Alpha-galactosidase production by Aspergillus oryzae in solid-state fermentation

Comparisons were made for alpha-galactosidase production using red gram plant waste (RGPW) with wheat bran (WB) and other locally available substrates using the fungus Aspergillus oryzae under solid-state fermentation (SSF). RGPW proved to be potential substrate for alpha-galactosidase production as it gave higher enzyme titers (3.4 U/g) compared to WB (2.7 U/g) and other substrates tested. Mixing WB with RGPW (1:1, w/w) resulted enhanced alpha-galactosidase yield. The volume of moistening agent in the ratio of 1:2 (w/v), pH 5.5 and 1 ml (1 x 10(6) spores) of inoculum volume and four days incubation were optimum for alpha-galactosidase production. Increase in substrate concentration (RGPW+WB) did not decrease enzyme yield in trays.     

Evaluation of culture conditions for cellulase production by two Trichoderma reesei mutants under solid-state fermentation conditions

Response surface methodology (RSM) was used to evaluate the effects of fermentation parameters for cellulase production by Trichoderma reesei QM9414 and T. reesei MCG77 in solid-state fermentation using rice bran as substrate. Initial pH, moisture content and temperature were optimized using filter paper activity (FPA) as response. Statistical analysis of the results for T. reesei QM9414 showed that only moisture content had significant effect on cellulase activity and had a linear effect on enzyme activity (maximum enzyme activities were obtained at 70% moisture content). The results for T. reesei MCG77 showed that temperature and moisture content were the most significant parameters for cellulase activity. The optimum cellulase production was in the temperature range of 25-30 degrees C and moisture content between 55% and 70%. After the optimization, the FPA in T. reesei MCG77 was increased by 2.5 folds compared to that of T. reesei QM9414.     

Improvement of the glaB promoter expressed in solid-state fermentation (SSF) of Aspergillus oryzae

The glucoamylase-encoding gene (glaB) promoter should be very useful for recombinant protein production in solid-state fermentation (SSF) of Aspergillus oryzae. A 97-bp fragment containing the cis-element of the glaB promoter was inserted into the glaA promoter, which was little expressed in SSF. The chimeric promoter showed about a 24-fold increase in promoter activity in SSF. Eight copies of the 97-bp fragment were tandemly fused with the glaB promoter. The improved promoter showed about a 4.6-fold increase in promoter activity in SSF. The glaB gene was overexpressed under control of the improved glaB promoter in SSF. Recombinant glucoamylase production reached about 1524 mg/kg-broth for 2 d. The improved glaB promoter should be very useful for overproduction of a recombinant protein in SSF of A. oryzae.     

Optimization of liquid-state fermentation conditions for the glyphosate degradation enzyme production of strain Aspergillus oryzae by ultraviolet mutagenesis

This study aimed to obtain strains with high glyphosate-degrading ability and improve the ability of glyphosate degradation enzyme by the optimization of fermentation conditions. Spore from Aspergillus oryzae A-F02 was subjected to ultraviolet mutagenesis. Single-factor experiment and response surface methodology were used to optimize glyphosate degradation enzyme production from mutant strain by liquid-state fermentation. Four mutant strains were obtained and named as FUJX 001, FUJX 002, FUJX 003, and FUJX 004, in which FUJX 001 gave the highest total enzyme activity. Starch concentration at 0.56%, GP concentration at 1,370?mg/l, initial pH at 6.8, and temperature at 30°C were the optimum conditions for the improved glyphosate degradation endoenzyme production of A. oryzae FUJX 001. Under these conditions, the experimental endoenzyme activity was 784.15?U/100?ml fermentation liquor. The result (784.15?U/100?ml fermentation liquor) was approximately 14-fold higher than that of the original strain. The result highlights the potential of glyphosate degradation enzyme to degrade glyphosate.     

里氏木霉与米根霉混合固态发酵产纤维素酶

以里氏木霉及米根霉单菌固态发酵为对象,考察不同混合发酵形式对里氏木霉与米根霉混合固态发酵产纤维素酶的影响。结果表明:同时接种里氏木霉与米根霉,试验考察的两菌种接种量比1∶1(以孢子个数计)及5∶1条件下,两菌未产生明显协同产酶作用。米根霉延时(24 h)接种且菌种量比5∶1以及米根霉延时(48 h)接种且菌种量比1∶1,2种发酵形式产酶情况类似,滤纸酶活(FPA)及羧甲基纤维素酶(CMCase)酶活相对米根霉单菌发酵有所提高,而β-葡萄糖苷酶(β-GA)酶活相对里氏木霉单菌固态发酵结束时分别增加4.66及4.40倍,可以发现两菌产生一定协同作用。在米根霉延时(48 h)接种且菌种量比5∶1的发酵形式下,FPA及CMCase在发酵第7天酶活分别达到44.04 IU/g、627.14 U/g(以1 g干曲计),分别是里氏木霉固态单菌发酵产酶达到稳定期时酶活的1.36和1.63倍,两菌产生了有效的协同作用。     

Acid protease production by solid-state fermentation using Aspergillus oryzae MTCC 5341: optimization of process parameters

