Xylanase Produced By Actinomycetes, Separation, Screening, And Polyphasic Taxonomic Study

Xylan is a kind of hemicellulose rich in broad-leaf tree and annuals cell walls which is predominantly linked byβ-1,4-xylose units in cellulose. Xylan is one of the most significant recyclable resources, which makes up 30% of total biomass in nature. The xylanase is a complex system of hydrolytic enzymes including endo-β-1,4-Xylanases, exo-β-1,4-Xylanases, andβ-D-Xylanase. This paper focuses on endo-β-1,4-xylanases. This study focuses on endo-β-1,4-Xylanases, exo-β-1,4-Xylanases.Actinomycetes isolation work was carried out and xylanase activity tests were followed, then some of these isolates were classified in this study. The novel actinomycetes that can produce xylanases were discoved in this study.Dilution plating method was employed to isolate actinobacterial strains in this study. The meadow soil and forest soil samples collected from XiShuangBanNa, Xingjiang, Qinghai, Thailand, and Laos were used in this study, and 16 media mainly contained xylan as the predominent carbon source and different nitrogen sources, which were supplemented with magnesium ion, potassium ion, calcium ion, phosphate, vitamins, essential trace & semi-trace elements were prepared. In order to discover novel xylanase-producing actinomycetes, selective inhibitors were also added to the isolation media in group and four sorts of soil pretreatment were used in this study. As a result, a total of 691 actinobacterial strains were isolated, including 429 rare actinomycetes which made up 62.1% of all strains.The activity of xylanase produced by 691 actinomycetes strains were tested using Azo-wheat arabinoxylan manufactured by Megazyme co. as the substrate. The activity of xylanase produced by 113 strains was greater than 10 IU/ml and reevaluated, among which 93 strains were Streptomyces-morphology actinomycetes, and 20 strains were rare actinomycetes-morphology actinomycetes. The activity of xylanase produced by Streptomyces sp. I09A-00123 was found highly greater than other strains. The conditions of fermentation and xylanase activity measurement were optimized preliminarily. The optimum reaction temperature, pH were 55℃, and 6.5, respectively. Xylanase activity retained over 98% after 30 min incubation in water bath at 40℃,58% after 30 min incubation in water bath at 55℃, while only 13% after 30 min incubation in water bath at 70℃. The optimum carbon source, nitrogen source, pH value, for strain I09A-00123 to produce xylanase were xylan, NH4NO3, and 6.5, respectively, and the optimum fermentation time were 60h at 28℃.16S rRNA gene sequencing result showed that the isolated xylanase-producing actinomycetes strains belonged to Streptomyces, Micromonospora, Actinomadura, Amycolatopsis, Pseudonocardia, Arthrobacter, Citricoccus, Agromyces, Actinopolyspora etc. Amycolatopsis and Actinopolyspora were found to produce xylanase for the first time. This study extends the research scope of xylanase-producing microbes.According to morphological characteristics and 16S rRNA gene sequencing results, five actinomycetes strains were selected to be taxonomy studied using polyphasic taxonomy approch. Two novel actinomycetes species were discovered and discribed based on morphological characteristics, physiological & biochemical tests, chemical analysis, and molecular taxonomy study. The strain CPCC 202699T was proposed as a novel species of the genus Amycolatopsis, with the name Amycolatopsis xylanica sp. nov. for which was isolated from the medium contained xylan as the main carbon and energy source. The related study on the strain CPCC 202699T has been published online in IJSEM. The taxonomy study on the strain CPCC 202695T was also submitted to IJSEM. Strain CPCC 202695T was proposed to representa a novel species in the genus Agromyces, based on the polyphasic taxonomyresults with the name Agromyces flavus sp. nov., for the colonies were yellow on the tested media. Taxonomic status of 1 Citricoccus strain and the other 2 Actinomadura strains were to be further confirmed according to the hybridization values with corresponding closest strains.Xylanase activity screening and actinomycetes taxonomy study were combined in this study. As a result, the novel actinomycetes strains that can produce xylanases were first discoved, which enlarge the study on application of actinomycetes.