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1.
Martin Cerff Alexander Scholz Tobias Käppler Kim E. Ottow Tim J. Hobley Clemens Posten 《Biotechnology and bioengineering》2013,110(8):2161-2172
In modern biotechnology proteases play a major role as detergent ingredients. Especially the production of extracellular protease by Bacillus species facilitates downstream processing because the protease can be directly harvested from the biosuspension. In situ magnetic separation (ISMS) constitutes an excellent adsorptive method for efficient extracellular protease removal during cultivation. In this work, the impact of semi‐continuous ISMS on the overall protease yield has been investigated. Results reveal significant removal of the protease from Bacillus licheniformis cultivations. Bacitracin‐functionalized magnetic particles were successfully applied, regenerated and reused up to 30 times. Immediate reproduction of the protease after ISMS proved the biocompatibility of this integrated approach. Six subsequent ISMS steps significantly increased the overall protease yield up to 98% because proteolytic degradation and potential inhibition of the protease in the medium could be minimized. Furthermore, integration of semi‐continuous ISMS increased the overall process efficiency due to reduction of the medium consumption. Process simulation revealed a deeper insight into protease production, and was used to optimize ISMS steps to obtain the maximum overall protease yield. Biotechnol. Bioeng. 2013; 110: 2161–2172. © 2013 Wiley Periodicals, Inc. 相似文献
2.
A new approach for in situ product removal from bioreactors is presented in which high-gradient magnetic separation is used. This separation process was used for the adsorptive removal of proteases secreted by Bacillus licheniformis. Small, non-porous bacitracin linked magnetic adsorbents were employed directly in the broth during the fermentation, followed by in situ magnetic separation. Proof of the concept was first demonstrated in shake flask culture, then scaled up and applied during a fed batch cultivation in a 3.7 L bioreactor. It could be demonstrated that growth of B. licheniformis was not influenced by the in situ product removal step. Protease production also remained the same after the separation step. Furthermore, degradation of the protease, which followed first order kinetics, was reduced by using the method. Using a theoretical modeling approach, we could show that protease yield in total was enhanced by using in situ magnetic separation. The process described here is a promising technique to improve overall yield in bio production processes which are often limited due to weak downstream operations. Potential limitations encountered during a bioprocess can be overcome such as product inhibition or degradation. We also discuss the key points where research is needed to implement in situ magnetic separation in industrial production. 相似文献
3.
Christoph K. Winkler Dorina Clay Esta van Heerden Kurt Faber 《Biotechnology and bioengineering》2013,110(12):3085-3092
Eleven flavoproteins from the old yellow enzyme family were found to catalyze the disproportionation (“dismutation”) of conjugated enones. Incomplete conversions, which were attributed to enzyme inhibition by the co‐product phenol could be circumvented via in situ co‐product removal by scavenging the phenol using the polymeric adsorbent MP‐carbonate. The optimized system allowed to reduce an alkene activated by ester groups in a “coupled‐substrate” approach via nicotinamide‐free hydrogen transfer with >90% conversion and complete stereoselectivity. Biotechnol. Bioeng. 2013;110: 3085–3092. © 2013 The Authors. Biotechnology and Bioengineering Published by Willey Periodicals, Inc. 相似文献
4.
After 24 h of chemical permeabilization with 20% (v/v) methanol at 25 °C, the amount of daidzein released from soybean seeds is 15 to 20% of the amount (0.0423 ± 0.0045 mg/g seed dry wt) obtained by physical grinding. With this chemical permeabilization condition, 70% of the permeabilized seeds are still able to germinate. The release of daidzein is enhanced to 33% with the addition of XAD-4 to 20% (v/v) methanol without affecting seed viability. © Rapid Science Ltd. 1998 相似文献
5.