Aspergillus oryzae MTCC 5341, when grown on wheat bran as substrate, produces several extracellular acid proteases. Production of the major acid protease (constituting 34% of the total) by solid-state fermentation is optimized. Optimum operating conditions obtained are determined as pH 5, temperature of incubation of 30°C, defatted soy flour addition of 4%, and fermentation time of 120 h, resulting in acid protease production of 8.64 × 10⁵ U/g bran. Response-surface methodology is used to generate a predictive model of the combined effects of independent variables such as, pH, temperature, defatted soy flour addition, and fermentation time. The statistical design indicates that all four independent variables have significant effects on acid protease production. Optimum factor levels are pH 5.4, incubation temperature of 31°C, 4.4% defatted soy flour addition, and fermentation time of 123 h to yield a maximum activity of 8.93 × 10⁵ U/g bran. Evaluation experiments, carried out to verify the predictions, reveal that A. oryzae produces 8.47 × 10⁵ U/g bran, which corresponds to 94.8% of the predicted value. This is the highest acid protease activity reported so far, wherein the fungus produces four times higher activity than previously reported [J Bacteriol 130(1): 48–56, 1977].     

棘孢曲霉固态发酵柚皮产柚苷酶的条件优化

【目的】以柚皮为原料,优化棘孢曲霉利用柑橘加工副产物固态发酵柚苷酶的条件。【方法】采用高效液相色谱法检测酶活力,通过单因素试验考察固水比、装样量、接种量、温度对柚苷酶发酵的影响,用正交试验优化发酵条件。【结果】单因素试验结果的显著性分析表明培养基的固水比、装样量和培养温度对柚苷酶产量有显著性影响,而接种量影响不显著;经正交试验确定的优化条件是：固水比1:1 (质量体积比),装样量5 g/250 mL三角瓶,温度为30 °C,接种1 mL孢子悬浮液,发酵8 d。在此优化条件下,柚苷酶酶活力为8.19 IU/g干物质,比初始培养基产柚苷酶活力提高7.38倍。【结论】通过对固水比、装样量和发酵温度进行优化,大幅度提高了棘孢曲霉固态发酵柑橘加工副产物的柚苷酶产量,为柚苷酶的生产提供了一种高产发酵工艺。     

米曲霉发酵纯化无患子皂苷的工艺研究

采用微生物发酵法对无患子皂苷水提取液进行纯化.比较了采用自然发酵、接种酵母菌发酵和接种米曲霉发酵纯化无患子皂苷的效果.结果表明,提取液不灭菌,接种米曲霉发酵纯化效果较为明显,优化后的发酵条件为:温度30℃、接种龄12 h、接种量为3％、摇床转速150 r/min,发酵7d后,皂苷含量稍有下降,但皂苷纯度可从48.71％提高到82.47％.米曲霉发酵法明显优于水提醇沉法、絮凝法和正丁醇萃取法.     

Production and purification of extracellular chitinases from Penicillium aculeatum NRRL 2129 under solid-state fermentation

Fourteen Penicillium strains have been screened on wheat bran–crude chitin mixture medium for extracellular chitinase production in solid-state fermentation. Under the experimental conditions tested, Penicillium aculeatum NRRL 2129 (=ATCC 10409) was selected as the best enzyme producer. The optimum incubation period for chitinase production by the potent organism was found to be 72 h. Chromatofocusing was performed as the first step in the purification scheme, but high amount of contaminating proteins interfered with the method. Hence, ion-exchange chromatography experiments were carried out followed by gel filtration to separate and isolate chitinase isoenzymes. Four major chitinase peaks of molecular weight 82.7, 44.6, 28.2 and 26.9 kDa were observed after gel filtration chromatography while, on SDS-PAGE, three protein bands of molecular weights 82.6, 33.9 and 29.1 kDa were identified. The purified enzyme showed optimal temperature and pH at 50 and 5.5 °C, respectively.     

Improvement of xylanase production in solid-state fermentation by alkali tolerant Aspergillus versicolor MKU3

AIMS: To optimize the media components for xylanase production by Aspergillus versicolor MKU3 in solid-state fermentation (SSF). METHODS AND RESULTS: Medium optimization was carried out using De Moe's fractional factorial design with seven components. Maximum production of xylanase 3249.9 U g(-1) was obtained in SSF with an optimized medium containing (g l(-1)): NaNO(3), 20; K(2)HPO(4), 20; MgSO(4), 10; FeSO(4), 0.001; KCl, 1; peptone, 10 and yeast extract, 10. Four components namely NaNO(3), MgSO(4), peptone and K(2)HPO(4) significantly increased the xylanase production by A. versicolor MKU3. CONCLUSIONS: Fractional factorial design was used to optimize the seven components in the fermentation medium for SSF. The optimized media increased xylanase production by 3.4-fold. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspergillus versicolor MKU3 produced maximum xylanase after two steps of media optimization under alkaline condition. This medium will be significant value for xylanase production in SSF.     

