Abstract
Pullulanase is an important starch-debranching enzyme mostly used in starch processing-related food industries. However, the levels of pullulanase produced from recombinant Bacillus subtilis, a Generally Recognized as Safe (GRAS) host, are generally limited. To enhance the activity of pullulanase, batch fermentation and fed-batch fermentation were systematically investigated. The overall purpose is to improve the fermentation yield by optimizing the feeding strategy in the fermentation process, thereby increasing the enzyme activity of pullulanase. Therefore, in this study, the feeding methods, the feeding ingredients, the feeding concentration, and pH values were studied in detail. The optimized fermentation conditions for pullulanase production from recombinant B. subtilis were determined as following: inoculum volume 7%, pH 6.5, the dissolved oxygen level 30%, and constant-rate feeding of 100 mL glucose solution (400 g L−1) in late logarithmic growth. The OD600 of recombinant B. subtilis and enzyme activity were 84.54 and 102.75 U mL−1, which were respectively 141% and 144% higher than that before optimization. These findings provided a prerequisite for further amplification of the fermentation system to obtain higher enzyme activity.
Highlights
Pullulanase (EC 3.2.1.41) is a class of debranching enzymes that catalyze the hydrolysis of alpha-1, 6-glucosidic linkages of unmodified substrates (Nakamura 1996, van der Maarel et al 2002)
During the batch fermentation process, the temperature and dissolved oxygen level were maintained at 37 °C and 30%, respectively, and pH value was natural
The recombinant strain concentration and enzyme activity of the feeding fermentations were better than the batch fermentation
Summary
Pullulanase (EC 3.2.1.41) is a class of debranching enzymes that catalyze the hydrolysis of alpha-1, 6-glucosidic linkages of unmodified substrates (Nakamura 1996, van der Maarel et al 2002). The capability of the pullulanase to decompose the branches makes it widely used in various fields, which emphasizes its leading role as one of the promising new varieties of amylases (Leathers 2003). Pullulanase is widely used in the different kinds of industrial fields. Pullulanase and amylase can improve the efficiency of starch saccharification and reduce the cost. The starch industry is Bacillus subtilis, as a GRAS strain, has many advantages and is used in a wide range of applications as expression host for production of food processing-related enzymes. There are many types and sources of pullulanase that have been previously reported, there are still some limitations in using B. subtilis system in respect of the yield and activity of this enzyme (Kang et al 2011, Singh et al 2010).
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