Enhancement of glucose oxidase production in batch cultivation of recombinant Saccharomyces cerevisiae: optimization of oxygen transfer condition

AIMS: To obtain an optimal combination of agitation speed and aeration rate for maximization of specific glucose oxidase (GOD) production in recombinant Saccharomyces cerevisiae, and to establish a correlation between kLa vis-à-vis oxygen transfer condition and specific glucose oxidase production. METHODS AND RESULTS: The oxygen transfer condition was manifested indirectly by manipulating the impeller speed and aeration rate in accordance with a Central Composite Rotatory Design (CCRD). The dissolved oxygen concentration and the volumetric oxygen transfer coefficient (kLa) were determined at corresponding combinations of impeller speed and aeration rate. The maximal specific extracellular glucose oxidase production (3.17 U mg-1 dry cell mass) was achieved when the initial dissolved oxygen concentration was 6.83 mg l-1 at the impeller speed of 420 rev min-1 and at the rate of aeration of 0.25 vvm. It was found out that while impeller speed had a direct effect on the production of enzyme, a correlation between kLa and specific GOD production could not be established. CONCLUSION: At the agitation speed of 420 rev min-1 and at 0.25 vvm aeration rate, the degree of turbulence and the dissolved oxygen concentration were thought to be optimal both for cellular growth and production of enzyme. SIGNIFICANCE AND IMPACT OF THE STUDY: The combined effect of agitation and aeration on recombinant glucose oxidase production in batch cultivation has not yet been reported in the literature. Therefore, this study gives an insight into the effect of these two important physical parameters on recombinant protein production. It also suggests that since there is no correlation between kLa and specific production of GOD, kLa should not be used as one of the scale-up parameters.     

Enhanced Production of carboxymethylcellulase by a marine bacterium,Bacillus velezensis A-68, by using rice hulls in pilot-scale bioreactor under optimized conditions for dissolved oxygen

The optimal conditions for the production of carboxymethylcellulase (CMCase) by Bacillus velezensis A-68 at a flask scale have been previously reported. In this study, the parameters involved in dissolved oxygen in 7 and 100 L bioreactors were optimized for the pilot-scale production of CMCase. The optimal agitation speed and aeration rate for cell growth of B. velezensis A-68 were 323 rpm and 1.46 vvm in a 7 L bioreactor, whereas those for the production of CMCase were 380 rpm and 0.54 vvm, respectively. The analysis of variance (ANOVA) implied that the highly significant factor for cell growth was the aeration rate, whereas that for the production of CMCase was the agitation speed. The optimal inner pressures for cell growth and the production of CMCase by B. velezensis A-68 in a 100 L bioreactor were 0.00 and 0.04 MPa, respectively. The maximal production of CMCase in a 100 L bioreactor under optimized conditions using rice hulls was 108.1 U/ml, which was 1.8 times higher than that at a flask scale under previously optimized conditions.     

Operating conditions optimization for (+)-terrein production in a stirred bioreactor by Aspergillus terreus strain PF-26 from marine sponge Phakellia fusca

Terrein is a fungal metabolite with application values in the fields of medicine, cosmetology, and agriculture. However, mass production of single configuration terrein is still a big challenge. In this study, operating factors such as inoculation, agitation speed, aeration rate, pH control, and nutrient feeding were preliminarily optimized to improve the (+)-terrein production in the 5-L stirred bioreactor from the marine sponge-derived fungus Aspergillus terreus PF-26. Spore inoculation, low agitation speed, and aeration rate were proved to be suitable for A. terreus PF-26 to produce (+)-terrein in the stirred bioreactor. At 50?rpm agitation speed and 0.33?vvm aeration rate, 2.68?g/L (+)-terrein was achieved by feeding twofold concentrated maltose and glucose medium on the sixth day and controlling pH at 4.5 from the fourth day. This study lays foundation for the mass production of (+)-terrein by the marine filamentous A. terreus strain PF-26 in the stirred bioreactor.     

