Bioprocess Biosyst Eng DOI 10.1007/s00449-014-1258-6
ORIGINAL PAPER
Improving the catalytic potential and substrate tolerance of Gibberella intermedia nitrilase by whole-cell immobilization Heng Li • Tao Yang • Jin-Song Gong Lei Xiong • Zhen-Ming Lu • Hui Li • Jin-Song Shi • Zheng-Hong Xu
•
Received: 8 May 2014 / Accepted: 11 July 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Comparative studies of immobilized and free cells of Gibberella intermedia CA3-1 in bioconversion of 3-cyanopyridine to nicotinic acid were performed. Entrapping method was chosen based on the advantages in enzymatic activity recovery, mechanical strength and preparation procedure. Four entrapment matrices were investigated and sodium alginate was screened to be the most suitable material. Maximal nitrilase activity of alginate immobilized cells was obtained under conditions of 2 % alginate, 0.6 % CaCl2, 0.4 g cell/g alginate, 1.8 mm bead size. The immobilized cells showed excellent substrate tolerance even when the 3-cyanopyridine concentration was 700 mM. The half-lives of immobilized cells at 30, 40 and 50 °C were 315, 117.5 and 10.9 h, respectively, correspondingly 1.4, 1.6 and 1.7-fold compared with that of the free cells. Efficient reusability of immobilized cells up to 28 batches was achieved and 205.7 g/(g dcw) nicotinic acid was obtained with 80.55 % enzyme activity preserved. Keywords Nicotinic acid 3-Cyanopyridine Immobilization Gibberella intermedia Nitrilase
H. Li and T. Yang have contributed equally to this article. Heng Li T. Yang J.-S. Gong L. Xiong Z.-M. Lu Hui Li J.-S. Shi Z.-H. Xu (&) School of Pharmaceutical Science, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, People’s Republic of China e-mail: [email protected] J.-S. Gong Z.-H. Xu The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, People’s Republic of China
Introduction Nitrilases (EC 3.5.5.1) have been recognized as a kind of potential and powerful biocatalysts for synthesizing high value-added carboxylic acids under mild conditions. It has been applied in formation of various industrially important organic acids, such as nicotinic acid, acrylic acid, mandelic acid and (S)-piperazine carboxylic acid [1]. With the properties of excellent chemo-, regio- and stereo-selectivity, biocatalysis mediated by nitrilases has now attracted considerable attention [2]. To date, many nitrilases from a variety of organisms have been sourced, including Pseudomonas sp. [3], Bacillus sp. [4], Rhodococcus sp. [5], Acinetobacter sp. [6], Klebsiella ozaenae [7], Alcaligenes faecalis [8], Aspergillus niger [9], Fusarium solani [10], among which mainly are bacteria. Studies on fungal nitrilase are relatively limited and the present knowledge about it is still insufficient. A fungus of G. intermedia CA3-1 was isolated from soil in our laboratory and it showed high activities toward nitriles for production of carboxylic acids [11]. As fungal nitrilase has distinct advantages over most bacterial nitrilases in terms of specific activity, selectivity and thermostability, it is necessary to carry forward the fungal nitrilase studies [10]. Inspite of the great synthetic potential of nitrilases, their utilizations as versatile biocatalysts are largely unexploited. Several nitrile biotransformation processes have now been conducted with isolated or purified enzymes [12, 13]. Putting aside their bioconversion activities, the purified nitrilases tend to be more susceptible to changes in operating conditions and substrate or product toxicity [1, 14]. In addition, the purified procedure is tedious and costly. The limitations of application of purified enzyme have promoted the use of whole-cell biocatalysts [15]. Whole-cell biotransformation processes have the advantage
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that the enzymes are retained in their natural environment, which could enhance stability. Besides, whole-cell bioprocesses can also reduce the economic costs. Immobilization of whole cells further improves the potential of biocatalyst for application. Biotransformation with immobilized cells performed superiorly in product separation, protection of cells against toxic substances and eliminating the costly processes of cell recovery over freecell bioconversion [16]. The universal immobilization approaches mainly included covalent binding, adsorption, entrapment (encapsulation) and cross-linking [17]. The disadvantage of adsorption was the relative weak binding force; cross-linking and covalent binding made serious damage to enzyme [18]. Entrapment was one of the most widely used techniques because the polymeric matrices can be made as beads with superior mechanical strength, rigidity and porosity characteristics [19, 20]. So far, several matrices have been applied in embedment of nitrilaseharboring microorganism, including cellulose [21], sodium alginate [22], agar [23], carrageenan [13], polyacrylamide [15], polyvinyl alcohol (PVA)–sodium alginate [13]. In the present study, G. intermedia CA3-1 was immobilized through the entrapment method. Four matrices which were the most common materials used for embedding were selected and evaluated. The most suitable immobilization matrix was screened out and the preparation and biotransformation conditions were determined. On the basis, the stability and reusability of immobilized cells were investigated.
suspension was introduced into the same volume of chitosan solution and mixed thoroughly to obtain a homogeneous cell/chitosan suspension. The mixture was extruded into 10 % (w/v) sodium polyphosphate solution using a syringe to form chitosan beads. The beads were washed with normal saline three times and stored at 4 °C in 10 mM sodium phosphate buffer with addition of 10 mM NaCl before being used. Entrapped in calcium alginate (Ca-alginate) beads The typical protocol of cell entrapment using calcium alginate was performed as follows: 2 mL cell suspension was mixed with 4 % (w/v) sodium alginate solution with a volume ratio of 1:1. The mixture was extruded into 0.6 % (w/v) calcium chloride solution via a syringe to form calcium alginate beads and immersed for 5 h at 4 °C. The beads were washed with normal saline three times and stored at 4 °C in 10 mM sodium phosphate buffer with addition of 10 mM NaCl and 20 mM CaCl2. The bead size varied with flow rates. Embedded in gelatin gel Gelatin was dissolved in distilled water to form a 4 % (w/v) solution at 80 °C. After cooled to 40 °C, 2 mL gelatin solution was mixed with the same volume of cell suspension, and allowed to cool. The resulting gel was cut into cubes (approximately 3 mm 9 3 mm 9 3 mm), washed and stored applying the same procedure used for chitosan immobilized cells.
