Background: The main objective of this study was to isolate and identify moderately halophilic bacteria that produce saline- tolerant PULLULANASE enzyme in hypersaline condition.Methods: The bacteria were isolated by saline culture from saline soils in Degh Biarjemand desert of Shahrod. Then moderately halophilic bacteria were isolated. On the next step, the isolated halophilic bacteria were analyzed for production of pullulanse enzyme. After that, they were identified by molecular and biochemical methods.Results: A total of 11 strains were isolated from Biarjemand desert of Shahrod. 9 out of the 11 halophilic bacteria were moderately halophilic and the two others were halotolerant, but no extreme halphilic bacteria were among the isolated strains. The halophilic bacteria producing hydrolytic enzymes were analyzed. 2 strains of the bacteria produced PULLULANASE which one of them was moderately halophilic bacterium. The halophilic bacterium producing pullulanse enzyme was identified concerning the 16S rRNA sequencing technique as well as it’s molecular and biochemical properties showed that the isolate belongs to the genus Bacillus.Conclusion: In this study, for the first time, the halophilic bacteria from Degh Biarjemand desert of Shahrod were isolated and analyzed for production of PULLULANASE enzyme and special halophilic bacteria were isolated. The isolated halophilic bacterium produced saline-tolerant PULLULANASE enzyme, was tolerant and active in hypersaline condition.

Identification and use of more efficient enzymes in the food and pharmaceutical industries is the focus of many researchers. The aim of this study was to search for a new bacterial strain capable of producing high levels of PULLULANASE applicable to biotechnology, the starch bioprocessing and food industries. A new pullulan hydrolyzing Bacillus strain was isolated and designated SDK2. Morphological and biochemical tests identified the strain as a putative Bacillus cereus strain, which was further characterized and confirmed through 16s rRNA sequencing, and was submitted to GeneBank, under the accession number FR6864500. Quantitative analysis of the strain’s PULLULANASE activity was carried out by the Dintrosalicyclic (DNS) acid-based assay. Thin layer chromatography (TLC) of the culture supernatant, identified the extracellular PULLULANASE as neoPULLULANASE. Effects of temperature and pH on PULLULANASE activity were also studied. The optimum conditions for enzyme activity, as represented by 60oC and a pH of 7, resulted in an activity of 13.43 U/ml, which is much higher than some of the previously reported activities. However, growth of B. cereus SDK2 was also observed at a pH range of 5 to 10, and temperatures of 30oC to 50oC. The effect of metal ions and reagents, such as Mg+2, Ca+2, Zn+2, Cu+2, Fe+2, Ni+2 on enzyme activity showed that Ca+2 ions increased pullulan activity, whereas the other ions and reagents inhibited PULLULANASE activity. The ability of B. cereus SDK2 to produce high levels of neoPULLULANASE stable at 60oC that can generate panose from pullulan, make this newly isolated strain a valuable source of debranching enzyme for biotechnology, the starch bioprocess and medical industries.

Production of ten hydrolytic enzymes was qualitatively studied on the haloarchaeal strains isolated from Aran-Bidgol hypersaline lake in the central desert area of Iran. A total of 293 haloarchea strains were selected among 300 extremely halophilic isolated prokaryotes. Accordingly, 142, 141, 128, 64, 38, 16, 7, 3 and 1 archaeal isolates were able to produce DNase, amylase, lipase, inulinase, PULLULANASE, protease, cellulase, chitinase and xylanase, respectively. None was able to produce pectinase activity. Combined hydrolytic activity was also detected in many strains. A total of 0.3 % of the strains showed 6 hydrolytic activities, 0.3 % of the strains had 5 hydrolytic activities, 5.4 % of the strains presented 4 hydrolytic activities, 25 % of the strains presented 3 hydrolytic activities, 28 % of the strains presented 2 hydrolytic activities and 18 % of the strains presented 1 hydrolytic activity. According to their phenotypic characteristics and comparative partial 16 S rRNA sequence analysis, the halophilic strains were all identified as members of family Halobacteriaceae within 12 different taxa from the following genera: Halorubrum, Haloarcula, Natrinema, Halovivax and Natronomonas. Most enzymes production rate was observed in the genera Halorubrum, Haloarcula and Natrinema whereas; there was not any detectable amount of enzyme production in the genera Halovivax and Natronomonas. The most hydrolytic isolate with 6 combinatorial enzyme production belonged to the genus Natrinema. This investigation showed that the extreme halophilic archaea from Aran-Bidgol lake are a potential source of hydrolytic enzyme under stress conditions and may have possess commercial value.

Starch debranching enzymes that merely hydrolyze α-(1→ 6) glycosidic linkages are classified into isoamylases (EC 3. 2. 1. 68) and PULLULANASEs (EC 3. 2. 1. 41). An exception to this definition is amyloPULLULANASE, a type of PULLULANASE that is capable of cleaving both α-(1→ 4) and α-(1→ 6) linkages. AmyloPULLULANASEs are in demand in liquid sugar industries to generate glucose and some other starch derivatives. PULLULANASEs can be used in conjunction with amylases to improve sugar availability during sugar syrup production. Here, a thermophilic Cohnella sp. A01 amyloPULLULANASE (EC 3. 2. 1. 41) gene, namely Coh4159, was PCR amplified and cloned in pET-26b(+) and transformed into BL21(DE3). Recombinant Coh4159 was heterologously expressed in the presence of 0. 5 mM IPTG and purified via affinity chromatography, and further characterized. Enzyme activity was demonstrated via zymogram analysis in the presence of pullulan. The enzyme had a hydrolytic effect on pullulan with Vmax = 2. 85 μ mol. min-1 and Km = 0. 5 mM. Temperature optima and pH were 60 ˚ C and 6. 0, in which the enzyme kept its activity at wide pH (4-9) and temperature (30-70 ˚ C) ranges. The recombinant enzyme kept 50% of its activity for 60 min, 100 min and 120 min when incubated at 80, 70 and 60 ˚ C, respectively. Amongst metal ions tested, Mn2+ and Ca2+ have improved the enzyme activity both at 5 and 10 mM. The results promise the capability of producing a commercial industrial enzyme well-suited to liquid sugar syrup industry specifications.