In situ butanol recovery from Clostridium acetobutylicum fermentations by expanded bed adsorption 
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下载免费PDF全文 Although butanol is a promising biofuel, its fermentative production suffers from inhibition caused by end product toxicity. The in situ removal of butanol from cultures via expanded bed adsorption offers an effective strategy for mitigating the effects of product toxicity while eliminating the need to clarify cultures via microfiltration. The hydrophobic polymer resin Dowex Optipore L‐493 was found to be both an effective butanol adsorbent and suitable for use in expanded bed adsorption. Recirculation rates through the adsorption column were strongly correlated with and ultimately controlled rates of butanol uptake from the media which, reaching as high as 41.1 g/L h, easily exceed those of its production in a typical fermentation. Vacuum application with vapor collection was found to be an effective means of adsorbent regeneration, with an average of 81% butanol recovery possible, with butanol concentrations in the cold trap reaching as high as 85.8 g/L. Integration of expanded bed adsorption with a fed‐batch Clostridium acetobutylicum ATCC 824 fermentation and its continuous operation for 38.5 h enabled the net production (i.e., in solution and adsorbed) of butanol and total solvent products at up to 27.2 and 40.7 g/L of culture, respectively, representing 2.2‐ and 2.3‐fold improvements over conventional batch culture. While adsorbent biofouling was found to be minimal, further investigation of biofouling in longer‐term studies will provide useful and further insight regarding the robustness of the process strategy. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:68–78, 2014 相似文献
6.
An integrated bioprocess for the production of the natural rose-like aroma compounds, 2-phenylethanol (2-PE) and 2-phenylethylacetate (2-PEAc), from L-phenylalanine (L-phe) with yeasts was investigated. The hydrophobicity of the products leads to product inhibition, which can be compensated by in situ product removal (ISPR). An organophilic pervaporation unit, equipped with a polyoctylmethylsiloxane (POMS) membrane, was coupled via a bypass to a bioreactor and proved to be a suitable technique for the in situ removal of high-boiling products from culture broth. With batch cultures of the thermotolerant yeast Kluyveromyces marxianus CBS 600 in a standard medium at 35 degrees C, the use of pervaporation resulted in a double 2-PE concentration (2.2 g/L) and 1.3 g/L 2-PEAc, which only accumulated transiently in low concentrations during cultivation without ISPR. Using a previously optimized medium, the variation of the temperature from 30 degrees C to 40 degrees C caused an increase in the total conversion yield from 63% to 79%, corresponding to total product concentrations of 5.23 and 5.85 g/L, respectively. In the 40 degrees C batch experiment, the volumetric productivity (2-PE + 2-PEAc) during the exponential phase was 5.2 mmol/L h. While for 2-PE, there is still potential for further optimization, the more hydrophobic 2-PEAc was nearly completely removed from the aqueous culture broth (enrichment factor >400), resulting in highly aroma-enriched permeates. Due to the temperature-correlated performance of the pervaporation, the bioconversion was still efficient even at 45 degrees C (conversion yield: 69%). Surprisingly, at 45 degrees C, the molar ratio of the two products inverted and 2-PEAc turned out to be the main product (4.0 g/L), which opens easy control of the reaction's selectivity by external means. Retrofitting the process with interim heating and cooling equipment to use different temperature levels for cultivation and pervaporation resulted in a decreased yield and product concentration caused by multiple stress factors. The medium composition affected the pervaporation efficiency with molasses acting detrimental. 相似文献
7.
Recombinant Escherichia coli JM101 was used for the in vivo biocatalytic synthesis of 3-tert-butyl- catechol. The bacterial strain synthesized the laboratory-evolved variant HbpA(T2) of 2-hydroxybiphenyl 3-monooxygenase (HbpA, EC 1.14.13.44) from Pseudomonas azelaica HBP1. The mutant enzyme HbpA(T2) is able to hydroxylate 2-tert-butylphenol to the corresponding catechol, a reaction that is not catalyzed by the wild-type enzyme. The biotransformation was performed in a 3-L bioreactor for 24 h. To mitigate the toxicity of the 2-tert-butylphenol starting material, we applied a limited substrate feed. Continuous in situ product removal with the hydrophobic resin Amberlite XAD-4 was used to separate the product from culture broth. In addition, binding to the resin stabilized the product, which was important because 3-tert-butylcatechol is very labile in aqueous solution. The productivity of the process was 63 mg L(-1) h(-1) so that after 24 h, 3.0 g of 3-tert-butylcatechol were isolated. Down-stream processing consisted of two steps. First, bound 2-tert-butylphenol and 3-tert-butylcatechol were eluted from Amberlite XAD-4 with methanol. Second, the two compounds were separated over neutral aluminum oxide, which selectively binds the produced catechol but not the phenol substrate. The final purity of 3-tert-butylcatechol was greater than 98%. 相似文献
8.
