World J Microbiol Biotechnol (2017) 33:176 DOI 10.1007/s11274-017-2341-3
ORIGINAL PAPER
Production of d-alanine from dl-alanine using immobilized cells of Bacillus subtilis HLZ-68 Yangyang Zhang1 · Xiangping Li1 · Caifei Zhang1 · Xiaodong Yu1 · Fei Huang2 · Shihai Huang1 · Lianwei Li1 · Shiyu Liu1
Received: 9 June 2017 / Accepted: 8 September 2017 / Published online: 13 September 2017 © Springer Science+Business Media B.V. 2017
Abstract Immobilized cells of Bacillus subtilis HLZ-68 were used to produce d-alanine from dl-alanine by asymmetric degradation. Different compounds such as polyvinyl alcohol and calcium alginate were employed for immobilizing the B. subtilis HLZ-68 cells, and the results showed that cells immobilized using a mixture of these two compounds presented higher l-alanine degradation activity, when compared with free cells. Subsequently, the effects of different concentrations of polyvinyl alcohol and calcium alginate on l-alanine consumption were examined. Maximum l-alanine degradation was exhibited by cells immobilized with 8% (w/v) polyvinyl alcohol and 2% (w/v) calcium alginate. Addition of 400 g of dl-alanine (200 g at the beginning of the reaction and 200 g after 30 h of incubation) into the reaction solution at 30 °C, pH 6.0, aeration of 1.0 vvm, and agitation of 400 rpm resulted in complete l-alanine degradation within 60 h, leaving 185 g of d-alanine in the reaction solution. The immobilized cells were applied for more than 15 cycles of degradation and a maximum utilization rate was achieved at the third cycle. d-alanine was easily extracted from the reaction solution using cation-exchange resin, and the chemical and optical purity of the extracted d-alanine was 99.1 and 99.6%, respectively.
* Shihai Huang [email protected] 1
State Key Laboratory for Conservation and Utilization of Subtropical Agro‑Bioresources, Guangxi University, Nanning 530004, Guangxi, China
High-tech Industrial Development Zone, Xixiangtang, Nanning 530004, Guangxi, China
2
Keywords Asymmetric degradation · Bacillus subtilis HLZ-68 · Calcium alginate · d-Alanine · Immobilization · Polyvinyl alcohol
Introduction Alanine (Ala) is one of the 20 basic amino acids that constitute human proteins and a significant part of the living body (Hoffer 2016). In general, alanine can be classified into d-alanine and l-alanine according to its molecular structure of chiral (Moozeh et al. 2015) and optical rotation (Radkov and Moe 2014). As d-alanine is an important source of organic chiral compounds, it has a wide range of applications in the pharmaceutical (Qiu et al. 2015) and food industries (Friedman 2010), such as in the production of new antibiotics, vitamin B6, and the sweetener Aclame anhydrous (Yuasa et al. 2001). With the widespread use of d-alanine in the food and pharmaceutical industries, the market demands for d-alanine have also increased. Currently, d-alanine production methods mainly consist of microbial fermentation (Umemura et al. 1990; Yamamoto et al. 2012), chemical asymmetric synthesis (Huang et al. 2015), and amino acylase separation (Takenaka et al. 2015; Wiese et al. 2001). However, the separation and purification of products obtained through microbial fermentation are very difficult owing to the complicated composition of the fermentation broth. Similarly, chemical synthesis is a complex process requiring chiral additives that are limited and expensive, and amino acylase separation method requires expensive enzyme preparation step, thus limiting the large-scale production of d-alanine. dl-Alanine contains 50% d-alanine and l-alanine isomers, respectively. It is possible to screen a microorganism containing l-amino acid oxidase without d-amino acid oxidase
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and capable of asymmetrically degrading dl-alanine to produce d-alanine (Umemura et al. 1992). In the present study, we screened a microorganism named HLZ-68 from soil, which exhibited much higher rate of l-alanine consumption than d-alanine consumption, and was identified as Bacillus subtilis. Immobilization method is a core technique in modern biotechnology and its industrial applications (Gotovtsev et al. 2015). The cells or enzymes fixed in an appropriate carrier (Krasňan et al. 2016) can not only be reused, but also promote easier separation of products from the reaction mixture, thus significantly improving the efficiency of the process. In the present study, B. subtilis HLZ-68 cells with the ability to perform asymmetric degradation of d-alanine and l-alanine were immobilized with polyvinyl alcohol and calcium alginate to effectively remove l-alanine from dlalanine so as to accumulate more d-alanine.