Fermentation strategies for the production of lipase by an indigenous isolate Burkholderia sp. C20

Burkholderia sp. C20 strain isolated from food wastes produces a lipase with hydrolytic activities towards olive oil. Fermentation strategies for efficient production of this Burkholderia lipase were developed using a 5-L bench top bioreactor. Critical factors affecting the fermentative lipase production were examined, including pH, aeration rate, agitation rate, and incubation time. Adjusting the aeration rate from 0.5 to 2 vvm gave an increase in the overall lipase productivity from 0.057 to 0.076 U/(ml h), which was further improved to 0.09 U/(ml h) by adjusting the agitation speed to 100 rpm. The production of Burkholderia lipase followed mixed growth-associated kinetics with a yield coefficient of 524 U/g-dry-cell-weight. The pH optimum for cell growth and lipase production was different at 7.0 and 6.0, respectively. Furthermore, stepwise addition of carbon substrate (i.e., olive oil) enhanced lipase production in both flask and bioreactor experiments.     

Production and characterization of Penicillium brasilianum pectinases with regard to industrial application

The objective of this study was to evaluate the production of pectinase by an isolated strain of Penicillium brasilianum in a bioreactor and to consider its potential for industrial applications (i.e. fruit juice). The optimization of production was achieved through experimental design. The maximum exo-polygalacturonase (Exo-PG) production in the bioreactor was 53.8?U mL^?1 under the conditions of 180?rpm, an aeration rate of 1.5 vvm, 30?°C, pH_initial of 5.5, 5?×?10⁶ spores mL^?1, 32?g L^?1 pectin, 10?g L^?1 of yeast extract and 0.5?g L^?1 magnesium sulfate and bioproduction for 36?h. The production of Exo-PG in the bioreactor was 1.3 times higher than that obtained in shake flasks, with aeration (1.5 vvm) and agitation (180?rpm) control. The crude enzyme complex, beyond the pectinolytic activity of Exo-PG (53.8?U mL^?1), also contained activity pectin methylesterase (6.0?U mL^?1) and pectin lyase (6.61?U mL^?1). At a crude enzyme complex with a concentration of 0.5% (v/v), viscosity of peach juice was reduced by 11.66%, turbidity was reduced by 13.71% and clarification was increased by 26.92%. Based on the present results, we can conclude that the new strain of isolated P. brasilianum produced high amounts of pectinases in a bioreactor with mechanical agitation, and has the potential to be applied to in the clarification of juices.     

Degradation of Benzene,Toluene and Xylene by Pseudomonas aeruginosa Engineered with the Vitreoscilla Hemoglobin Gene

This study concerns the potential use of Pseudomonas aeruginosa expressing the Vitreoscilla hemoglobin gene for the degradation of important harmful aromatic compounds such as benzene, toluene, and xylene (BTX). The use of these compounds by both strains was determined as the production of cell mass (viable cell number) in a minimal medium containing any one of the BTX compounds as the sole carbon and energy source. Furthermore, the BTX degradation capability of both strains was monitored by measuring the production of 3‐methylcatechol, a common intermediate. For the cells of the logarithmic phase, which were grown at high aeration/high agitation or low aeration/low agitation, the engineered strain showed a better growth rate than the host strain. With the benzene in the medium, the recombinant strain exhibited a higher (up to 4‐fold) cell density than the parental wild‐type strain at this phase. In contrast, regarding the cells of the late stationary phase under high aeration/high agitation conditions, the host strain had generally higher viable cell numbers than the recombinant strain. At this phase this difference was, however, less significant under the conditions of low aeration/low agitation. Similarly, in toluene containing medium (at high aeration/high agitation) the recombinant strain showed a higher cell density which was from a 15‐fold to almost one order of magnitude greater than its parental strain during the logarithmic phase where the cell density of P. aeruginosa remained nearly constant. Contrary to the results with benzene and toluene, both strains exhibited similar growth characteristics when they were grown in the presence of xylene. The positive effect of the oxygen uptake by the recombinant system on the BTX metabolizing activity was also apparent in a high accumulation of 3‐methylcatechol in the cultures of the recombinant strain. At certain points of incubation, the hemoglobin expressing strain showed a significantly (p < 0.05) higher 3‐methylcatechol accumulation than the host strain. These results demonstrated the possible potential of the Vitreoscilla hemoglobin as an efficient oxygen uptake system for the bioremediation of some compounds of environmental concern.     

