An efficient strategy overcoming the bottleneck in Candida parapsilosis catalyzing stereoinversion from (R)‐1‐phenyl‐1, 2‐ethanediol to the corresponding counterpart

BACKGROUND: Microbial stereoinversion has been widely used for the biosynthesis of numerous chiral compounds. However, little work has been done to improve the efficiency of microbial stereoinversion. This study investigated the bottleneck in the deracemization of 1‐phenyl‐1,2‐ethanediol (PED), and then the efficiency and the sustainability of biocatalyst was improved significantly by using a strategy. RESULTS: When (S)‐PED concentration exceeded 17.5 g L^?1, it strongly inhibited deracemization. Furthermore, the deficiency of NADPH regeneration also limited such reaction. To overcome these limitations, extractive biocatalysis was developed using adsorbent resin NKII combined with xylose addition for cofactor regeneration. Compared with the initial reaction condition, which only afforded (S)‐PED with 35% optical purity after the first batch reaction at 30 g L^?1 substrate concentration, the cells in the new system could be reused three times and the optical purity remained at a high level of 95%. CONCLUSION: Product inhibition and coenzyme regeneration had a significant effect on catalytic activity of Candida parapsilosis. By using a resin and D‐xylose, the efficiency and reusability of whole‐cell catalyst can be considerably improved, which would be helpful for effective synthesis of high value chiral intermediates. Copyright © 2009 Society of Chemical Industry     

Determination of agitation and aeration conditions for scale-up of cellulolytic enzymes production by <Emphasis Type="Italic">Trichoderma inhamatum</Emphasis> KSJ1

10 to 35 L jar fermentation scale-up cultures were performed to determine the optimum agitation and aeration rates in the cellulolytic enzymes production culture by Trichoderma inhamatum KSJ1. The optimum agitation rate in the 35 L jar fermenter was provisionally determined to be 150 rpm by using a geometrically resembled scale up method from the 10 L jar fermenter. The optimum aeration rate was determined to be 0.5 vvm by applying the mean values of superficial velocity and vvm. The DO (Dissolved Oxygen) concentration of the culture liquid was maintained below the critical DO concentration (2.336 mg/L) at 150 rpm in the 35 L jar fermenter. To increase the DO above the critical DO concentration, the agitation rate was increased from 150 to 200 rpm, with the aeration rate maintained at 0.5 vvm. As a result, the DO was maintained above critical DO concentration. The OUR (Oxygen Uptake Rate) and k _L a values were 0.91 mg-DO/L·min and 11.1 hr⁻¹, respectively. The amylase and FPase (filter paper activity) activities were 4.48 and 0.74 U/mL, respectively, in the 35 L jar fermenter, which was comparable to that in the 10 L fermenter (4.2 and 0.5 U/mL, respectively). Therefore, the scale-up conditions, 0.5 vvm and 200 rpm, were concluded to be the optimum aeration and agitation rates in the 35 L jar fermenter.     

Combined Effect of Agitation/Aeration and Fed‐Batch Strategy on Ubiquin‐ one‐10 Production by Pseudomonas diminuta

The effects of aeration rate and agitation speed on ubiquinone‐10 (CoQ10) submerged fermentation in a stirred‐tank reactor using Pseudomonas diminuta NCIM 2865 were investigated. CoQ10 production, biomass formation, glycerol utilization, and volumetric mass transfer coefficient (k_La) were affected by both aeration and agitation. An agitation speed of 400 rpm and aeration rate of 0.5 vvm supported the maximum production (38.56 mg L^–1) of CoQ10 during batch fermentation. The fermentation run supporting maximum production had an k_La of 27.07 h^–1 with the highest specific productivity and CoQ10 yield of 0.064 mg g^–1h^–1 and 0.96 mg g^–1 glycerol, respectively. Fermentation kinetics performed under optimum aeration and agitation showed the growth‐associated constant (a = 5.067 mg g^–1) to be higher than the nongrowth‐associated constant (β = 0.0242 mg g^–1h^–1). These results were successfully utilized for the development of fed‐batch fermentation, which increased the CoQ10 production from 38.56 mg L^–1 to 42.85 mg L^–1.     