Materials and methods Microorganism Bacillus subtilis HLZ-68 cells were obtained from Guangxi University, China. Preparation of cell suspension The B. subtilis HLZ-68 cells were cultured in a 5-L fermentation jar (Micro DCU-Twin-system, Germany) containing 3-L of fermentation medium for 24 h at 30 °C with an aeration of 1.0 vvm and agitation of 600 rpm. The pH of the fermentation medium was maintained at 6.0 using 5 M H2SO4. The fermentation medium was composed of (w/v) 2% dl-alanine, 0.5% yeast extract, 0.2% K 2HPO4, 0.09% MgSO4·7H2O, and 0.02% C aCl2. The cells were harvested by centrifugation (6500 g for 10 min) and washed twice thoroughly with deionized water prior to use. Preparation of immobilized cells The B. subtilis HLZ-68 cells were immobilized using polyvinyl alcohol and sodium alginate. A certain amount of sodium alginate (3 g) was dissolved in boiling water, sterilized by autoclaving at 121 °C for 15 min, and cooled to 40–45 °C. Subsequently, the bacterial suspension was resuspended in 9 g/L NaCl and thoroughly mixed with the sterile sodium alginate solution. The concentration of sodium alginate in the final mixture was 3% (w/v) and the amount of B. subtilis cells was 1 × 108 CFU/mL, respectively. Using a peristaltic pump, the mixture was dropped into 0.3 M calcium chloride from a 10-cm height to achieve crosslinking and obtain equal-size polymeric beads (3 mm) of calcium alginate. The resulting beads were cured at 4 °C for 6 h prior to filtration
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through a sieve, and rinsed three or four times with sterile distilled water to eliminate excess calcium ions and free cells (Darah et al. 2015; Kim et al. 2015). A similar method was used to immobilize the B. subtilis HLZ-68 cells using 10% (w/v) polyvinyl alcohol and 3% (w/v) boric acid (crosslinking agent) (Hu and Yang 2015). For immobilizing the B. subtilis HLZ-68 cells using a mixture of polyvinyl alcohol and sodium alginate, 8% (w/v) polyvinyl alcohol and 2.5% (w/v) sodium alginate were used, along with the crosslinker, 2.5% (w/v) calcium chloride and 3% (w/v) boric acid solution (Wu et al. 2012). Degradation of l‑alanine from dl‑alanine After preparing immobilized cells using the three aforementioned methods, the ability of these cells to degrade l-alanine from dl-alanine was investigated in a 5-L fermentation jar. In brief, 500 g of the immobilized cells were suspended in 2-L reaction solution containing approximately 200 g of dl-alanine. The composition of the reaction solution was as follows (g/L): KH2PO4, 5; K 2HPO4, 2.5; Mg2SO4·7H2O, 2; CaCl2, 0.1; and polyether antifoam agent, 0.2. The reaction was performed for 30 h at 30 °C with an aeration of 1.0 vvm and agitation of 400 rpm. The pH of the reaction solution was maintained at 6.0 by using 5 M H2SO4. The control comprised B. subtilis HLZ-68 free cells (4.0 × 107 CFU/mL) incubated under the same reaction conditions. Optimization of calcium alginate and polyvinyl alcohol matrix For the preparation of beads with optimal l-isomer catabolism activity coupled with good mechanical strength, various concentrations of sodium alginate (0.5–3% w/v) and polyvinyl alcohol (6–11% w/v) were examined. Repeated batch degradation Repeated batch degradation was conducted with immobilized cells for 60 h. Initially, 200 g of dl-alanine were added to the reaction mixture at the beginning of the reaction, and after 30 h of incubation, an additional 200 g of dl-alanine (dissolved in a small amount of sterile distilled water) were added to the reaction solution and the reaction was continued for 30 h. At the end of each degradation cycle, the immobilized bacterial beads were recovered by filtration through a sieve, thoroughly cleaned with sterile water, and added to fresh reaction solution, and the degradation was continued. Extraction of d‑alanine from the degradation mixture After 60 h of catabolism, the final reaction mixture, in which all l-alanine had been removed, was centrifuged and the
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supernatant was extracted. The pH of the supernatant was adjusted to 4.0 using H 2SO4 before passing through a column filled with 732-type strong acid cation-exchange resin (Hems et al. 2010). Subsequently, the ion-exchange column was rinsed with sterile water to remove impurities and d-alanine was eluted with 2 M NH4OH. The collected eluate was discolored with activated charcoal, and the decolorized solution was concentrated in vacuum until d-alanine crystallized. Analytical methods
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cells were capable of eliminating l-alanine in dl-alanine within 35 h, but only achieved 4.5% decrease in d-alanine over the subsequent 25 h. Therefore, based on these characteristics, we immobilized the B. subtilis HLZ-68 cells to achieve asymmetric degradation and produce d-alanine from dl-alanine. Immobilization of B. subtilis HLZ‑68 cells on different matrices
The results of degradation of different isomers of alanine by free B. subtilis HLZ-68 cells resuspended in the reaction medium (1 × 108 CFU/mL) are shown in Fig. 1. Under the same conditions, the bacterial cells presented varied utilization of alanine isomers. The bacterial cells exhibited highest l-alanine degradation activity, showing complete l-alanine consumption in 60 h. In contrast, d-alanine degradation by the bacterial cells was extremely low, with only 8% d-alanine catabolism in 60 h. Similarly, the B. subtilis HLZ-68
The immobilized cells prepared with different matrices were spherical and had a diameter of 3 mm, and the percentage of cells immobilized in the beads was 94.32%, as measured by blood count. Comparative data of l-alanine degradation by free B. subtilis HLZ-68 cells and those immobilized with different carriers are shown in Fig. 2. The l-alanine consumption rate exhibited by polyvinyl alcohol immobilized cells increased to 63.63% during the third cycle and remained almost unchanged in the next two cycles. In contrast, the l-alanine degradation rate presented by sodium alginate immobilized cells increased to 82.17% and then decreased to 45.97%. However, cells immobilized with a mixture of polyvinyl alcohol and sodium alginate displayed the highest l-alanine utilization rate of 86.98%, which remained constant. Under the same conditions, the l-alanine degradation rate demonstrated by free cells was 71.31%. Therefore, polyvinyl alcohol–sodium alginate was considered to be the best matrix for asymmetric degradation of dl-alanine to produce d-alanine, and cells immobilized with polyvinyl alcohol–sodium alginate were used for further optimization studies.
Fig. 1 Degradation of different isomers of alanine by B. subtilis HLZ-68. The free cells were suspended in 2.0 L of reaction solution (1 × 108 CFU/mL) containing 200 g of d-alanine, l-alanine, or dl-alanine in a 5-L jar fermentor. The reaction was performed for 60 h at 30 °C with an aeration of 1.0 vvm and agitation of 400 rpm. The pH of the reaction solution was maintained at 6.0 by using 5 M H2SO4
Fig. 2 Degradation of l-alanine by free and immobilized B. subtilis HLZ-68 cells. Degradation of l-alanine was initiated by the addition of 200 g of dl-alanine into 2 L of reaction solution containing equal amount of free (4.0 × 107 CFU/mL) and immobilized cells (500 g) in a 5-L jar fermentor. The reaction was performed for 30 h at 30 °C with pH 6.0, aeration of 1.0 vvm, and agitation of 400 rpm
The alanine concentration was determined by spectrophotometry using ninhydrin (Barbehenn 2010). The d-alanine and l-alanine concentrations in the mixed solution were measured using d-amino acid oxidase (Pilone 2000) and l-amino acid oxidase (Yu and Qiao 2012), respectively.
