Proizvodnja nove endoglukanaze, tolerantne na povišenu temperaturu i povećanu pH-vrijednost, od isplativih poljoprivrednih otpadaka s pomoću novoizoliranog soja Bacillus sp. NZ

Abstract

Highly alkalitolerant and moderately thermotolerant Bacillus sp. NZ, isolated from degrading lignocellulose, produced a novel highly thermotolerant and alkalitolerant endoglucanase. Maximum activity of endoglucanase was observed in the medium with the initial pH=9 (920 U/L) after 72 h of fermentation with shaking (250 rpm) at 45 °C. Growth of the organism and enzyme production displayed a precise relationship, showing maxima after 72–96 h of fermentation; however, the organism formed maximum biomass at pH=8, but produced the highest enzyme titre at pH=9. Among the examined crude substrates, wheat bran gave maximum activity of endoglucanase (1960–2280 U/L), and was followed by wheat straw (1480–1760 U/L), filter paper (1360–1450 U/L) and sawdust (1160 U/L). Soybean meal served as a suitable nitrogen source for good production of endoglucanase. The enzyme showed activity over a broad pH range (5–10), with maximum at pH=9–10 (2240–2290 U/L). Endoglucanase was highly active (2210–2300 U/L) over a broad range of temperature (50–100 °C), with maximum activity at 50 °C (2290 U/L) and at 90 °C (2300 U/L). Significantly high activity was observed even at 100 °C (91.3 %). The enzyme was highly stable at temperatures from 60 to 90 °C for a period of 30 min. Endoglucanase was purified 23-fold using ammonium sulphate precipitation. Substantial activity and stability of endoglucanase at elevated temperatures and at pH extremes indicate a potential for its successful application in industries.Iz razgrađene lignocelulozne biomase izoliran je soj Bacillus sp. NZ, iznimno tolerantan na povećanje pH-vrijednosti i umjereno otporan na povišenu temperaturu. S pomoću toga soja proizvedena je nova endoglukanaza, čija je aktivnost i nadalje bila velika pri povišenoj temperaturi i povećanoj pH-vrijednosti. Maksimalna aktivnost endoglukanaze od 920 U/L uočena je u podlozi početne pH=9, nakon 72 sata uzgoja na tresilici (250 rpm) pri 45 ºC. Maksimalni rast soja i proizvodnja enzima postignuti su nakon 72-96 h uzgoja. Iako je najviši titar u enzimskom testu bio pri pH=9, maksimalna je količina biomase proizvedena pri pH=8. Najveća je aktivnost endoglukanaze (1960-2280 U/L) ostvarena primjenom podloge od pšeničnih mekinja, zatim slame žitarica (1480-1760 U/L), filtrirnog papira (1360-1450 U/L) i piljevine (1160 U/L). Sojina je sačma bila prikladan izvor dušika za uspješnu proizvodnju endoglukanaze. Enzimska je aktivnost bila od 2210 do 2300 U/L u širokom rasponu pH-vrijednosti (5-10), a najveća pri pH=9-10 (2240-2290 U/L), te pri velikom temperaturnom rasponu (50-100 ºC). Maksimalna aktivnost enzima postignuta je na 50 (2290 U/L) i 90 ºC (2300 U/L), a bila je velika čak i pri 100 ºC (91,3 %). Enzim je pokazao dobru stabilnost tijekom 30 min na temperaturama od 60 do 90 ºC. Pročišćen je 23 puta pomoću taloga amonijeva sulfata. Značajna aktivnost i stabilnost endoglukanaze pri povišenim temperaturama i povećanoj pH-vrijednosti omogućuje njezinu uspješnu primjenu u industriji