Chitinolytic enzymes from Clostridium aminovalericum: activity screening and purification.
A strain isolated from the feces of takin was identified as Clostridium aminovalericum. In response to various types of chitin used as growth substrates, the bacterium produced a complete array of chitinolytic enzymes: chitinase ('endochitinase'), exochitinase, beta-N-acetylglucosaminidase, chitosanase and chitin deacetylase. The highest activities of chitinase (536 pkat/mL) and exochitinase (747 pkat/mL) were induced by colloidal chitin. Fungal chitin also induced high levels of these enzymes (463 pkat/mL and 502 pkat/mL, respectively). Crab shell chitin was the best inducer of chitosanase activity (232 pkat/mL). The chitinolytic enzymes of this strain were separated from culture filtrate by ion-exchange chromatography on the carboxylic sorbent Polygran 27. At pH 4.5, some isoforms of the chitinolytic enzymes (30% of total enzyme activity) did not bind to Polygran 27. The enzymes were eluted under a stepwise pH gradient (pH 5-8) in 0.1 mol/L phosphate buffer. At merely acidic pH (4.5-5.5), the adsorbed enzymes were co-eluted. However, at pH close to neutral values, the peaks of highly purified isoforms of exochitinases and chitinases were isolated. The protein and enzyme recovery reached 90%.
Simůnek
J
J
Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 142 20 Prague, Czechia. simunek@iapg.cas.cz
Tishchenko
G
G
Rozhetsky
K
K
Bartonová
H
H
Kopecný
J
J
Hodrová
B
B
D000118:Acetylglucosaminidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000581:Amidohydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003013:Clostridium / Q000201:enzymology* / Q000302:isolation & purification / Q000378:metabolism; D004790:Enzyme Induction; D005243:Feces / Q000382:microbiology; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006041:Goats / Q000382:microbiology; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D007527:Isoenzymes; D002686:Chitin; D006026:Glycoside Hydrolases; C019868:chitosanase; D002688:Chitinases; D000118:Acetylglucosaminidase; D000581:Amidohydrolases; C019866:chitin deacetylase