碱性果胶酶的生物制造及其在纺织工业清洁生产中的应用研究进展

以下综述了碱性果胶酶的生物制造及其在纺织工业清洁生产中的应用研究进展。微生物发酵法是目前生产碱性果胶酶的主要方式,枯草芽孢杆菌是碱性果胶酶工业发酵生产中效果较好的野生菌株。影响发酵法生产碱性果胶酶的主要因素有:底物浓度及其流加方式、细胞浓度、搅拌转速、通气速率、pH、温度等。构建基因工程菌为碱性果胶酶的发酵生产开辟了一条有效途径,其中重组毕赤酵母的产酶水平最高,在10吨发酵罐上酶活达1305U/mL。碱性果胶酶可用于棉织物前处理的精练工艺,与传统高温碱煮相比,具有保护纤维、提高精练效率、降低能耗和污染的优势。通过分子定向进化技术对碱性果胶酶进行分子改造,使其催化特性更加适合于纺织精练工艺,进而实现纺织工业的清洁生产是未来的研究重点和热点。     

Enhanced production of carboxymethylcellulase of a marine microorganism, Bacillus subtilis subsp. subtilis A-53 in a pilot-scaled bioreactor by a recombinant Escherichia coli JM109/A-53 from rice bran

A gene encoding the carboxymethylcellulase (CMCase) of a marine bacterium, Bacillus subtilis subsp. subtilis A-53, was cloned in Escherichia coli JMB109 and the recombinant strain was named as E. coli JMB109/A-53. The optimal conditions of rice bran, ammonium chloride, and initial pH of the medium for cell growth, extracted by Design Expert Software based on response surface methodology, were 100.0 g/l, 7.5 g/l, and 7.0, respectively, whereas those for production of CMCase were 100.0 g/l, 7.5 g/l, and 8.0. The optimal temperatures for cell growth and the production of CMCase by E. coli JM109/A-53 were found to be and 40 and 35 °C, respectively. The optimal agitation speed and aeration rate of a 7 l bioreactor for cell growth were 400 rpm and 1.5 vvm, whereas those for production of CMCase were 400 rpm and 0.5 vvm. The optimal inner pressure for cell growth was 0.06 MPa, which was the same as that for production of CMCase. The production of CMCase by E. coli JM109/A-53 under optimized conditions was 880.2 U/ml, which was 2.9 times higher than that before optimization. In this study, rice bran and ammonium chloride were developed as carbon and nitrogen source for production of CMCase by a recombinant E. coli JM109/A-53 and the productivity of E. coli JM109/A-53 was 5.9 times higher than that of B. subtilis subp. subtilis A-53.     

Enhancement of extracellular glucose oxidase production in pH-stat feed-back controlled fed-batch culture of recombinant Saccharomyces cerevisiae

Glucose oxidase (GOD) from Aspergillus niger was expressed in Saccharomyces cerevisiae under the regime of GAL-10 promoter and GAL-7 terminator of S. cerevisiae and -amylase signal sequence of Aspergillus oryzae. The enhancement of the expression level was achieved in pH-stat feed-back controlled fed-batch culture. The highest titre of extracellular GOD was 199 U/ml which marked two fold improvement over the batch (95 U/ml) and 28% above that of non-feed back controlled fed-batch (154 U/ml) operation. © Rapid Science Ltd. 1998     

Recombinant Production of an Inulinase in a Saccharomyces cerevisiae gal80 Strain

