Biokemijska karakterizacija rekombinantne ksilanaze iz bakterije Bacillus tequilensis BT21 i njezina primjena u proizvodnji ksilobioze iz poljoprivrednih otpadaka

Bacterial strain Bacillus tequilensis BT21 isolated from marine sediments was found to produce extracellular xylanase. The xynBT21 gene encoding xylanase enzyme was cloned and expressed in Escherichia coli. The gene encoded a protein consisting of 213 amino acid residues with calculated molecular mass of 23.3 kDa. Purified recombinant xylanase had optimum activity at 60 °C and pH=6. The enzyme was highly stable in alkaline pH, at pH=7 it remained 100 % active for 24 h, while its activity increased at pH=8 and 9 during incubation. B. tequilensis BT21 xylanase had alkaline pI of 9.4 and belongs to glycosyl hydrolase family 11. The mode of action of XynBT21 on beechwood xylan and xylooligosaccharides was studied. It hydrolysed xylooligosaccharides and beechwood xylan yielding mainly xylobiose (X2) with a small amount of xylose (X1), indicating that XynBT21 was probably an endo-acting xylanase. Enzymatic hydrolysis using wheat bran as a substrate revealed that xylanase reported here has the potential to produce xylobiose from wheat bran. Xylooligosaccharides, especially xylobiose, have strong bifidogenic properties and are increasingly used as a prebiotic. This is the first report that describes this novel xylanase enzyme from marine B. tequilensis BT21 used for the release of xylobiose from wheat bran.Utvrđeno je da soj bakterije Bacillus tequilensis BT21, izoliran iz morskog sedimenta, može proizvesti izvanstaničnu ksilanazu. Gen xynBT21 za kodiranje ksilanaze kloniran je i eksprimiran u bakteriji Escherichia coli, gdje je kodirao protein molekularne mase od 23,3 kDa, koji sadržava 213 aminokiselinskih ostataka. Optimalna aktivnost pročišćene rekombinantne ksilanaze postignuta je pri temperaturi od 60 °C i pH=6. Enzim je bio izuzetno stabilan pri alkalnim pH vrijednostima. Pri pH=7 aktivnost mu je bila 100 % tijekom 24 sata, dok se tijekom inkubacije pri pH=8 i 9 aktivnost enzima povećala. Ksilanaza iz B. tequilensis imala je alkalnu pI vrijednost od 9,4; a pripada obitelji glikozilnih hidrolaza 11. Ispitano je djelovanje ksilanaze XynBT21 na ksilan iz bukve i ksilooligosaharide. Njihovom hidrolizom dobivena je pretežno ksilobioza (X2) uz manju količinu ksiloze (X1), zbog čega je zaključeno da je XynBT21 vjerojatno endoksilanaza. Enzimskom hidrolizom pšeničnih mekinja potvrđeno je da ksilanaza može proizvesti ksilobiozu na toj podlozi. Ksilooligosaharidi, osobito ksilobioza, imaju snažna bifidogena svojstva, pa se sve češće primjenjuju kao prebiotici. Ovo je prvi rad koji opisuje primjenu nove ksilanaze iz morske bakterije B. tequilensis BT21 za oslobađanje ksilobioze iz pšeničnih mekinja

Recombinant Xylanase from Bacillus tequilensis BT21: Biochemical Characterisation and Its Application in the Production of Xylobiose from Agricultural Residues

Bacterial strain Bacillus tequilensis BT21 isolated from marine sediments was found to produce extracellular xylanase. The xynBT21 gene encoding xylanase enzyme was cloned and expressed in Escherichia coli. The gene encoded a protein consisting of 213 amino acid residues with calculated molecular mass of 23.3 kDa. Purified recombinant xylanase had optimum activity at 60 °C and pH=6. The enzyme was highly stable in alkaline pH, at pH=7 it remained 100 % active for 24 h, while its activity increased at pH=8 and 9 during incubation. B. tequilensis BT21 xylanase had alkaline pI of 9.4 and belongs to glycosyl hydrolase family 11. The mode of action of XynBT21 on beechwood xylan and xylooligosaccharides was studied. It hydrolysed xylooligosaccharides and beechwood xylan yielding mainly xylobiose (X2) with a small amount of xylose (X1), indicating that XynBT21 was probably an endo-acting xylanase. Enzymatic hydrolysis using wheat bran as a substrate revealed that xylanase reported here has the potential to produce xylobiose from wheat bran. Xylooligosaccharides, especially xylobiose, have strong bifidogenic properties and are increasingly used as a prebiotic. This is the first report that describes this novel xylanase enzyme from marine B. tequilensis BT21 used for the release of xylobiose from wheat bran