 5g xylan/l in the culture medium. pH 4.0 was optimal for endoxylanase production, while -xylosidase production was maximum at pH 5.5. Temperatures in the range of 23–28°C stimulated enzyme production. The endoxylanase activity in the crude culture filtrate was greatest at 50°C and pH around 5.0. The optimum pH and temperature for -xylosidase activity were 5.5 and 50°C respectively.     

Heterologous expression of <Emphasis Type="Italic">Thermobifida fusca</Emphasis> thermostable alpha-amylase in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> and its application in boiling stable resistant sago starch preparation

A gene encoding the thermostable α-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced, and cloned into Yarrowia lipolytica P01g host strain using the vector pYLSC1 allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular amylase production, as high as 730 U/l in the Hinton flask culture broth. It is higher than that observed in P. pastoris expression system and E. coli expression system. The purified amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis and this agrees with the predicted size based on the nucleotide sequence. About 70% of the original activity remained after heat treatment at 60°C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and 60°C, respectively. The purified amylase exhibited a high level of activity with raw sago starch. After 72-h treatment, the DP _w of raw sago starch obviously decreased from 830,945 to 237,092. The boiling stable resistant starch content of the sago starch increased from 8.3 to 18.1%. The starch recovery rate was 71%.     

Optimization of culture conditions for the production of rifamycin oxidase by Curvularia lunata

Maximum activity (8.9 IU/ml) of rifamycin oxidase in Curvularia lunata, grown in shake-flask culture at 28°C and pH 6.5, was after 96 h. Nearly all the glucose was used in 72 h. An initial culture pH of 6.5 and 28°C were optimum for the growth and enzyme production. Among various carbon and organic nitrogen sources, carboxymethylcellulose and peptone were the most effective for enzyme yield. The rate of enzyme production was enhanced when yeast extract was also added to the medium. The optimum medium for the production of rifamycin oxidase contained 10 g each of yeast extract, peptone and carboxymethylcellulose/l and 0.04% (NH₄)₂SO₄.The author is with the Biochemical Engineering Research and Process Development Centre, Institute of Microbial Technology, Post Box 1304, Sector 39-A, Chandigarh 160 014, India