Screening, Identification and Fermentation Optimization of Aminotransferase-producing Strain / 微生物学通报

A strain named as WJ44 with capability of producing branched-chain aminotransferase was isolated from the soil, the enzyme activity is especially high when used leucine as a substrate. According to the physiological characteristics, biochemical characteristics and 16S rRNA sequence of WJ44, the strain was identified as Bacillus cereus. The optimal carbon source and nitrogen source were also determined as 20 g/L glucose, 15 g/L tryptone and 5 g/L beef extract, respectively. The optimal proportion of other component in the medium is corn steep liquor 15 g/L, KH2PO4 3 g/L, MgSO4?7H2O 0.5 g/L. The optimum original pH value is 7.0, the cultural temperature is 37℃, liquid volume is 20 mL/250 mL, the shaker’s rotating speed is 200 r/min. The highest leucine transferase activity of 45.787 U/mL was achieved after 10 h under the opti- mal condition, and the dry weight of cell is 8.643 g/L, increased by 54% and 10% respectively. This result shows that WJ44 is a branched-chain aminotransferase-production strain with fine capability.

Improvement on the activity of microbial transglutaminase with Streptomyces hygroscopicus by the addition of surfactant CTAB / 生物工程学报

Effect of CTAB addition on the accumulation of microbial transglutaminase (MTG) with Streptomyces hygroscopicus was investigated. The results showed that the addition of CTAB enhanced MTG accumulation, and the optimal addition time and concentration of CTAB were 32 h and 1%. The maximum MTG activity in the culture broth was 5.04 u/mL and increased by 21.8% compared with the control. With the addition of CTAB, pro-MTG was activated to become MTG. CTAB could enhance the production of pro-MTG by relieving the product inhibition, and the accumulation of MTG was improved.

Acetate production by acidification-homoacetogenesis two-phase coupling process: effect of initial pH / 生物工程学报

The effect of initial pH values on acetate production was studied in the acidification-homoacetogenesis two-phase coupling system using glucose as the substrate and the heat-treated and activated anaerobic sludge as the inoculum. Substrate degradation, product yield and pH variation during fermentation were examined at various initial pH values (5, 6, 7, 8, 9, 10 and 11). The results show that initial pH values affect volatile fatty acids and ethanol production not only in the acidification phase itself but also in the homoacetogenesis phase. Ethanol-type fermentation mainly takes place at initial pH 5 while butyrate-type fermentation at initial pH 6 and 7. But acetate is the dominant product at initial pH 8-11. The optimal initial pH value is 10 for acetate production in the two-phase coupling system. At initial pH 8-11, ethanol concentration is highest at the beginning of acidification, but there is a subsequent decline as ethanol is converted to acetate as a result of further metabolism of the microbes.

Purification and characterization of a monofunctional catalase from an alkaliphilic Bacillus sp. F26 / 生物工程学报

An alkaline catalase has been purified and characterized from a slightly halophilic and alkaliphilic bacterium Bacillus sp. F26. The purification was performed with a four step procedure consisting of ammonium sulfate precipitation, ion exchange, gel filtration and hydrophobic interaction chromatography, and finally achieved a 58.5-fold-purifying over the crude extract. The purified catalase was composed of two identical subunits with a native molecular mass of 140 kD. The native enzyme showed the typical Soret band appearing at 408 nm. The pyridine hemochrome spectrum indicated the presence of protoheme IX as the prosthetic group. The apparent Km value for enzyme activity on H2O2 was calculated to be 32.5 mmol/L. The activity of this catalase was not reduced by dithionite but was strongly inhibited by cyanide, azide, and 3-amino-1,2,4-triazole (the specific inhibitor of monofunctional catalase). No peroxidase activity of this enzyme was detected when using o-dianisidine, diaminobenzidine (DAB) and p-phenylenediamine as electron donor. Moreover, the N-terminal sequence of this catalase exhibited substantial similarity to the monofunctional catalase subgroup rather than catalase-peroxidase or Mn-catalase one. Therefore, we characterize the purified catalase as a monofunctional catalase. Besides, this monofunctional catalase was thermosensitive and its activity exhibited pH-independent over pH 5-9 but showed a sharp maximum at pH 11. An activity half-life of approximately 49 h was measured when the enzyme was incubated at 20 degrees C and pH 11. To our knowledge, pH 11 is the most alkaline condition for optimum catalysis and enzyme stability among the catalases reported up to now. Furthermore, this monofunctional catalase also showed excellent halo-alkali-stability with a half-life of approximately 90 h at 0.5 mol/L NaCl and pH 10.5. On the other hand, so far as we know, the characterized catalase is the first dimeric monofunctional catalase from alkaliphiles and is also the first monofunctional catalase derived from a natural soda lake, which could partially reflect the oxidative stress response in the corresponding environment.