Buque-Taboada EM Straathof AJ Heijnen JJ van der Wielen LA 《Biotechnology and bioengineering》2004,86(7):795-800
In situ product crystallization was investigated for solid product crystals that were obtained during fermentation. The model reaction was the asymmetric reduction of 4-oxoisophorone (OIP) using baker's yeast (S. cerevisiae) as a biocatalyst. The target product was 6R-dihydro-oxoisophorone (DOIP), also known as levodione, a key intermediate in carotenoid synthesis. DOIP was degraded by baker's yeast mainly to (4S,6R)-actinol, an unwanted byproduct in the process. Actinol formation reached up to 12.5% of the initial amount of OIP in the reactor during a batch process. However, better results were obtained when the dissolved DOIP concentration was controlled using an integrated fermentation-crystallization process because: (a) actinol formation was reduced to 4%; and (b) DOIP crystal formation in the reactor was avoided. DOIP productivity was improved by 50% and its selectivity was raised from 87% to 96% relative to the batch process. In the integrated process, most of the product was recovered as pure crystals; this may already minimize, if not eliminate, the need for organic solvents in the final purification steps. An almost sixfold reduction in biocatalyst consumption per kilogram product was achieved, which also can contribute to the minimization of waste streams. 相似文献
9.
Extractive fermentation (or in situ product removal (ISPR)) is an operational method used to combat product inhibition in fermentations. To achieve ISPR, different separation techniques, modes of operation and physical reactor configurations have been proposed. However, the relative paucity of industrial application necessitates continued investigation into reactor systems. This article outlines a bioreactor designed to facilitate in situ product extraction and recovery, through adapting the reaction volume to include a settler and solvent extraction and recycle section. This semipartition bioreactor is proposed as a new mode of operation for continuous liquid‐liquid extractive fermentation. The design is demonstrated as a modified bench‐top fermentation vessel, initially analysed in terms of fluid dynamic studies, in a model two‐liquid phase system. A continuous abiotic simulation of lactic acid (LA) fermentation is then demonstrated. The results show that mixing in the main reaction vessel is unaffected by the inserted settling zone, and that the size of the settling tube effects the maximum volumetric removal rate. In these tests the largest settling tube gave a potential continuous volumetric removal rate of 7.63 ml/min; sufficiently large to allow for continuous product extraction even in a highly productive fermentation. To demonstrate the applicability of the developed reactor, an abiotic simulation of a LA fermentation was performed. LA was added to reactor continuously at a rate of 33ml/h, while continuous in situ extraction removed the LA using 15% trioctylamine in oleyl alcohol. The reactor showed stable LA concentration of 1 g/L, with the balance of the LA successfully extracted and recovered using back extraction. This study demonstrates a potentially useful physical configuration for continuous in situ extraction. 相似文献
10.
Whole lyophilized cells of an Escherichia coli overexpressing the alcohol dehydrogenase (ADH-'A') from Rhodococcus ruber DSM 44541 were used for the asymmetric reduction of ketones to secondary alcohols. The recycling of the required nicotinamide cofactor (NADH) was achieved in a coupled-substrate process. In the course of the reaction the ketone is reduced to the alcohol and the hydrogen donor 2-propanol is oxidized to acetone by one enzyme. This leads to a thermodynamic equilibrium between all four components determining the maximum achievable conversion. To overcome this limitation an in situ product removal technique (ISPR) for the application with whole cells was developed. In this method the most volatile compound is separated from the reaction vessel by an air flow resulting in a shift of the equilibrium towards the desired secondary alcohol. The so-called stripping process represents a simple and efficient method to overcome the thermodynamic limitation in biocatalytic reactions. Employing this method, the conversion of selected biotransformations was increased up to completeness. 相似文献
11.
