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Enzymatic synthesis of 3-hydroxypropionic acid at high productivity by using free or immobilized cells of recombinant Escherichia coli
Journal of Molecular Catalysis B: Enzymatic 129 (2016) 37–42
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Enzymatic synthesis of 3-hydroxypropionic acid at high productivity by using free or immobilized cells of recombinant Escherichia coli Shanshan Yu a,b,1 , Peiyuan Yao b,1 , Jianjiong Li b , Jie Ren b , Jing Yuan b , Jinhui Feng b , Min Wang a , Qiaqing Wu b,∗ , Dunming Zhu b,∗ a Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China b National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin 300308, China
a r t i c l e
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Article history: Received 25 December 2015 Received in revised form 29 February 2016 Accepted 26 March 2016 Available online 14 April 2016 Keywords: Nitrilase Immobilization 3-Hydroxypropionic acid Hydrolysis Enzyme catalysis
a b s t r a c t 3-Hydroxypropionic acid (3-HP) is an important platform chemical for organic synthesis and high performance polymers. Despite a wealth of reports related to 3-HP biosynthesis in microorganisms, its industrial application still requires further research because of low titer and productivity. Herein an effective enzymatic method for the synthesis of 3-HP was achieved by using free or immobilized recombinant Escherichia coli BL21(DE3) cells harboring a nitrilase gene from environmental sample (NIT190). Under the optimal conditions (100 mmol/L Tris-HCl buffer, pH 8.0, 30 ◦ C), the maximum substrate concentration which could be completely hydrolyzed by using free cells within 24 h was 4.5 mol/L (319.5 g/L). Furthermore, immobilization of the whole cells enhanced their substrate tolerance (up to 7.0 mol/L), stability, and reusability. The immobilized cells could be reused for up to 30 batches, and 70% of enzyme activity was retained after 74 batches in distilled water. The titer (184.7 g/L) and productivity (36.9 g/(L h)) were obtained by isolation and purification of 3-HP from the first 30 batches. These results demonstrate that the immobilized cells have potential industrial application for the synthesis of 3-HP. © 2016 Elsevier B.V. All rights reserved.
1. Introduction 3-Hydroxypropionic acid (3-HP) is a versatile building block in the synthesis of many industrially valuable chemicals, such as acrylic acid, 1,3-propandiol, malonic acid, propiolactone, and other valuable products [1–4]. Its polymerized form, poly (3-HP) has great potential applications in biodegradable materials due to its outstanding material characteristics [5]. In addition, 3-HP showed selective nematicidal activity against the plant-parasitic nematode Meloidogyne incognita and no antimicrobial, cytotoxic or phytotoxic effects [6]. Thus 3-HP was ranked the third position in the top value-added platform chemicals from biomass by the US Department of Energy in 2004 [7]. Owing to its broad applications, 3-HP has attracted increasing attention. Conventional chemical syntheses of 3-HP involve hydrolysis of 3-hydroxypropionitrile (3-HPN) [8] or propiolactone [9], hydration of acrylic acid [10], and oxidation of 1,3-propanediol [11], levulinic
∗ Corresponding authors. E-mail addresses: wu [email protected] (Q. Wu), zhu [email protected] (D. Zhu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.molcatb.2016.03.011 1381-1177/© 2016 Elsevier B.V. All rights reserved.
acid [12] or allyl alcohol [13]. Although excellent results have been obtained by catalytic chemical processes, there is growing interest in the development of bioprocesses for its production because of milder reaction conditions and fewer byproducts. Studies of the biological production of 3-HP began from early 2000s [14]. Several archaea were found in which 3-HP was an intermediary compound during 3-HP cycle, such as Sulfolobus, Acidianus, and Metallosphaera spp. [15]. 3-HP was also produced by some bacteria, such as Lactobacillus sp., Desulfovibrio fructosovorans, Klebsiella pneumoniae and Chloroflexus aurantiacus [15,16]. However, low yield and productivity by wild strains could not afford to meet global market demand that had been estimated at 3.63 million tons per year in 2008 [17]. A number of recombinant strains of Escherichia coli [18–20], yeast [21–23], Pseudomonas denitrificans [16], Lactobacillus reuteri [24] and K. pneumonia [25] were developed to largely accumulate 3HP with glycerol or glucose as the starting material. Although the productivity of 3-HP from glycerol by CoA-independent pathway recently increased quickly, various limiting factors affecting the process availability still remain and require further researches, such as yield, the availability of enzymes, cofactor and toleration of an excess of substrate or high concentration of product [14].
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Currently, the hydrolysis of 3-HPN should offer an effective approach for the synthesis of 3-HP. However, the chemical hydrolysis of 3-HPN requires strongly basic conditions and high temperature, and produces hypersaline waste water. Biocatalysis offers a “greener” alternative with mild reaction conditions and excellent selectivity. Klempier et al. reported that 100 mmol/L of 3-HPN could be hydrolyzed to 3-HP by immobilized nitrilase in 24 h and the yield was 63% [26]. Bramucci et al. demonstrated that Comamonas testosteroni 5-MGAM-4D and Comamonas testosteroni 22-1 cells could catalyze the hydrolysis of 3-HPN to 3-HP with a maximum substrate concentration of 1.0 mol/L after 15 h [27]. The low substrate concentrations have limited their application on the industrial scale. This study is aimed at developing a commercially feasible process for the production of 3-HP, which has been achieved by using free or immobilized recombinant E. coli cells harboring a nitrilase gene, with high activity for the hydrolysis of 3-HPN at high substrate concentration. 2. Materials and methods 2.1. Materials The recombinant strains used in current study were maintained in our laboratory. 3-Hydroxypropionamide was prepared as described in the literature (for 1 H and 13 C NMR see Figs. S1 and 2 in the Supporting information) and used as standard sample for GC analysis [28]. 3-Hydroxypropionitrile was purchased from SigmaAldrich, 3-hydroxypropionic acid was purchased from TCI (Tokyo Chemical Industry Co., Ltd.,). Sodium alginate was purchased from Sinopharm Chemical Reagent Co., Ltd. The other chemicals were purchased from commercial sources. The High Performance Liquid Chromatography (HPLC) analysis was performed with an Agilent 1200 series HPLC system. The GC analysis was performed on an Agilent 7890A gas chromatography (GC) system. 1 H and 13 C NMR spectra were recorded on a Bruker AVANCE-III 400 MHz NMR spectrometer. 