Isolation and primary screening of the best xylanase-producing Bacillusspecies from Caspian sea sediments

Background and Objective: Xylanase enzyme (xylanase), which is one of the important structuralcomponents of plant cell walls, can cause the decomposition of hemicellulose. The production of thisenzyme has also been reported by fungi, bacteria, seaweed, and snails. Research shows that the mainsource of industrial xylanase is filamentous fungi and bacteria, which are widely used in the paper, textile,biofuel, and food industries. Therefore, the aim of this research is to isolate the best Bacillus species fromthe water sediments of the Caspian Sea and to optimize the production of the highest amount of xylanaseenzyme.methods: In this research, ۱۰ samples of water sediments (mud and silt) from the Caspian Sea werecollected in the Nowshahr region and transferred to the laboratory under sterile conditions and atemperature of ۴ degrees Celsius. In order to further strengthen and grow, each sample was cultivatedindependently in LB liquid medium. Then the heat treatment technique was used to destroy the vegetativecells and the remaining spores, and then serial dilution of ۹ tubes and the pour plate method were alsoperformed. For the purpose of primary screening of xylanase-producing species in the genus Bacillus,obtained by the pour plate method, they were spotted cultured on the specific medium of xylan agar.Following the observation of the enzyme halo around the colonies, the second stage of screening wereperformed using the well method. At this stage, Gram and spore staining, as well as the catalase test, wasperformed to ensure the isolation and screening of the Bacillus genus. Finally, to select the best specieswith the highest halo diameter of enzyme production, the enzyme activity measurement technique ofdinitrosalicylic acid (DNS) was used according to the standard method of Bailey & Miller. Also, byincubating the selected sample at ۱۶۰ rpm and ۳۷°C Celsius for ۲۴ hours, the optimization of xylanaseenzyme production was also evaluated.Findings: In this research, out of ۱۰۰ Bacillus species (aerobic gram-positive, spore-bearing, and catalasepositive) isolated, ۳۰ species were selected in the primary screening stage and ۱۵ species in the secondstage that had the largest diameter of the enzyme halo. However, during screening and incubation at ۱۶۰rpm and ۳۷ degrees Celsius for ۲۴ hours, a Bacillus species (sample no. ۴) with a halo diameter of ۲۴ mmwas selected as the best enzyme producer. The results of measuring the level of enzyme activity by thestandard dinitrosalicylic acid method also show that this sample at ۴۵ degrees Celsius with an acidity of ۹ and optical absorption (OD = ۰.۱۲۷), the highest amount of xylanase enzyme production activity (۶IU/ml) /۷۶) showed.Conclusion: Considering the importance of xylanase and its industrial applications, the search forbacterial and fungal sources capable of producing this enzyme is a priority. Therefore, in the presentstudy, the isolation of xylanase-producing Bacillus species from the Caspian Sea shows that the newnative ecosystems of Iran can have countless capabilities for enzyme-producing microbial species.Another strong point of this research is to find a native species of the country with a high potential forenzyme production in order to carry out additional research in the future, in order to achieve enzymeproduction processes on an industrial and commercial scale

Keywords:

Xylanase enzyme , Bacillus , Dinitrosalicylic acid (DNS) technique