Stark D Kornmann H Münch T Sonnleitner B Marison IW von Stockar U 《Biotechnology and bioengineering》2003,83(4):376-385
A novel in situ product removal (ISPR) method that uses microcapsules to extract inhibitory products from the reaction suspension is introduced into fermentation technology. More specifically, L-phenylalanine (L-Phe) was transformed by Saccharomyces cerevisiae to 2-phenylethanol (PEA), which is inhibitory toward the yeast. In order to continuously remove PEA from the vicinity of the cells, the reaction suspension was brought into contact with capsules of 2.2-mm diameter that had a hydrophobic core of dibutyl sebacate and an alginate-based wall. This novel process combines the advantages of a normal in situ extraction process (fast mass transfer and simple process set-up) with the benefits of a membrane-based process (reduction of the solvent toxicity and avoidance of stable emulsions). In particular, the microbial cells are shielded from the phase toxicity of the organic solvent by a hydrogel layer surrounding the organic core. By placing the microcapsules into the fermenter, the final overall concentration of PEA in a fed-batch culture was increased from 3.8 to 5.6 g/L because a part of the inhibitory product dissolved in the dibutyl sebacate core. In another fermentation experiment, the capsules were placed in a fluidized bed that was connected via a loop to the fermenter. In addition, the fluidized bed was connected via a second loop to a back-extractor to regenerate the capsules. By alternating the extraction and back-extraction cycles, it was possible to limit the PEA concentration of the fed-batch culture in the fermenter to 2.4 g/L while producing important quantities of PEA that accumulated in an external reservoir. 相似文献
12.
13.
van den Berg C Wierckx N Vente J Bussmann P de Bont J van der Wielen L 《Biotechnology and bioengineering》2008,100(3):466-472
The sustainable production of fine/bulk chemicals is often hampered by product toxicity and inhibition to the producing micro-organisms. Consequently, the product must be removed from the micro-organisms' environment. To achieve this, so-called solvent-impregnated resins (SIRs) as well as commercial resins have been added to a Pseudomonas putida S12TPL fermentation that produces phenol as a model compound from glucose. The SIRs contained an ionic liquid which extracts phenol effectively. It was observed that the addition of these particles resulted in an increased phenol production of more than a fourfold while the commercial resin (XAD-4) which is widely used in aromatic removal from aqueous phases, only gave a 2.5-fold increase in volumetric production. 相似文献
14.
Schroën CG Nierstrasz VA Bosma R Kroon PJ Tjeerdsma PS DeVroom E VanderLaan JM Moody HM Beeftink HH Janssen AE Tramper J 《Biotechnology and bioengineering》2002,80(2):144-155
Integrated process concepts for enzymatic cephalexin synthesis were investigated by our group, and this article focuses on the integration of reactions and product removal during the reactions. The last step in cephalexin production is the enzymatic kinetic coupling of activated phenylglycine (phenylglycine amide or phenylglycine methyl ester) and 7-aminodeacetoxycephalosporanic acid (7-ADCA). The traditional production of 7-ADCA takes place via a chemical ring expansion step and an enzymatic hydrolysis step starting from penicillin G. However, 7-ADCA can also be produced by the enzymatic hydrolysis of adipyl-7-ADCA. In this work, this reaction was combined with the enzymatic synthesis reaction and performed simultaneously (i.e., one-pot synthesis). Furthermore, in situ product removal by adsorption and complexation were investigated as means of preventing enzymatic hydrolysis of cephalexin. We found that adipyl-7-ADCA hydrolysis and cephalexin synthesis could be performed simultaneously. The maximum yield on conversion (reaction) of the combined process was very similar to the yield of the separate processes performed under the same reaction conditions with the enzyme concentrations adjusted correctly. This implied that the number of reaction steps in the cephalexin process could be reduced significantly. The removal of cephalexin by adsorption was not specific enough to be applied in situ. The adsorbents also bound the substrates and therewith caused lower yields. Complexation with beta-naphthol proved to be an effective removal technique; however, it also showed a drawback in that the activity of the cephalexin-synthesizing enzyme was influenced negatively. Complexation with beta-naphthol rendered a 50% higher cephalexin yield and considerably less byproduct formation (reduction of 40%) as compared to cephalexin synthesis only. If adipyl-7-ADCA hydrolysis and cephalexin synthesis were performed simultaneously and in combination with complexation with beta-naphthol, higher cephalexin concentrations also were found. In conclusion, a highly integrated process (two reactions simultaneously combined with in situ product removal) was shown possible, although further optimization is necessary. 相似文献
15.
