Enhancement of pullulanase production from recombinant Bacillus subtilis by optimization of feeding strategy and fermentation conditions

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 OD 600 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.

Microbial community and metabolic function analysis of cigar tobacco leaves during fermentation

“…To account for potential biases between samples with uneven sequencing depth, all read counts from the remaining samples were rarefied to the read depth of the sample with the lowest read number. Specifically, bacterial and fungal communities in each sample were rarefied to 25,762 (the minimum number of bacterial sequences) and 68,080 sequences (the minimum number of fungal sequences), respectively (Glowska et al, 2020 ; Huang et al, 2020 ; Schloss & Handelsman, 2006 ); 154,572 valid bacterial sequences and 408,480 valid fungal sequences remained for subsequent analysis (Table A1 in Appendix 2). Illumina sequence analysis indicated 1,041 bacterial OTUs and 270 fungal OTUs with 97% similarity.…”

Pullulanase: unleashing the power of enzyme with a promising future in the food industry

“…In this context, various authors reported the enhanced production of pullulanase from recombinant Bacillus subtilis ( Zhang et al, 2020b ; Pang et al, 2020 ). Efficient enzyme and metabolite-producing strains produced by CRISPR engineering exist today, highlighting the technology’s enormous potential.…”

“…NM-DCM-1 ( Mesbah and Wiegel, 2018 ) and Lactobacillus plantarum L137 ( Kim et al, 2009 ) showed a comparable increase in enzymatic activity after the deletion of the non-conserved C-terminus. Zhang et al (2020b) also studied the susceptiblility to turn inactive at 50°C with a 50% reduction in its specific activity observed due to N-terminal truncation in case of pullulanase PulPB1. Another strategy of Structure/sequence-guided consensus has been proven, logically easier to create variations and mutant libraries that will promote enzyme-directed evolution and save time on experiments.…”

Properties of a neutral, thermally stable and surfactant-tolerant pullulanase from worker termite gut-dwelling Bacillus safensis as potential for industrial applications

“…In line for various industrial and commercial applications of pullulan, its degradation and enzymatic conversion is highly required, so that variety of useful products could be obtained. However, this complex structure could only be broken down by specific glucanase amylolytic enzyme called pullulanase, a debranching enzyme ( Da Cruz, 2013 ; Zhang et al., 2020 ).…”

“…Debranching enzymes are classified into indirect and direct branching enzymes. Amylo-1,6 glucosidases, indirect debranching enzymes produced by animals and yeast are only capable of hydrolysing a 1,6 - α branch point while pullulanases and isoamylases, classes of direct debranching enzymes found in plants and bacteria have been observed to effectively break α-1,6 glucosidic bond of unmodified substrate ( Hii et al., 2012 ; Zhang et al., 2020 ). Therefore, pullulanase (pullulan α-glucano hydrolase; EC 3.2.1.41) has been highly preferred in industrial processes because of its capability to be involved in saccharification of starch, amylopectin, pullulan and related polysaccharides in combination with other amylolytic enzymes and subsequently yield hydrolysis of α-1, 6 bonds in the polysaccharides with the production of maltose, maltotriose and fructose ( Wu et al., 2015 ; Ma et al., 2015 ; Dakhmouche et al., 2021 ).…”