Results Degradation of different isomers of alanine
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Optimization of polyvinyl alcohol concentration Polyvinyl alcohol is a good embedding material because it is non-toxic, inexpensive, and easy to obtain, and its mechanical strength and stability evidently improves after chemical crosslinking or hardening treatment (He et al. 2011). The immobilized cells prepared with sodium alginate (2.5%, w/v) and different concentrations of polyvinyl alcohol (6.0–11.0%, w/v). The results of the analysis of the effects of polyvinyl alcohol concentration on l-alanine degradation are shown in Fig. 3. The mechanical strength of the immobilized bacterial beads appeared to be dependent on the concentration of polyvinyl alcohol used to formulate the gel capsules. The consumption rate of l-alanine increased to 28% when the polyvinyl alcohol concentration was increased from 6 to 8% (w/v). However, further increase in the concentration of polyvinyl alcohol to 9 and 11% (w/v) reduced the l-alanine consumption rate to merely 72.1 and 61.9%, respectively, when compared with that noted with 8% (w/v) polyvinyl alcohol concentration (85.0%). This decrease in the degradation of l-alanine could be owing to the diffusional resistance of the beads, and therefore, 8% (w/v) polyvinyl alcohol was employed for subsequent analyses. Optimization of sodium alginate concentration Polyvinyl alcohol gel particles have a strong tendency to aggregate, causing adhesion between the particles and making them difficult to separate. An effective solution to this problem is the addition of sodium alginate, which forms calcium alginate gel, a porous mesh structure that can
Fig. 3 Effect of polyvinyl alcohol concentration (6.0–11.0%, w/v) on degradation of l-alanine. The immobilized cells (500 g wet weight) prepared with different concentrations of polyvinyl alcohol (6.0– 11.0%, w/v) were suspended in 2 L reaction solution containing 200 g dl-alanine in a jar fermentor. The reaction was performed for 30 h at 30 °C with pH 6.0, aeration of 1.0 vvm, and agitation of 400 rpm
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improve the mass transfer performance of the carrier(Ying et al. 2012). Therefore, in the present study, the immobilized cells prepared with polyvinyl alcohol (8.0%, w/v) and different concentrations of sodium alginate (1.0–3.0%, w/v), and their effects on l-alanine degradation was examined (Fig. 4). Higher or lower concentrations of sodium alginate had an adverse effect on the catabolism of l-alanine, with maximum l-alanine degradation rate obtained with 2.0% (w/v) sodium alginate (96%). It must be noted that when the concentration of sodium alginate is higher than 2%, the internal crosslinking of the carrier is close, which reduces the mass transfer performance of the immobilized cells. Besides, when the mixed matrix becomes very viscous, the immobilized cells in the cross-linking agent form a long strip, rather than beads, which could also adversely affect degradation. Considering the degradation efficiency and rigidity, 2.0% (w/v) sodium alginate was concluded to be optimal for the production of ideal immobilized bacterial beads. Time course of l‑alanine degradation by immobilized cells The time course of alanine degradation by immobilized cells prepared with 8% (w/v) polyvinyl alcohol and 2% (w/v) calcium alginate was studied under the standard reaction conditions, and the results are shown in Fig. 5. To avoid the inhibitory effect of high concentrations of dl-alanine on the degradation activity of the immobilized cells, dlalanine was added in two batches into the reaction mixture. In other words, one part of dl-alanine (200 g) was added
Fig. 4 Effect of sodium alginate concentration (1.0–3.0%, w/v) on the degradation of l-alanine. The immobilized cells (500 g of wet weight) prepared with different concentrations of sodium alginate (1.0–3.0%, w/v) were suspended in 2 L of reaction solution containing 200 g of dl-alanine in a jar fermentor. The reaction was performed for 30 h at 30 °C with pH 6.0, aeration of 1.0 vvm, and agitation of 400 rpm
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Fig. 5 Time course of l-alanine degradation by immobilized cells. The immobilized cells (500 g of wet weight) were suspended in 2 L of reaction solution containing 200 g of dl-alanine in a jar fermentor. After 30 h of incubation, 200 g of dl-alanine dissolved in small amount of distilled water were added. The reaction was performed at 30 °C with pH 6.0, aeration of 1.0 vvm, and agitation of 400 rpm
Fig. 6 Repeated batch degradation of l-alanine by immobilized cells. The immobilized cells (500 g of wet weight) and dl-alanine (200 g) were mixed with 2 L of reaction solution, and after 30 h, additional dl-alanine (200 g) was added. Each cycle was performed for 60 h in a jar fermentor at 30 °C, 400 rpm, and aeration of 1.0 vvm. The pH of the reaction solution was maintained at 6.0 using 5 M H2SO4
to the reaction mixture at the beginning of the reaction and the other part (200 g) was added after 30 h of incubation, when most of the initially added l-isomer had disappeared. As the reaction proceeded, the l-alanine content decreased rapidly, whereas d-alanine slightly decreased throughout the reaction. At 55 h, l-alanine was almost completely degraded, while 92 g/L d-alanine remained in the reaction solution.
Besides, the chemical purity of the isolated and purified d-alanine was 99.1% and the optical purity was 99.6%.
