Identification, isolation of bacteria from the hot spring and the purification of ß-galactosidase in Alicyclobacillus acidocaldarius subspecies Rittmanii

The morphological, physiological, biochemical characteristics, as well as lipid and fatty acid composition, quinone types and 16S rRNA sequence analyses of bacteria isolated from Taşlıdere (Batman) and Kös (Bingöl) hot springs were carried out in order to identify the isolates. Moreover, ? -galactosidase enzyme, which has biotechnological importance, of a thermophilic Alicyclobacillus acidocaldarius subsp. rittmannii isolated from Antarctica was purified and some properties of this enzyme were studied.The isolate named as KG5 isolated from Kös hot spring was rod-shaped, Gram-positive, facultatively anaerobic, motile and endospores are oval and centrally located. KG5 grows at at a temperature of 15-45 ? C (optimum 40 ? C ??? which has been considered as mesophilic strain. KG5 for its catalase and glucose utilisation being negative was found to be different from other Bacillus cereus strains.The fatty acid profiles of KG5 and B. cereus were also found to be similar. In both bacteria, nC16:0 is major fatty acid, while iC15:0 is the second most aboundant fatty acid. However, KG5 was found to possess nC18:0 much more than B.cereus. Moreover, 16S rRNA sequence of KG5 was similar to B. cereus at a ratio of 100 %, Bacillus thrungiensis at 99.9 % and Bacillus anthracis at 99.8 %.The isolate named as KG9 isolated from Taşlıdere hot spring was rod-shaped, Gram-positive, facultatively anaerobic, motile and endospores are oval at subterminal location. KG9 grows at at a temperature of 30-55 ? C (optimum 50 ? C ??? which has been considered as moderatively thermophilic strain. KG9 was found to be very similar to Bacillus licheniformis. KG9 is a halotolerant strain which grows in 15 % salt. The fatty acid profiles of KG9 and B. licheniformis showed that major fatty acid is iC15:0. However, nC18:0 was found in KG9, but not in other B. licheniformis strains. 16S rRNA sequence of KG9 was 99 % similar to B.licheniformis.The isolate named as KG8 also isolated from Taşlıdere hot spring was rod-shaped, Gram-positive, facultatively anaerobic, motile and endospores are oval at subterminal location. KG9 grows at a temperature of 35-65 ? C (optimum 55 ? C ??? which has been considered as moderatively thermophilic strain. The fatty acid profile of KG8 indicated that this strain was a member of Anoxybacillus and 16S rDNA sequence of KG8 was found to be similar to Anoxybacillus kamchatkensis at a ratio of 99 %, A.flavithermus, A. ayderensis and A. gonensi? at 98 % and A. pushchiensis at 97 %.A. kamchatkensis, of which 16S rDNA sequence is 99 % similar to KG8, is catalase-negative, starch hydrolysis-negative and grows at an optimum temperature of 60 ? C, while KG8 is catalase (+), starch hydrolysis (+) and grows at an optimum temperature of 55 ? C. Although the similarity of 16S rRNA sequence is high for both strains, KG8 is probably different species due to the differences mentioned above. The homology obtained by DNA-DNA hybridisation with A. kamchatkensis will reveal whether this isolate is a new species or a subspecies.The lipid analysis showed that three strains resemble bacils for their glicolipid, phospholipid and aminolipid content. KG5 possessed phospho-glicolipid, compared to other two strains. In addition, quinone types were studied for identification, which all had MK types. Further investigation showed quinone type for KG8 to be MK-7.Because of the potantial use of extremophile species as enzyme sources, the industrially important, intracellular ? -galactosidase of Alicyclobacillus acidocaldarius subsp. rittmanii previously isolated from Antarctica was purified and some properties of this enzyme were studied.The homogenous enzyme had a specific activity of 113 U/mg protein, with a fold purification of 163 and a yield of 8%. The Km value for ONPG were determined as 8.9mM in the purified ? -galactosidase from A. acidocaldarius subsp. rittmannii. The pH and temperature optima for the purified enzyme are 6.0 and 65 ?C, respectivelyThe molecular mass of the purified enzyme determined by gel filtration on FPLC (native) and SDS-PAGE (under denatured conditions) was 165 and 76 kDa, respectively. It is clear that the ? -galactosidase from A. acidocaldarius subsp. rittmannii is very likely to belong to GH-42, according to its molecular weight, the organism isolated from, and its weak or no binding ability to an affinity column. The results of gel filtration and SDS-PAGE demonstrate that A. acidocaldarius subsp. rittmannii ? -galactosidase is oligomeric (dimeric or trimeric; native mass 165 kDa), composed of similar or identical subunits (mass 76 kDa).The optima of pH and temperature, both constitutive synthesis and inducibility of A. acidocaldarius subsp. rittmannii ? -galactosidase indicate that this enzyme may be suitable for hydrolysis of lactose in milk and sweet whey. Further study is needed in order to evaluate A. acidocaldarius subsp. rittmannii ? -galactosidase for use in milk and whey processing.The starch and casein hydrolysis-positive, catalase-positve characteristics of KG9, casein and gelatine hydrolysis positive characteristics of KG5 and starch hydrolysis-positive characteristics of KG8 showed that these strains can be used as sources of industrially important enzymes (amylase, protease etc.). Particularly the thermophilic species, KG8 and KG9 deserve more attention in this regard.