2.2. Selection of the nitrilase The nitrilases were expressed in E. coli BL21(DE3) cells and the recombinant strains were cultured in the Luria-Bertani (LB) medium (containing adequate antibiotic) at the optimized conditions (Table S1). Cells were harvested by centrifugation, washed once with sodium chloride (0.9%) and cryopreserved at −20 ◦ C. The biotransformation was performed by using 100 g/L (wet cells weight) resting cells in 1.0 mL reaction mixtures, which contained 1.0 mol/L 3-HPN in potassium phosphate buffer (100 mmol/L, pH 8.0). The reaction mixtures were incubated at 30 ◦ C and 200 rpm for 6 h. 2.3. Preparation of whole cell biocatalysts and enzyme activity assay The plasmid pET-32a-Nit190 was transformed into E. coli BL21 (DE3) cells and the recombinant strains were grown in 4 mL LB medium containing 100 g/mL ampicillin at 37 ◦ C with shaking 200 rpm for 6–8 h. The culture was inoculated into 800 mL LB medium with 100 g/mL ampicillin, which was then incubated at the same conditions. When the optical density at 600 nm (OD600) was 0.6–0.8, the gene expression was induced by adding of 0.1 mmol/L isopropyl -d-1-thiogalactopyranoside (IPTG) for 12 h at 25 ◦ C. The cells were harvested by centrifugation, washed once with 0.9% NaCl solution and stored at −20 ◦ C for use. The activity of the free whole cells or immobilized cells was assayed by using 3-HPN (1 mol/L) as substrate. A suspension (20 mg/mL wcw) of cells in 1 mL Tris-HCl buffer (100 mmol/L, pH
8.0) or immobilized cells containing equal amounts of wet cells in 10 mL Tris-HCl buffer (100 mmol/L, pH 8.0) was incubated with the substrate at 30 ◦ C with 200 rpm shaking for 10 min. A simple (500 L) of the reaction mixture was taken and quenched with 200 L HCl (1.0 mol/L) and 300 L distilled water. The concentration of 3-HP was measured by HPLC analysis. One unit of the enzyme activity was defined as the amount of whole cells producing 1 mol of 3-HP per minute. 2.4. Analytical methods Aliquots (100 L) were taken at different intervals, and the reactions were terminated by adding 200 L HCl (1.0 mol/L) and 700 L distilled water. The yields were determined by HPLC analysis performed on an Agilent 1200 series HPLC system with an Eclipse XDB-C18 column and monitored at wavelength of 210 nm with a UV detector. The eluent was a mixture of phosphoric acid solution (0.05%) and methanol (95/5, v/v), column temperature was 30 ◦ C and flow rate was 0.8 mL/min. Under these conditions, retention time of 3-HP was 2.6 min (Fig. S3 in the Supporting information). Aliquots (100 L) were drawn and quenched by adding 100 L HCl (6 mol/L) and 800 L ethyl acetate. The mixture was centrifuged at 12,000g for 1 min, and the organic phase was used to measure the residual substrate and 3-hydroxypropionamide by GC analysis after dried over anhydrous sodium sulfate. GC analysis was performed using a CP-ChiraSil-DEX CB column (25 m × 0.25 mm × 0.25 m) with helium as the carrier gas and flame ionization detector. The injector and detector temperature was set at 220 ◦ C, and the oven temperature was controlled as follows: 50 ◦ C (3 min) −20 ◦ C/min −180 ◦ C (4 min). The retention times for standard samples of 3-HPN and 3-hydroxypropionamide were 7.12 and 9.41 min, respectively. No 3-hydroxypropionamide was detected in the reaction mixture. 2.5. Immobilization of whole cells Freshly harvested cells (4%, w/v) were suspended in a NaCl solution (0.9%), and sodium alginate (3%, w/v) was dissolved in a NaCl solution (0.9%). Once the alginate solution was cooled to room temperature, the suspension cells were added into the solution and thoroughly mixed. The mixture was then added dropwise from a peristaltic pump to a stirred solution of calcium chloride (2%, w/v) which was precooled in the ice water. After stirring for 4 h, the formed beads were filtered and washed three times with distilled water. Part of those was stored in fresh calcium chloride solution (2%, w/v) at 4 ◦ C and the other was used for chemical cross-linking. The alginate beads were stirred in distilled water containing 2% (v/v) of PEI (50%) for 20 min at the room temperature. After filtered and washed three times with distilled water, the beads were stirred in 2% (v/v) of glutaraldehyde solution (25%) and filtered immediately after 30 s. The chemically cross-linked alginate beads were washed three times with distilled water and stored in fresh calcium chloride solution (2%, w/v) at 4 ◦ C for use in the biotransformation reaction [29]. 2.6. Optimization of biotransformation conditions For the optimization of reaction conditions, 1.0 mL (for free cells) or 10 mL (for immobilized cells) of the reaction mixture was used with variations as follows. The concentration of free cells or immobilized cells which contained equal amount of wet cells was 20 g/L, and the substrate concentration was 1.0 mol/L for optimization of pH, temperature, and thermal stability. Four kinds of buffers were used to determine the optimal pH, including citrate buffer (100 mmol/L, pH 5.0–6.0), phosphate buffer (100 mmol/L, pH 6.0–8.0), Tris-HCl buffer (100 mmol/L, pH 7.0–9.0), and glycineNaOH buffer (100 mmol/L, pH 9.0–10.0), at 30 ◦ C for 1 h. The
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temperatures, ranging from 25 to 60 ◦ C, were investigated at the optimal pH, and thermal stability was investigated by incubating whole cells at 30, 37, or 45 ◦ C for 12 h. concentration range of 3-HPN tested was The 3.0 mol/L–5.0 mol/L for free cells and 3.0 mol/L–8.0 mol/L for immobilized cells with 0.5 or 1.0 mol/L intervals at the optimal conditions. 2.7. Batch reaction by free or immobilized cells To evaluate the reusability of the immobilized cells, two kinds of reaction media, Tris-HCl buffer and distilled water, were used for batch reaction. The reaction was performed with free or immobilized cells (containing 20 g/L of wet cells) suspending in 10 mL of reaction medium contained 3.0 mol/L of substrate and 20 mmol/L of CaCl2 at 30 ◦ C for 5 h. After each reaction, the beads were washed by distilled water and transferred to fresh reaction mixture. Repeated batch reaction was carried out under the same conditions as the first cycle. 2.8. Isolation and identification of 3-HP The isolation of 3-HP was performed as the following procedure: 6.0 mol/L HCl was added into the combined reaction mixture of the first 30 batches to acidify to pH 1.0–2.0. The mixture was extracted three times with three times volume of ethyl acetate. The organic phase was separated and dried over anhydrous sodium sulphate. The desired product was obtained as pale yellow oil by removal of the solvent. 1 H NMR (400 MHz, DMSO-d6 ): ␦ 3.62 (t, J = 6.4 Hz, 2 H), 2.35 (t, J = 6.4 Hz, 2 H). 13 C NMR (100 MHz, DMSO-d6 ): ␦ 173.60, 57.50, 38.19 (Figs. S4 and 5 in the Supporting information).