Materials and methods Entrapped in PVA beads Microorganism, medium and cultivation conditions Gibberella intermedia CA3-1 harboring nitrilase was used in this study. This microorganism was cultured in 250-mL Erlenmeyer flasks containing 50 mL growth medium at 30 °C and 220 rpm for 48 h. The optimized medium contained (g/L): glucose 10, yeast extract 7.5, potassium dihydrogen phosphate 2.72, sodium chloride 1.16, ferrous sulfate 0.03 and caprolactam 3.39, pH 7.0. After 48 h cultivation, the cells were harvested by centrifugation for 10 min at 12,000 rpm, 4 °C. Then the cells were washed with normal saline and resuspended in the same buffer to obtain a cell suspension of 16.0 g dry cell weight (dcw)/L. Immobilization procedures Entrapped in chitosan beads 4 % (w/v) chitosan solution was prepared with 2 % (w/v) acetic acid aqueous solution as the solvent. Then 2 mL cell
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4 % (w/v) PVA solution was prepared and mixed with the same volume of cell suspension. The mixture was immediately extruded dropwise into saturated boric acid solution to form PVA beads and immersed at 4 °C for 5 h. Then the beads were washed and stored applying the same procedure used for chitosan immobilized cells. Characterization of immobilized cell Mechanical strength of immobilization matrix Hundred immobilized beads (or cubes) and 20 glass particles were added simultaneously into a 100-mL shake flask with 20 mL PBS buffer (pH 7.0). After 24 h stirring (200 rpm), the ratio of well-preserved pellets by morphologic observation was calculated to estimate the mechanical strength of the immobilization matrix [13]. All assays were performed in triplicate and the average values were given.
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Enzymatic activity recovery after immobilization Enzymatic activity recovery was determined by measuring the enzyme activity before and after cell immobilization. The parameter of enzymatic activity recovery was calculated with the following formula: Enzymatic activity recovery ð%Þ ¼ ðaimm =afree Þ 100 where aimm and afree represented the specific activity of the immobilized cell and free cell, respectively. Scanning electron microscopy (SEM) The internal structure of immobilized cells was observed employing a scanning electron microscope (SEM, Hitachi S-4800). The sample was cross-linked by glutaric dialdehyde, dehydrated with graded ethanol and then freezedried. The mounted samples were gold coated and examined by SEM at an accelerated voltage of 1.0 kV [15]. Enzyme assay The biotransformation of 3-cyanopyridine to nicotinic acid was employed as the model reaction. The standard reaction mixture (10 mL) contained 100 mM 3-cyanopyridine, 10 mM sodium phosphate buffer and a certain amount of whole cells with the same amount of cell usage, respectively, for both immobilized and free cells. Biotransformation process was carried out at 30 °C for 30 min with stirring. The reaction was stopped by addition of 2.0 M HCl. The reaction mixture was centrifuged and the concentrations of nicotinic acid and 3-cyanopyridine in the supernatant were determined by high-performance liquid chromatography system (HPLC, Dionex U3000) with a C18-column (Waters, 4.6 9 150 mm, 5 lm) at 268 nm. The mobile phase consisted of acetonitrile and water (60:40 v/v) with addition of 0.01 % phosphorus acid. The flow rate was 1.0 mL/min. One unit of activity was defined as the amount of cells used for the formation of 1 lmol nicotinic acid per min under the above assay conditions. All assays were performed in triplicate and the average values were given. Influences of temperature, pH and substrate concentration on enzyme activity The influence of temperature on biocatalytic activity for immobilized and free cells was investigated through detecting the residual enzyme activity. The biotransformation process was carried out between 25 and 60 °C for 30 min with stirring. The influences of pH (ranging from 3.0 to 11.0) and substrate concentration (ranging from 100
to 700 mM) for both immobilized and free cells under otherwise standard conditions were studied. Thermal stability study To investigate the thermal stability of immobilized cells, studies were carried out by pre-incubating the biocatalysts in normal saline without substrate at set temperature. Samples were taken at regular intervals to detect the residual enzymatic activity for determining the half-life. The natural logarithm of residual enzymatic activity [ln(RA)] was plotted against time. The half-lives of free and immobilized cells at each temperature were calculated by extrapolating from the slope of the individual line denoted as KDeact [11]. Repeated batch bioconversion of 3-cyanopyridine by immobilized and free cells To measure the reusabilities of the immobilized and free cells, repeated batch bioconversion of 3-cyanopyridine to nicotinic acid with the substrate concentration of 200 mM was performed at 30 °C with stirring. After each cycle of 60-min conversion, the beads and free cells were washed three times with normal saline and reused in a subsequent cycle.
Results and discussion Preparation of immobilized cells Screening for entrapment matrix Entrapment method was employed to enhance the applicability of whole cell biocatalysts. Various immobilization materials were screened based on enzymatic activity recovery, mechanical strength and preparation complexity (Table 1). The inner microscopical structures of immobilized cells were observed with SEM. Scanning electron microscopy photographs showed that cells were randomly distributed in the solid matrix and there were plenty of microspores in the four different gels, especially in chitosan gel, which was supposed to be beneficial to mass transfer. Plenty of channels in Ca-alginate beads were also observed (Fig. 1a), which proved Caalginate was an ‘‘egg-box’’ porous matrix [24]. Chitosan entrapped cells showed the highest enzymatic activity recovery with 87.51 %, followed by gelatin (82.09 %) and alginate (79.11 %). Boric acid used during the PVA immobilization process damaged the cells enclosed in the matrix [25], which resulted in the great loss in enzymatic activity. Nevertheless, mechanical strength rendered a
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Bioprocess Biosyst Eng Table 1 Screening of immobilization materials based on enzymatic activity recovery, mechanical strength and preparation complexity Immobilized materials
Shape
Preparation complexity
Enzymatic activity recovery (%)
Ratio of well-preserved pellets (%)
Mechanical strength
Free cells
–
–
100.0a
–
–
Chitosan
Bead
***
87.51
49.1
??
Alginate
Bead
**
79.11
75.8
???
Gelatin gel
Square
***
82.09
61.3
??
PVA
Bead
****
67.81
93.2
????
‘‘–’’ meant it was not considered, ‘‘*’’ indicated the difficulty of preparation, measured by the preparation time and procedure of the immobilized cells, ‘‘?’’ indicated the mechanical strength of immobilization cells measured by the ratio of well-preserved pellets a
The activity of free cells was considered as a control
completely reverse trend. Through the data of ratio of wellpreserved pellets, it could be seen that only 49 % of chitosan entrapped cells maintained their original morphology, whereas 75 and 93 % for alginate and PVA, respectively. Obviously, chitosan and gelatin beads had poor mechanical strength, whereas PVA and alginate beads were much better. However, PVA beads were found to be inclined to adhere with each other during preparation, which limited its stability. Evaluated by enzymatic activity recovery, mechanical strength and complexity of preparation procedure, alginate was finally selected for further studies, which has been widely utilized as a favorable matrix for cell/enzyme immobilization [26, 27].
increased between 0.1 and 0.6 %. Lower concentration of CaCl2 (less than 0.2 %) led to cell leakage and low mechanical strength. Further increase in CaCl2 concentration significantly enhanced the mechanical strength of immobilized beads whereas the nitrilase activity decreased. The influences of alginate and CaCl2 concentrations were attributed to the diffusion limitation effect of the immobilization matrix. High concentration of alginate or CaCl2 is intended to form compact matrix during immobilization process, which improved the diffusion restriction of substrate and products [28]. The optimized concentration of alginate and CaCl2 were then determined to be 2 and 0.6 %, respectively.
