Cloning and extracellular expression of a raw starch digesting α‐amylase (Blamy‐I) and its application in bioethanol production from a non‐conventional source of starch

Abstract

The aim of this study was to clone and efficiently express a raw starch-digesting α-amylase enzyme in the culture media and also to investigate the potential application of this recombinant enzyme in the digestion of non-conventional raw starch for bioethanol production. A raw starch digesting α-amylase gene isolated from Bacillus licheniformis strain AS08E was cloned and extracellularly expressed in E. coli cells using the native signal peptide. The mature recombinant α-amylase (Blamy-I) consisting of 483 amino acid residues was found to be homogenous with a mass of 55.3 kDa (by SDS-PAGE analysis) and a predicted pI of 6.05. Structural and functional analysis of Blamy-I revealed the presence of an extra Ca(2+) -binding region between the A and C domains responsible for higher thermostability of this enzyme. The statistical optimization of E. coli culture conditions resulted in an approximately eightfold increase in extracellular expression of Blamy-I as compared to its production under non-optimized conditions. Blamy-I demonstrated optimum enzyme activity at 80 °C and pH 10.0, and efficiently hydrolyzed raw starch isolated from a non-conventional, underutilized jack fruit seeds. Further utilization of this starch for bioethanol production using Blamy-I and Saccharomyces cerevisiae also proved to be highly promising.

Enhanced extracellular raw starch-degrading α-amylase production in Bacillus subtilis by promoter engineering and translation initiation efficiency optimization

“…The AmyZ3 activity produced by Bacillus amyloliquefaciens BAZ3-16 with the in vitro methylation was 288.70 U/mL [ 41 ]. Using E. coli BL21 as the host, the recombinant Blamy-I activity reached 409.5 U/mL [ 43 ], which was the highest RSDA activity in shake flasks fermentation reported in the current literature as far as we know. In comparison, the RSDA activity of strain BZd343 was 1691.1 U/mL, which was 4.12-fold higher than that of the recombinant strain E. coli BL21 [ 43 ].…”

Finding out various potentials and possibilities of jackfruit seed and its usage in the industry: a review

“…They are crucial as sizing and finishing agents in paper and textiles for non-food usage, whereas they are utilised in foods to thicken, gel, or otherwise control texture (Mukprasirt & Sajjaanantakul 2004), (Noor et al 2014), (Tulyathan et al 2002), (Dutta et al 2011), (Madruga et al 2014), (S Castro et al 2023, (Zhang et al 2021a(Zhang et al , 2021b(Zhang et al , 2021c Food products Jackfruit seeds are used for making various food products such as extruded products (cookies, noodles, etc. ), drinks etc (Brahma & Ray, 2022), (Kumari et al 2022), ), (Ortega-González et al 2022, (Yudhistira 2022) Bioethanol Bioethanol is an alcohol created through microbial fermentation, primarily from carbohydrates provided by sugar-or starch-bearing plants such as corn, sugarcane, sweet sorghum, or lignocellulosic biomass (Ginting et al 2020), (Bahlawan et al 2022), (Arif et al 2018), (Roy et al 2015), (Ochaikul et al 2012), (Kumar et al 2011), (Minh et al 2022), (Chongkhong et al 2012) Oil/Biodiesel Oil/Biodiesel has been utilized throughout the years as a cooking fuel and vehicle fuel…”

Raw starch degrading α-amylases: an unsolved riddle

“…Efficient extracellular expression in Escherichia coli is nowadays possible due to different strategies with various signal peptide sequences. Several recent RSDA were expressed this way for example by using OmpA [51] or native signal peptide sequence [52]. We reported recently a new approach based on the fusion of recombinant precursor to the signal peptide of DsbA for overexpression and secretion of fully active α-amylase originated from Bacillus licheniformis ATCC 9945a (BliAmy) [53] by targeting recombinant precursors to the co-translational signal recognition-particle dependent pathway [54].…”