Purification, characterisation and application of α‐l‐rhamnosidase from Penicillium citrinum MTCC‐8897

Summary An α-l-rhamnosidase secreted by Penicillium citrinum MTCC-8897 has been purified to homogeneity from the culture filtrate of the fungal strain using ammonium sulphate precipitation and cation-exchange chromatography on carboxymethyl cellulose. The sodium dodecyl sulphate/polyacrylamide gel electrophoresis analysis of the purified enzyme gave a single protein band corresponding to the molecular mass 51.0 kDa. The native polyacrylamide gel electrophoresis also gave a single protein band confirming the enzyme purity. The Km and Vmax values of the enzyme for p-nitrophenyl α-l-rhamnopyranoside were 0.36 mm and 22.54 μmole min−1 mg−1, respectively, and kcat value was 17.1 s−1 giving kcat/Km value of 4.75 × 104 m−1 s−1. The pH and temperature optima of the enzyme were 7.0 and 60 °C, respectively. The purified enzyme liberated l-rhamnose from naringin, rutin, hesperidin and wine, indicating that it has biotechnological application potential for the preparation of l-rhamnose and other pharmaceutically important compounds from natural glycosides containing terminal α-l-rhamnose and also in the enhancement of wine aroma.

[1]  S. Yadava,et al.  α-L-Rhamnosidase from Aspergillus flavus MTCC-9606 isolated from lemon fruit peel , 2011 .

[2]  P. Yadav,et al.  α-l-Rhamnosidase: A review , 2010 .

[3]  J. Breccia,et al.  Cold-active α-l-rhamnosidase from psychrotolerant bacteria isolated from a sub-Antarctic ecosystem , 2007 .

[4]  M. D. Busto,et al.  Immobilization of naringinase from Aspergillus niger CECT 2088 in poly(vinyl alcohol) cryogels for the debittering of juices , 2007 .

[5]  L. Kang,et al.  The microbiological transformation of steroidal saponins by Curvularia lunata , 2005 .

[6]  Chunzhi Zhang,et al.  Purification and Characterization of Dioscin-α- L -rhamnosidase from Pig Liver , 2005 .

[7]  M. Puri,et al.  Purification and characterization of naringinase from a newly isolated strain of Aspergillus niger 1344 for the transformation of flavonoids , 2005 .

[8]  J. Iborra,et al.  Immobilization of naringinase on glycophase-coated porous glass , 1985, Biotechnology Letters.

[9]  K. Dong-Hyun,et al.  Purification and Characterization of Quercitrin-Hydrolyzing α-L-Rhamnosidase from Fusobacterium K-60, a Human Intestinal Bacterium , 2005 .

[10]  V. Křen,et al.  Generation of an α‐L‐rhamnosidase library and its application for the selective derhamnosylation of natural products , 2004 .

[11]  G. Ellenrieder,et al.  Hydrolytic properties of crude α‐L‐rhamnosidases produced by several wild strains of mesophilic fungi , 2002, Letters in applied microbiology.

[12]  P. R. Kulkarni,et al.  Enzymic debittering of Indian grapefruit (Citrus paradisi) juice , 2002 .

[13]  Chunzhi Zhang,et al.  Purification and Characterization of Ginsenoside-a-L-Rhamnosidase , 2002 .

[14]  J. Visser,et al.  Purification and Characterization of Two Different α-l-Rhamnosidases, RhaA and RhaB, fromAspergillus aculeatus , 2001, Applied and Environmental Microbiology.

[15]  Barbagallo,et al.  A simple method for purifying glycosidases: alpha-l-rhamnopyranosidase from Aspergillus niger to increase the aroma of Moscato wine. , 2000, Enzyme and microbial technology.

[16]  P. Manzanares,et al.  Purification and characterization of an α‐ l‐rhamnosidase from Aspergillus nidulans , 2000 .

[17]  M. Puri,et al.  Production, purification, and characterization of the debittering enzyme naringinase. , 2000, Biotechnology Advances.

[18]  Michikatsu Sato,et al.  Purification and Characterization of an α-L-Rhamnosidase from Pichia angusta X349 , 2000, Bioscience, biotechnology, and biochemistry.

[19]  V. Zverlov,et al.  The thermostable α‐l‐rhamnosidase RamA of Clostridium stercorarium: biochemical characterization and primary structure of a bacterial α‐l‐rhamnoside hydrolase, a new type of inverting glycoside hydrolase , 2000, Molecular microbiology.

[20]  K. Watanabe,et al.  Purification and characterization of intracellular α-l-rhamnosidase from Pseudomonas paucimobilis FP2001 , 2000, Archives of Microbiology.

[21]  S. Kawai,et al.  Characterization of alpha-L-rhamnosidase of Bacillus sp. GL1 responsible for the complete depolymerization of gellan. , 1999, Archives of biochemistry and biophysics.

[22]  K. Murata,et al.  alpha-L-rhamnosidase of Sphingomonas sp. R1 producing an unusual exopolysaccharide of sphingan. , 1998, Bioscience, biotechnology, and biochemistry.

[23]  D. Kim,et al.  Purification and characterization of alpha-L-rhamnosidase from Bacteroides JY-6, a human intestinal bacterium. , 1996, Biological & pharmaceutical bulletin.

[24]  P. Pifferi,et al.  Kinetic and immobilization studies on fungal glycosidases for aroma enhancement in wine , 1994 .

[25]  H. Tsen,et al.  Fiber entrapment of naringinase from Penicillium sp. and application to fruit juice debittering , 1989 .

[26]  J. Brillouet,et al.  Sequential enzymic hydrolysis of potentially aromatic glycosides from grape , 1988 .

[27]  C. Shackleton,et al.  Hydrolysis of dietary flavonoid glycosides by strains of intestinal Bacteroides from humans. , 1987, The Biochemical journal.

[28]  J. Iborra,et al.  A method for assaying the rhamnosidase activity of naringinase. , 1985, Analytical biochemistry.

[29]  F. Pittner,et al.  Preparation of prunin with the help of immobilized naringinase pretreated with alkaline buffer , 1984 .

[30]  Patrick J. Williams,et al.  Studies on the hydrolysis of Vitis vinifera monoterpene precursor compounds and model monoterpene .beta.-D glucosides rationalizing the monoterpene composition of grapes , 1982 .

[31]  A. Olson,et al.  Hydrolysis of high levels of naringin in grapefruit juice using a hollow fiber naringinase reactor , 1981 .

[32]  R. Bourbouze,et al.  Rhamnodiastase et α-l-rhamnosidase de Fagopyrum esculentum , 1975 .

[33]  Y. Kurosawa,et al.  -L-rhamnosidases of the liver of Turbo cornutus and Aspergillus niger. , 1973, Journal of biochemistry.

[34]  K. Weber,et al.  The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. , 1969, The Journal of biological chemistry.

[35]  H. Suzuki.  Hydrolysis of flavonoid glycosides by enzymes (rhamnodiastase from Rhamnus and other sources. , 1962, Archives of biochemistry and biophysics.

[36]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.