Yewang Zhang Dengchao Li Shulai Liu Qingxun Song 《Biocatalysis and Biotransformation》2013,31(1):59-64
Enzymatic synthesis of cefaclor by penicillin acylase (PA) was carried out under kinetic control with in situ product removal (ISPR). We present a continuous acyl donor feeding strategy for enzymatic reactions. Using this strategy, the conversion of the antibiotic nucleus was improved from 65 to 91%, and the hydrolysis of phenylglycine methyl ester (PGME) was decreased. Side product (phenylglycine) production was less than half of that in the control batch. The ratio of synthesis to hydrolysis (S/H) in the process was kept stable for longer and at a higher level than in the control. This is a practical method for enzymatic synthesis of cefaclor. 相似文献
16.
Yewang Zhang Dongzhi Wei Dengchao Li Shulai Liu Qingxun Song 《Biocatalysis and Biotransformation》2007,25(1):59-64
Enzymatic synthesis of cefaclor by penicillin acylase (PA) was carried out under kinetic control with in situ product removal (ISPR). We present a continuous acyl donor feeding strategy for enzymatic reactions. Using this strategy, the conversion of the antibiotic nucleus was improved from 65 to 91%, and the hydrolysis of phenylglycine methyl ester (PGME) was decreased. Side product (phenylglycine) production was less than half of that in the control batch. The ratio of synthesis to hydrolysis (S/H) in the process was kept stable for longer and at a higher level than in the control. This is a practical method for enzymatic synthesis of cefaclor. 相似文献
17.
Lindegaard ML Olivecrona G Christoffersen C Kratky D Hannibal J Petersen BL Zechner R Damm P Nielsen LB 《Journal of lipid research》2005,46(11):2339-2346
Placenta expresses various lipase activities. However, a detailed characterization of the involved genes and proteins is lacking. In this study, we compared the expression of endothelial lipase (EL) and LPL in human term placenta. When placental protein extracts were separated by heparin-Sepharose affinity chromatography, the EL protein eluted as a single peak without detectable phospholipid or triglyceride (TG) lipase activity. The major portion of LPL protein eluted slightly after EL. This peak also had no lipase activity and most likely contained monomeric LPL. Fractions eluting at a higher NaCl concentration contained small amounts of LPL protein (most likely dimeric LPL) and had substantial TG lipase activity. In situ hybridization studies showed EL mRNA expression in syncytiotrophoblasts and endothelial cells and LPL mRNA in syncytiotrophoblasts. In contrast, immunohistochemistry showed EL and LPL protein associated with both cell types. In mouse placentas, lack of LPL expression resulted in increased EL mRNA expression. These results suggest that the cellular expression of EL and LPL in human placenta is different. Nevertheless, the two lipases might have overlapping functions in the mouse placenta. Our data also suggest that the major portions of both proteins are stored in an inactive form in human term placenta. 相似文献
18.
Chao Zhong Christiane Luley-Goedl Bernd Nidetzky 《Biotechnology and bioengineering》2019,116(9):2146-2155
Soluble cellodextrins (linear β-1,4-d -gluco-oligosaccharides) have interesting applications as ingredients for human and animal nutrition. Their bottom-up synthesis from glucose is promising for bulk production, but to ensure a completely water-soluble product via degree of polymerization (DP) control (DP ≤ 6) is challenging. Here, we show biocatalytic production of cellodextrins with DP centered at 3 to 6 (~96 wt.% of total product) using coupled cellobiose and cellodextrin phosphorylase. The cascade reaction, wherein glucose was elongated sequentially from α-d -glucose 1-phosphate (αGlc1-P), required optimization and control at two main points. First, kinetic and thermodynamic restrictions upon αGlc1-P utilization (200 mM; 45°C, pH 7.0) were effectively overcome (53% → ≥90% conversion after 10 hrs of reaction) by in situ removal of the phosphate released via precipitation with Mg2+. Second, the product DP was controlled by the molar ratio of glucose/αGlc1-P (∼0.25; 50 mM glucose) used in the reaction. In optimized conversion, soluble cellodextrins in a total product concentration of 36 g/L were obtained through efficient utilization of the substrates used (glucose: 98%; αGlc1-P: ∼80%) after 1 hr of reaction. We also showed that, by keeping the glucose concentration low (i.e., 1–10 mM; 200 mM αGlc1-P), the reaction was shifted completely towards insoluble product formation (DP ∼9–10). In summary, this study provides the basis for an efficient and product DP-controlled biocatalytic synthesis of cellodextrins from expedient substrates. 相似文献
19.
