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KMID : 0545120170270020277
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Journal of Microbiology and Biotechnology
2017 Volume.27 No. 2 p.277 ~ p.288
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Purification and Properties of Extracellular Lipases with Transesterification Activity and 1,3-Regioselectivity from Rhizomucor miehei and Rhizopus oryzae
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Tako Miklos
Kotogan Alexandra
Papp Tamas
Kadaikunnan Shine
Alharbi Naiyf S.
Vagvolgyi Csaba
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Abstract
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Rhizomucor miehei NRRL 5282 and Rhizopus oryzae NRRL 1526 can produce lipases with high synthetic activities in wheat bran-based solid-state culture. In this study, the purification and biochemical characterization of the lipolytic activities of these lipases are presented. SDSPAGE indicated a molecular mass of about 55 and 35 kDa for the purified R. miehei and Rh. oryzae enzymes, respectively. p-Nitrophenyl palmitate (pNPP) hydrolysis was maximal at 40°C and pH 7.0 for the R. miehei lipase, and at 30°C and pH 5.2 for the Rh. oryzae enzyme. The enzymes showed almost equal affinity to pNPP, but the Vmax of the Rh. oryzae lipase was about 1.13 times higher than that determined for R. miehei using the same substrate. For both enzymes, a dramatic loss of activity was observed in the presence of 5 mM Hg2+, Zn2+, or Mn2+, 10 mM N-bromosuccinimide or sodium dodecyl sulfate, and 5-10% (v/v) of hexanol or butanol. At the same time, they proved to be extraordinarily stable in the presence of nhexane, cyclohexane, n-heptane, and isooctane. Moreover, isopentanol up to 10% (v/v) and propionic acid in 1 mM concentrations increased the pNPP hydrolyzing activity of R. miehei lipase. Both enzymes had 1,3-regioselectivity, and efficiently hydrolyzed p-nitrophenyl (pNP) esters with C8-C16 acids, exhibiting maximum activity towards pNP-caprylate (R. miehei) and pNP-dodecanoate (Rh. oryzae). The purified lipases are promising candidates for various biotechnological applications.
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KEYWORD
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Zygomycetes, extracellular lipase, enzyme purification, lipolysis characterization, synthetic activity
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