Elsevier

Process Biochemistry

Volume 43, Issue 7, July 2008, Pages 758-764
Process Biochemistry

Optimization of β-alanine production from β-aminopropionitrile by resting cells of Rhodococcus sp. G20 in a bubble column reactor using response surface methodology

https://doi.org/10.1016/j.procbio.2008.03.002Get rights and content

Abstract

Resting cells of Rhodococcus sp. G20 were used for the transformation of β-aminopropionitrile to β-alanine, an important beta amino acid. A 23 central composite experimental design was performed with the purpose of optimizing the β-alanine production in a bubble column reactor with 200 mL working volume using response surface methodology (RSM). The individual and interactive effects of three independent variables (cells loading, substrate concentration, airflow rate) on β-alanine production were investigated. A quadratic polynomial predictive model was obtained after statistical analysis to predict the optimum biotransformation conditions. The optimum bioconversion conditions of β-aminopropionitrile in a batch operation for β-alanine production were as follows: cells loading of 16.50 gww/200 mL, substrate concentration of 1.29% (v/v), and airflow rate of 86.56 L/h, under which an overall 40.6% increase in productivity of β-alanine was obtained. The influences of the temperature and pH on the conversion were also studied, and the optimums were 30 °C and pH 7.5. The measured activation energy (Ea) was found to be 22,199 J/mol, thus indicating the presence of diffusional resistance.

Introduction

Nitriles are widely used in organic synthesis as precursors for compounds such as amides and organic acids. However, chemical conversion of nitriles presents several problems: reactions require either strongly acidic or basic media; energy consumption is high; and unwanted by-products (toxic substances or large amount of salts) are formed [1]. Nitrile-hydrolyzing enzymes, including nitrilase (EC3.5.5.1) [2], [3] that transforms the nitriles directly into acids, nitrile hydratase (EC4.2.1.84) [4], [5] and amidase (EC3.5.1.4) [6], [7] that convert nitriles into acids following a two-step reaction via amides as intermediate, have great potential as catalysts for converting nitriles to higher value amides and acids on an industrial scale.

β-Alanine is the only naturally occurring beta amino acid, which are amino acids in which the amino group is at the β-position from the carboxylate group. The IUPAC name for β-alanine would be 3-aminopropionic acid. Unlike its normal counterpart, l-α-alanine, β-alanine has no chiral center. As a component of the naturally occurring peptides carnosine and anserine, and also of pantothenic acid (Vitamin B-5) which itself is a component of coenzyme A [8], β-alanine plays an important role in fine chemical and pharmaceutical synthesis.

Currently, the methods used by the industrial production of β-alanine are mainly concentrated on chemical conversion. The treatment of acrylonitrile with ammonia under heat and pressure could synthesize β-alanine [9]. The process for formation of β-alanine from β-aminopropionitrile in the presence of barium hydroxide under heat is also a chemical method [10]. However, there was only one report relating to β-alanine production from β-aminopropionitrile by microorganisms, which were Alcaligenes sp. OMT-MY14 and Aminobacter aminobrance ATCC 23314 [11].

Rhodococcus spp. are members of the nocardioforms and actinomycetes, and belong to a group of bacteria with a diverse spectrum of carbon and energy compounds [12], [13]. To our knowledge, there were a lot of reports relating to nitrilase, nitrile hydratase, and amidase produced by Rhodococcus spp., for example, the nitrile hydratase and amidase of Rhodococcus equi TG328 have been used to produce 2-aryl propionic acids from the corresponding nitriles [14]; Rhodococcus sp. N-774 was selected as efficient catalysts for the production of acrylamide [15], [16]; Rhodococcus erythropolis NCIMB 11540 was found to have a highly active nitrile hydratase/amidase enzyme system which could accept the nitrile function of alpha-hydroxynitriles (cyanohydrins) as substrates [17]; Rhodococcus rhodochrous J1 was found to have a more powerful ability to produce acrylamide, and could produce acrylic acid and methacrylic acid when induced [18], [19]; Rhodococcus sp. ZJUT-N595 could convert glycolonitrile to glycolic acid [20]. This paper reports on the β-aminopropionitrile bioconversion into β-alanine using resting cells of microorganism, which has been identified as Rhodococcus sp. based on the characteristics of morphology, physiology and biochemical tests, Biolog GP2 identification, and 16S rDNA sequence analysis. As far as we know, this is the first report of biotransformation of β-aminopropionitrile to β-alanine by Rhodococcus sp.