Addition of TCA cycle intermediates enhances pyruvate production / 生物工程学报

The capability of utilizing the intermediates of TCA-cycle as the sole carbon source by the multi-vitamin auxotrophic yeast Torulopsis glabrata CCTCC M202019 under the conditions of vitamins limitation was demonstrated. Furthermore, the colony numbers grown on medium supplemented with glucose, acetate and one of the intermediates of TCA-cycle was higher than that of medium used glucose and acetate or medium used one of the intermediates of TCA-cycle carbon source. Among the intermediates of TCA-cycle used in this study, oxaloacetate was the best carbon source for the yeast and it was found that its presence stimulated the conversion of acetate to acetyl-CoA. In batch fermentation with glucose medium, the addition of 10 g/L of oxaloacetate improved the dry cell weight from 11.8 g/L to 13.6 g/L, and the productivity of pyruvate from 0.96 g x L(-1) x h(-1) to 1.19 g x L(-1) x h(-1), a 24% increase after 56 h growth. The yield of pyruvate on glucose was also improved as well, from 0.63 g/g to 0.66 g/g.

Thermo-alkali-stable catalase from Thermoascus aurantiacus and its potential use in textile bleaching process / 生物工程学报

The effect of growth and fermentation conditions on the production of catalase by T. aurantiacus WSH 03-01 was investigated in shaking flasks. Catalase activity reached 1594 u/mL when the culture was grown on a complex carbon source containing 20 g/L dextrin and 1% (V/V) ethanol, which was 23% higher than the sum produced on 20 g/L dextrin and 1% (V/V) ethanol, respectively. It was concluded that dextrin might act as a major carbon source in the complex, while ethanol was rather a stimulator than a carbon source. The stimulation effect of ethanol on catalase production was postulated to be two aspects; catalase-dependent alcohol metabolism is activated by acute alcohol, thus more catalase need to be synthesized for that use, named direct induction. As for indirect induction, which may result from little amount of H2O2 generation in process of NADH regeneration in respiratory chain. Peptone was shown to be a favorable nitrogen source for catalase production and its optimum concentration was found to be 10 g/L. Catalase production by T. aurantiacus WSH 03-01 was further improved by optimizing the initial pH, volume of medium in flasks as well as the concentration of external H2O2. Under the optimum culture conditions, the activity of catalase reached 2762 u/mL, which was nearly 6.8 times higher than that of the initiate conditions. Furthermore, the potential application of this novel catalase in the treatment of textile bleaching effluents was evaluated. Thermo-and alkaline stability of this catalase was compared with the commercial available catalases produced from bovine and Aspergillus niger. The crude enzyme from T. aurantiacus WSH 03-01 showed stronger stabilities at (70 degrees C, 80 degrees C, 90 degrees C) and (pH 9.0, pH 10.0, pH 11.0) than the other two types of catalases, indicating a great application potential in the clean production process of textile industry.

Effects of nutrient conditions and fed-batch culture on CoQ10 production by Rhizobium radiobacter WSH2601 / 生物工程学报

The effects of the variables, including the concentrations of glucose, sucrose, corn steep liquor (CSL) and peptone, and the conditions of fed-batch culture, on CoQ10 fermentation by Rhizobium radiobacter WSH2601 were assessed. The results showed that the optimum concentrations of glucose, sucrose, CSL and peptone were 30 g/L, 40 g/L, 11 g/L and 16 g/L respectively. Addition of CSL and tomato juice stimulated the cell growth. CSL, L-methionine and isopentyl alcohol efficiently increased the biosynthesis of CoQ10. In a 7L fermentor, the fed-batch culture improved both cell growth and CoQ10 production compared to a batch culture; and suitable mixed feeding of CSL and sucrose enhanced CoQ10 yield to 52.4 mg/L. The DCW reached 26.4 g/L, an increase of 53% in comparison to that without feeding, and an increase of 24% to that feeding with sucrose only. The C/B value reached 2.38 mg/g(DCW), representing an increase of 33% to that of without feeding, and an increase of 26% to that of feeding with sucrose only.