Fig. 1. Effects of pH on the activity of 3-HPN to 3-HP by using free or immobilized recombinant Escherichia coli cells. The buffers were citrate buffer (100 mmol/L, pH 5.0–6.0), phosphate buffer (100 mmol/L, pH 6.0–8.0), Tris-HCl buffer (100 mmol/L, pH 7.0–9.0), and glycine-NaOH buffer (100 mmol/L, pH 9.0–10.0). The reaction activity in Tris-HCl buffer (100 mmol/L, pH 8.0) at 30 ◦ C for 1 h was regarded as 100%. The relative activity of free cells ( ), alginate beads ( ) and GA/PEI-Cross-Linked cells ( ) was determined by the standard assay procedure.
3. Results and discussion 3.1. Nitrilases selection The biotransformation of 3-HPN by recombination E.coli cells harboring 15 nitrilase genes available in our laboratory was performed at a substrate concentration of 1.0 mol/L and the results showed that four nitrilases could catalyze the hydrolysis of 3HPN to 3-HP by HPLC analysis (Table S1 in the Supporting information). However, when the substrate concentration was increased to 2.5 mol/L, only Nit190 could completely convert 3HPN into 3-HP. The residual substrate and possible by-product 3-hydroxypropionamide were not detected in the product by GC analysis. The nitrilase NIT190 was a mutant from environmental microorganism which could convert 3-hydroxyglutaronitrile to (R)-4-cyano-3-hydroxybutyric acid at 3.0 mol/L substrate concentration [30,31]. We wondered whether NIT190 would be a suitable biocatalyst used to develop a highly efficient bioprocess for synthesis of 3-HP from 3-HPN. Thus the recombinant E.coli BL21 (DE3) whole cells expressing Nit190 gene was selected for further detailed studies. SDS-PAGE analysis of cell-free lysate was shown in Fig. S6 in the Supplementary information and the specific activity was about 584 U/g wcw. 3.2. Characterization of free and immobilized E. coli cells expressing nitrilase NIT190 Effects of pH and temperature on the NIT190 enzyme activity were investigated at a substrate concentration of 1.0 mol/L by using 20 g/L wet free cells or immobilized cells that contained the same amount of wet cells. As shown in Fig. 1, the optimal pH values for free cells and immobilized cells were 8.0 and 9.0, respectively. The immobilized cells were more stable in a wide pH range (from 6.0 to 10.0) than the free cells. Almost no activity was
Fig. 2. Effects of reaction temperature on the activity of 3-HPN to 3-HP by using free or immobilized recombinant Escherichia coli cells. The temperature range was 25–60 ◦ C. The reaction activity in Tris-HCl buffer (100 mmol/L, pH 8.0) at 45 ◦ C for 1 h was regarded as 100%. The relative activity of free cells ( ), alginate beads ( ) and GA/PEI-Cross-Linked cells ( ) was determined by the standard assay procedure.
detected for both free cells and immobilized cells below pH 5.0. This is slightly different from the pH profile of the purified NIT190 toward 3-hydroxyglutaronitrile, which possesses a relatively narrow working pH range with optimum from pH 7.5–8.0 [31]. The free cells and immobilized cells exhibited the maximum relative activities at 45 ◦ C and 50 ◦ C, respectively (Fig. 2). The thermal stability was investigated by measuring the remaining activity of free or immobilized whole cells after incubation at different temperatures for 12 h, and the initial activity without incubation was defined as 100%. The results indicated that the free cells retained 88% of activity at 30 ◦ C for 12 h, and almost no activity was observed at 45 ◦ C after 12 h (Fig. 3). Immobilized cells were more stable than free cells, and GA/PEI-Cross-Linked cells showed the highest thermostability. The relative activities of immobilized cells remained 100% at 30 ◦ C and 37 ◦ C, and more than 70% at 50 ◦ C. For an optimal combination of enzyme activity and
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Fig. 3. The thermal stability of free or immobilized recombinant Escherichia coli cells in the temperature range of 30–50 ◦ C for 12 h. The reaction activity of no incubation in Tris-HCl buffer (100 mmol/L, pH 8.0) at 30 ◦ C for 1 h was regarded as 100%. The relative activity of free cells ( ), alginate beads ( ) and GA/PEI-Cross-Linked cells ( ) was determined by the standard assay procedure.
thermostability, the following biotransformations were performed at pH 8.0 and 30 ◦ C. The effect of substrate concentration on the yield of 3-HP was performed by using free cells with 3.0 mol/L–5.0 mol/L of 3-HPN and immobilized cells with 3.0 mol/L–8.0 mol/L of 3-HPN under the optimal conditions. As shown in Fig. 4, up to 4.5 mol/L of 3HPN was completely hydrolyzed by using free cells within 24 h. Further increase of the substrate concentration to 5.0 mol/L inhibited the reaction, and the yield of 3-HP was only 70% after 24 h when using free cells. However, complete hydrolysis of 7.0 mol/L (497 g/L) of 3-HPN was observed within 24 h when using immobilized cells. It took 12 h for the complete hydrolysis of 4.0 mol/L of 3-HPN with free cells, whereas only 6 h was required when immobilized cells were used. It is no doubt that immobilized cells possess higher enzyme activity and tolerate higher substrate concentration than free cells. Similar results were reported that immobilization of the nitrilase and nitrile hydratase could enhance thermostability of enzymes and reduce substrate inhibition [32,33]. 3.3. Reusability of the free and immobilized whole cells Higher substrate concentration is beneficial for separation of product, thus the reactions were performed with 6.0 mol/L of 3HPN at the optimal conditions for 10 h. A sharp reduction of reaction rate was observed after two batches (Fig. 5(a)). This could be attributed to partial activity loss of the enzyme, rather than the immobilized beads broken down or cells leakage from the beads, since no such phenomenon were revealed by SEM analysis of the beads before and after the reactions (Figs. S7 and 8 in the Supporting information). Probably, the high concentration of 3-HP or 3-HPN has irreversible inhibitory effects on the nitrilase. When the substrate concentration was decreased to 3.0 mol/L, the reaction time was reduced to 5 h. As shown in Fig. 5(b), the product yield using GA/PEI-cross-linked cells began to decrease after 30 batches, whereas 19 batches for calcium alginate embedding cells and 2 batches for free cells. Moreover, the product yield after 52 cycles of reaction was still over 70% by using GA/PEI-cross-linked cells. These results suggested that the GA/PEI-cross-linked cells are quite stable and feasible for industrial application. Adoption of an appropriate substrate concentration not only increased the reusability of the immobilized cells, but also could reduce the cost of production.