Scaling up the Bioconversion of Cheese Whey Permeate into Fungal Oil by Mucor circinelloides

The bioconversion of hydrolyzed whey permeate into an oil-rich biomass by Mucor circinelloides was scaled up from 250 mL to 4 L with the use of an aerated stirred tank bioreactor. Biomass production and oil accumulation were strongly influenced by agitation speed (99–451 rpm) and aeration rate (0.29–1.70 vvm). Higher agitation and aeration rates (e.g., >400 rpm, >1 vvm), resulted in significantly higher biomass yield due to increased oxygen transfer capabilities and better mixing. Additionally, oil accumulation in the fungal biomass was related to high agitation (>400 rpm), while aeration (0.5–1.5 vvm) had no significant effect within the range evaluated. The predictive model was validated at the optimal conditions of 450 rpm and 1 vvm. Maximum biomass yield of 10.7 g L⁻¹ and lipid content of 32% dry biomass were achieved during 120 hours of fermentation. Simultaneous optimization of agitation and aeration in a bioreactor was found to not only improve fungal growth but also lipid content (24% vs. 32%), lipid yield (2.2 vs. 3.1 g L⁻¹) and γ-linoleic production (73–464 mg L⁻¹) compared to that of shake-flask. This study resulted in a scaled-up and optimized fermentation process that increased production of M. circinelloides biomass for subsequent use as raw material for food, feed, and fuel applications. This signifies a starting point for further studies aimed at assessing the development of a fully functioning fungi-to-food/fuel system on an industrial scale for several agricultural streams.     

Comparison of lipase production by Geotrichum candidum in stirring and airlift fermenters

The production of lipase by Geotrichum candidum in both, stirred tank and airlift bioreactors were compared. G candidum an imperfect filamentous fungus, grows well in liquid medium, and produces a lipase with specific affinity for long‐chain fatty acids with cis‐9 double bonds but, lipase production is generally not efficient because the optimum medium composition and fermentation conditions are not known. Response surface methodology was used to optimize the agitation speed (100–500 rpm) and aeration (0.2–1.8 vvm) for production of lipase by G candidum in a bench‐scale stirred fermenter. A Central Composite Rotatable Design (CCRD) was used to optimize lipase activity and productivity. Lipase production in an airlift fermenter was also studied with aeration ranging from 1 to 3 vvm. A previously optimized culture medium containing 3.58% of peptone, 0.64% of soy oil and an initial pH of 7.0, was used in the experiments, incubating at 30°C. In the stirred reactor the optimum conditions of agitation and aeration for lipase production and productivity were 300 rpm and 1 vvm, leading to an activity of 20 U cm^?3 in 54 h of fermentation and 0.3900 (U cm^?3 h^?1) of productivity. The best aeration condition in the airlift fermenter was 2.5 vvm, which yielded similar lipase activity after 30 h of fermentation, resulting in a productivity of 0.6423 (U cm^?3 h^?1). In the absence of mechanical agitation similar lipase yields were achieved but in less time, resulting in productivity, about 60% greater than in a stirred fermenter; the lower energy demand for the same lipase yield offers economic advantages. Copyright © 2004 Society of Chemical Industry     

长川霉素发酵条件的优化

考察了发酵过程中接种、培养基、搅拌速度、通气量对长川霉素(Changchuanmycin)发酵的影响，通过实验确定了250L发酵罐的批式发酵条件为，以5％～10％的接种量在36～40h时接种，搅拌速度发酵前期为200rpm，然后逐步提高至400rpm；通气量发酵前期为0．6vvm，中期为1．0vvm，后期为0．6vvm；发酵过程中溶氧控制在30％以上，可以使发酵单位达到380μg/ml。     

13‐Hydroxy‐9Z,11E‐Octadecadienoic Acid Production by Recombinant Cells Expressing Burkholderia thailandensis 13‐Lipoxygenase