The inulinase gene (INU1) from Kluyveromyces marxianus NCYC2887 strain was overexpressed by using GAL10 promotor in a △gal80 strain of Saccharomyces cerevisiae. The inulinase gene lacking the original signal sequence was fused in-frame to mating factor alpha signal sequence for secretory expression. Use of the △gal80 strain allowed the galactose-free induction of inulinase expression using a glucose-only medium. Shake flask cultivation in YPD medium produced 34.6 U/ml of the recombinant inulinase, which was approximately 13-fold higher than that produced by K. marxianus NCYC2887. It was found that the use of the △gal80 strain improved the expression of inulinase in the recombinant S. cerevisiae in both the aerobic and the anaerobic condition by about 2.9- and 1.7-fold, respectively. 5 L fed-batch fermentation using YPD medium was performed under aerobic condition with glucose feeding, which resulted in the inulinase production of 31.7 U/ml at OD600 of 67. Ethanol fermentation of dried powder of Jerusalem artichoke, an inulin-rich biomass, was also performed using the recombinant S. cerevisiae expressing INU1 and K. marxianus NCYC2887. Fermentation in a 5L scale fermentor was carried out at an aeration rate of 0.2 vvm, an agitation rate of 300 rpm, and the pH was controlled at 5.0. The temperature was maintained at 30degrees C and 37degrees C, respectively, for the recombinant S. cerevisiae and K. marxianus. The maximum productivities of ethanol were 59.0 and 53.5 g/L, respectively.     

High-content screening of <Emphasis Type="Italic">Aspergillus niger</Emphasis> with both increased production and high secretion rate of glucose oxidase

Objectives

To develop a rapid, dual-parameter, plate-based screening process to improve production and secretion rate of glucose oxidase simultaneously in Aspergillus niger.

Results

A morphology engineering based on CaCO₃ was implemented, where the yield of GOD by A. niger was increased by up to 50%. Analysis of extracellular GOD activity was achieved in 96-well plates. There was a close negative correlation between the total GOD activity and its residual glucose of the fermentation broth. Based on this, a rapid, plate-based, qualitative analysis method of the total GOD activity was developed. Compared with the conventional analysis method using o-dianisidine, a correlation coefficient of ?0.92 by statistical analysis was obtained.

Conclusion

Using this dual-parameter screening method, we acquired a strain with GOD activity of 3126 U l^?1, which was 146% higher than the original strain. Its secretion rate of GOD was 83, 32% higher than the original strain.

     

Optimization of culture conditions for glucose oxidase production by a Penicillium chrysogenum SRT 19 strain

The enzyme glucose oxidase (GOD) has been used for a variety of biotechnological applications in food and pharmaceutical industries. In this study, the optimization of extracellular GOD production was carried out in a Penicillium chrysogenum SRT 19 strain isolated from contaminated and decaying cheese samples. Maximum GOD production was attained at pH 6 and 20°C in fermentation broth after 72 h of incubation. The effects of metal ions and sugars were screened for the induction of higher GOD production. The results revealed that glucose and lactose give the highest production of enzyme (0.670 and 0.552 U/mL, respectively) as compared with other sugars (sucrose, cellulose, mannitol and fructose). Out of the seven metal ions studied, CaCO₃ (1.123 U/mL) and FeSO₄ (0.822 U/mL) act as modulators, while MgSO₄ (0.535 U/mL), CuSO₄ (0.498 U/mL), HgCl₂ (0.476 U/mL), ZnSO₄ (0.457 U/mL) and BaSO₄ (0.422 U/mL) yield lower production. The study therefore suggests that a strain of P. chrysogenum SRT 19 can be used as a new strain for GOD production.     