The results obtained in the reuse of GA/PEI-cross-linked cells in Tris-HCl buffer (100 mmol/L, pH 8.0) encouraged us to further simplify the process. Compared to the Tris-HCl buffer, distilled water is not only cheaper, but also facilitates the product purification. Therefore, the hydrolysis of 3-HPN was carried out in distilled water at different substrate concentrations using GA/PEI-crosslinked cells as the biocatalyst. The results demonstrated that the conversion of 3-HPN in distilled water was similar to that in TrisHCl buffer, and 6.0 mol/L of substrate was completely converted to product within 10 h (Fig. 4(d)). Batch reactions were performed in distilled water containing 3.0 mol/L of substrate at the optimal conditions over 5 h for each batch. As shown in Fig. 5(b), conversions were kept above 99% before 30 batches, and 70% conversion was obtained after 74 batches. 3-HP accumulated from the first 30 batches was isolated as slightly yellow oil (55.42 g, 68.4% yield) by extraction with ethyl acetate. The low isolation yield was due to the high solubility of product in water, and the product isolation procedure should be optimized to reduce the product loss. The bio-production of 3-HP from different substrates or by different biocatalysts have been reported, and the yield, titer and productivity from recent reports are listed in Table 1 for comparison [18,25–27,34–39]. The process of using glycerol as substrate has been improved continuously, and the highest titer (71.1 g/L) and productivity (1.8 g/(L h)) achieved to date is through systematic engineering of an aldehyde dehydrogenase, GabD4, in glycerol metabolism and recombinant expression of B12-dependent glycerol dehydratase and mutant GabD4 E209Q/E269Q [38]. However, addition of Vitamin B12, an expensive cofactor, would increase the production cost. The process developed in current study achieved much higher titer (184.7 g/L) and productivity (36.9 g/(L h)) calculated with the isolated product from 30 cycles. A similar process was developed by using immobilized C. testosteroni 5-MGAM-4D cells to convert 3-HPN to 3-HP. The reaction was performed with 1 M substrate concentration at 15 ◦ C and the volumetric productivity of the initial reaction was 23 g 3-HP/(L h) (determined by HPLC analysis) [34]. The transformation was catalyzed by a nitrile hydratase and an amidase. Such two-step transformation requires the match of the specific activities of the two enzymes, otherwise the intermediate, 3-hydroxypropionamide, would accumulate in the reaction mixture, which increased the difficulty of product isolation. 4. Conclusion A highly substrate-tolerant nitrilase was discovered for the synthesis of 3-HP. The resting cells of the recombinant E. coli strain harboring this nitrilase gene was immobilized on calcium alginate, which had increased the toleration of higher temperature, higher substrate concentration. The maximum substrate concentration of complete conversion was up to 7.0 mol/L at the optimal conditions (pH 8.0 and 30 ◦ C) in 24 h by using calcium alginate embedding and chemically cross-linking immobilized cells. The hydrolysis of 3-HPN was completed in 5 h at the substrate concentration of 3.0 mol/L. At this substrate concentration GA/PEI-cross-linked cells retained 70% of residual nitrilase activity after 74 batches in distilled water, while no activity loss was observed up to 30 batches. These results indicate that GA/PEI-cross-linked cells are quite stable and feasible for application in the industrial production of 3-HP from 3-HPN, which is readily available from the ring-opening of ethylene oxide with cyanide. Acknowledgement This work was financially supported by the National Natural Science Foundation of China (No. 21572261), Key Project of Tianjin Municipal Science and Technology Support Program(No.
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Fig. 4. Effects of substrate concentration on the yield of 3-HP by using (A) free cells, (B) immobilized cells on alginate beads, (C) GA/PEI-Cross-Linked cells in Tris-HCl (100 mmol/L, pH 8.0) buffer and (D) GA/PEI-Cross-Linked cells in distilled water. The yield was determined by HPLC analysis.
Fig. 5. Reusability of the free and immobilized cells with a substrate concentration of (A) 6.0 mol/L and (B) 3.0 mol/L. (A) Alginate beads ( ), GA/PEI-Cross-Linked cells in Tris-HCl (100 mmol/L, pH 8.0) buffer ( ) (B) free cells ( ), alginate beads ( ), GA/PEI-Cross-Linked cells in Tris-HCl (100 mmol/L, pH 8.0) buffer ( ) and GA/PEI-Cross-Linked cells in distilled water ( ). The yield was determined by HPLC analysis.
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Table 1 Comparison of 3-HP production in this study with recent systems reported in literatures. Strain
Substrate
Yield (%)a
Rhodococcus sp. Comamonas testosteroni 22-1 or Comamonas testosteroni 5-MGAM-4D Immobilized Comamonas testosteroni 5-MGAM-4D Rhodococcus erythropolis Recombinant E. coli Recombinant Klebsiella pneumoniae glpKdhaT Recombinant E. coli Pseudomonas denitrificans3hpdh 3hibdhIV Recombinant E. coli Recombinant E. coli Recombinant E. coli
3-HPN 3-HPN
63b 100
a b
3-HPN acrylic acid glycerol glycerol glycerol glycerol glycerol 3-hydroxy propionaldehyde 3-HPN
44 35 50 91 78 68 68b
Titer (g/L)
Productivity (g/(L h))
5.67 90
0.24 6.0
17.5 31 22.3 57.3 2.88 71.1 1.1 184.7
23.0 1.22 0.43 0.46 1.59 0.24 1.8 0.06 36.9
Reference Klempier et al. [26] Bramucci et al. [27]
Hann et al. [34] Lee et al. [35] Raj et al. [18] Ashok et al. [25] Kim et al. [36] Zhou et al. [37] Chu et al. [38] Sabet-Azad et al. [39] This study
The yields were determined by HPLC analysis. Isolated yield.