Reusability of the gel matrix The continuous degradation of l-alanine by immobilized cells prepared with 8% (w/v) polyvinyl alcohol and 2% (w/v) calcium alginate was examined in a fermenter (Fig. 6). The highest catabolism activity of the immobilized cells was noted in the third cycle, followed by gradual decrease in the consumption activity with the increase in the number of degradation cycles. The immobilized cells consistently maintained a high l-alanine utilization rate of more than 90% from the third to eighth cycle, with each degradation cycle lasting for 60 h. After 15 successive degradation cycles under these conditions, the immobilized cells maintained 49% of degradation activity. Isolation of d‑alanine and its optical purity d-Alanine was isolated from the reaction mixture by using a simple procedure (see “Materials and methods” section). After cation exchange and drying, 185 g of d-alanine were obtained, resulting in a yield of 92.5%. The specific rota= −14.5◦ at 20 °C. tion of the obtained d-alanine was [𝛼]20 D
Discussion Cell immobilization is a useful technique in modern biotechnology and its industrial applications. B. subtilis HLZ-68 possesses l-amino acid oxidase and not d-amino acid oxidase and can carry out asymmetric oxidative degradation of l-alanine from dl-alanine. Besides, as this B. subtilis strain did not exhibit racemase activity and could not transform l-alanine into d-alanine, and just consumes l-alanine, leaving d-alanine, it was immobilized in the present study to produce d-alanine. Polyvinyl alcohol and sodium alginate are commonly used immobilized carrier matrices. Polyvinyl alcohol has good mechanical strength, but poor mass transfer performance, and the polyvinyl alcohol gel particles have a strong tendency to aggregate. The immobilization blocks prepared with polyvinyl alcohol could aggregate and reduce the diffusion of substrate and oxygen into the matrices, thus lowering the degradation ability of the immobilized cells, when compared with that of free cells. A similar effect has also been reported by Al-Zuhair and El-Naas (2011). In contrast, sodium alginate presents good mass transfer performance, but low mechanical strength (Garay-Flores et al. 2014). In the present study, blocks prepared with calcium alginate were unable to retain their shape during repeated batch catabolism and disintegrated after three cycles. As a result, the degradation activity of the immobilized cells first
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increased and then decreased. However, when cells were immobilized with a mixture of polyvinyl alcohol and sodium alginate, the carrier exhibited high mechanical strength and good mass transfer performance. Consequently, the l-alanine utilization activity of these immobilized cells was higher than that of free cells and did not decrease owing to the breakdown of the beads. A similar effect has also been reported elsewhere (Dave and Madamwar 2006). Therefore, polyvinyl alcohol–sodium alginate was considered to be the best matrix for asymmetric degradation of dl-alanine to produce d-alanine. Besides, to improve the degradation activity of the immobilized cells, the effect of polyvinyl alcohol and sodium alginate concentrations on the immobilized cells was examined. The results showed that the immobilized cells presented the highest l-alanine consumption activity when the concentrations of polyvinyl alcohol and sodium alginate were increased to 8% (w/v) and 2% (w/v), respectively. Furthermore, to increase the production of d-alanine, 200 g of dl-alanine were added to the reaction mixture after the consumption of l-alanine, and the final concentration of d-alanine obtained was 92.5 g/L. This method not only improved the utilization of the immobilized cells, but also satisfied the industrial production requirements. Moreover, under these conditions, the immobilized cells were able to continuously degrade l-alanine over 15 cycles. An increase in the degradation activity of the immobilized cells during the early cycles could be owing to appropriate adaptation and growth of the cells in the microenvironment. Accordingly, with the increasing number of cycles, the immobilized cells may gradually break and the internal embedding of cells may progressively weaken, thus decreasing l-alanine utilization. Similar observations have been explicitly reported by Mohapatra et al. (2007). After cation exchange and drying, the chemical and optical purity of the obtained d-alanine was 99.1 and 99.6%, respectively, which meet the industrial production requirements. In conclusion, the results of the present study showed that polyvinyl alcohol and calcium alginate are promising compounds for the immobilization of B. subtilis HLZ-68 cells for d-alanine production from dl-alanine by asymmetric degradation. When compared with other microbial processes, immobilized cells can be employed for repeated batch consumption of l-alanine from dl-alanine, and the subsequent separation and purification process is simple. In addition, polyvinyl alcohol and sodium alginate are inexpensive and nontoxic, and the immobilized bacteria can be used for large-scale production of d-alanine from dl-alanine. To the best of our knowledge, this study is the first report on bacterial cell immobilization for d-alanine production. Acknowledgements This work was financially support by the Science Research and Technology Development Project of Nanning (20125193) and Wuzou (201201023).
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References Al-Zuhair S, El-Naas M (2011) Immobilization of Pseudomonas putida in PVA gel particles for the biodegradation of phenol at high concentrations. Biochem Eng J 56:46–50 Barbehenn RV (2010) Measurement of protein in whole plant samples with ninhydrin. J Sci Food Agric 69:353–359 Darah I, Nisha M, Lim SH (2015) Polygalacturonase production by calcium alginate immobilized Enterobacter aerogenes NBO2 cells. Appl Biochem Biotechnol 175:2629–2636 Dave R, Madamwar D (2006) Esterification in organic solvents by lipase immobilized in polymer of PVA–alginate–boric acid. Process Biochem 41:951–955 Friedman M (2010) Origin, microbiology, nutrition, and pharmacology of D-amino acids. Chem Biodivers 7:1491 Garay-Flores RV, Segura-Ceniceros EP, De León-Gámez R, Balvantín-García C, Martínez-Hernández JL, Betancourt-Galindo R, Ramírez ARP, Aguilar CN, Ilyina A (2014) Production of glucose oxidase and catalase by Aspergillus niger free and immobilized in alginate-polyvinyl alcohol beads. J Gen Appl Microbiol 60:262 Gotovtsev PM et al (2015) Immobilization of microbial cells for biotechnological production: modern solutions and promising technologies. Appl Biochem Microbiol 51:792–803 He SY, Lin YH, Hou KY, Hwang SC (2011) Degradation of dimethyl-sulfoxide-containing wastewater using airlift bioreactor by polyvinyl-alcohol-immobilized cell beads. Bioresour Technol 102:5609–5616 Hems BA, Page JK, Waller JG (2010) The use of ion exchange resins for separation of basic amino acids. J Chem Technol Biotechnol 67:77–80 Hoffer LJ (2016) Human Protein and Amino Acid Requirements. Jpen J Parenter