Influences of sodium alginate and calcium chloride concentration
Influences of cell loading and bead size
The concentrations of sodium alginate and calcium chloride play significant roles in the enzyme activity retained for immobilized cells. Their effects on the activity of immobilized cells were investigated. As shown in Fig. 2a, the nitrilase activity of alginate entrapped cells increased with the increase in alginate concentration from 1 to 2 % (w/v). The maximal enzyme activity was obtained with the alginate concentration of 2 %. When the concentration of alginate further increased, the nitrilase activity of alginate entrapped cells decreased. It was observed that beads prepared by low concentration of sodium alginate were rather soft, which were easily damaged in biotransformation process as a result of low mechanical strength. Besides, when the concentration of sodium alginate was higher than 2.5 %, high viscosity of alginate caused difficulty in beads formation, and the beads formed were not regular spherical. This was consistent with former studies [4, 13]. Similar trend was found during optimization of calcium chloride concentration (Fig. 2b). The enzyme activity and the rigidity of alginate entrapped cells were both improved as the CaCl2 concentration
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The influence of cell loading on the biocatalytic performance of alginate entrapped cells was examined. As shown in Fig. 3, the conversion of 3-cyanopyridine increased gradually with the increase in cell loading. However, beyond continuous increase in conversion, there was a drop in the specific activity of alginate entrapped cells. The highest specific activity was achieved when the cell loading was 0.4 g cell/g alginate. When the cell loading exceeded 0.4 g cell/g alginate, the specific activity decreased, indicating that more cell loading is not more favorable for substrate transformation. The same phenomenon was discovered by Kaul et al. [15] and revealed by studying on the variation in the effectiveness of the immobilized cells. Effectiveness is a parameter reflecting the mass transfer effect. It has been reported that higher cell loading leads to the decrease in effectiveness. The resulting diffusion restriction to the substrate becomes the rate controlling step. Besides, the influence of bead size was also studied. It could be found that the relative activity of alginate entrapped cells decreased with the increase in bead size (Fig. 4). Maximum relative activity was obtained with a
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Fig. 1 SEM images of different kinds of immobilized beads containing Gibberella intermedia CA3-1: a general view of Ca-alginate beads, b Ca-alginate beads, c gelatin gel immobilization, d PVA gel beads, e CS gel beads
bead diameter of 1.8 mm. This could also be assigned to the diffusion limited effect. Kaul et al. [15] have measured the effectiveness and diffusion coefficient of alginate immobilized beads. Both the parameters display the decreasing trends with the increase in bead size, which imply that larger beads tend to have larger diffusion limitation. In addition, it was also examined that large beads had a strong tendency to swell, which is consistent with the study of Chen et al. [29].
Optimization of bioconversion conditions Influences of temperature and pH The influence of temperature on the bioconversion of 3-cyanopyridine was studied from 25 to 60 °C. For the immobilized and free cells, the maximal relative activities were both obtained at 40 °C (Fig. 5a). Comparatively, the cells entrapped in alginate displayed slightly higher activity
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Fig. 4 Effect of bead size on nitrilase activity of alginate immobilized cells
Fig. 2 Effects of concentration of alginate (a) and calcium chloride (b) on nitrilase activity of alginate immobilized cells
Fig. 3 Effect of cell loading on nitrilase activity of alginate immobilized cells: conversion (filled squares), specific activity (unfilled squares)
at high temperature, indicating immobilization plays an important role in keeping the cells from denaturation under higher temperature [15, 30].
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Fig. 5 Influences of temperature (a) and pH (b) on nitrilase activity: free cells (unfilled squares), alginate immobilized cells (unfilled circles)
For enzymatic reactions, buffer pH would affect the enzyme activity and product selectivity. The enzyme activity was examined with the pH value of 3.0–11.0. The
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maximum activity of free cells were found to be at pH 7.0, whereas 8.0 for immobilized cells (Fig. 5b). The shift is mainly ascribed to the structural changes induced by pH variation [31]. It has been reported that the carboxylic parts of alginate could attract and enrich H? around them [32]. Hence to balance the excess H?, weak alkaline buffer was needed and favorable for substrate transformation for immobilized cells. Influence of substrate concentration Substrate concentration significantly affects the substrate conversion and enzyme activity, especially for the substrate with certain toxicity to cells, such as 3-cyanopyridine [33]. Sharma et al. [34] found that the concentration of 3-cyanopyridine exceeding 300 mM displayed inhibition effect. The effects of initial concentration of 3-cyanopyridine on the activity of the immobilized and free cells were analyzed with the substrate concentration of 100–700 mM (Fig. 6). Generally, along with the increase in 3-cyanopyridine concentration, the activity of immobilized and free cells both decreased rapidly with the time needed for total conversion prolonged. Detailed comparison revealed that the free cells were more active with shorter time for total conversion when 3-cyanopyridine concentration was below 300 mM. As the concentration increased to 300 mM, the trend turned out to be just the opposite. The time required for total conversion was 100 min for immobilized cells whereas 120 min for free cells. This time gap between immobilized and free cells was further widen as 3-cyanopyridine concentration increased. When
Fig. 6 Effect of 3-cyanopyridine concentration on nitrilase activity, hollow symbols free cells, solid symbols alginate immobilized cells: 100 mM (unfilled squares, filled squares), 200 mM (unfilled circles, filled circles), 300 mM (unfilled triangles, filled triangles), 400 mM (unfilled diamonds, filled diamonds), 500 mM (unfilled inverted triangles, filled inverted triangles), 600 mM (unfilled stars, filled stars), 700 mM (unfilled right arrowhead, filled right arrowhead)