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Journal of Molecular Catalysis B: Enzymatic journal homepage: www.elsevier.com/locate/molcatb
Enzymatic synthesis of 3-hydroxypropionic acid at high productivity by using free or immobilized cells of recombinant Escherichia coli Shanshan Yu a,b,1 , Peiyuan Yao b,1 , Jianjiong Li b , Jie Ren b , Jing Yuan b , Jinhui Feng b , Min Wang a , Qiaqing Wu b,∗ , Dunming Zhu b,∗ a Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China b National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin 300308, China
a r t i c l e
i n f o
Article history: Received 25 December 2015 Received in revised form 29 February 2016 Accepted 26 March 2016 Available online 14 April 2016 Keywords: Nitrilase Immobilization 3-Hydroxypropionic acid Hydrolysis Enzyme catalysis
a b s t r a c t 3-Hydroxypropionic acid (3-HP) is an important platform chemical for organic synthesis and high performance polymers. Despite a wealth of reports related to 3-HP biosynthesis in microorganisms, its industrial application still requires further research because of low titer and productivity. Herein an effective enzymatic method for the synthesis of 3-HP was achieved by using free or immobilized recombinant Escherichia coli BL21(DE3) cells harboring a nitrilase gene from environmental sample (NIT190). Under the optimal conditions (100 mmol/L Tris-HCl buffer, pH 8.0, 30 ◦ C), the maximum substrate concentration which could be completely hydrolyzed by using free cells within 24 h was 4.5 mol/L (319.5 g/L). Furthermore, immobilization of the whole cells enhanced their substrate tolerance (up to 7.0 mol/L), stability, and reusability. The immobilized cells could be reused for up to 30 batches, and 70% of enzyme activity was retained after 74 batches in distilled water. The titer (184.7 g/L) and productivity (36.9 g/(L h)) were obtained by isolation and purification of 3-HP from the first 30 batches. These results demonstrate that the immobilized cells have potential industrial application for the synthesis of 3-HP. © 2016 Elsevier B.V. All rights reserved.
1. Introduction 3-Hydroxypropionic acid (3-HP) is a versatile building block in the synthesis of many industrially valuable chemicals, such as acrylic acid, 1,3-propandiol, malonic acid, propiolactone, and other valuable products [1–4]. Its polymerized form, poly (3-HP) has great potential applications in biodegradable materials due to its outstanding material characteristics [5]. In addition, 3-HP showed selective nematicidal activity against the plant-parasitic nematode Meloidogyne incognita and no antimicrobial, cytotoxic or phytotoxic effects [6]. Thus 3-HP was ranked the third position in the top value-added platform chemicals from biomass by the US Department of Energy in 2004 [7]. Owing to its broad applications, 3-HP has attracted increasing attention. Conventional chemical syntheses of 3-HP involve hydrolysis of 3-hydroxypropionitrile (3-HPN) [8] or propiolactone [9], hydration of acrylic acid [10], and oxidation of 1,3-propanediol [11], levulinic
∗ Corresponding authors. E-mail addresses: wu [email protected] (Q. Wu), zhu [email protected] (D. Zhu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.molcatb.2016.03.011 1381-1177/© 2016 Elsevier B.V. All rights reserved.
acid [12] or allyl alcohol [13]. Although excellent results have been obtained by catalytic chemical processes, there is growing interest in the development of bioprocesses for its production because of milder reaction conditions and fewer byproducts. Studies of the biological production of 3-HP began from early 2000s [14]. Several archaea were found in which 3-HP was an intermediary compound during 3-HP cycle, such as Sulfolobus, Acidianus, and Metallosphaera spp. [15]. 3-HP was also produced by some bacteria, such as Lactobacillus sp., Desulfovibrio fructosovorans, Klebsiella pneumoniae and Chloroflexus aurantiacus [15,16]. However, low yield and productivity by wild strains could not afford to meet global market demand that had been estimated at 3.63 million tons per year in 2008 [17]. A number of recombinant strains of Escherichia coli [18–20], yeast [21–23], Pseudomonas denitrificans [16], Lactobacillus reuteri [24] and K. pneumonia [25] were developed to largely accumulate 3HP with glycerol or glucose as the starting material. Although the productivity of 3-HP from glycerol by CoA-independent pathway recently increased quickly, various limiting factors affecting the process availability still remain and require further researches, such as yield, the availability of enzymes, cofactor and toleration of an excess of substrate or high concentration of product [14].
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Currently, the hydrolysis of 3-HPN should offer an effective approach for the synthesis of 3-HP. However, the chemical hydrolysis of 3-HPN requires strongly basic conditions and high temperature, and produces hypersaline waste water. Biocatalysis offers a “greener” alternative with mild reaction conditions and excellent selectivity. Klempier et al. reported that 100 mmol/L of 3-HPN could be hydrolyzed to 3-HP by immobilized nitrilase in 24 h and the yield was 63% [26]. Bramucci et al. demonstrated that Comamonas testosteroni 5-MGAM-4D and Comamonas testosteroni 22-1 cells could catalyze the hydrolysis of 3-HPN to 3-HP with a maximum substrate concentration of 1.0 mol/L after 15 h [27]. The low substrate concentrations have limited their application on the industrial scale. This study is aimed at developing a commercially feasible process for the production of 3-HP, which has been achieved by using free or immobilized recombinant E. coli cells harboring a nitrilase gene, with high activity for the hydrolysis of 3-HPN at high substrate concentration. 2. Materials and methods 2.1. Materials The recombinant strains used in current study were maintained in our laboratory. 3-Hydroxypropionamide was prepared as described in the literature (for 1 H and 13 C NMR see Figs. S1 and 2 in the Supporting information) and used as standard sample for GC analysis [28]. 3-Hydroxypropionitrile was purchased from SigmaAldrich, 3-hydroxypropionic acid was purchased from TCI (Tokyo Chemical Industry Co., Ltd.,). Sodium alginate was purchased from Sinopharm Chemical Reagent Co., Ltd. The other chemicals were purchased from commercial sources. The High Performance Liquid Chromatography (HPLC) analysis was performed with an Agilent 1200 series HPLC system. The GC analysis was performed on an Agilent 7890A gas chromatography (GC) system. 1 H and 13 C NMR spectra were recorded on a Bruker AVANCE-III 400 MHz NMR spectrometer. 