Linoleate 13‐lipoxygenase from Burkholderia thailandensis was expressed in Escherichia coli for the production of 13‐hydroxyoctadecadienoic acid (13‐HODE), an antiseptic emulsifier. Linoleate 13‐lipoxygenase in cells had higher thermal stability than the purified enzyme. To increase 13‐HODE production, recombinant cells were permeabilized by solvents, detergents, salts, and other chemicals. The enzymatic activity in cells was the highest for permeabilized cells treated with 0.5 M NaCl among the permeabilizers tested. The optimal reaction conditions for the production of 13‐HODE from linoleic acid by permeabilized cells treated with 0.5 M NaCl were at pH 7.5, 25 °C, 20 g/l linoleic acid, 15 g/l cells, 0.15 mM Cu²⁺, and 6 % (v/v) methanol in a 100‐ml baffled flask containing a 5‐ml working volume with agitation at 200 rpm. Under these conditions, permeabilized cells produced 15.8 g/l 13‐HODE after 30 min with a conversion yield of 79 % (w/w) and a productivity of 31.6 g/l/h. The conversion yield and productivity of permeabilized cells for 13‐HODE production were higher than those of purified and crude enzymes as well as nonpermeabilized cells. Therefore, permeabilized cells were efficient biocatalysts for 13‐HODE production. To the best of our knowledge, this is the first report of the production of 13‐HODE using cells.     

Highly Enantioselective Phase‐Transfer Catalytic α‐Alkylation of α‐tert‐Butoxycarbonyllactams: Construction of β‐Quaternary Chiral Pyrrolidine and Piperidine Systems

A new enantioselective α‐alkylation of α‐tert‐butoxycarbonyllactams for the construction of β‐quaternary chiral pyrrolidine and piperidine core systems is reported. α‐Alkylations of N‐methyl‐α‐tert‐butoxycarbonylbutyrolactam and N‐diphenylmethyl‐α‐tert‐butoxycarbonylvalerolactam under phase‐transfer catalytic conditions (solid potassium hydroxide, toluene, −40 °C) in the presence of (S,S)‐3,4,5‐trifluorophenyl‐3,3′,5,5′‐tetrahydro‐2,6‐bis(3,4,5‐trifluorophenyl)‐4,4′‐spirobi[4H‐dinaphth[2,1‐c:1′,2′‐e]azepinium] bromide [(S,S)‐NAS Br] (5 mol%) afforded the corresponding α‐alkyl‐α‐tert‐butoxycarbonyllactams in very high chemical (up to 99%) and optical yields (up to 98% ee). Our new catalytic systems provide attractive synthetic methods for pyrrolidine‐ and piperidine‐based alkaloids and chiral intermediates with β‐quaternary carbon centers.     

Effect of oxygen transfer rates on alcohols production by Candida guilliermondii cultivated on soybean hull hydrolysate

BACKGROUND: In this research the use of soybean hull hydrolysate (SHH) as substrate for xylitol and ethanol production using an osmotolerant strain of Candida guilliermondii was studied. The production of alcohols was investigated in batch cultivations in which the variable parameter was the volumetric oxygen mass transfer coefficient (k_La) obtained from three different conditions of air supply: anaerobic (150 rpm, no aeration); microaerobic (300 rpm, 1 vvm), and aerobic (600 rpm, 2 vvm), corresponding to k_La values of 0; 8; and 46 h^?1, respectively. RESULTS: SHH, although presenting a very high osmotic pressure (1413 mOsm kg^?1), was completely metabolized under aerobic conditions with high biomass productivities of 0.49 g cells (L h)^?1, with little formation of ethanol. Xylitol was produced under microaeration, with product yield of 0.22 g g^?1 xylose, with the formation of glycerol as a by‐product. No xylose was metabolized under anaerobic conditions, but ethanol was produced from hexoses with high product yields of 0.5 g g^?1. CONCLUSION: These results suggest that the hydrolysis of soybean hull and its conversion to ethanol and other alcohols could be an important use of this agro‐industrial waste, which could be used for biofuel, xylitol or biomass production, depending on the aeration conditions of the cultures. Copyright © 2008 Society of Chemical Industry     

