Improve Production of Pullulanase of Bacillus subtilis in Batch and Fed-Batch Cultures
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Improve Production of Pullulanase of Bacillus subtilis in Batch and Fed-Batch Cultures Fanqiang Meng 1 & Xiaoyu Zhu 1 & Haizhen Zhao 1 & Fengxia Lu 1 & Yingjian Lu 2 & Zhaoxin Lu 1 Received: 28 July 2020 / Accepted: 11 September 2020/ # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Pullulanase is a debranching enzyme that cleaves explicitly α-1,6 glycosidic bonds, which is widely used in starch saccharification, production of glucose, maltose, and bioethanol. The thermal-resistant pullulanase is isolated from a variety of microorganisms; however, the lack of industrial production of pullulanase has hindered the transformation of the laboratory to industry. In this study, the expensive maltose syrup and soybean meal powder were replaced with cheap corn starch and corn steep liquor, exhibiting 440 U/mL of pullulanase in shake flasks by changing the C/N value and the total energy of the medium. Subsequently, the cultivation conditions were explored in a 50-L and 50-m3 bioreactor. In batch culture, the pullulanase activity reached 896 U/mL, while it increased to 1743 U/mL in fed-batch culture by controlling the dissolved oxygen, pH, reducing sugar content, and temperature. Remarkably, the cultivation volume was enlarged to 50 m3 based on the technical parameters of fed-batch culture. The industrial production of pullulanase was successful, and the activity achieved 1546 U/mL. When the product was stored at room temperature (25 °C) for 6 months, the pullulanase activity was over 90%. The half-lives at 60 and 80 °C were 119.45 h and 51.18 h, respectively, which satisfied the industrial application requirements of pullulanase. Keywords Pullulanase . Industrial production . Batch and fed-batch . Thermal stability
* Zhaoxin Lu [email protected]
1
College of Food Science and Technology, Nanjing Agriculture University, 1 Weigang, Nanjing 210095, China
2
College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 21003, China
Applied Biochemistry and Biotechnology
Introduction With the ever-expanding global population, the use of fossil fuels is increasing, causing severe environmental pollution [1, 2]. Based on the concerns about environmental protection and sustainable development, it is better to use bioethanol fuel instead of fossil fuels. Therefore, the efficient transformation of inexpensive biomass resources into ethanol has attracted a great deal of attention [3–5]. In the USA, bioethanol production reaches 45.6 million tons annually, accounting for 10.2% of the total gasoline consumption. Brazil produces 21.89 million tons of bioethanol annually, which exceeds 40% of gasoline consumption [6]. Starch is one of the most abundant biomass on the planet and the primary raw material for the production of bioethanol. Common corn starch contains about 70% amylopectin; even high-amylose corn starch also contains 30% amylopectin. However, glucoamylase hydrolyzes α-1,4 glycosidic bonds of amylose, but cannot hydrolyze α-1,6 glycosidic bonds of amylopectin [7
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