Enter Nutr 40:460 Hu J, Yang Q (2015) Microbial degradation of di-n-butyl phthalate by Micrococcus sp. immobilized with polyvinyl alcohol. Desalination Water Treat 56:2457–2463 Huang Y, Pathirana C, Ye Q, Palaniswamy V (2015) Non-enzymatic transformation of dl -glyceraldehyde, 1,3-dihydroxyacetone, and pyruvaldehyde with primary amine to the same dl -alanine derivatives. Tetrahedron Lett 56:4516–4519 Kim Y, Koo BS, Lee HC, Yoon Y (2015) Improved production of isomaltulose by a newly isolated mutant of Serratia sp. cells immobilized in calcium alginate. Can J Microbiol 61:193–199 Krasňan V, Stloukal R, Rosenberg M, Rebroš M (2016) Immobilization of cells and enzymes to L entiKats®. Appl Microbiol Biotechnol 100:2535–2553 Mohapatra PKD, Mondal KC, Pati BR (2007) Production of tannase by the immobilized cells of Bacillus licheniformis KBR6 in Caalginate beads. J Appl Microbiol 102:1462–1467 Moozeh K, So SM, Chin J (2015) Catalytic stereoinversion of L-Alanine to deuterated D-Alanine. Angew Chem 54:9381 Pilone MS (2000) D-amino acid oxidase: new findings. Cell Mol Life Sci Cmls 57:1732–1747 Qiu W et al (2015) d-Alanine metabolism is essential for growth and biofilm formation of Streptococcus mutans. Mol Oral Microbiol 31:435–444 Radkov AD, Moe LA (2014) Bacterial synthesis of D-amino acids. Appl Microbiol Biotechnol 98:5363 Takenaka T, Ito T, Miyahara I, Hemmi H, Yoshimura T (2015) A new member of MocR/GabR-type PLP-binding regulator of d-alanyld-alanine ligase in Brevibacillus brevis. Febs J 282:4201 Umemura I, Yanagiya K, Komatsubara S, Sato T, Tosa T (1990) d -Alanine production by using asymmetric degrading activity of Candida maltosa. Ann N Y Acad Sci 613:659–662 Umemura I, Yanagiya K, Komatsubara S, Sato T, Tosa T (1992) d -Alanine production from dl -alanine by Candida maltosa with
World J Microbiol Biotechnol (2017) 33:176 asymmetric degrading activity. Appl Microbiol Biotechnol 36:722–726 Wiese A, Syldatk C, Mattes R, Altenbuchner J (2001) Organization of genes responsible for the stereospecific conversion of hydantoins to α-amino acids in Arthrobacter aurescens DSM 3747. Arch Microbiol 176:187–196 Wu Z et al (2012) Characterization of the nitrobenzene-degrading strain Pseudomonas sp. a3 and use of its immobilized cells in the treatment of mixed aromatics wastewater. World J Microbiol Biotechnol 28:2679 Yamamoto S et al (2012) Overexpression of genes encoding glycolytic enzymes in Corynebacterium glutamicum enhances glucose
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ORIGINAL PAPER
Production of d-alanine from dl-alanine using immobilized cells of Bacillus subtilis HLZ-68 Yangyang Zhang1 · Xiangping Li1 · Caifei Zhang1 · Xiaodong Yu1 · Fei Huang2 · Shihai Huang1 · Lianwei Li1 · Shiyu Liu1
Received: 9 June 2017 / Accepted: 8 September 2017 / Published online: 13 September 2017 © Springer Science+Business Media B.V. 2017
Abstract Immobilized cells of Bacillus subtilis HLZ-68 were used to produce d-alanine from dl-alanine by asymmetric degradation. Different compounds such as polyvinyl alcohol and calcium alginate were employed for immobilizing the B. subtilis HLZ-68 cells, and the results showed that cells immobilized using a mixture of these two compounds presented higher l-alanine degradation activity, when compared with free cells. Subsequently, the effects of different concentrations of polyvinyl alcohol and calcium alginate on l-alanine consumption were examined. Maximum l-alanine degradation was exhibited by cells immobilized with 8% (w/v) polyvinyl alcohol and 2% (w/v) calcium alginate. Addition of 400 g of dl-alanine (200 g at the beginning of the reaction and 200 g after 30 h of incubation) into the reaction solution at 30 °C, pH 6.0, aeration of 1.0 vvm, and agitation of 400 rpm resulted in complete l-alanine degradation within 60 h, leaving 185 g of d-alanine in the reaction solution. The immobilized cells were applied for more than 15 cycles of degradation and a maximum utilization rate was achieved at the third cycle. d-alanine was easily extracted from the reaction solution using cation-exchange resin, and the chemical and optical purity of the extracted d-alanine was 99.1 and 99.6%, respectively.
* Shihai Huang [email protected] 1
State Key Laboratory for Conservation and Utilization of Subtropical Agro‑Bioresources, Guangxi University, Nanning 530004, Guangxi, China
High-tech Industrial Development Zone, Xixiangtang, Nanning 530004, Guangxi, China
2
Keywords Asymmetric degradation · Bacillus subtilis HLZ-68 · Calcium alginate · d-Alanine · Immobilization · Polyvinyl alcohol
Introduction Alanine (Ala) is one of the 20 basic amino acids that constitute human proteins and a significant part of the living body (Hoffer 2016). In general, alanine can be classified into d-alanine and l-alanine according to its molecular structure of chiral (Moozeh et al. 2015) and optical rotation (Radkov and Moe 2014). As d-alanine is an important source of organic chiral compounds, it has a wide range of applications in the pharmaceutical (Qiu et al. 2015) and food industries (Friedman 2010), such as in the production of new antibiotics, vitamin B6, and the sweetener Aclame anhydrous (Yuasa et al. 2001). With the widespread use of d-alanine in the food and pharmaceutical industries, the market demands for d-alanine have also increased. Currently, d-alanine production methods mainly consist of microbial fermentation (Umemura et al. 1990; Yamamoto et al. 2012), chemical asymmetric synthesis (Huang et al. 2015), and amino acylase separation (Takenaka et al. 2015; Wiese et al. 2001). However, the separation and purification of products obtained through microbial fermentation are very difficult owing to the complicated composition of the fermentation broth. Similarly, chemical synthesis is a complex process requiring chiral additives that are limited and expensive, and amino acylase separation method requires expensive enzyme preparation step, thus limiting the large-scale production of d-alanine. dl-Alanine contains 50% d-alanine and l-alanine isomers, respectively. It is possible to screen a microorganism containing l-amino acid oxidase without d-amino acid oxidase
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and capable of asymmetrically degrading dl-alanine to produce d-alanine (Umemura et al. 1992). In the present study, we screened a microorganism named HLZ-68 from soil, which exhibited much higher rate of l-alanine consumption than d-alanine consumption, and was identified as Bacillus subtilis. Immobilization method is a core technique in modern biotechnology and its industrial applications (Gotovtsev et al. 2015). The cells or enzymes fixed in an appropriate carrier (Krasňan et al. 2016) can not only be reused, but also promote easier separation of products from the reaction mixture, thus significantly improving the efficiency of the process. In the present study, B. subtilis HLZ-68 cells with the ability to perform asymmetric degradation of d-alanine and l-alanine were immobilized with polyvinyl alcohol and calcium alginate to effectively remove l-alanine from dlalanine so as to accumulate more d-alanine.