3-cyanopyridine concentration increased to 500 mM and above, the free cells could even not transform the substrate completely. Both the immobilized and free cells could not transform the substrate completely at the concentration of 700 mM. With lower 3-cyanopyridine concentration, the diffusion limitation effect of the matrix led to longer time for total conversion. Nevertheless, when the substrate concentration increased, the toxicity of 3-cyanopyridine gradually manifested and the transformation capacity of free cells decreased, which was mainly ascribed to the damage of cells. Detailed effects need to be investigated. This indicated that immobilization was predominant in cell protection, especially when the substrate concentration was high [14]. It was found that diffusion of substrate from solutions to the beads was not easy [35], and the substrate in immobilized cells was transformed continuously. The concentration of toxic substrate around the immobilized cells was thus lower than the solution which led to the improvement of substrate tolerance. Thermal stability of alginate entrapped cells The thermal stability of immobilized cells was evaluated by preincubating at 30, 40 and 50 °C, respectively (Fig. 7). Although the free cells of G. intermedia have displayed excellent thermal stability [11], immobilization in this study could further improve its stability by 1.4, 1.6 and 1.7fold compared with the free ones. The half-lives of immobilized cells at 30, 40 and 50 °C were calculated to be 315, 117.5 and 10.9 h, respectively. Evidently, the half-life of immobilized cells was enhanced greatly through immobilization. This indicates the immobilization matrix is
Fig. 7 Thermostability of alginate immobilized cells: 30 °C (unfilled squares), 40 °C (unfilled circles), 50 °C (unfilled triangles)
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supposed to preserve the suitable microenvironment for cells and make the cells insensitive to temperature variations, which resulted in the improvement of thermostability of immobilized cells. Similar phenomenon was also observed by Zhang et al. [30]. Repeated batch bioconversion of 3-cyanopyridine Repeated batch mode of 3-cyanopyridine transformation was performed to examine and compare the operation stability of immobilized and free cells (Fig. 8). Continuous batch conversion was operated with 3-cyanopyridine concentration of 200 mM and 60 min for each batch. Probably because of the toxicity of 3-cyanopyridine and the gradual decrease of enzymatic activity, the biocatalysts cannot be reused for many times. In case of free cells, the reaction system was run for 10 consecutive batches to produce 72.2 g/(g dcw) nicotinic acid with 82.45 % enzyme activity reserved. In contrast, under the same transformation conditions, the reaction system of immobilized cells was run for 28 consecutive batches to produce 205.7 g/(g dcw) nicotinic acid with 80.55 % enzyme activity retained. Therefore, the immobilized cells performed with much better operation stability than that of free cells. Up to now, biotransformation of 3-cyanopyridine to nicotinic acid mainly focused on transformation using free cells. Sharma et al. reported the biosynthesis of nicotinic acid by hyperinduced Nocardia globerula NHB-2. With hyperinduced resting cells corresponding to 10 U/mL nitrilase activity (1.5 (g dcw)/L), a total of 123.11 g nicotinic acid was produced with the production capacity of 82 g/(g dcw) in a fed batch reaction on 1 L scale [34]. The highest throughput of nicotinic acid reported hitherto was 98.45 g/(g dcw) [36]. Biotransformation of 3-cyanopyridine with immobilized cells has been applied in manufacturing nicotinamide in Lonza. 3-Cyanopyridine was
continuously transformed with Rhodococcus rhodochrous J1 cells which were immobilized in polyacrylamide. This bio-hydrolysis course was carried out under mild aqueous conditions with high product selectivity, which was much simpler than chemical route [37]. The great catalytic potential of Ca-alginate immobilized G. intermedia CA3-1 could be seen in the preparation of nicotinic acid. Conclusions Immobilization of whole cells offers an alternative to improve the stability and reusability of G. intermedia CA3-1 harboring nitrilase. With the entrapment method, the immobilization matrices were screened and alginate was selected out for its advantages in enzymatic activity recovery, mechanical strength and preparation complexity. After the preparation conditions were determined, the effects of biotransformation conditions on enzyme activity were investigated. The optimum buffer pH for immobilized cells was 8.0, which was slightly more alkaline than the free cells. The optimum temperature was almost the same for both immobilized and free cells, but the immobilized cells displayed a little more insensitive to high temperature. Data of half-lives of immobilized cells at different temperatures gave further evidences. The immobilized cells showed excellent substrate tolerance properties with cell protection effect. With the concentration of 3-cyanopyridine at 200 mM, efficient biocatalyst recycling was achieved as a result of immobilization with 28 consecutive batches and 205.7 g/(g dcw) nicotinic acid obtained. Compared with the free cells, the number of recycle batches was substantially improved for immobilized catalyst and thus it supported the outstanding nicotinic acid production in this study. This study fulfills the requirement for low-cost production of nicotinic acid due to its prominent advantages of reusability and thermostability over free cells. It offers a foundation for large-scale bioconversion of 3-cyanopyridine to nicotinic acid with cell immobilization. Continuous transformation mode and large-scale application with immobilized cells are currently in progress. Acknowledgments This work was supported by the National Natural Science Foundation of China (21206055), Natural Science Foundation of Jiangsu Province (BK2012127, BK20140133), and National High Technology Research and Development Program of China (No. 2012AA022204C).
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Fig. 8 Reuse of free (unfilled squares) and alginate immobilized (unfilled circles) cells
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22. Wang YS, Zheng RC, Xu JM, Liu ZQ, Cheng F, Feng ZH, Liu LL, Zheng YG, Shen YC (2010) Enantioselective hydrolysis of (R)-2,2-dimethylcyclopropane carboxamide by immobilized cells of an R-amidase-producing bacterium, Delftia tsuruhatensis CCTCC M 205114, on an alginate capsule carrier. J Ind Microbiol Biotechnol 37:503–510 23. Nigam VK, Khandelwal AK, Gothwal RK, Mohan MK, Choudhury B, Vidyarthi AS, Ghosh P (2009) Nitrilase-catalysed conversion of acrylonitrile by free and immobilized cells of Streptomyces sp. J Biosci 34:21–26 24. Lee KH, Choi IS, Kim YG, Yang DJ, Bae HJ (2011) Enhanced production of bioethanol and ultrastructural characteristics of reused Saccharomyces cerevisiae immobilized calcium alginate beads. Bioresour Technol 102:8191–8198 25. Takei T, Ikeda K, Ijima H, Kawakami K (2011) Fabrication of poly (vinyl alcohol) hydrogel beads crosslinked using sodium sulfate for microorganism immobilization. Process Biochem 46:566–571 26. Pai O, Banoth L, Ghosh S, Chisti Y, Banerjee UC (2014) Biotransformation of 3-cyanopyridine to nicotinic acid by free and immobilized cells of recombinant Escherichia coli. Process Biochem 49:655–659 27. Cheng YM, Ma L, Deng C, Xu ZH, Chen JH (2014) Effect of PEG-mediated pore forming on Ca-alginate immobilization of nitrilase-producing bacteria Pseudomonas putida XY4. Bioprocess Biosyst Eng 37:1–6 28. El-Hadi AA, El-Minofi HA (2012) Enhanced