Improvement of Bioconversion of Vitamin D3 into Calcitriol by Actinomyces hyovaginalis through Protoplast Fusion and Enzyme Immobilization


  • Ahmad M. Abbas Department of Microbiology and Immunology, Faculty of Pharmacy, Ain Shams University (ASU)
  • Khaled M. Aboshanab Department of Microbiology and Immunology, Faculty of Pharmacy, Ain Shams University (ASU)
  • Walid F. Elkhatib Department of Microbiology and Immunology, Faculty of Pharmacy, Ain Shams University (ASU)
  • Mohammad M. Aboulwafa Department of Microbiology and Immunology, Faculty of Pharmacy, Ain Shams University (ASU)
  • Nadia A. Hassouna Department of Microbiology and Immunology, Faculty of Pharmacy, Ain Shams University (ASU)



Bioconversion, protoplast fusion, immobilization, vitamin D3, calcitriol, Actinomyces hyovaginalis


Protoplast fusion and enzyme immobilization techniques were applied to increase calcitriol production from vitamin D3 using Actinomyces hyovaginalis, a local isolate recovered from Egyptian soil, that has a potential bioconversion activity of vitamin D3 into calcitriol. A total of sixteen protoplast hybrids, formed between Actinomyces hyovaginalis isolate and two Bacillus species (B. thuringiensis and B. weihenstephanensis) were screened for vitamin D3 bioconversion activity. Compared to wild type isolate, four hybrids (formed between Actinomyces hyovaginalis isolate and B. thuringiensis) were found to preserve the bioconversion activity; out of which, three hybrids coded V2B, V3B and V8A exhibited higher calcitriol production. The hybrids coded V2B and V8A produced, per 1 L culture medium, about 0.5 and 0.4 mg calcitriol corresponding to 350% and 280%, respectively, increase compared to the wild type isolate. Among different alginate concentrations applied, immobilization of cell lysate of Actinomyces hyovaginalis isolate using 2% alginate showed 140% increase in calcitriol production from vitamin D3 compared to the free cell lysate. Activity of the immobilized form was preserved for five repetitive uses over a period of 15 days but with a 50% decline in production occurring at the fifth use.