Materials and methods Microorganism Bacillus subtilis HLZ-68 cells were obtained from Guangxi University, China. Preparation of cell suspension The B. subtilis HLZ-68 cells were cultured in a 5-L fermentation jar (Micro DCU-Twin-system, Germany) containing 3-L of fermentation medium for 24 h at 30 °C with an aeration of 1.0 vvm and agitation of 600 rpm. The pH of the fermentation medium was maintained at 6.0 using 5 M H2SO4. The fermentation medium was composed of (w/v) 2% dl-alanine, 0.5% yeast extract, 0.2% K 2HPO4, 0.09% MgSO4·7H2O, and 0.02% C aCl2. The cells were harvested by centrifugation (6500 g for 10 min) and washed twice thoroughly with deionized water prior to use. Preparation of immobilized cells The B. subtilis HLZ-68 cells were immobilized using polyvinyl alcohol and sodium alginate. A certain amount of sodium alginate (3 g) was dissolved in boiling water, sterilized by autoclaving at 121 °C for 15 min, and cooled to 40–45 °C. Subsequently, the bacterial suspension was resuspended in 9 g/L NaCl and thoroughly mixed with the sterile sodium alginate solution. The concentration of sodium alginate in the final mixture was 3% (w/v) and the amount of B. subtilis cells was 1 × 108 CFU/mL, respectively. Using a peristaltic pump, the mixture was dropped into 0.3 M calcium chloride from a 10-cm height to achieve crosslinking and obtain equal-size polymeric beads (3 mm) of calcium alginate. The resulting beads were cured at 4 °C for 6 h prior to filtration
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through a sieve, and rinsed three or four times with sterile distilled water to eliminate excess calcium ions and free cells (Darah et al. 2015; Kim et al. 2015). A similar method was used to immobilize the B. subtilis HLZ-68 cells using 10% (w/v) polyvinyl alcohol and 3% (w/v) boric acid (crosslinking agent) (Hu and Yang 2015). For immobilizing the B. subtilis HLZ-68 cells using a mixture of polyvinyl alcohol and sodium alginate, 8% (w/v) polyvinyl alcohol and 2.5% (w/v) sodium alginate were used, along with the crosslinker, 2.5% (w/v) calcium chloride and 3% (w/v) boric acid solution (Wu et al. 2012). Degradation of l‑alanine from dl‑alanine After preparing immobilized cells using the three aforementioned methods, the ability of these cells to degrade l-alanine from dl-alanine was investigated in a 5-L fermentation jar. In brief, 500 g of the immobilized cells were suspended in 2-L reaction solution containing approximately 200 g of dl-alanine. The composition of the reaction solution was as follows (g/L): KH2PO4, 5; K 2HPO4, 2.5; Mg2SO4·7H2O, 2; CaCl2, 0.1; and polyether antifoam agent, 0.2. The reaction was performed for 30 h at 30 °C with an aeration of 1.0 vvm and agitation of 400 rpm. The pH of the reaction solution was maintained at 6.0 by using 5 M H2SO4. The control comprised B. subtilis HLZ-68 free cells (4.0 × 107 CFU/mL) incubated under the same reaction conditions. Optimization of calcium alginate and polyvinyl alcohol matrix For the preparation of beads with optimal l-isomer catabolism activity coupled with good mechanical strength, various concentrations of sodium alginate (0.5–3% w/v) and polyvinyl alcohol (6–11% w/v) were examined. Repeated batch degradation Repeated batch degradation was conducted with immobilized cells for 60 h. Initially, 200 g of dl-alanine were added to the reaction mixture at the beginning of the reaction, and after 30 h of incubation, an additional 200 g of dl-alanine (dissolved in a small amount of sterile distilled water) were added to the reaction solution and the reaction was continued for 30 h. At the end of each degradation cycle, the immobilized bacterial beads were recovered by filtration through a sieve, thoroughly cleaned with sterile water, and added to fresh reaction solution, and the degradation was continued. Extraction of d‑alanine from the degradation mixture After 60 h of catabolism, the final reaction mixture, in which all l-alanine had been removed, was centrifuged and the
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supernatant was extracted. The pH of the supernatant was adjusted to 4.0 using H 2SO4 before passing through a column filled with 732-type strong acid cation-exchange resin (Hems et al. 2010). Subsequently, the ion-exchange column was rinsed with sterile water to remove impurities and d-alanine was eluted with 2 M NH4OH. The collected eluate was discolored with activated charcoal, and the decolorized solution was concentrated in vacuum until d-alanine crystallized. Analytical methods
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cells were capable of eliminating l-alanine in dl-alanine within 35 h, but only achieved 4.5% decrease in d-alanine over the subsequent 25 h. Therefore, based on these characteristics, we immobilized the B. subtilis HLZ-68 cells to achieve asymmetric degradation and produce d-alanine from dl-alanine. Immobilization of B. subtilis HLZ‑68 cells on different matrices
The results of degradation of different isomers of alanine by free B. subtilis HLZ-68 cells resuspended in the reaction medium (1 × 108 CFU/mL) are shown in Fig. 1. Under the same conditions, the bacterial cells presented varied utilization of alanine isomers. The bacterial cells exhibited highest l-alanine degradation activity, showing complete l-alanine consumption in 60 h. In contrast, d-alanine degradation by the bacterial cells was extremely low, with only 8% d-alanine catabolism in 60 h. Similarly, the B. subtilis HLZ-68
The immobilized cells prepared with different matrices were spherical and had a diameter of 3 mm, and the percentage of cells immobilized in the beads was 94.32%, as measured by blood count. Comparative data of l-alanine degradation by free B. subtilis HLZ-68 cells and those immobilized with different carriers are shown in Fig. 2. The l-alanine consumption rate exhibited by polyvinyl alcohol immobilized cells increased to 63.63% during the third cycle and remained almost unchanged in the next two cycles. In contrast, the l-alanine degradation rate presented by sodium alginate immobilized cells increased to 82.17% and then decreased to 45.97%. However, cells immobilized with a mixture of polyvinyl alcohol and sodium alginate displayed the highest l-alanine utilization rate of 86.98%, which remained constant. Under the same conditions, the l-alanine degradation rate demonstrated by free cells was 71.31%. Therefore, polyvinyl alcohol–sodium alginate was considered to be the best matrix for asymmetric degradation of dl-alanine to produce d-alanine, and cells immobilized with polyvinyl alcohol–sodium alginate were used for further optimization studies.