bioconversion of lactose by immobilized cells of Bacillus subtilis using different matrices. Wudpecker J Agric Res 1:415–423 29. Chen J, Zheng YG, Shen YC (2008) Biosynthesis of pmethoxyphenylacetic acid from p-methoxyphenylacetonitrile by immobilized Bacillus subtilis ZJB-063. Process Biochem 43:978–983 30. Zhang YW, Prabhu P, Lee JK (2010) Alginate immobilization of recombinant Escherichia coli whole cells harboring L-arabinose isomerase for L-ribulose production. Bioprocess Biosyst Eng 33:741–748 31. Lu D, Zhang Y, Niu S, Wang L, Lin S, Wang C, Ye W, Yan C (2012) Study of phenol biodegradation using Bacillus amyloliquefaciens strain WJDB-1 immobilized in alginate–chitosan– alginate (ACA) microcapsules by electrochemical method. Biodegradation 23:209–219 32. Chung TP, Tseng HY, Juang RS (2003) Mass transfer effect and intermediate detection for phenol degradation in immobilized Pseudomonas putida systems. Process Biochem 38:1497–1507 33. Graham D, Pereira R, Barfield D, Cowan D (2000) Nitrile biotransformations using free and immobilized cells of a thermophilic Bacillus spp. Enzyme Microb Tech 26:368–373 34. Sharma NN, Sharma M, Bhalla TC (2011) An improved nitrilasemediated bioprocess for synthesis of nicotinic acid from 3-cyanopyridine with hyperinduced Nocardia globerula NHB-2. J Ind Microbiol Biotechnol 38:1235–1243 35. Tanaka H, Matsumura M, Veliky IA (1984) Diffusion characteristics of substrates in Ca-alginate gel beads. Biotechnol Bioeng 26:053–058 36. Prasad S, Misra A, Jangir VP, Awasthi A, Raj J, Bhalla TC (2006) A propionitrile-induced nitrilase of Rhodococcus sp. NDB 1165 and its application in nicotinic acid synthesis. World J Microbiol Biotechnol 23:345–353 37. Shaw NM, Robins KT, Kiener A (2003) Lonza: 20 years of biotransformations. Adv Synth Catal 345:425–435
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ORIGINAL PAPER
Improving the catalytic potential and substrate tolerance of Gibberella intermedia nitrilase by whole-cell immobilization Heng Li • Tao Yang • Jin-Song Gong Lei Xiong • Zhen-Ming Lu • Hui Li • Jin-Song Shi • Zheng-Hong Xu
•
Received: 8 May 2014 / Accepted: 11 July 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Comparative studies of immobilized and free cells of Gibberella intermedia CA3-1 in bioconversion of 3-cyanopyridine to nicotinic acid were performed. Entrapping method was chosen based on the advantages in enzymatic activity recovery, mechanical strength and preparation procedure. Four entrapment matrices were investigated and sodium alginate was screened to be the most suitable material. Maximal nitrilase activity of alginate immobilized cells was obtained under conditions of 2 % alginate, 0.6 % CaCl2, 0.4 g cell/g alginate, 1.8 mm bead size. The immobilized cells showed excellent substrate tolerance even when the 3-cyanopyridine concentration was 700 mM. The half-lives of immobilized cells at 30, 40 and 50 °C were 315, 117.5 and 10.9 h, respectively, correspondingly 1.4, 1.6 and 1.7-fold compared with that of the free cells. Efficient reusability of immobilized cells up to 28 batches was achieved and 205.7 g/(g dcw) nicotinic acid was obtained with 80.55 % enzyme activity preserved. Keywords Nicotinic acid 3-Cyanopyridine Immobilization Gibberella intermedia Nitrilase
H. Li and T. Yang have contributed equally to this article. Heng Li T. Yang J.-S. Gong L. Xiong Z.-M. Lu Hui Li J.-S. Shi Z.-H. Xu (&) School of Pharmaceutical Science, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, People’s Republic of China e-mail: [email protected] J.-S. Gong Z.-H. Xu The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, People’s Republic of China
Introduction Nitrilases (EC 3.5.5.1) have been recognized as a kind of potential and powerful biocatalysts for synthesizing high value-added carboxylic acids under mild conditions. It has been applied in formation of various industrially important organic acids, such as nicotinic acid, acrylic acid, mandelic acid and (S)-piperazine carboxylic acid [1]. With the properties of excellent chemo-, regio- and stereo-selectivity, biocatalysis mediated by nitrilases has now attracted considerable attention [2]. To date, many nitrilases from a variety of organisms have been sourced, including Pseudomonas sp. [3], Bacillus sp. [4], Rhodococcus sp. [5], Acinetobacter sp. [6], Klebsiella ozaenae [7], Alcaligenes faecalis [8], Aspergillus niger [9], Fusarium solani [10], among which mainly are bacteria. Studies on fungal nitrilase are relatively limited and the present knowledge about it is still insufficient. A fungus of G. intermedia CA3-1 was isolated from soil in our laboratory and it showed high activities toward nitriles for production of carboxylic acids [11]. As fungal nitrilase has distinct advantages over most bacterial nitrilases in terms of specific activity, selectivity and thermostability, it is necessary to carry forward the fungal nitrilase studies [10]. Inspite of the great synthetic potential of nitrilases, their utilizations as versatile biocatalysts are largely unexploited. Several nitrile biotransformation processes have now been conducted with isolated or purified enzymes [12, 13]. Putting aside their bioconversion activities, the purified nitrilases tend to be more susceptible to changes in operating conditions and substrate or product toxicity [1, 14]. In addition, the purified procedure is tedious and costly. The limitations of application of purified enzyme have promoted the use of whole-cell biocatalysts [15]. Whole-cell biotransformation processes have the advantage
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that the enzymes are retained in their natural environment, which could enhance stability. Besides, whole-cell bioprocesses can also reduce the economic costs. Immobilization of whole cells further improves the potential of biocatalyst for application. Biotransformation with immobilized cells performed superiorly in product separation, protection of cells against toxic substances and eliminating the costly processes of cell recovery over freecell bioconversion [16]. The universal immobilization approaches mainly included covalent binding, adsorption, entrapment (encapsulation) and cross-linking [17]. The disadvantage of adsorption was the relative weak binding force; cross-linking and covalent binding made serious damage to enzyme [18]. Entrapment was one of the most widely used techniques because the polymeric matrices can be made as beads with superior mechanical strength, rigidity and porosity characteristics [19, 20]. So far, several matrices have been applied in embedment of nitrilaseharboring microorganism, including cellulose [21], sodium alginate [22], agar [23], carrageenan [13], polyacrylamide [15], polyvinyl alcohol (PVA)–sodium alginate [13]. In the present study, G. intermedia CA3-1 was immobilized through the entrapment method. Four matrices which were the most common materials used for embedding were selected and evaluated. The most suitable immobilization matrix was screened out and the preparation and biotransformation conditions were determined. On the basis, the stability and reusability of immobilized cells were investigated.