Production of antifungal lipopeptide from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in submerged fermentation using shake flask and fermentor

Optimization of the production of antifungal lipopeptide, iturin A, by B. subtilis was carried out in submerged cultural fermentation. In a shake-flask experiment, response surface methodology (RSM) was employed to optimize the cultivation conditions of Bacillus subtilis S3 for the enhancement of iturin A production. The optimal conditions for iturin A production obtained from RSM were pH 6.0, 0.93% maltodextrin, 1.11% glucose, 0.72% corn steep powder (C.S.P), 1.5 mM MgSO₄, 0.75 mM KH₂PO₄, rotation speed 180 rpm and area of aeration 4.35 cm². 57% increase of iturin A productivity (from 47.19 mg/L to 74.22 mg/L) was observed using the one-factor-at -a-time technique; however, 180% increase of iturin A productivity (from 47.19 mg/L to 132.23 mg/L) was observed with RSM. The iturin A production was further studied in a 5-L fermentor with a variation of agitation speed, aeration and baffles. In the 5-L fermentor, it was found that increased agitation speed improved the growth of B. subtilis and the production of iturin A. Aeration at 2 vvm gave excellent production of iturin A (175.52 mg/L) at 6 d of fermentation. The addition of baffles in the fermentor has significantly influenced the oxygen mass transfer coefficient (KLa) and iturin A production.     

Optimization of pullulan production from synthetic medium by Aureobasidium pullulans in a stirred tank reactor by response surface methodology

The production of pullulan from synthetic medium by Aureobasidium pullulans P56 in a stirred tank fermenter was investigated. The kinetics of polysaccharide, pullulan and biomass production was determined. Response surface methodology was used to investigate the effects of three factors (initial sugar concentration, aeration rate and agitation speed) on the concentration of pullulan in batch cultures of A pullulans. In the experiments, the range of values used for the three variables described were; 30–70 g dm^?3 initial sugar concentration, 200–600 rpm agitation speed and 1.0–3.0 vvm aeration rate. No previous work has used statistical analysis in determining the interactions among these variables in pullulan production. Results of the statistical analysis showed that the fit of the model was good in all cases. Aeration rate, agitation speed and sugar concentration had a strong linear effect on pullulan concentration. Moreover, pullulan concentration was significantly influenced by the negative quadratic effects of the given variables and by their positive or negative interactions with the exception that the interaction between agitation speed and aeration rate was insignificant (P > 0.05). Maximum pullulan concentration of 17.2 g dm^?3 was obtained at the optimum levels of process variables (initial sugar concentration 51.4 g dm^?3, aeration rate 2.36 vvm, agitation speed 345.3 rpm). These values were obtained by fitting of the experimental data to the model equation. Scanning electron microscope (SEM) photographs of polysaccharide particles containing different concentrations of pullulan were also taken to observe the morphological differences of the samples. Copyright © 2005 Society of Chemical Industry     

Optimization of the production of chitosan from beet molasses by response surface methodology

Chitosan was produced by Rhizopus oryzae 00.4367 in shake flask culture and a stirred tank fermenter. Synthetic medium, treated and untreated beet molasses were used as cultivation media in shake flask cultures. In the stirred tank fermenter, the cultivation media were synthetic medium and untreated beet molasses. Shake flask culture containing untreated molasses with a sugar concentration of 40 g dm^?3 produced the maximum chitosan yield (961 mg dm^?3). Chitosan concentration reached its maximum value at the late exponential growth phase of R oryzae. In all experiments almost 8–10% of biomass and 32–38% of alkali‐insoluble material was extracted as chitosan. A central composite design was employed to determine the optimum values of process variables (aeration rate, agitation speed and initial sugar concentration) leading to maximum chitosan concentration in the stirred tank fermenter. In all cases, the fit of the model was found to be good. Aeration rate, agitation speed and initial sugar concentration had a strong linear effect on chitosan concentration. Moreover, the concentration of chitosan was significantly influenced by the negative quadratic effects of the given variables and by their positive or negative interactions. A maximum chitosan concentration of 1109.32 mg dm^?3 was obtained in untreated molasses medium containing an initial sugar concentration of 45.37 g dm^?3 with an aeration rate and agitation speed of 2.10 vvm and 338.93 rpm, respectively. Copyright © 2004 Society of Chemical Industry     