Fig. 1 Degradation of different isomers of alanine by B. subtilis HLZ-68. The free cells were suspended in 2.0 L of reaction solution (1 × 108 CFU/mL) containing 200 g of d-alanine, l-alanine, or dl-alanine in a 5-L jar fermentor. The reaction was performed for 60 h at 30 °C with an aeration of 1.0 vvm and agitation of 400 rpm. The pH of the reaction solution was maintained at 6.0 by using 5 M H2SO4
Fig. 2 Degradation of l-alanine by free and immobilized B. subtilis HLZ-68 cells. Degradation of l-alanine was initiated by the addition of 200 g of dl-alanine into 2 L of reaction solution containing equal amount of free (4.0 × 107 CFU/mL) and immobilized cells (500 g) in a 5-L jar fermentor. The reaction was performed for 30 h at 30 °C with pH 6.0, aeration of 1.0 vvm, and agitation of 400 rpm
The alanine concentration was determined by spectrophotometry using ninhydrin (Barbehenn 2010). The d-alanine and l-alanine concentrations in the mixed solution were measured using d-amino acid oxidase (Pilone 2000) and l-amino acid oxidase (Yu and Qiao 2012), respectively.
Results Degradation of different isomers of alanine
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Optimization of polyvinyl alcohol concentration Polyvinyl alcohol is a good embedding material because it is non-toxic, inexpensive, and easy to obtain, and its mechanical strength and stability evidently improves after chemical crosslinking or hardening treatment (He et al. 2011). The immobilized cells prepared with sodium alginate (2.5%, w/v) and different concentrations of polyvinyl alcohol (6.0–11.0%, w/v). The results of the analysis of the effects of polyvinyl alcohol concentration on l-alanine degradation are shown in Fig. 3. The mechanical strength of the immobilized bacterial beads appeared to be dependent on the concentration of polyvinyl alcohol used to formulate the gel capsules. The consumption rate of l-alanine increased to 28% when the polyvinyl alcohol concentration was increased from 6 to 8% (w/v). However, further increase in the concentration of polyvinyl alcohol to 9 and 11% (w/v) reduced the l-alanine consumption rate to merely 72.1 and 61.9%, respectively, when compared with that noted with 8% (w/v) polyvinyl alcohol concentration (85.0%). This decrease in the degradation of l-alanine could be owing to the diffusional resistance of the beads, and therefore, 8% (w/v) polyvinyl alcohol was employed for subsequent analyses. Optimization of sodium alginate concentration Polyvinyl alcohol gel particles have a strong tendency to aggregate, causing adhesion between the particles and making them difficult to separate. An effective solution to this problem is the addition of sodium alginate, which forms calcium alginate gel, a porous mesh structure that can
Fig. 3 Effect of polyvinyl alcohol concentration (6.0–11.0%, w/v) on degradation of l-alanine. The immobilized cells (500 g wet weight) prepared with different concentrations of polyvinyl alcohol (6.0– 11.0%, w/v) were suspended in 2 L reaction solution containing 200 g dl-alanine in a jar fermentor. The reaction was performed for 30 h at 30 °C with pH 6.0, aeration of 1.0 vvm, and agitation of 400 rpm
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improve the mass transfer performance of the carrier(Ying et al. 2012). Therefore, in the present study, the immobilized cells prepared with polyvinyl alcohol (8.0%, w/v) and different concentrations of sodium alginate (1.0–3.0%, w/v), and their effects on l-alanine degradation was examined (Fig. 4). Higher or lower concentrations of sodium alginate had an adverse effect on the catabolism of l-alanine, with maximum l-alanine degradation rate obtained with 2.0% (w/v) sodium alginate (96%). It must be noted that when the concentration of sodium alginate is higher than 2%, the internal crosslinking of the carrier is close, which reduces the mass transfer performance of the immobilized cells. Besides, when the mixed matrix becomes very viscous, the immobilized cells in the cross-linking agent form a long strip, rather than beads, which could also adversely affect degradation. Considering the degradation efficiency and rigidity, 2.0% (w/v) sodium alginate was concluded to be optimal for the production of ideal immobilized bacterial beads. Time course of l‑alanine degradation by immobilized cells The time course of alanine degradation by immobilized cells prepared with 8% (w/v) polyvinyl alcohol and 2% (w/v) calcium alginate was studied under the standard reaction conditions, and the results are shown in Fig. 5. To avoid the inhibitory effect of high concentrations of dl-alanine on the degradation activity of the immobilized cells, dlalanine was added in two batches into the reaction mixture. In other words, one part of dl-alanine (200 g) was added
Fig. 4 Effect of sodium alginate concentration (1.0–3.0%, w/v) on the degradation of l-alanine. The immobilized cells (500 g of wet weight) prepared with different concentrations of sodium alginate (1.0–3.0%, w/v) were suspended in 2 L of reaction solution containing 200 g of dl-alanine in a jar fermentor. The reaction was performed for 30 h at 30 °C with pH 6.0, aeration of 1.0 vvm, and agitation of 400 rpm
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Fig. 5 Time course of l-alanine degradation by immobilized cells. The immobilized cells (500 g of wet weight) were suspended in 2 L of reaction solution containing 200 g of dl-alanine in a jar fermentor. After 30 h of incubation, 200 g of dl-alanine dissolved in small amount of distilled water were added. The reaction was performed at 30 °C with pH 6.0, aeration of 1.0 vvm, and agitation of 400 rpm
Fig. 6 Repeated batch degradation of l-alanine by immobilized cells. The immobilized cells (500 g of wet weight) and dl-alanine (200 g) were mixed with 2 L of reaction solution, and after 30 h, additional dl-alanine (200 g) was added. Each cycle was performed for 60 h in a jar fermentor at 30 °C, 400 rpm, and aeration of 1.0 vvm. The pH of the reaction solution was maintained at 6.0 using 5 M H2SO4
to the reaction mixture at the beginning of the reaction and the other part (200 g) was added after 30 h of incubation, when most of the initially added l-isomer had disappeared. As the reaction proceeded, the l-alanine content decreased rapidly, whereas d-alanine slightly decreased throughout the reaction. At 55 h, l-alanine was almost completely degraded, while 92 g/L d-alanine remained in the reaction solution.