Efficient secretion of lipase r27RCL in Pichia pastoris by enhancing the disulfide bond formation pathway in the endoplasmic reticulum

The lipase r27RCL from Rhizopus chinensis CCTCC M201021 was heterologously expressed in Pichia pastoris GS115 by simultaneous co-expression with two secretion factors ERO1p and PDI involved in the endoplasmic reticulum (ER). Compared to the expression of the lipase alone (12,500 U/ml), co-expression with these two proteins resulted in the production of larger total quantities of enzymes. The largest increase was seen when the combined ERO1p/PDI system was co-expressed, resulting in approximately 30 % higher enzyme yields (16,200 U/ml) than in the absence of co-expressed secretion factors. The extracellular protein concentration of the recombinant strain Co XY RCL-5 reached 9.39 g/l in the 7-l fermentor. Simultaneously, the fermentation time was also shortened by about 8 h compared to that of the control. The substrate-specific consumption rate (Qs) and the product-specific production rate (Qp) were both investigated in this research. In conclusion, the space–time yield was improved by co-expression with ERO1p and PDI. This is a potential strategy for high level expression of other heterologous proteins in P. pastoris.     

Rotating simplex method of optimization of physical parameters for higher production of extracellular pectinases in bioreactor

Rotating simplex method of optimization has been successfully used to determine the optimal conditions for extracellular pectinase production by Aspergillus niger in batch bioreactor. Maximum production of 1.132 U of polymethylgalacturonase, 1.313 U of polygalacturonase and 0.0091 U of pectinlyase was obtained at pH (controlled). 3.38; agitation rate, 483 rpm and aeration rate, 2.12 vvm. These conditions were found to be more suitable for pectinase production.     

Statistical optimization of glucose oxidase production from Aspergillus niger NRC9 under submerged fermentation using response surface methodology

Response surface methodology (RSM), employing the fractional factorial design (FFD) was used to optimize the fermentation medium for the production of glucose oxidase (GOD) from a marine isolate (NRC9) of Aspergillus niger under submerged fermentation. The design was employed by selecting glucose, CaCO_3, ammonium phosphate and MgSO₄ concentrations as model factors by ‘one variable at a time’ experiment. A second-order quadratic model and response surface method showed that the optimum concentrations (g/l) glucose, 100; CaCO₃, 25; (NH₄)₂HPO₄, 1.8 and 0.4 of MgSO₄, resulted in an improvement of GOD production (170?±?0.88 U/ml) as compared to the initial level (109.81?±?1.38 U/ml) after four days of incubation at 200 rpm and 30 °C, whereas its predicted value obtained by the quadratic model was 164.36 U/ml. Analysis of variance (ANOVA) showed a high coefficient of determination value (R ²) of 0.967, ensuring a satisfactory adjustment of the quadratic model with the experimental data. This is the first report on production of glucose oxidase from a marine fungal isolate, Aspergillus niger NRC9, using statistical experimental design and response surface methodology in optimization of its production under submerged fermentation.     

Effect of the aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor: mathematical modeling

The effect of aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor was investigated. B. trispora formed hyphae, zygophores and zygospores during the fermentation. The zygospores were the morphological form responsible for β-carotene production. Both aeration and agitation significantly affected β-carotene concentration, productivity, biomass and the volumetric mass transfer coefficient (K_La). The highest β-carotene concentration (1.5 kg m⁻³) and the highest productivity (0.08 kg m⁻³ per day) were obtained at low impeller speed (150 rpm) and high aeration rate (1.5 vvm). Also, maximum productivity (0.08 kg m⁻³ per day) and biomass dry weight (26.4 kg m⁻³) were achieved at high agitation speed (500 rpm) and moderate aeration rate (1.0 vvm). Conversely, the highest value of K_La (0.33 s⁻¹) was observed at high agitation speed (500 rpm) and high aeration rate (1.5 vvm). The experiments were arranged according to a central composite statistical design. Response surface methodology was used to describe the effect of impeller speed and aeration rate on the most important fermentation parameters. In all cases, the fit of the model was found to be good. All fermentation parameters (except biomass concentration) were strongly affected by the interactions among the operation variables. β-Carotene concentration and productivity were significantly influenced by the aeration, agitation, and by the positive or negative quadratic effect of the aeration rate. Biomass concentration was principally related to the aeration rate, agitation speed, and the positive or negative quadratic effect of the impeller speed and aeration rate, respectively. Finally, the volumetric mass transfer coefficient was characterized by the significant effect of the agitation speed, while the aeration rate had a small effect on K_La.     