Control strategy of pH,dissolved oxygen concentration and stirring speed for enhancing β‐poly (malic acid) production by Aureobasidium pullulans ipe‐1

BACKGROUND: β‐poly(malic acid) (PMLA) can be used as a pro‐drug or for a drug‐delivery system. Effects of pH, dissolved oxygen concentration (DO) and stirring speed were investigated to improve PMLA production by A. pullulans ipe‐1. RESULTS: The strain produced a high PMLA concentration when pH and DO remained at about 6.0 and above 70%, respectively, and the yeast‐like cells were the main PMLA producers. To further promote PMLA production, the cultivation could be divided into three phases. In phase I, cell growth was accelerated by maintaining high DO (>70%) with a constant stirring speed of 800 rpm. In phase II, PMLA production was increased by controlling DO at 70% using the automatically controlled stirring speed. In phase III, PMLA production on per gram of glucose (Y_p/s) was enhanced by keeping DO at 70%, and using a low stirring speed to decrease cell growth. Compared with batch cultures, a higher PMLA yield was obtained with this strategy, i.e. PMLA production and Y_p/s increased by 15% and 18%, respectively. CONCLUSION: Control strategies for pH, DO and stirring speed provide a good reference for process development and optimization of PMLA production. © 2012 Society of Chemical Industry     

Rhodium‐Catalyzed Asymmetric Hydroformylation of 1,1‐Disubstituted Allylphthalimides: A Catalytic Route to β3‐Amino Acids

The high enantioselective rhodium‐catalyzed hydroformylation of 1,1‐disubstituted allylphthalimides has been developed. By employing chiral ligand 1,2‐bis[(2S,5S)‐2,5‐diphenylphospholano]ethane [(S,S)‐Ph‐BPE], a series of β³‐aminoaldehydes can be prepared with up to 95% enantioselectivity. This asymmetric procedure provides an efficient alternative route to prepare chiral β³‐amino acids and alcohols.     

Phase‐Transfer‐Catalyzed Olefin Isomerization/α‐Alkylation of α‐Alkynylcrotonates as a Route for 1,4‐Enynes

The phase‐transfer‐catalyzed alkylation of α‐alkynylcrotonates was developed as a means to provide 1,4‐enynes deconjugated by an all‐carbon quaternary center. Extension to the asymmetric version using the chiral phase‐transfer catalyst (S)‐ 3 provided the alkylated compounds with up to 87% ee.     

A comprehensive study of lipase production by Yarrowia lipolytica CECT 1240 (ATCC 18942): from shake flask to continuous bioreactor

BACKGROUND: Lipases are commercially important enzymes, and the development and optimization of their production processes are of great interest. The diversity of behaviours between strains stresses the need for research on this topic, especially when bioreactor culture is considered. The study of a continuous operating mode is especially attractive, since very scarce information is available on its application to microbial lipases production. RESULTS: Lipase production in submerged cultures of Yarrowia lipolytica CECT 1240 (ATCC 18 942) has been investigated. Significant lipolytic activity (over 700 U dm^?3), mostly extracellular and membrane‐bound, was obtained in shake flasks using medium supplemented with olive oil. The culture was carried out in air‐lift and stirred tank bench‐scale bioreactors and the latter was selected. The influence of aeration and agitation rates was assessed in batch cultures, and agitation from 400–700 rpm and low aeration rates (i.e. 0.2 vvm) are recommended. Batch, fed‐batch and continuous operation were investigated, and regular enzyme production (up to 600 U dm^?3) was achieved with the latter. CONCLUSION: Lipase production by the selected strain was successfully carried out in shake flasks and bench‐scale bioreactors. After studying batch, fed‐batch and continuous processes, continuous culture in a stirred tank bioreactor was found best in terms of regular enzyme production, exceptionally good operational stability and good fitting of the results to mathematical models. Copyright © 2009 Society of Chemical Industry     