suspension was introduced into the same volume of chitosan solution and mixed thoroughly to obtain a homogeneous cell/chitosan suspension. The mixture was extruded into 10 % (w/v) sodium polyphosphate solution using a syringe to form chitosan beads. The beads were washed with normal saline three times and stored at 4 °C in 10 mM sodium phosphate buffer with addition of 10 mM NaCl before being used. Entrapped in calcium alginate (Ca-alginate) beads The typical protocol of cell entrapment using calcium alginate was performed as follows: 2 mL cell suspension was mixed with 4 % (w/v) sodium alginate solution with a volume ratio of 1:1. The mixture was extruded into 0.6 % (w/v) calcium chloride solution via a syringe to form calcium alginate beads and immersed for 5 h at 4 °C. The beads were washed with normal saline three times and stored at 4 °C in 10 mM sodium phosphate buffer with addition of 10 mM NaCl and 20 mM CaCl2. The bead size varied with flow rates. Embedded in gelatin gel Gelatin was dissolved in distilled water to form a 4 % (w/v) solution at 80 °C. After cooled to 40 °C, 2 mL gelatin solution was mixed with the same volume of cell suspension, and allowed to cool. The resulting gel was cut into cubes (approximately 3 mm 9 3 mm 9 3 mm), washed and stored applying the same procedure used for chitosan immobilized cells.
Materials and methods Entrapped in PVA beads Microorganism, medium and cultivation conditions Gibberella intermedia CA3-1 harboring nitrilase was used in this study. This microorganism was cultured in 250-mL Erlenmeyer flasks containing 50 mL growth medium at 30 °C and 220 rpm for 48 h. The optimized medium contained (g/L): glucose 10, yeast extract 7.5, potassium dihydrogen phosphate 2.72, sodium chloride 1.16, ferrous sulfate 0.03 and caprolactam 3.39, pH 7.0. After 48 h cultivation, the cells were harvested by centrifugation for 10 min at 12,000 rpm, 4 °C. Then the cells were washed with normal saline and resuspended in the same buffer to obtain a cell suspension of 16.0 g dry cell weight (dcw)/L. Immobilization procedures Entrapped in chitosan beads 4 % (w/v) chitosan solution was prepared with 2 % (w/v) acetic acid aqueous solution as the solvent. Then 2 mL cell
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4 % (w/v) PVA solution was prepared and mixed with the same volume of cell suspension. The mixture was immediately extruded dropwise into saturated boric acid solution to form PVA beads and immersed at 4 °C for 5 h. Then the beads were washed and stored applying the same procedure used for chitosan immobilized cells. Characterization of immobilized cell Mechanical strength of immobilization matrix Hundred immobilized beads (or cubes) and 20 glass particles were added simultaneously into a 100-mL shake flask with 20 mL PBS buffer (pH 7.0). After 24 h stirring (200 rpm), the ratio of well-preserved pellets by morphologic observation was calculated to estimate the mechanical strength of the immobilization matrix [13]. All assays were performed in triplicate and the average values were given.
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Enzymatic activity recovery after immobilization Enzymatic activity recovery was determined by measuring the enzyme activity before and after cell immobilization. The parameter of enzymatic activity recovery was calculated with the following formula: Enzymatic activity recovery ð%Þ ¼ ðaimm =afree Þ 100 where aimm and afree represented the specific activity of the immobilized cell and free cell, respectively. Scanning electron microscopy (SEM) The internal structure of immobilized cells was observed employing a scanning electron microscope (SEM, Hitachi S-4800). The sample was cross-linked by glutaric dialdehyde, dehydrated with graded ethanol and then freezedried. The mounted samples were gold coated and examined by SEM at an accelerated voltage of 1.0 kV [15]. Enzyme assay The biotransformation of 3-cyanopyridine to nicotinic acid was employed as the model reaction. The standard reaction mixture (10 mL) contained 100 mM 3-cyanopyridine, 10 mM sodium phosphate buffer and a certain amount of whole cells with the same amount of cell usage, respectively, for both immobilized and free cells. Biotransformation process was carried out at 30 °C for 30 min with stirring. The reaction was stopped by addition of 2.0 M HCl. The reaction mixture was centrifuged and the concentrations of nicotinic acid and 3-cyanopyridine in the supernatant were determined by high-performance liquid chromatography system (HPLC, Dionex U3000) with a C18-column (Waters, 4.6 9 150 mm, 5 lm) at 268 nm. The mobile phase consisted of acetonitrile and water (60:40 v/v) with addition of 0.01 % phosphorus acid. The flow rate was 1.0 mL/min. One unit of activity was defined as the amount of cells used for the formation of 1 lmol nicotinic acid per min under the above assay conditions. All assays were performed in triplicate and the average values were given. Influences of temperature, pH and substrate concentration on enzyme activity The influence of temperature on biocatalytic activity for immobilized and free cells was investigated through detecting the residual enzyme activity. The biotransformation process was carried out between 25 and 60 °C for 30 min with stirring. The influences of pH (ranging from 3.0 to 11.0) and substrate concentration (ranging from 100
to 700 mM) for both immobilized and free cells under otherwise standard conditions were studied. Thermal stability study To investigate the thermal stability of immobilized cells, studies were carried out by pre-incubating the biocatalysts in normal saline without substrate at set temperature. Samples were taken at regular intervals to detect the residual enzymatic activity for determining the half-life. The natural logarithm of residual enzymatic activity [ln(RA)] was plotted against time. The half-lives of free and immobilized cells at each temperature were calculated by extrapolating from the slope of the individual line denoted as KDeact [11]. Repeated batch bioconversion of 3-cyanopyridine by immobilized and free cells To measure the reusabilities of the immobilized and free cells, repeated batch bioconversion of 3-cyanopyridine to nicotinic acid with the substrate concentration of 200 mM was performed at 30 °C with stirring. After each cycle of 60-min conversion, the beads and free cells were washed three times with normal saline and reused in a subsequent cycle.
