Alkaline and Thermostable Xylanases of Potential Industrial Importance from Alkaliphiles
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Date
1998-12
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Addis Ababa University
Abstract
Three xylanase producing alkaliphilic bacterial strains designated AR-009, AR-135 and
AR-22-1, were isolated from Lake Arenguadie, an alkaline soda lake in Ethiopia. AR-
009 and AR-135 were identified as strains of the genus Bacillus and Micrococcus,
respectively, while AR-22-1 remains unidentified. The enzyme from all the 3 strains
were purified following standard protein purification procedures, and the molecular
weight of each enzyme was estimated using SDS-PAGE.
Two xylanases designated as xylA and xylB, having molecular weights of 23 and 48
kD, respectively, were purified from the cell free culture supernatant of Bacillus sp.
AR-009. XylA was optimally active at pH 9 while xylB showed optimum activity in
the pH range of 9 to 10 and stable from pH 5 to 11. The optimum temperature for the
activity of xylA was 70°C at pH 8 and 60°C at pH 9. On the other hand xylB was
optiImilly active at 70°C at pH 8 and 75°C at pH 9. Both enzymes showed good
stability at 60°C and at a pH of 8 and 9.
AR-135 xylanase was optimally active at 55°C and a pH of 7.5 - 9.0. Over 60% of
the maximum activity was displayed at pH 11. Its thermal stability above 40°C was
very low. The optimum temperature and pH for the activity of AR-22-1 xylanase was
70°C and 8.0 to 9.5, respectively. The enzyme was stable in a broad pH range and
showed good stability up to 60°C at pH 8 and 9
Agar immobilized cells of Bacillus sp. AR-009 were used for xylanase production with
a view of finding cheap ways of enzyme production. In a batch culture maximum
enzyme production was observed after 48 h and remained high up to 72 h. In repeated
batch cultivation immobilized cells produced appreciable level of xylanase activity in 7
consecutive batches with out any significant decline in productivity. For continuous
xylanase production immobilized cells were packed in a jacketed glass coluIlUl and
sterile medium was continuously pumped. A stable continuous production of xylanase
was observed over a period of one month. The volumetric productivity of the
continuous culture was 17 fold higher than the batch culture using free cells.
Another method of enzyme production investigated was the use of solid state
fennentation. Bacillus sp. AR-009 produced high level of xylanase activity when
grown using solid state fermentation (SSF) with wheat bran serving as a substrate.
Xylanase production was highest at a wheat bran to moisture ratio of between 1:0.5 to
1: 1.5 and a Na2COJ concentration of 10% (w/w). No significant effect was observed
on xylanase production when wheat bran is supplemented with peptone, try one, and
yeast extract, thus avoiding the need to supplement wheat bran with expensive media.
The ability of the organism to produce high titre xylanase activity at alkaline pH and
lower wheat bran to moisture ratio could have a potential advantage in minimising the
risk of contamination. In addition, because the enzyme can be extracted using
minimum volume of liquid, the cost of down stream processing during product
upgrading and the cost of waste treatment steps can be greatly reduced. The use of SSF
for the production of xylanase by Bacillus sp. AR-009 could therefore lead to
substantial reduction in the over all cost of enzyme production.
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Biology