Optically Active 4‐Substituted (S)‐2‐Phenyl‐2‐oxazolines

(R)‐4‐Hydroxymethyl‐2‐phenyl‐2‐oxazoline (R)‐ 1 ) was prepared from (L)‐serine. The respective tosylate ((S)‐ 2 ) was converted into sulfides (S)‐ 4 and (S)‐ 5 , and sulfone (S)‐ 6 , useful starting materials for the elaboration of additional chiral centers. A previously reported [ α]_D ²⁵ value for (R)‐ 4 is corrected.     

Production and properties of 7,10,12‐trihydroxy‐8(E)‐octadecenoic acid from ricinoleic acid conversion by Pseudomonas aeruginosa

This study was undertaken to produce a novel antifungal compound, 7,10,12‐trihydroxy‐8(E)‐octadecenoic acid (7,10,12‐THOD), in large quantities for determining its properties by use of crude ricinoleic acid as converted by Pseudomonas aeruginosa NRRL B‐23260. A useful reactor process was developed by employing a unique aeration mechanism and a new agitation device. The aeration mechanism involved a filtered airflow constantly supplied from the top through two ports on the headplate at 220 mL/min and at varied rates as needed and regulated through a bottom sparger, to control foaming. The agitation device combined a marine impeller and an open turbine to overcome the early phase of oxygen depletion and enhance production yields. A yield of 40% was achieved after 77 h of reaction when down‐stream processing of the bioproduct was most suitable. 7,10,12‐THOD was crystallized from ethyl acetate extracts of the reactor broth following a sequential cooling process to ?20 °C. The melting point of 7,10,12‐THOD was 94.5–95.0 °C. The ¹H‐ and ¹³C‐NMR signals of all protons and carbons in the 2(E)‐ene‐1,4,6‐triol moiety of 7,10,12‐THOD were now assigned, also with the aid of various 2D techniques. 7,10,12‐THOD was found to exhibit a unique surface‐active property. This study provides basic information and process technology for potential new uses of 7,10,12‐THOD.     

Enhanced production of carboxymethylcellulase by Cellulophaga lytica LBH-14 in pilot-scale bioreactor under optimized conditions involved in dissolved oxygen

The optimal conditions for the production of carboxymethylcellulase (CMCase) by Cellulophaga lytica LBH-14 at flask scale has been previously reported. In this study, we optimized the parameters involved in dissolved oxygen in 7 and 100 L bioreactors for pilot-scaled production of CMCase by C. lytica LBH-14. The optimal conditions of agitation speed and aeration rate for cell growth in 7 L bioreactors were 395 rpm and 0.98 vvm, whereas those for production of CMCase were 357 rpm and 0.55 vvm. The optimal inner pressures for cell growth and production of CMCase by C. lytica LBH-14 in 100 L bioreactors were 0.00 and 0.06MPa, respectively. The production of CMCase under an optimized inner pressure was 1.38 times higher than that without an inner pressure. The maximal production of CMCase by C. lytica under optimized conditions at pilot scale using rice bran and ammonium chloride was 153.6 U/mL, which was 1.39 times higher than that at flask scale.     

Improving k La determination in fungal fermentation,taking into account electrode response time

An alternative way for determining the oxygen mass transfer coefficient, k_La, based upon the traditional dynamic method, is proposed. The oxygen material balance equation in the liquid phase is integrated after insertion of the oxygen probe response time (first order type), and k_La values are determined by employing Marquardt's algorithm, considering as a weighting factor the model's sensitivity with respect to k_La. Bench‐scale fermentations of Aspergillus awamori, performed under different agitation (300–700 rpm) and aeration conditions (0.2–0.6 vvm), were utilized for calculating k_La values (0.0283–0.0874 s⁻¹), employing three methods: two so‐called traditional, the gas balancing and the dynamic methods, and the one proposed here. The latter method is shown to be as reliable as the aforementioned methods but is easier to apply when the oxygen level in the reactor is above the critical value. © 2000 Society of Chemical Industry