Results and discussion Preparation of immobilized cells Screening for entrapment matrix Entrapment method was employed to enhance the applicability of whole cell biocatalysts. Various immobilization materials were screened based on enzymatic activity recovery, mechanical strength and preparation complexity (Table 1). The inner microscopical structures of immobilized cells were observed with SEM. Scanning electron microscopy photographs showed that cells were randomly distributed in the solid matrix and there were plenty of microspores in the four different gels, especially in chitosan gel, which was supposed to be beneficial to mass transfer. Plenty of channels in Ca-alginate beads were also observed (Fig. 1a), which proved Caalginate was an ‘‘egg-box’’ porous matrix [24]. Chitosan entrapped cells showed the highest enzymatic activity recovery with 87.51 %, followed by gelatin (82.09 %) and alginate (79.11 %). Boric acid used during the PVA immobilization process damaged the cells enclosed in the matrix [25], which resulted in the great loss in enzymatic activity. Nevertheless, mechanical strength rendered a
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Bioprocess Biosyst Eng Table 1 Screening of immobilization materials based on enzymatic activity recovery, mechanical strength and preparation complexity Immobilized materials
Shape
Preparation complexity
Enzymatic activity recovery (%)
Ratio of well-preserved pellets (%)
Mechanical strength
Free cells
–
–
100.0a
–
–
Chitosan
Bead
***
87.51
49.1
??
Alginate
Bead
**
79.11
75.8
???
Gelatin gel
Square
***
82.09
61.3
??
PVA
Bead
****
67.81
93.2
????
‘‘–’’ meant it was not considered, ‘‘*’’ indicated the difficulty of preparation, measured by the preparation time and procedure of the immobilized cells, ‘‘?’’ indicated the mechanical strength of immobilization cells measured by the ratio of well-preserved pellets a
The activity of free cells was considered as a control
completely reverse trend. Through the data of ratio of wellpreserved pellets, it could be seen that only 49 % of chitosan entrapped cells maintained their original morphology, whereas 75 and 93 % for alginate and PVA, respectively. Obviously, chitosan and gelatin beads had poor mechanical strength, whereas PVA and alginate beads were much better. However, PVA beads were found to be inclined to adhere with each other during preparation, which limited its stability. Evaluated by enzymatic activity recovery, mechanical strength and complexity of preparation procedure, alginate was finally selected for further studies, which has been widely utilized as a favorable matrix for cell/enzyme immobilization [26, 27].
increased between 0.1 and 0.6 %. Lower concentration of CaCl2 (less than 0.2 %) led to cell leakage and low mechanical strength. Further increase in CaCl2 concentration significantly enhanced the mechanical strength of immobilized beads whereas the nitrilase activity decreased. The influences of alginate and CaCl2 concentrations were attributed to the diffusion limitation effect of the immobilization matrix. High concentration of alginate or CaCl2 is intended to form compact matrix during immobilization process, which improved the diffusion restriction of substrate and products [28]. The optimized concentration of alginate and CaCl2 were then determined to be 2 and 0.6 %, respectively.
Influences of sodium alginate and calcium chloride concentration
Influences of cell loading and bead size
The concentrations of sodium alginate and calcium chloride play significant roles in the enzyme activity retained for immobilized cells. Their effects on the activity of immobilized cells were investigated. As shown in Fig. 2a, the nitrilase activity of alginate entrapped cells increased with the increase in alginate concentration from 1 to 2 % (w/v). The maximal enzyme activity was obtained with the alginate concentration of 2 %. When the concentration of alginate further increased, the nitrilase activity of alginate entrapped cells decreased. It was observed that beads prepared by low concentration of sodium alginate were rather soft, which were easily damaged in biotransformation process as a result of low mechanical strength. Besides, when the concentration of sodium alginate was higher than 2.5 %, high viscosity of alginate caused difficulty in beads formation, and the beads formed were not regular spherical. This was consistent with former studies [4, 13]. Similar trend was found during optimization of calcium chloride concentration (Fig. 2b). The enzyme activity and the rigidity of alginate entrapped cells were both improved as the CaCl2 concentration
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The influence of cell loading on the biocatalytic performance of alginate entrapped cells was examined. As shown in Fig. 3, the conversion of 3-cyanopyridine increased gradually with the increase in cell loading. However, beyond continuous increase in conversion, there was a drop in the specific activity of alginate entrapped cells. The highest specific activity was achieved when the cell loading was 0.4 g cell/g alginate. When the cell loading exceeded 0.4 g cell/g alginate, the specific activity decreased, indicating that more cell loading is not more favorable for substrate transformation. The same phenomenon was discovered by Kaul et al. [15] and revealed by studying on the variation in the effectiveness of the immobilized cells. Effectiveness is a parameter reflecting the mass transfer effect. It has been reported that higher cell loading leads to the decrease in effectiveness. The resulting diffusion restriction to the substrate becomes the rate controlling step. Besides, the influence of bead size was also studied. It could be found that the relative activity of alginate entrapped cells decreased with the increase in bead size (Fig. 4). Maximum relative activity was obtained with a
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Fig. 1 SEM images of different kinds of immobilized beads containing Gibberella intermedia CA3-1: a general view of Ca-alginate beads, b Ca-alginate beads, c gelatin gel immobilization, d PVA gel beads, e CS gel beads
bead diameter of 1.8 mm. This could also be assigned to the diffusion limited effect. Kaul et al. [15] have measured the effectiveness and diffusion coefficient of alginate immobilized beads. Both the parameters display the decreasing trends with the increase in bead size, which imply that larger beads tend to have larger diffusion limitation. In addition, it was also examined that large beads had a strong tendency to swell, which is consistent with the study of Chen et al. [29].
Optimization of bioconversion conditions Influences of temperature and pH The influence of temperature on the bioconversion of 3-cyanopyridine was studied from 25 to 60 °C. For the immobilized and free cells, the maximal relative activities were both obtained at 40 °C (Fig. 5a). Comparatively, the cells entrapped in alginate displayed slightly higher activity
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Fig. 4 Effect of bead size on nitrilase activity of alginate immobilized cells
Fig. 2 Effects of concentration of alginate (a) and calcium chloride (b) on nitrilase activity of alginate immobilized cells
Fig. 3 Effect of cell loading on nitrilase activity of alginate immobilized cells: conversion (filled squares), specific activity (unfilled squares)
at high temperature, indicating immobilization plays an important role in keeping the cells from denaturation under higher temperature [15, 30].