Contribution of aerial hyphae of Aspergillus oryzae to respiration in a model solid-state fermentation system

Oxygen transfer is for two reasons a major concern in scale-up and process control in industrial application of aerobic fungal solid-state fermentation (SSF): 1) heat production is proportional to oxygen uptake and it is well known that heat removal is one of the main problems in scaled-up fermenters, and 2) oxygen supply to the mycelium on the surface of or inside the substrate particles may be hampered by diffusion limitation. This article gives the first experimental evidence that aerial hyphae are important for fungal respiration in SSF. In cultures of A. oryzae on a wheat-flour model substrate, aerial hyphae contributed up to 75% of the oxygen uptake rate by the fungus. This is due to the fact that A. oryzae forms very abundant aerial mycelium and diffusion of oxygen in the gas-filled pores of the aerial hyphae layer is rapid. It means that diffusion limitation in the densely packed mycelium layer that is formed closer to the substrate surface and that has liquid-filled pores is much less important for A. oryzae than was previously reported for R. oligosporus and C. minitans. It also means that the overall oxygen uptake rate for A. oryzae is much higher than the oxygen uptake rate that can be predicted in the densely packed mycelium layer for R. oligosporus and C. minitans. This would imply that cooling problems become more pronounced. Therefore, it is very important to clarify the physiological role of aerial hyphae in SSF.     

黑曲霉AF-98固体发酵产纤维素酶的产酶条件研究

通过单因子及正交试验,对黑曲霉AF-98固体发酵产纤维素酶的产酶条件进行了探讨。其优化的产酶条件为:甘蔗渣3g,麸皮2g,加含尿素为0.15%的Mandels营养液25mL(加水比1:5),调初始pH5.0,28℃发酵72h。在此优化条件下,纤维素酶活力可达7.56u/g干曲。     

Agar plate growth studies of Rhizopus oligosporus and Aspergillus oryzae to determine their suitability for solid-state fermentation

Summary Colony radial growth rates of Rhizopus oligosporus and Aspergillus oryzae were compared under various conditions on agar plates containing cassava starch. Both organisms grew well on cassava starch as their sole source of carbon and energy, although growth was stimulated by the addition of yeast extract and peptone. Neither organism utilized ungelatinized starch effectively. The optimum initial pH for R. oligosporus was 7, although good growth was obtained at pH 5 when ammonium sulfate was partially replaced by urea. A. oryzae grew well over a range of initial pH values from 5 to 8. Growth of R. oligosporus was inhibited by NaCl concentrations above 0.5% (w/v) while A. oryzae was unaffected up to 4% NaCl. The best colony radial growth rate obtained for R. oligosporus was 1.01 mm/h, which was far superior to that obtained for A. oryzae (0.29 mm/h). R. oligosporus was chosen as the more suitable organism for future studies of the protein enrichment of cassava by solid-state fermentation.     

Water and glucose gradients in the substrate measured with NMR imaging during solid-state fermentation with Aspergillus oryzae

Gradients inside substrate particles cannot be prevented in solid-state fermentation. These gradients can have a strong effect on the physiology of the microorganisms but have hitherto received little attention in experimental studies. We report gradients in moisture and glucose content during cultivation of Aspergillus oryzae on membrane-covered wheat-dough slices that were calculated from (1)H-NMR images. We found that moisture gradients in the solid substrate remain small when evaporation is minimized. This is corroborated by predictions of a diffusion model. In contrast, strong glucose gradients developed. Glucose concentrations just below the fungal mat remained low due to high glucose uptake rates, but deeper in the matrix glucose accumulated to very high levels. Integration of the glucose profile gave an average concentration close to the measured average content. On the basis of published data, we expect that the glucose levels in the matrix cause a strong decrease in water activity. The results demonstrate that NMR can play an important role in quantitative analysis of water and glucose gradients at the particle level during solid-state fermentation, which is needed to improve our understanding of the response of fungi to this nonconventional fermentation environment.     

Optimization of pretreatment and fermentation conditions for production of extracellular cellulase complex using sugarcane bagasse

Sugarcane bagasse (SCB), a lignocellulosic byproduct of juice extraction from sugarcane, is rich in cellulose (40-42%). This could be used as a substrate for the production of cellulase complex. Fermentation conditions were optimized for production of cellulase complex (CMCase, Cellulobiase and FPase) by wild type Trichoderma sp. using sugarcane bagasse as sole carbon source. Alkaline treatment (2% NaOH) of bagasse (AlSCB) was found suitable for the production of reducing sugar over the acidic pretreatment method. After 5 days of incubation period, 5% substrate concentration at pH 5.0 and 400C resulted in maximum production of CMCase (0.622 U), while maximum (3.388 U) production of cellulobiase was obtained at 300C. The CMCase was precipitated and purified to the extent of 59.06 fold by affinity chromatography with 49.09% recovery. On 12% SDS-PAGE, a single band corresponding to 33 kDa was observed. The Km and Vmax for CMCase from Trichoderma was found 507.04 mg/ml and 65.32 mM/min, respectively. The enzyme exhibited maximum activity at 300C at pH-5.0 (0.363 U) and was stable over range of 20-60°C and pH 5.0-7.5.