2.2. Selection of the nitrilase The nitrilases were expressed in E. coli BL21(DE3) cells and the recombinant strains were cultured in the Luria-Bertani (LB) medium (containing adequate antibiotic) at the optimized conditions (Table S1). Cells were harvested by centrifugation, washed once with sodium chloride (0.9%) and cryopreserved at −20 ◦ C. The biotransformation was performed by using 100 g/L (wet cells weight) resting cells in 1.0 mL reaction mixtures, which contained 1.0 mol/L 3-HPN in potassium phosphate buffer (100 mmol/L, pH 8.0). The reaction mixtures were incubated at 30 ◦ C and 200 rpm for 6 h. 2.3. Preparation of whole cell biocatalysts and enzyme activity assay The plasmid pET-32a-Nit190 was transformed into E. coli BL21 (DE3) cells and the recombinant strains were grown in 4 mL LB medium containing 100 g/mL ampicillin at 37 ◦ C with shaking 200 rpm for 6–8 h. The culture was inoculated into 800 mL LB medium with 100 g/mL ampicillin, which was then incubated at the same conditions. When the optical density at 600 nm (OD600) was 0.6–0.8, the gene expression was induced by adding of 0.1 mmol/L isopropyl -d-1-thiogalactopyranoside (IPTG) for 12 h at 25 ◦ C. The cells were harvested by centrifugation, washed once with 0.9% NaCl solution and stored at −20 ◦ C for use. The activity of the free whole cells or immobilized cells was assayed by using 3-HPN (1 mol/L) as substrate. A suspension (20 mg/mL wcw) of cells in 1 mL Tris-HCl buffer (100 mmol/L, pH
8.0) or immobilized cells containing equal amounts of wet cells in 10 mL Tris-HCl buffer (100 mmol/L, pH 8.0) was incubated with the substrate at 30 ◦ C with 200 rpm shaking for 10 min. A simple (500 L) of the reaction mixture was taken and quenched with 200 L HCl (1.0 mol/L) and 300 L distilled water. The concentration of 3-HP was measured by HPLC analysis. One unit of the enzyme activity was defined as the amount of whole cells producing 1 mol of 3-HP per minute. 2.4. Analytical methods Aliquots (100 L) were taken at different intervals, and the reactions were terminated by adding 200 L HCl (1.0 mol/L) and 700 L distilled water. The yields were determined by HPLC analysis performed on an Agilent 1200 series HPLC system with an Eclipse XDB-C18 column and monitored at wavelength of 210 nm with a UV detector. The eluent was a mixture of phosphoric acid solution (0.05%) and methanol (95/5, v/v), column temperature was 30 ◦ C and flow rate was 0.8 mL/min. Under these conditions, retention time of 3-HP was 2.6 min (Fig. S3 in the Supporting information). Aliquots (100 L) were drawn and quenched by adding 100 L HCl (6 mol/L) and 800 L ethyl acetate. The mixture was centrifuged at 12,000g for 1 min, and the organic phase was used to measure the residual substrate and 3-hydroxypropionamide by GC analysis after dried over anhydrous sodium sulfate. GC analysis was performed using a CP-ChiraSil-DEX CB column (25 m × 0.25 mm × 0.25 m) with helium as the carrier gas and flame ionization detector. The injector and detector temperature was set at 220 ◦ C, and the oven temperature was controlled as follows: 50 ◦ C (3 min) −20 ◦ C/min −180 ◦ C (4 min). The retention times for standard samples of 3-HPN and 3-hydroxypropionamide were 7.12 and 9.41 min, respectively. No 3-hydroxypropionamide was detected in the reaction mixture. 2.5. Immobilization of whole cells Freshly harvested cells (4%, w/v) were suspended in a NaCl solution (0.9%), and sodium alginate (3%, w/v) was dissolved in a NaCl solution (0.9%). Once the alginate solution was cooled to room temperature, the suspension cells were added into the solution and thoroughly mixed. The mixture was then added dropwise from a peristaltic pump to a stirred solution of calcium chloride (2%, w/v) which was precooled in the ice water. After stirring for 4 h, the formed beads were filtered and washed three times with distilled water. Part of those was stored in fresh calcium chloride solution (2%, w/v) at 4 ◦ C and the other was used for chemical cross-linking. The alginate beads were stirred in distilled water containing 2% (v/v) of PEI (50%) for 20 min at the room temperature. After filtered and washed three times with distilled water, the beads were stirred in 2% (v/v) of glutaraldehyde solution (25%) and filtered immediately after 30 s. The chemically cross-linked alginate beads were washed three times with distilled water and stored in fresh calcium chloride solution (2%, w/v) at 4 ◦ C for use in the biotransformation reaction [29]. 2.6. Optimization of biotransformation conditions For the optimization of reaction conditions, 1.0 mL (for free cells) or 10 mL (for immobilized cells) of the reaction mixture was used with variations as follows. The concentration of free cells or immobilized cells which contained equal amount of wet cells was 20 g/L, and the substrate concentration was 1.0 mol/L for optimization of pH, temperature, and thermal stability. Four kinds of buffers were used to determine the optimal pH, including citrate buffer (100 mmol/L, pH 5.0–6.0), phosphate buffer (100 mmol/L, pH 6.0–8.0), Tris-HCl buffer (100 mmol/L, pH 7.0–9.0), and glycineNaOH buffer (100 mmol/L, pH 9.0–10.0), at 30 ◦ C for 1 h. The
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temperatures, ranging from 25 to 60 ◦ C, were investigated at the optimal pH, and thermal stability was investigated by incubating whole cells at 30, 37, or 45 ◦ C for 12 h. concentration range of 3-HPN tested was The 3.0 mol/L–5.0 mol/L for free cells and 3.0 mol/L–8.0 mol/L for immobilized cells with 0.5 or 1.0 mol/L intervals at the optimal conditions. 2.7. Batch reaction by free or immobilized cells To evaluate the reusability of the immobilized cells, two kinds of reaction media, Tris-HCl buffer and distilled water, were used for batch reaction. The reaction was performed with free or immobilized cells (containing 20 g/L of wet cells) suspending in 10 mL of reaction medium contained 3.0 mol/L of substrate and 20 mmol/L of CaCl2 at 30 ◦ C for 5 h. After each reaction, the beads were washed by distilled water and transferred to fresh reaction mixture. Repeated batch reaction was carried out under the same conditions as the first cycle. 2.8. Isolation and identification of 3-HP The isolation of 3-HP was performed as the following procedure: 6.0 mol/L HCl was added into the combined reaction mixture of the first 30 batches to acidify to pH 1.0–2.0. The mixture was extracted three times with three times volume of ethyl acetate. The organic phase was separated and dried over anhydrous sodium sulphate. The desired product was obtained as pale yellow oil by removal of the solvent. 1 H NMR (400 MHz, DMSO-d6 ): ␦ 3.62 (t, J = 6.4 Hz, 2 H), 2.35 (t, J = 6.4 Hz, 2 H). 13 C NMR (100 MHz, DMSO-d6 ): ␦ 173.60, 57.50, 38.19 (Figs. S4 and 5 in the Supporting information).