Bioreactors are used as a means of supporting or immobilizing, and hence applying, the biocatalysts in the biotransformation systems [21]. There are several types of bioreactors designed for bioconversion, for example, packed bed bioreactor [22], external-loop fluidized bed airlift bioreactor [23], stirred tank reactor [24], membrane bioreactor [25], microlitre/millilitre shaken bioreactor [26]. A bubble column reactor was tentatively selected for our experimental investigation of β-aminopropionitrile-converting with batch operations, and in the process of conducting such an experiment, we found that the duration of β-aminopropionitrile conversion was short and there was no risk of microbial contamination due to lack of culture medium.

Response surface methodology (RSM), an empirical modeling technique used to estimate the relationship between a set of controllable experimental factors and observed results [27], is an effective tool for optimizing the process. This method has been successfully applied in the optimization of medium compositions, conditions of enzymatic hydrolysis, and fermentation processes [28], [29], [30], [31].

In the present study, response surface method was applied to study the combined effects of various factors such as cells loading, substrate concentration, and airflow rate, which would affect the process of β-aminopropionitrile bioconversion into β-alanine, to obtain optimum β-alanine productivity. In addition, the influences of temperature and pH on the bioconversion, the time course experiments, and the stability of system were also studied. This study will assist further in determining the suitable bioconversion conditions for industrial production.

Section snippets

Microorganism

A bacterial strain namely G20, which could convert β-aminopropionitrile into β-alanine, was isolated and screened from soil samples collected mainly from hill area in Hangzhou. Based on the characteristics of morphology, physiology and biochemical tests, Biolog GP2 identification, and 16S rDNA sequence analysis, strain G20 was identified as Rhodococcus sp. The bacterial colonies on the plate appeared round, dry, convex and pale orange-yellow in color after 48 h incubation. The cells were long

Predictive model of regression

The values of response variable (productivity of β-alanine) obtained under the different experimental conditions designed by RSM and the analysis of variance (ANOVA) for the predictive quadratic model are shown in Table 2, Table 3, respectively.

The analysis was done using coded values. When the regression model is determined with coded values of the variables, the size of each coefficient gives a direct measurement of the importance of each effect [32].

The second-order response surface model

Conclusion

Response surface methodology was employed to evaluate the effects of cells loading, substrate concentration, and airflow rate on the productivity of β-alanine, which was produced in a bubble column reactor catalyzed by Rhodococcus sp. G20. The best conditions were: cells loading 16.50 gww/200 mL, substrate concentration 1.29% (v/v), and airflow rate 86.56 L/h. The influences of the temperature and pH on the conversion were also studied, and the optimums were 30 °C and pH 7.5. The measured

Acknowledgements

This work was supported by Major Basic Research Development Program of China (973 Program) (No. 2007CB714306), Fund of the National High Technology Research and Development Program of China (863 Program) (No. 2006AA02Z241) and Doctor Program of High Education of China (No. 20051033701).

References (38)

  • S.K. Psomas et al.

    Optimization study of xanthan gum production using response surface methodology

    Biochem Eng J

    (2007)
  • Y.L. Gao et al.

    Statistical prediction of effects of food composition on reduction of Bacillus subtilis As 1,1731 spores suspended in food matrices treated with high pressure

    J Food Eng

    (2007)
  • J.B. Joshi

    Computational flow modelling and design of bubble column reactors

    Chem Eng Sci

    (2001)
  • M. Cantarella et al.

    Use of a UF-membrane reactor for controlling selectively the nitrile hydratase-amidase system in Microbacterium imperiale CBS 498-74 resting cells case study: benzonitrile conversion

    Enzyme Microb Technol

    (2006)
  • M. Cantarella et al.

    A study in UF-membrane reactor on activity and stability of nitrile hydratase from Microbacterium imperiale CBS 498-74 resting cells for propionamide production

    J Mol Catal B-Enzym

    (2004)
  • T. Nagasawa et al.

    Interrelations of chemistry and biotechnology. 6. Microbial-production of commodity chemicals

    Pure Appl Chem

    (1995)
  • V. Vejvoda et al.

    Mild hydrolysis of nitriles by Fusarium solani strain O1

    Folia Microbiol

    (2006)
  • Y.G. Zheng et al.

    Isolation, identification and characterization of Bacillus subtilis ZJB-063, a versatile nitrile-converting bacterium

    Appl Microbiol Biotechnol

    (2008)
  • A. Inoue et al.

    Asymmetric synthesis of L-alpha-methylcysteine with the amidase from Xanthobacter flavus NR303

    Adv Synth Catal

    (2005)
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