Cloning and optimized expression of a GH-11 xylanase from Fusarium oxysporum in Pichia pastoris

The endo-1,4-β-xylanase gene xyn11a from Fusarium oxysporum, member of the fungal glycosyl hydrolase (GH) family 11, was cloned and expressed in Pichia pastoris. The mature xylanase gene, which generates after the excision of one intron and the secreting signal peptide, was placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPICZαC. The final construction was integrated into the genome of the methylotrophic yeast P. pastoris X33 and the ability to produce xylanase activity was evaluated in flask cultures. Recombinant P. pastoris efficiently secreted xylanase into the medium and produced high level of enzymatic activity (110 U/ml) after 216 hours of growth, under methanol induction. To achieve higher enzyme production, the influence of initial pH, methanol concentration, agitation and flask design was evaluated. Under optimum culture conditions, production of the recombinant xylanase increased by 50%, reaching a final yield of 170 U/ml, underpinning aeration as the most important factor in improving enzyme production.     

Enhancement of thermostable lipase production by a genotypically identified extremophilic Bacillus subtilis NS 8 in a continuous bioreactor

Bacillus strain NS 8, a lipase-producing bacterium isolated from a Malaysian hot spring, is able to tolerate a broad range of temperature and pH, which makes it beneficial for this study. It generated PCR products with molecular weight of 1,532 bp, and the 16S rRNA sequence analysis identified it as Bacillus subtilis with accession number AB110598. It showed a 71% similarity index with B. subtilis using Biolog Microstation System. Its lipase production was optimized using a shake flask system by changing the physical (agitation speed, pH and temperature) and nutritional (nitrogen, carbon and minerals) factors. The most suitable combination of the basal medium for lipase production was 2.5% olive oil (carbon), 1.5% peptone (nitrogen), 0.1% MgSO(4) (mineral) at an optimum temperature of 50°C, pH 7.5 and 150 rpm agitation, giving an enzyme yield of 4.23 U/ml. Statistical optimization using response surface methodology was carried out. An optimum lipase production of 5.67 U/ml was achieved when olive oil concentration of 3%, peptone 2%, MgSO(4)·7H(2)O 0.2% and an agitation rate of 200 rpm were combined. Lipase production was further carried out inside a 2-liter bioreactor, which yielded an enzyme activity of 14.5 U/ml after 15 h of incubation.     

Augmented cellulase production by Bacillus subtilis strain MU S1 using different statistical experimental designs

The production of cellulase by Bacillus subtilis MU S1, a strain isolated from Eravikulam National Park, was optimized using one-factor-at-a-time (OFAT) and statistical methods. Physical parameters like incubation temperature and agitation speed were optimized using OFAT and found to be 40?°C and 150?rpm, respectively, whereas, medium was optimized by statistical tools. Plackett-Burman design (PBD) was employed to screen the significant variables that highly influence cellulase production. The design showed carboxymethyl cellulose (CMC), yeast extract, NaCl, pH, MgSO₄ and NaNO₃ as the most significant components that affect cellulase production. Among these CMC, yeast extract, NaCl and pH showed positive effect whereas MgSO₄ and NaNO₃ were found to be significant at their lower levels. The optimum levels of the components that positively affect enzyme production were determined using response surface methodology (RSM) based on central composite design (CCD). Three factors namely CMC, yeast extract and NaCl were studied at five levels whilst pH of the medium was kept constant at 7. The optimal levels of the components were CMC (13.46?g/l), yeast extract (8.38?g/l) and NaCl (6.31?g/l) at pH 7. The maximum cellulase activity in optimized medium was 566.66?U/ml which was close to the predicted activity of 541.05?U/ml. Optimization of physical parameters and medium components showed an overall 3.2-fold increase in activity compared to unoptimized condition (179.06?U/ml).