Improving amylase production of Xanthomonas campestris TISTR 840 and its effects on xanthan gum production

Abstract

Raw cassava starch was used as a carbon source to produce xanthan gum by Xanthomonas campestris because its availability in Thailand. The first experiment was done in 5 liter bioreactor with 3 XOL medium containing 1% gelatinized cassava starch that was heat til the soution temperature reach to 80℃. The incubation condition was controlled at pH = 7.0, 30℃, aeration rate 0.5 vvm, and stirred at 200 rpm. It was found that the bacteria could hydrolyze starch by secreted amylase. The highest amylase activity, 0.350 U/ml was foun at 16 hour of cultivation and xanthan gum yield was 9.06 g/l. However, using gelatinized starch gave high impurity, the small molecule substance, n crude xanthan gum. There was 48.97% of impurity was found in the crude xanthan gum. From this reason, 1% of raw cassava starch was used instead of gelatinized form. The result showed amylase activity (0.2777 U/ml) was lower than cultured in gelatinized starch medium. Moreover, using raw starch gave 4 folds lower of xanthan gum yield (2.23 g/l) than using gelatinized form. However, the impurity found in crude xanthan gum produced from raw starch was 25.95%, it was significantly lower than culturing in gelatinized starch. Morover, producton of xanthan gum by raw cassava starch needed peptone as a growth factor. The variation of raw starch content by adjusted C:N ratio in the system and aeration rate were used to improved ability to produced amylase of X. campestrs. The C:N ratio was adjusted as 10:1, 20:1, and 30:1, and aeration rate at 0.0, 0.5 and 1.0 vvm were used in the Central Composite Design experiment. The result showed that increasing of C:N ratio together with aeration rate increased amylase production of the bacteria and the anthan gum was also increased. The optimum condition for highest amylase and xanthan gum production was in the same pint at C:N = 30 and applied aeration rate at 1.0 vvm. In this condition, the X. campestris produced highest amylase activity and xanthan gum at 0.344 U/ml and 7.947 g/l, respectively. Mutation of X. campestris with ethyl methansulfonate (EMS) was also used to improve new strain that could overproduce amylase. The mutant strain was selected by starch agar plate and cultured in 5 liter bioreactor to compare the ability of amylase and xanthan gum production with wild-type strain. It was found that the mutant could produce xanthan gum with significantly higher than the wild-type. The mutant and wildtype produced 5.97 and 4.31 g/l of xanthan gum, respectively.