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Fig. 5 Influences of temperature (a) and pH (b) on nitrilase activity: free cells (unfilled squares), alginate immobilized cells (unfilled circles)
For enzymatic reactions, buffer pH would affect the enzyme activity and product selectivity. The enzyme activity was examined with the pH value of 3.0–11.0. The
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maximum activity of free cells were found to be at pH 7.0, whereas 8.0 for immobilized cells (Fig. 5b). The shift is mainly ascribed to the structural changes induced by pH variation [31]. It has been reported that the carboxylic parts of alginate could attract and enrich H? around them [32]. Hence to balance the excess H?, weak alkaline buffer was needed and favorable for substrate transformation for immobilized cells. Influence of substrate concentration Substrate concentration significantly affects the substrate conversion and enzyme activity, especially for the substrate with certain toxicity to cells, such as 3-cyanopyridine [33]. Sharma et al. [34] found that the concentration of 3-cyanopyridine exceeding 300 mM displayed inhibition effect. The effects of initial concentration of 3-cyanopyridine on the activity of the immobilized and free cells were analyzed with the substrate concentration of 100–700 mM (Fig. 6). Generally, along with the increase in 3-cyanopyridine concentration, the activity of immobilized and free cells both decreased rapidly with the time needed for total conversion prolonged. Detailed comparison revealed that the free cells were more active with shorter time for total conversion when 3-cyanopyridine concentration was below 300 mM. As the concentration increased to 300 mM, the trend turned out to be just the opposite. The time required for total conversion was 100 min for immobilized cells whereas 120 min for free cells. This time gap between immobilized and free cells was further widen as 3-cyanopyridine concentration increased. When
Fig. 6 Effect of 3-cyanopyridine concentration on nitrilase activity, hollow symbols free cells, solid symbols alginate immobilized cells: 100 mM (unfilled squares, filled squares), 200 mM (unfilled circles, filled circles), 300 mM (unfilled triangles, filled triangles), 400 mM (unfilled diamonds, filled diamonds), 500 mM (unfilled inverted triangles, filled inverted triangles), 600 mM (unfilled stars, filled stars), 700 mM (unfilled right arrowhead, filled right arrowhead)
3-cyanopyridine concentration increased to 500 mM and above, the free cells could even not transform the substrate completely. Both the immobilized and free cells could not transform the substrate completely at the concentration of 700 mM. With lower 3-cyanopyridine concentration, the diffusion limitation effect of the matrix led to longer time for total conversion. Nevertheless, when the substrate concentration increased, the toxicity of 3-cyanopyridine gradually manifested and the transformation capacity of free cells decreased, which was mainly ascribed to the damage of cells. Detailed effects need to be investigated. This indicated that immobilization was predominant in cell protection, especially when the substrate concentration was high [14]. It was found that diffusion of substrate from solutions to the beads was not easy [35], and the substrate in immobilized cells was transformed continuously. The concentration of toxic substrate around the immobilized cells was thus lower than the solution which led to the improvement of substrate tolerance. Thermal stability of alginate entrapped cells The thermal stability of immobilized cells was evaluated by preincubating at 30, 40 and 50 °C, respectively (Fig. 7). Although the free cells of G. intermedia have displayed excellent thermal stability [11], immobilization in this study could further improve its stability by 1.4, 1.6 and 1.7fold compared with the free ones. The half-lives of immobilized cells at 30, 40 and 50 °C were calculated to be 315, 117.5 and 10.9 h, respectively. Evidently, the half-life of immobilized cells was enhanced greatly through immobilization. This indicates the immobilization matrix is
Fig. 7 Thermostability of alginate immobilized cells: 30 °C (unfilled squares), 40 °C (unfilled circles), 50 °C (unfilled triangles)
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supposed to preserve the suitable microenvironment for cells and make the cells insensitive to temperature variations, which resulted in the improvement of thermostability of immobilized cells. Similar phenomenon was also observed by Zhang et al. [30]. Repeated batch bioconversion of 3-cyanopyridine Repeated batch mode of 3-cyanopyridine transformation was performed to examine and compare the operation stability of immobilized and free cells (Fig. 8). Continuous batch conversion was operated with 3-cyanopyridine concentration of 200 mM and 60 min for each batch. Probably because of the toxicity of 3-cyanopyridine and the gradual decrease of enzymatic activity, the biocatalysts cannot be reused for many times. In case of free cells, the reaction system was run for 10 consecutive batches to produce 72.2 g/(g dcw) nicotinic acid with 82.45 % enzyme activity reserved. In contrast, under the same transformation conditions, the reaction system of immobilized cells was run for 28 consecutive batches to produce 205.7 g/(g dcw) nicotinic acid with 80.55 % enzyme activity retained. Therefore, the immobilized cells performed with much better operation stability than that of free cells. Up to now, biotransformation of 3-cyanopyridine to nicotinic acid mainly focused on transformation using free cells. Sharma et al. reported the biosynthesis of nicotinic acid by hyperinduced Nocardia globerula NHB-2. With hyperinduced resting cells corresponding to 10 U/mL nitrilase activity (1.5 (g dcw)/L), a total of 123.11 g nicotinic acid was produced with the production capacity of 82 g/(g dcw) in a fed batch reaction on 1 L scale [34]. The highest throughput of nicotinic acid reported hitherto was 98.45 g/(g dcw) [36]. Biotransformation of 3-cyanopyridine with immobilized cells has been applied in manufacturing nicotinamide in Lonza. 3-Cyanopyridine was
continuously transformed with Rhodococcus rhodochrous J1 cells which were immobilized in polyacrylamide. This bio-hydrolysis course was carried out under mild aqueous conditions with high product selectivity, which was much simpler than chemical route [37]. The great catalytic potential of Ca-alginate immobilized G. intermedia CA3-1 could be seen in the preparation of nicotinic acid. Conclusions Immobilization of whole cells offers an alternative to improve the stability and reusability of G. intermedia CA3-1 harboring nitrilase. With the entrapment method, the immobilization matrices were screened and alginate was selected out for its advantages in enzymatic activity recovery, mechanical strength and preparation complexity. After the preparation conditions were determined, the effects of biotransformation conditions on enzyme activity were investigated. The optimum buffer pH for immobilized cells was 8.0, which was slightly more alkaline than the free cells. The optimum temperature was almost the same for both immobilized and free cells, but the immobilized cells displayed a little more insensitive to high temperature. Data of half-lives of immobilized cells at different temperatures gave further evidences. The immobilized cells showed excellent substrate tolerance properties with cell protection effect. With the concentration of 3-cyanopyridine at 200 mM, efficient biocatalyst recycling was achieved as a result of immobilization with 28 consecutive batches and 205.7 g/(g dcw) nicotinic acid obtained. Compared with the free cells, the number of recycle batches was substantially improved for immobilized catalyst and thus it supported the outstanding nicotinic acid production in this study. This study fulfills the requirement for low-cost production of nicotinic acid due to its prominent advantages of reusability and thermostability over free cells. It offers a foundation for large-scale bioconversion of 3-cyanopyridine to nicotinic acid with cell immobilization. Continuous transformation mode and large-scale application with immobilized cells are currently in progress. Acknowledgments This work was supported by the National Natural Science Foundation of China (21206055), Natural Science Foundation of Jiangsu Province (BK2012127, BK20140133), and National High Technology Research and Development Program of China (No. 2012AA022204C).
References
Fig. 8 Reuse of free (unfilled squares) and alginate immobilized (unfilled circles) cells
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