The microorganisms can convert biomass components like cellulose and hemicellulose into chemical substances. Ustilago maydis can grow on Xylan and release certain chemicals that are of biotechnologically beneficial. This study focuses on identifying the enzyme endo-1,4-beta-xylanase involved in degradation of Xylan.
Hemicellulose constitutes Xylan, which occupies about 35 percent of total plant dry weight. Hemicellulases can hydrolyze 1,4-beta-D-glycosidic linkages in the Xylan chain. As the xylanases form xylooligosaccharides, beta-xylosidases will cleave the oligomeric fragments into xylose. Many cellulolytic enzymes will have the ability to degrade xylans as well. Enzymes degrading xylans are released by a variety of aerobic and anaerobic fungi and bacteria as well as by the arthropods, protozoa, algae and gastropods.
The common microbes that can degrade Xylan are Aspergillus or Trichoderma species. Xylanases can hydrolyze lignocellulosic biomass into sugars which can be transformed into chemicals. Ustilago maydis has the biotechnological potential of producing chemicals like itaconic acid, glycolipids, succinic acid and malic acid. U.maydis belongs to basidiomycetes group and is a phytopathogenic fungus. It is known to cause maize smut disease. It is observed that the xylanolytic ability of U.maydis is lesser than that of Fusarium graminaerium and Aspergillus niger.
The enzymes of U.maydis are not characterized till now and this study endorses the capacity of U.maydis MB215 in reducing Xylan, by identifying a xylanase enzyme in the organism that is encoded by um06350.
In this study, Ustilago maydis strain MB215 (DSM 17144) was used which is grown in YEPS medium. The cell densities of the fungus were measured as their absorbance values at 600nm using Unico spectrophotometer 1201. The dry weight of the fungus is determined using Macherey-Nagel paper MN218B. The reducing sugar concentration was evaluated using the Nelson Somogyi assay and their absorbances were read at 520nm. Xylose standard curve was used for measuring the reducing sugars. The degradation products of Xylan were analyzed using thin layer chromatography (TLC).
To identify the proteins present in the U.maydis culture broth, the culture was centrifuged. The supernatant was lyophilized and dissolved in TrisHCl. To purify the protein, the solution is subjected to SDS PAGE.
A gene construct for gene replacement was built with genomic sequence of U.maydis 521 to upset umxyn11A by reverse genetics. Hygromycin resistant complete replacement construct was transformed into Ustilago maydis MB215 protoplasts. The integration of the gene is tested by Southern blot analysis.
Results of study
The medium supplemented with Xylan was used to grow U.maydis MB215 and the growth was found to have stopped after 28hours. The maximum absorbance of the culture was 8.18+/-0.07, dry weight of the cell was 3.82+/-0.09 g/liter. The maximum rate of growth was 0.1 per hour. The growth cessation was probably due to the drop in pH from 5.63 to 3.37. The concentration of reducing sugars was 3.07+/-0.01 g/liter after 16hours period. The sugar concentration reduced quickly after 28hours to a value of 0.96+/-0.03 g/liter.
The hydrolysis products of Xylan degradation were identified successfully through TLC. In the initial phase of the growth, all the Xylo-oligosaccharides were detected while after 25hours, Xylo-triose was alone observed to be present in the supernatant. It shows the activity of endo-xylanase. The absence of xylobiose and xylose, probably was their usage in biomass production.
There was a gel-precipitate formed in centrifugation after 25hours of growth when the reducing sugars were dropped simultaneously. In TLC, the high molecular weight spot was also absent at the bottom. The precipitate was absent in the case of controls grown on xylose nor they were present in the Xylan containing medium without fungal inoculation. Hence, it is understood that the precipitate resulted from the changes made in the Xylan, by Ustilago maydis MB215. The precipitate was known to be consisting of glucopyranosyluronic acid formed after Xylan degradation.
In an important study by Muller et al., it is revealed that 12 open reading frames are present in U.maydis secretome that are involved in Xylan degradation. They are endo-1,4-beta-xylanase, alpha-L-arabinofuranosidases, beta-galactosidases, acetylxylan esters, and arabinoxylan arabinfuranohydrolase. The analysis of MALDI-TOF on U.maydis MB215 culture grown in Xylan, established that endo-1,4-betaxylanase encoded by um06350.1 was available in the supernatant accompanied by certain biomass degrading enzymes.
Deletion of um06350 in U.maydis
The activity of xylanase (umxyn11A gene) in U.maydis is confirmed by removing the concerned open reading frame and checking the Xylan degrading capacity of the organism. The rate of growth of the mutant was reduced to .09 per hour. The final absorbance of the deletion mutant was 6.78 and the final cell dry weight was 4.5+/-0.04 g/liter compared to that of the wild type. The drop in pH was delayed by 3 hours when compared to the time taken for the pH drop in the wildtype.
The concentration of reducing sugars was 1.95+/-0.03 g/liter observed to have resulted 9 hours later than the time taken in the wildtype. The release of Xylo-oligosaccharides was also slower by the mutant than in the wild type. The deletion mutant could show significant change in the rate of Xylan degradation. The TLC analysis also revealed the production of small oligosaccharides by the deletion mutant. The rate of growth of mutant on xylose was same as that of the wild type. These results establish the assumption that the removed open reading frame was associated with the Xylan degradation and not involved in the xylose uptake processes or in growth processes.
Elena Geiser, Nick Wierckx, Martin Zimmermannand Lars M Blank. Identification of an endo-1,4-beta-xylanase of Ustilago maydis. BMC Biotechnology. 2013, 13: 59.
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