Purification and partial characterization of milk-clotting enzyme extracted from glutinous rice wine mash liquor

Abstract

Glutinous rice wine mash liquor is a traditional food of south of China and its ability to coagulate the milk has been proved. The aim of this work was to extract milk-clotting enzyme from glutinous rice wine mash liquor. A partial purified extract of enzyme was obtained by fractional precipitation with (NH 4 ) 2 SO 4 . The fractions obtained by precipitation, 40-90% possessed the milk-clotting activity (MCA) (145.72 U/mg). The 40-90% (NH 4 ) 2 SO 4 fraction was further purified by sephadex G-100 and DEAE-sephadex A-50 with MCA (4,360±50 U/mg), which was confirmed by SDS-PAGE that showed only one band with a molecular mass of 36.0 kDa. Highest MCA was attained at 36 o C. The enzyme was completely inactivated by heating for 20 min at 60 o C. The MCA increased with the decreasing of milk pH from 8.0 to 5.5, and it was active at the wide range of pH 1 to 7. The metal ions Mg 2+ , Ca 2+ , Ba 2+ , Mn 2+ , Al 3+ , Fe 2+ had a very clear function to accelerate milk coagulation whereas Na + and K + decelerated the activity slightly. The curd effect of the milk-clotting enzyme has primarily been studied.

Purification and properties of a milk-clotting enzyme produced by Bacillus amyloliquefaciens D4

“…In contrast, Mn 2+ had a significant stimulatory effect on milkclotting activity, whereas 1 mM Li + and 1 mM Mg 2+ promoted the activity slightly, but had no effect when present at concentrations of only 5 mM or 10 mM. These results were similar to those reported by Wang et al [23], who reported that Na + and K + inhibited the enzyme activity in glutinous rice wine mash liquor. In contrast, Na + and K + had no effect on the activity of milk-clotting enzyme from Bacillus polymyxa B-17 [31].…”

“…2(b)). This result was similar to what is reported for the milk-clotting enzymes from Mucor miehei [22], Rhizopus oryzae [1] and glutinous rice wine mash liquor [23]. Like calf rennet, the purified enzyme from B. amyloliquefaciens D4 had a higher level of milk-clotting activity in the acidic range.…”

A two-stage oxygen supply control strategy for enhancing milk-clotting enzyme production by Bacillus amyloliquefaciens

“…However, this method is ethically arguable and has low productivity. Thus, research has focused on the identification of alternative milk-clotting enzymes (MCEs) among animals, plants and microorganisms as calf rennet substitutes [2][3][4][5][6][7][8].…”

Production of a Milk-Clotting Enzyme by Glutinous Rice Fermentation and Partial Characterization of the Enzyme

“…Wang et al . () obtained a rennet‐like enzyme from glutinous rice wine mash liquor with a molecular weight of 36.0 kDa and they concluded that the enzyme was probably produced by moulds from the rice starter. Whether the MCE in this study was produced by molds present in the rice starter during glutinous rice fermentation needs to be further confirmed.…”

“…In addition, genetically engineered enzymes (Vallejo et al 2012) have been explored. However, currently available MCEs still cannot meet the increasing worldwide demand for cheese production and many researchers continue to search for different sources of MCEs, including those produced by rice fermentation (Su et al 2007;Wang et al 2009).…”