Kinetic models for the effect of temperature on batch glutathione fermentation by Candida utilis / 生物工程学报

Glutathione (L-gamma-glutamyl-L-cysteinylglycine), one of the major non-protein thiol compounds, is widely distributed in living cells and plays an important role in maintaining the normal redox environment of cells as an antioxidant. In the production of glutathione by fermentation, temperature is one of the most important environmental factors that affect the yield and the productivity of glutathione. Here the effect of temperature, varied from 24 degrees C to 32 degrees C, on the batch fermentation of glutathione in a 7 L stirred fermenter by Candida utilis WSH 02-08 was investigated. It was found that cell growth was hastened along with the increase of temperature. The maximum dry cell weight was achieved approximately 16 g/L under various temperatures, as soon as the glucose was exhausted. The effect of temperature on glutathione production was different from that on cell growth: the lower the temperature, the higher the glutathione production, i.e. the maximum glutathione concentration at 32 degrees C (235 mg/L) was only 75% and 64% of that at 30 degrees C and 26 degrees C, respectively. The maximum average specific growth rate (0.13 h(-1)) was achieved at 30 degrees C while the maximum glutathione concentration (366 mg x L(-1)) and the maximum intracellular glutathione content (2.3%) were obtained at 26 degrees C. Therefore, the optimum temperatures for cell growth and glutathione production are quite different in the batch fermentation. A modified Logistic equation was successfully applied to estimate the kinetics of cell growth. The maximum specific growth rate and the substrate inhibition constant, calculated from this equation, were both increased along with the temperature. In addition, the glutathione fermentation by C. utilis WSH 02-08 under various temperatures was proven to be a partial growth-associated process by estimating the process with the Luedeking-Piret equation. Based on the estimated parameon the estimated parameters, the effect of temperature on the kinetics of cell growth was further studied. An equation, dX / dt = [0.0224(T + 1.7)]2 X(1-X/Xmax) / 1 + S/ {8.26 x 10(6) x exp [-31477/R/(T+273)]}, was developed and applied to interlink the relationship between biomass concentration and temperature as well as substrate concentration in the batch glutathione fermentation. The experiment results showed that this model could predict the growth pattern very well.

Effects of dissolved oxygen and pH on Candida utilis batch fermentation of glutathione / 生物工程学报

The effects of dissolved oxygen (DO) and pH on glutathione batch fermentation by Candida utilis WSH-02-08 in a 7 liters stirred fermentor were investigated. It was shown that DO concentration is an important factor in glutathione production. With the initial glucose concentration of 30 g/L and a 5 L/min air flow rate, and the agitation rate less than 250 r/min, the DO concentration was not sufficient to satisfy the oxygen requirement during the fermentation. With an agitation rate of more than 300 r/min, the cell growth and glutathione production were enhanced significantly, with the dry cell mass and glutathione production were 20% and 25% higher than that at 200 r/min. When C. utilis WSH 02-08 was cultivated in a batch process without pH control, cell growth and glutathione production were inhibited, likely due to a dramatic decrease in the pH. Intracellular glutathione leakages were observed when the pH was 1.5 or less. To assess the effect of pH on glutathione production, six batch processes controlled at pH 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5 were conducted. The yield was highest at pH 5.5, when the dry cell mass and yield were 27% and 95% respectively higher than fermentation without pH control. The maximal intracellular glutathione content (2.15 %) was also achieved at the pH. To improve our understandings on the effect of pH on the batch glutathione production, a modified Logistic equation and Luedeking-Piret equation were used to simulate cell growth and glutathione production, respectively, under different pH. Based on the parameters obtained by the nonlinear estimation, kinetic analysis was performed to elucidate the effect of pH on the batch glutathione production. The process controlled at pH 5.5 was proven to be the best due to the higher value of K(I) (substrate inhibitory constant in the Logistic equation), lower value of a and higher value of beta (slope and intercept in the Luedeking-Piret equation, respectively).

CaCO3 stimulates alpha-ketoglutarate accumulation during pyruvate fermentation by Torulopsis glabrata / 生物工程学报