Fig. 1. Effects of pH on the activity of 3-HPN to 3-HP by using free or immobilized recombinant Escherichia coli cells. The buffers were citrate buffer (100 mmol/L, pH 5.0–6.0), phosphate buffer (100 mmol/L, pH 6.0–8.0), Tris-HCl buffer (100 mmol/L, pH 7.0–9.0), and glycine-NaOH buffer (100 mmol/L, pH 9.0–10.0). The reaction activity in Tris-HCl buffer (100 mmol/L, pH 8.0) at 30 ◦ C for 1 h was regarded as 100%. The relative activity of free cells ( ), alginate beads ( ) and GA/PEI-Cross-Linked cells ( ) was determined by the standard assay procedure.
3. Results and discussion 3.1. Nitrilases selection The biotransformation of 3-HPN by recombination E.coli cells harboring 15 nitrilase genes available in our laboratory was performed at a substrate concentration of 1.0 mol/L and the results showed that four nitrilases could catalyze the hydrolysis of 3HPN to 3-HP by HPLC analysis (Table S1 in the Supporting information). However, when the substrate concentration was increased to 2.5 mol/L, only Nit190 could completely convert 3HPN into 3-HP. The residual substrate and possible by-product 3-hydroxypropionamide were not detected in the product by GC analysis. The nitrilase NIT190 was a mutant from environmental microorganism which could convert 3-hydroxyglutaronitrile to (R)-4-cyano-3-hydroxybutyric acid at 3.0 mol/L substrate concentration [30,31]. We wondered whether NIT190 would be a suitable biocatalyst used to develop a highly efficient bioprocess for synthesis of 3-HP from 3-HPN. Thus the recombinant E.coli BL21 (DE3) whole cells expressing Nit190 gene was selected for further detailed studies. SDS-PAGE analysis of cell-free lysate was shown in Fig. S6 in the Supplementary information and the specific activity was about 584 U/g wcw. 3.2. Characterization of free and immobilized E. coli cells expressing nitrilase NIT190 Effects of pH and temperature on the NIT190 enzyme activity were investigated at a substrate concentration of 1.0 mol/L by using 20 g/L wet free cells or immobilized cells that contained the same amount of wet cells. As shown in Fig. 1, the optimal pH values for free cells and immobilized cells were 8.0 and 9.0, respectively. The immobilized cells were more stable in a wide pH range (from 6.0 to 10.0) than the free cells. Almost no activity was
Fig. 2. Effects of reaction temperature on the activity of 3-HPN to 3-HP by using free or immobilized recombinant Escherichia coli cells. The temperature range was 25–60 ◦ C. The reaction activity in Tris-HCl buffer (100 mmol/L, pH 8.0) at 45 ◦ C for 1 h was regarded as 100%. The relative activity of free cells ( ), alginate beads ( ) and GA/PEI-Cross-Linked cells ( ) was determined by the standard assay procedure.
detected for both free cells and immobilized cells below pH 5.0. This is slightly different from the pH profile of the purified NIT190 toward 3-hydroxyglutaronitrile, which possesses a relatively narrow working pH range with optimum from pH 7.5–8.0 [31]. The free cells and immobilized cells exhibited the maximum relative activities at 45 ◦ C and 50 ◦ C, respectively (Fig. 2). The thermal stability was investigated by measuring the remaining activity of free or immobilized whole cells after incubation at different temperatures for 12 h, and the initial activity without incubation was defined as 100%. The results indicated that the free cells retained 88% of activity at 30 ◦ C for 12 h, and almost no activity was observed at 45 ◦ C after 12 h (Fig. 3). Immobilized cells were more stable than free cells, and GA/PEI-Cross-Linked cells showed the highest thermostability. The relative activities of immobilized cells remained 100% at 30 ◦ C and 37 ◦ C, and more than 70% at 50 ◦ C. For an optimal combination of enzyme activity and
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Fig. 3. The thermal stability of free or immobilized recombinant Escherichia coli cells in the temperature range of 30–50 ◦ C for 12 h. The reaction activity of no incubation in Tris-HCl buffer (100 mmol/L, pH 8.0) at 30 ◦ C for 1 h was regarded as 100%. The relative activity of free cells ( ), alginate beads ( ) and GA/PEI-Cross-Linked cells ( ) was determined by the standard assay procedure.
thermostability, the following biotransformations were performed at pH 8.0 and 30 ◦ C. The effect of substrate concentration on the yield of 3-HP was performed by using free cells with 3.0 mol/L–5.0 mol/L of 3-HPN and immobilized cells with 3.0 mol/L–8.0 mol/L of 3-HPN under the optimal conditions. As shown in Fig. 4, up to 4.5 mol/L of 3HPN was completely hydrolyzed by using free cells within 24 h. Further increase of the substrate concentration to 5.0 mol/L inhibited the reaction, and the yield of 3-HP was only 70% after 24 h when using free cells. However, complete hydrolysis of 7.0 mol/L (497 g/L) of 3-HPN was observed within 24 h when using immobilized cells. It took 12 h for the complete hydrolysis of 4.0 mol/L of 3-HPN with free cells, whereas only 6 h was required when immobilized cells were used. It is no doubt that immobilized cells possess higher enzyme activity and tolerate higher substrate concentration than free cells. Similar results were reported that immobilization of the nitrilase and nitrile hydratase could enhance thermostability of enzymes and reduce substrate inhibition [32,33]. 3.3. Reusability of the free and immobilized whole cells Higher substrate concentration is beneficial for separation of product, thus the reactions were performed with 6.0 mol/L of 3HPN at the optimal conditions for 10 h. A sharp reduction of reaction rate was observed after two batches (Fig. 5(a)). This could be attributed to partial activity loss of the enzyme, rather than the immobilized beads broken down or cells leakage from the beads, since no such phenomenon were revealed by SEM analysis of the beads before and after the reactions (Figs. S7 and 8 in the Supporting information). Probably, the high concentration of 3-HP or 3-HPN has irreversible inhibitory effects on the nitrilase. When the substrate concentration was decreased to 3.0 mol/L, the reaction time was reduced to 5 h. As shown in Fig. 5(b), the product yield using GA/PEI-cross-linked cells began to decrease after 30 batches, whereas 19 batches for calcium alginate embedding cells and 2 batches for free cells. Moreover, the product yield after 52 cycles of reaction was still over 70% by using GA/PEI-cross-linked cells. These results suggested that the GA/PEI-cross-linked cells are quite stable and feasible for industrial application. Adoption of an appropriate substrate concentration not only increased the reusability of the immobilized cells, but also could reduce the cost of production.