Besides, the chemical purity of the isolated and purified d-alanine was 99.1% and the optical purity was 99.6%.
Reusability of the gel matrix The continuous degradation of l-alanine by immobilized cells prepared with 8% (w/v) polyvinyl alcohol and 2% (w/v) calcium alginate was examined in a fermenter (Fig. 6). The highest catabolism activity of the immobilized cells was noted in the third cycle, followed by gradual decrease in the consumption activity with the increase in the number of degradation cycles. The immobilized cells consistently maintained a high l-alanine utilization rate of more than 90% from the third to eighth cycle, with each degradation cycle lasting for 60 h. After 15 successive degradation cycles under these conditions, the immobilized cells maintained 49% of degradation activity. Isolation of d‑alanine and its optical purity d-Alanine was isolated from the reaction mixture by using a simple procedure (see “Materials and methods” section). After cation exchange and drying, 185 g of d-alanine were obtained, resulting in a yield of 92.5%. The specific rota= −14.5◦ at 20 °C. tion of the obtained d-alanine was [𝛼]20 D
Discussion Cell immobilization is a useful technique in modern biotechnology and its industrial applications. B. subtilis HLZ-68 possesses l-amino acid oxidase and not d-amino acid oxidase and can carry out asymmetric oxidative degradation of l-alanine from dl-alanine. Besides, as this B. subtilis strain did not exhibit racemase activity and could not transform l-alanine into d-alanine, and just consumes l-alanine, leaving d-alanine, it was immobilized in the present study to produce d-alanine. Polyvinyl alcohol and sodium alginate are commonly used immobilized carrier matrices. Polyvinyl alcohol has good mechanical strength, but poor mass transfer performance, and the polyvinyl alcohol gel particles have a strong tendency to aggregate. The immobilization blocks prepared with polyvinyl alcohol could aggregate and reduce the diffusion of substrate and oxygen into the matrices, thus lowering the degradation ability of the immobilized cells, when compared with that of free cells. A similar effect has also been reported by Al-Zuhair and El-Naas (2011). In contrast, sodium alginate presents good mass transfer performance, but low mechanical strength (Garay-Flores et al. 2014). In the present study, blocks prepared with calcium alginate were unable to retain their shape during repeated batch catabolism and disintegrated after three cycles. As a result, the degradation activity of the immobilized cells first
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increased and then decreased. However, when cells were immobilized with a mixture of polyvinyl alcohol and sodium alginate, the carrier exhibited high mechanical strength and good mass transfer performance. Consequently, the l-alanine utilization activity of these immobilized cells was higher than that of free cells and did not decrease owing to the breakdown of the beads. A similar effect has also been reported elsewhere (Dave and Madamwar 2006). Therefore, polyvinyl alcohol–sodium alginate was considered to be the best matrix for asymmetric degradation of dl-alanine to produce d-alanine. Besides, to improve the degradation activity of the immobilized cells, the effect of polyvinyl alcohol and sodium alginate concentrations on the immobilized cells was examined. The results showed that the immobilized cells presented the highest l-alanine consumption activity when the concentrations of polyvinyl alcohol and sodium alginate were increased to 8% (w/v) and 2% (w/v), respectively. Furthermore, to increase the production of d-alanine, 200 g of dl-alanine were added to the reaction mixture after the consumption of l-alanine, and the final concentration of d-alanine obtained was 92.5 g/L. This method not only improved the utilization of the immobilized cells, but also satisfied the industrial production requirements. Moreover, under these conditions, the immobilized cells were able to continuously degrade l-alanine over 15 cycles. An increase in the degradation activity of the immobilized cells during the early cycles could be owing to appropriate adaptation and growth of the cells in the microenvironment. Accordingly, with the increasing number of cycles, the immobilized cells may gradually break and the internal embedding of cells may progressively weaken, thus decreasing l-alanine utilization. Similar observations have been explicitly reported by Mohapatra et al. (2007). After cation exchange and drying, the chemical and optical purity of the obtained d-alanine was 99.1 and 99.6%, respectively, which meet the industrial production requirements. In conclusion, the results of the present study showed that polyvinyl alcohol and calcium alginate are promising compounds for the immobilization of B. subtilis HLZ-68 cells for d-alanine production from dl-alanine by asymmetric degradation. When compared with other microbial processes, immobilized cells can be employed for repeated batch consumption of l-alanine from dl-alanine, and the subsequent separation and purification process is simple. In addition, polyvinyl alcohol and sodium alginate are inexpensive and nontoxic, and the immobilized bacteria can be used for large-scale production of d-alanine from dl-alanine. To the best of our knowledge, this study is the first report on bacterial cell immobilization for d-alanine production. Acknowledgements This work was financially support by the Science Research and Technology Development Project of Nanning (20125193) and Wuzou (201201023).
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