A large amount of alpha-ketoglutarate (alpha-KG) (6.8 g/L) was accumulated in flask culture when CaCO3 was used as a buffering agent in the production of pyruvate by multi-vitamin auxotrophic yeast Torulopsis glabrata CCTCC M202019. In a 5 L jar-fermentor, less alpha-KG (1.3 g/L) was produced when NaOH was used to adjust the pH, while more alpha-KG (11.5 g/L) detected when CaCO3 was used as the buffer. In the latter case, the molar carbon ratio of pyruvate to alpha-KG (C(PYR)/ CalphaKG) was similar to that obtained in flask culture, suggesting the accumulation of alpha-ketoglutarate was related to the addition of CaCO3. Furthermore, it was found that: (1) delaying the addition time of CaCO3 decreased the a-ketoglutarate formation but increased C(PYR)/ C(alphaKG); and (2) under vitamin limitation conditions increasing the concentration of CaCO3 led to an increased a-KG accumulation at the expenses of pyruvate. To study which ions in CaCO3 was responsible for the accumulation of alpha-KG, the effects of different pH buffers on the a-KG accumulation were studied. The level of alpha-KG was found to correlate with the levels of both Ca2+ and CO3(2-), with Ca2+ played a dominant role and CO3(2-) played a minor role. To find out which pathway was responsible for the accumulation of alpha-KG, the effects of biotin and thiamine on alpha-KG accumulation was investigated. The increase in biotin concentration led to an increase in alpha-KG accumulation and a decrease in C(PYR)/ C(alpha-KG), while the levels of alpha-KG and C(PYR)/C(alphaKG) were not affected by thiamine concentration. The activity of pyruvate carboxylase was increased as much as 40% when the medium was supplemented with Ca2+ . On the other hand, the activity of the pyruvate dehydrogenase complex was unaffected by the presence of Ca2+. To conclude, the higher level of a-KG was caused by higher activity of pyruvate carboxylase stimulated by Ca2+, with CO3(2-) served as the substrate of the reaction.

Formation of polyhydroxyalkanoates during the dual-nutrient-limited zone by Ralstonia eutropha / 生物工程学报

Polyhydroxyalkanoates (PHAs) are a class of polyesters biosynthesized by microorganisms (esp. Ralstonia eutropha) under an unbalanced growth condition, and which are supposed to partly take the place of traditional plastics made from petroleum in the near future since they are harmless to the environment and biodegradable. Organic acids (mainly butyrate, lactate, propionate and acetate) produced from anaerobic digested food wastes, industrial wastes and sewage may be used as cheap carbon sources since the large amounts of the above wastes disposed by industry and family each year. In order to better understand the process of PHAs formation with acids as carbon sources, so as to increase the yields of PHAs. Biosynthesis of PHAs by R. eutropha during the dual nutrient-limitation-zone was investigated with mixed organic acids (the mass ratio of the four component acids was butyrate: propionate: acetate: lactate = 3: 3: 1: 1, which was simulated as once the result of anaerobic digestion of food wastes) as carbon sources and (NH4)2 SO4 as nitrogen source. Two different manners of maintaining the dual-nutrient-limitation zone were adopted by feeding mixed acids and (NH4 )2SO4 at determined rates to the fermentation culture which were free of carbon sources (manner A) or nitrogen sources (manner B) firstly. The results suggest that, first of all, the meaning of the limitation of mixed acids or (NH4)2 SO4 does not mean to limit the supply of them, but mean to feed as more as possible of carbon and nitrogen sources in order to meet the cell growth and PHAs formation of R. eutropha by the largest extent. However, it's indispensable to make the residual concentration of carbon and nitrogen sources as low as possible since organic acids are inhibitive to the cell growth, and most importantly, only under the presence of nitrogen during the PHAS formation period of the fermentation could R. eutropha produce more PHAs than any other unbalanced growth condition. Secondly, with the increase of the width of the dual-nutrient-limitation zone, the yield of PHAs would also increase, it suggest that most of the PHAs were biosynthesized during the dual-nutrient-limitation zone. Finally, in contrast with the dual-nutrient-limitation manner of limiting the nitrogen source at first (manner B), the dual-nutrient-limitation manner of limiting the carbon source at first (manner A) was more favorable for the production of PHAs, and the maximum production of PHAs of these two manners are 3.72 g/L and 2.55 g/L, respectively. It may be because that PHAs formation required enzymes could not be well developed when R. eutropha grow under the state of nitrogen limitation from the beginning of fermentation. Besides, yield of PHAs produced by the dual-nutrient-limitation fermentation is larger than that of the single-nutrient-limitation batch culture. Therefore, it seems that to increase the output of PHAs production, the strategy of maintaining as wide as possible the width of dual-nutrient (C, N)-limitation zone may be effective.

Progress in biotechnological production of pyruvic acid / 生物工程学报

Pyruvate, an important organic acid, is widely used in the industries of pharmaceuticals, chemicals, agrochemicals, food additives and so on. Compared with the chemical method, biotechnological production of pyruvic acid is an alternative approach because of the low cost and high product quality. In this article, biosynthesis of pyruvate, including direct fermentative production and resting cell method as well as enzymatic method, was discussed. Furthermore, a comparison of these different methods was proposed. Since, a multi-vitamin auxotrophic strain of Torulopsis glabrata is the most competitive strain for industrial production of pyruvate, emphasis was therefore placed on the development of strains screening and fermentation optimization. Finally, some suggestions were put forward to improve the research in this field in the near future.