The results obtained in the reuse of GA/PEI-cross-linked cells in Tris-HCl buffer (100 mmol/L, pH 8.0) encouraged us to further simplify the process. Compared to the Tris-HCl buffer, distilled water is not only cheaper, but also facilitates the product purification. Therefore, the hydrolysis of 3-HPN was carried out in distilled water at different substrate concentrations using GA/PEI-crosslinked cells as the biocatalyst. The results demonstrated that the conversion of 3-HPN in distilled water was similar to that in TrisHCl buffer, and 6.0 mol/L of substrate was completely converted to product within 10 h (Fig. 4(d)). Batch reactions were performed in distilled water containing 3.0 mol/L of substrate at the optimal conditions over 5 h for each batch. As shown in Fig. 5(b), conversions were kept above 99% before 30 batches, and 70% conversion was obtained after 74 batches. 3-HP accumulated from the first 30 batches was isolated as slightly yellow oil (55.42 g, 68.4% yield) by extraction with ethyl acetate. The low isolation yield was due to the high solubility of product in water, and the product isolation procedure should be optimized to reduce the product loss. The bio-production of 3-HP from different substrates or by different biocatalysts have been reported, and the yield, titer and productivity from recent reports are listed in Table 1 for comparison [18,25–27,34–39]. The process of using glycerol as substrate has been improved continuously, and the highest titer (71.1 g/L) and productivity (1.8 g/(L h)) achieved to date is through systematic engineering of an aldehyde dehydrogenase, GabD4, in glycerol metabolism and recombinant expression of B12-dependent glycerol dehydratase and mutant GabD4 E209Q/E269Q [38]. However, addition of Vitamin B12, an expensive cofactor, would increase the production cost. The process developed in current study achieved much higher titer (184.7 g/L) and productivity (36.9 g/(L h)) calculated with the isolated product from 30 cycles. A similar process was developed by using immobilized C. testosteroni 5-MGAM-4D cells to convert 3-HPN to 3-HP. The reaction was performed with 1 M substrate concentration at 15 ◦ C and the volumetric productivity of the initial reaction was 23 g 3-HP/(L h) (determined by HPLC analysis) [34]. The transformation was catalyzed by a nitrile hydratase and an amidase. Such two-step transformation requires the match of the specific activities of the two enzymes, otherwise the intermediate, 3-hydroxypropionamide, would accumulate in the reaction mixture, which increased the difficulty of product isolation. 4. Conclusion A highly substrate-tolerant nitrilase was discovered for the synthesis of 3-HP. The resting cells of the recombinant E. coli strain harboring this nitrilase gene was immobilized on calcium alginate, which had increased the toleration of higher temperature, higher substrate concentration. The maximum substrate concentration of complete conversion was up to 7.0 mol/L at the optimal conditions (pH 8.0 and 30 ◦ C) in 24 h by using calcium alginate embedding and chemically cross-linking immobilized cells. The hydrolysis of 3-HPN was completed in 5 h at the substrate concentration of 3.0 mol/L. At this substrate concentration GA/PEI-cross-linked cells retained 70% of residual nitrilase activity after 74 batches in distilled water, while no activity loss was observed up to 30 batches. These results indicate that GA/PEI-cross-linked cells are quite stable and feasible for application in the industrial production of 3-HP from 3-HPN, which is readily available from the ring-opening of ethylene oxide with cyanide. Acknowledgement This work was financially supported by the National Natural Science Foundation of China (No. 21572261), Key Project of Tianjin Municipal Science and Technology Support Program(No.
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Fig. 4. Effects of substrate concentration on the yield of 3-HP by using (A) free cells, (B) immobilized cells on alginate beads, (C) GA/PEI-Cross-Linked cells in Tris-HCl (100 mmol/L, pH 8.0) buffer and (D) GA/PEI-Cross-Linked cells in distilled water. The yield was determined by HPLC analysis.
Fig. 5. Reusability of the free and immobilized cells with a substrate concentration of (A) 6.0 mol/L and (B) 3.0 mol/L. (A) Alginate beads ( ), GA/PEI-Cross-Linked cells in Tris-HCl (100 mmol/L, pH 8.0) buffer ( ) (B) free cells ( ), alginate beads ( ), GA/PEI-Cross-Linked cells in Tris-HCl (100 mmol/L, pH 8.0) buffer ( ) and GA/PEI-Cross-Linked cells in distilled water ( ). The yield was determined by HPLC analysis.
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S. Yu et al. / Journal of Molecular Catalysis B: Enzymatic 129 (2016) 37–42
Table 1 Comparison of 3-HP production in this study with recent systems reported in literatures. Strain
Substrate
Yield (%)a
Rhodococcus sp. Comamonas testosteroni 22-1 or Comamonas testosteroni 5-MGAM-4D Immobilized Comamonas testosteroni 5-MGAM-4D Rhodococcus erythropolis Recombinant E. coli Recombinant Klebsiella pneumoniae glpKdhaT Recombinant E. coli Pseudomonas denitrificans3hpdh 3hibdhIV Recombinant E. coli Recombinant E. coli Recombinant E. coli
3-HPN 3-HPN
63b 100
a b
3-HPN acrylic acid glycerol glycerol glycerol glycerol glycerol 3-hydroxy propionaldehyde 3-HPN
44 35 50 91 78 68 68b
Titer (g/L)
Productivity (g/(L h))
5.67 90
0.24 6.0
17.5 31 22.3 57.3 2.88 71.1 1.1 184.7
23.0 1.22 0.43 0.46 1.59 0.24 1.8 0.06 36.9
Reference Klempier et al. [26] Bramucci et al. [27]
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The yields were determined by HPLC analysis. Isolated yield.
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