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

Morphological and molecular evidence for first records and range extension of the Japanese seahorse, Hippocampus mohnikei (Bleeker 1853) in a bay-estuarine system of Goa, central west coast of India.

Accurate information of taxonomy and geographic range of seahorse species (genus Hippocampus) is the first step in preparing threat assessments and designing effective conservation measures. Here, we report first records and a range extension of the Japanese seahorse, Hippocampus mohnikei (Bleeker, 1853) from the Mandovi estuarine ecosystem of Goa, central west coast of India (CWCI) based on morphological and molecular analyses. The morphometric and meristic traits, particularly short snout (29-35% head length), double cheek spine, low coronet, long tail (51.2-57.9% of standard length), 11 trunk rings, 37-39 tail rings, 15-16 dorsal and 12-14 fin rays observed in four collected specimens matched with the reported key diagnostic morphological criteria of vouchered specimens of H. mohnikei. The seahorse mitochondrial cytochrome oxidase subunit I (COI) and cytochrome b (Cyt b) genes were partially sequenced for conclusive genetic identification of the species under study. Molecular analysis showed that all four individuals clustered together suggesting a monophyletic lineage. Using the maximum similarity with GenBank database, maximum likelihood network and subsequent morphological analysis, the identity of the collected seahorse species was reconfirmed as H. mohnikei. With this new report, the geographic range of H. mohnikei extended significantly to the west from its previously known range. This new sighting of H. mohnikei could indicate a long-distance dispersal facilitated by the prevailing oceanic circulation in the Indo-Pacific region or increased habitat suitability in bay-estuarine systems of Goa, CWCI. Comparison of the pair-wise genetic distances (Kimura 2-parameter) based on COI and Cyt b sequences revealed that the specimens examined in this study are genetically closer to H. mohnikei populations from Vietnam and Thailand than they are to those in Japan and China. To test the hypothesis whether H. mohnikei are vagrants or previously unreported established population, long-term inter-annual sampling and analyses are warranted