Hermo L Chung S Gregory M Smith CE Wang SP El-Alfy M Cyr DG Mitchell GA Trasler J 《Molecular reproduction and development》2008,75(4):565-577
Hormone-sensitive lipase (HSL, Lipe, E.C.3.1.1.3) functions as a triglyceride and cholesteryl esterase, supplying fatty acids, and cholesterol to cells. Gene-targeted HSL-deficient (HSL(-/-)) mice reveal abnormal spermatids and are infertile at 24 weeks after birth. The purpose of this study was to follow the evolution of spermatid abnormalities as HSL(-/-) mice age, characterize sperm motility in older HSL(-/-) mice, and determine if mice expressing a human testicular HSL transgene (HSL(-/-)ttg) produce normal motile sperm. In situ hybridization indicated that HSL is expressed exclusively in steps 5-16 spermatids, but not in Sertoli cells. In HSL(-/-) mice, abnormalities were evident in step 16 spermatids at 5 weeks after birth, with defects progressively increasing in spermatids with age. The defects included multinucleation of spermatids, abnormal shapes and a reduction of elongating spermatids. In older HSL(-/-) mice, sperm counts appeared reduced by 42%, but this value was lower because samples were compromised by the presence of small degenerating germ cells in addition to sperm, both of which appeared of similar size and density. Sperm motility was dramatically reduced with only 11% classified as motile in HSL(-/-) mice compared to 76-78% of sperm in wild-type and HSL(-/-)ttg mice. Sperm morphology, counts, and motility were normal in HSL(-/-)ttg mice, as was their fertility. Collectively, the data indicate that HSL deficiency results in abnormal spermatid development with defects arising at 5 weeks of age and progressively increasing at later ages. HSL(-/-) mice also show a dramatic reduction in sperm counts and motility and are infertile. 相似文献
20.
Jorge H. Santoyo-Garcia Laura E. Walls Marissa Valdivia-Cabrera Koray Malcı Nestor Jonguitud-Borrego Karen J. Halliday Leonardo Rios-Solis 《Biotechnology and bioengineering》2023,120(8):2160-2174
In situ product recovery is an efficient way to intensify bioprocesses as it can perform adsorption of the desired natural products in the cultivation. However, it is common to use only one adsorbent (liquid or solid) to perform the product recovery. For this study, the use of an in situ product recovery method with three combined commercial resins (HP-20, XAD7HP, and HP-2MG) with different chemical properties was performed. A new yeast strain of Saccharomyces cerevisiae was engineered using CRISPR Cas9 (strain EJ2) to deliver heterologous expression of oxygenated acetylated taxanes that are precursors of the anticancer drug Taxol ® (paclitaxel). Microscale cultivations using a definitive screening design (DSD) were set to get the best resin combinations and concentrations to retrieve high taxane titers. Once the best resin treatment was selected by the DSD, semi-continuous cultivation in high throughput microscale was performed to increase the total taxanes yield up to 783 ± 33 mg/L. The best T5α-yl Acetate yield obtained was up to 95 ± 4 mg/L, the highest titer of this compound ever reported by a heterologous expression. It was also observed that by using a combination of the resins in the cultivation, 8 additional uncharacterized taxanes were found in the gas chromatograms compared to the dodecane overlay method. Lastly, the cell-waste reactive oxygen species concentrations from the yeast were 1.5-fold lower in the resin's treatment compared to the control with no adsorbent aid. The possible future implications of this method could be critical for bioprocess intensification, allowing the transition to a semi-continuous flow bioprocess. Further, this new methodology broadens the use of different organisms for natural product synthesis/discovery benefiting from clear bioprocess intensification advantages. 相似文献