[1]DeLuca HF, Schnoes HK.Vitamin D: recent advances. Annu Rev Biochem 1983; 52:411-439.
[2]Kametani T, Furuyama H. Synthesis of vitamin D3 and related compounds. Med Res Rev 1987; 7: 147-171.
[3]Sasaki J, Mikami A, Mizoue K, Omura S. Transformation of 25- and l?-hydroxyvitamin D3 to l?, 25-dihydroxyvitamin D3 by using Streptomyces sp. Isolates. Appl Environ Microbiol 1991; 57: 2841-2846.
[4]Sasaki J, Miyazaki A, Saito M, et al.Transformation of vitamin D3 to 1?,25-dihydroxyvitamin D3 via 25-hydroxyvitamin D3 using Amycolata sp. isolates. Appl Microbiol Biotechnol 1992; 38: 152-157.
[5]Takeda K, Asou T, Matsuda A, et al. Application of cyclodextrin to microbial transformation of Vitamin D3 to 25-hydroxyvitamin D3 and 1?,25-dihydroxyvitamin D3. J Ferment Bioeng 1994; 78: 380-382.
[6]Kang D, Lee H, Park J, Bang J, Hong S, Kim T. Optimization of culture conditions for the bioconversion of vitamin D3 to 1?, 25-dihydroxyvitamin D3 using Pseudonocardia autotrophicaID 9302. Biotechnol Bioprocess Eng 2006; 11: 408-413.
[7]Fujii Y, Kabumoto H, Nishimura K, et al. Purification, characterization, and directed evolution study of a vitamin D3hydroxylase from Pseudonocardia autotrophica. Biochem Biophys Res Commun 2009; 385: 170-175.
[8]Yasutake Y, Fujii Y, Nishioka T, Cheon WK, Arisawa A, Tamura T. Structural evidence for enhancement of sequential vitamin D3 hydroxylation activities by directed evolution of cytochrome P450 vitamin D3 hydroxylase. J Biol Chem 2010;285: 31193-31201.
[9]Kang DJ, Im JH, Kang JH, Ki KH. Bioconversion of vitamin D3 to calcifediol by using resting cells of Pseudonocardia sp. Biotechnol Lett 2015; 37: 1895-1904.
[10]Luo J, Jiang F, Fang W, Lu Q. Optimization of bioconversion conditions for vitamin D3 to 25-hydroxyvitamin D using Pseudonocardia autotrophica CGMCC5098. Bicatal Biotransfor 2016.
[11]Abass AM, Aboshanab KM, Aboulwafa MM, Hassouna NA. Actinomyces hyovaginalis: A novel bacterial isolate with transforming activity of vitamin D3 to 1?, 25-dihydroxyvitamin D3. J Am Science 2011; 7: 230-237.
[12]Abass AM, Aboulwafa MM, Aboshanab KM, Hassouna NA.Optimization of culture conditions for transformation of vitamin D3 to calcitriol by Actinomyces hyovaginalis isolate A11-2. Arch Clinical microb 2011; 2: 1-15.
[13]Miller JH. Experiments in molecular genetics. New York: Cold Spring Harbor Laboratory press 1972.
[14]Sakr MM, Aboulwafa MM, Aboshanab KM, Hassouna NA. Screening and preliminary characterization of quenching activities of soil Bacillus isolates against acyl homoserine lactones of clinically isolated Pseudomonas aeruginosa. Malays J Microbiol 2014; 10: 80-91.
[15]Sambrook J, Russell D. Molecular Cloning: a Laboratory Manual, 3rd ed. New York: Cold Spring Harbor Laboratory press 2001.
[16]Okanishi M, Suzuki K, Umezawa H. Formation and reversion of streptomycete protoplasts: cultural conditions and morphological study. J Gen Microbiol 1974; 80: 389-400.
[17]Hopwood DA, Wright HM.Bacterial protoplast fusion: recombination in fused protoplasts of Streptomyces coelicolor. Mol Gen Genet 1978; 162: 307-317.
[18]Babcock MJ, Kendrick KE. Cloning of DNA involved in sporulation of Streptomyces griseus. J Bacteriol 1988; 170: 2802-2808.
[19]Klein J, Wagner F. Methods for the immobilization of microbial cells. Appl Biochem Bioeng 1983; 4:11-51.
[20]Singh S, Gogoi BK, Bezbaruah RL. Calcium alginate as a support material for immobilization of L-amino acid oxidase isolated from Aspergillus fumigatus. IIOAB J 2012; 3: 7-11.
[21]Srivastava PK, Kayastha AM. Srinivasan Characterization of gelatin- immobilized pigeonpea urease and preparation of a new urea biosensor. Biotechnol Appl Biochem 2001; 34: 55-62.
[22]Pithawala K, Mishra N, Bahadur A. Immobilization of urease in alginate, paraffin and lac. J Serb Chem Soc 2010; 75: 175-183.
[23]Evans DA. Agricultural applications of plant protoplast fusion. Nat Biotechnol 1983; 1: 253-261.
[24]Yari S, Inanlou DN, Yari F, Salech M, Farahound B, Akbarzadeh A. Effects of protoplast fusion on ?-endotoxin production in Bacillus thuringiensis spp CH 141. Iran Biomed J 2002; 6: 25-29.
[25]Pasha C, Kuhad RC, Rao CV.Isolate improvement of thermotolerant Saccharomyces cerevesie VS3 isolate for better utilization of lignocellulosic substrates. J Appl Microbiol 2007; 103: 1480-1489.
[26]Gokhale DV, Puntambekar US, Deobagkar DN. Protoplast fusion: A tool for intergeneric transfer in bacteria. Biotechnol Adv 1993; 11: 199-217.
[27]Aly NA, Teixeira da silva JA, Soliman EA. Intergeneric protoplast fusion by combining genes to improve lipase and alpha amylase enzyme activities. In: Teixeira da silva JA, editor. Volume 5 (Special Issue 2). Dynamic Biochemistry, Process Biotechnology and Molecular Biology. Ontario: Lifescience Global 2011; pp. 28-34.
[28]Kumakura M, Kaetsu I. Immobilization of cellulase using porous polymer matrix. J Appl Polymer Sci 2003; 29: 2713-2718.