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Fusion of agarase and neoagarobiose hydrolase for mono-sugar production from agar
Alkotaini, B.,Han, N. S.,Kim, B. S. Springer Science + Business Media 2017 Applied microbiology and biotechnology Vol.101 No.4
<P>In enzymatic saccharification of agar, endo- and exo-agarases together with neoagarobiose hydrolase (NABH) are important key enzymes for the sequential hydrolysis reactions. In this study, a bifunctional endo/exo-agarase was fused with NABH for production of mono-sugars (d-galactose and 3,6-anhydro-l-galactose) from agar using only one fusion enzyme. Two fusion enzymes with either bifunctional agarase (Sco3476) or NABH (Zg4663) at the N-terminus, Sco3476-Zg4663 (SZ) and Zg4663-Sco3476 (ZS), were constructed. Both fusion enzymes exhibited their optimal agarase and NABH activities at 40 and 35 A degrees C, respectively. Fusions SZ and ZS enhanced the thermostability of the NABH activity, while only fusion SZ showed a slight enhancement in the NABH catalytic efficiency (K (cat)/K (M)) from 14.8 (mg/mL)(-1) s(-1) to 15.8 (mg/mL)(-1) s(-1). Saccharification of agar using fusion SZ resulted in 2-fold higher mono-sugar production and 3-fold lower neoagarobiose accumulation when compared to the physical mixture of Sco3476 and Zg4663. Therefore, this fusion has the potential to reduce enzyme production cost, decrease intermediate accumulation, and increase mono-sugar yield in agar saccharification.</P>
Alkotaini, B.,Han, N.S.,Kim, B.S. IPC Science and Technology Press ; Elsevier Scienc 2016 Enzyme and microbial technology Vol.93 No.-
<P>Recently, Microbulbifer thertnotolerans JAMB-A94 endo-beta-agarase I was expressed as catalytic domain (GH16) without a carbohydrate-binding module (CBM). In this study, we successfully constructed different fusions of GH16 with its original CBM6 and CBM13 derived from Catenovulum agarivorans. The optimum temperature and pH for fusions GH16-CBM6, GH16-CBM13, GH16-CBM6-CBM13 and GH16-CBM13-CBM6 were similar to GH16, at 55 degrees C and pH 7. All the constructed fusions significantly enhanced the GH16 affinity (Km) and the catalytic efficiency (Kcat/Km) toward agar. Among them, GH16-CBM6-CBM13 exhibited the highest agarolytic activity, for which Km decreased from 3.67 to 2.11 mg/mL and Kcat/Km increased from 98.6 (mg/mL)(-1) sec(-1) to 400.6 (mg/mL)(-1) sec(-1). Moreover, all fusions selectively increased GH16 binding ability to agar, in which the highest binding ability of 95% was obtained with fusion GH16-CBM6-CBM13. Melted agar was prehydrolyzed with GH16-CBM6-CBM13, resulting in a degree of liquefaction of 45.3% and reducing sugar yield of 14.2%. Further addition of Saccharophagus degradans agarolytic enzymes resulted in mono-sugar yields of 35.4% for galactose and 31.5% for 3,6-anhydro-L-galactose. There was no pH neutralization step required and no 5-hydroxymethylfurfural detected, suggesting the potential of a new enzymatic prehydrolysis process for efficient production of bio-products such as biofuels. (C) 2016 Elsevier Inc. All rights reserved.</P>
Anoth Maharjan,Bassam Alkotaini,김범수 한국생물공학회 2018 Biotechnology and Bioprocess Engineering Vol.23 No.1
Bifunctional cellulase (glycoside hydrolase 5, GH5) from Bacillus sp. D04 having both endo- and exoglucanase activities was fused with two types of carbohydrate binding modules (CBMs). CBM3 from Bacillus sp. D04 and CBM9 from Thermotoga maritima Xyn10A were added to GH5 to hydrolyze microcrystalline cellulose (Avicel) as well as water-soluble cellulose (carboxymethyl cellulose, CMC). The optimum temperature of GH5 was 50oC, while it increased to 60oC for the fusion GH5-CBM3 and GH5-CBM9, indicating that addition of CBM increased the thermostability of the enzyme. Addition of CBM3 and CBM9 enhanced the GH5 affinity (KM), for which KM decreased from 104 to 33.9 ~ 35.1 mg/mL for CMC, and from 115 to 55.5 ~ 80.3 mg/mL for Avicel, respectively. The catalytic efficiency (kcat/KM) also increased from 4.80 to 5.36 ~ 6.46 (mL/mg)/sec for CMC, and from 1.77 to 2.40 ~ 4.45 (mL/mg)/sec for Avicel, respectively, by addition of CBM3 and CBM9.
Beom Soo Kim,Bassam Alkotaini,Hyunseok Koo 한국화학공학회 2016 Korean Journal of Chemical Engineering Vol.33 No.5
Polyhydroxyalkanoates (PHAs) are linear polyesters synthesized by microbial fermentation of various substrates. PHAs are accumulated in microbial cells in order to store carbon and energy for future use. We used acid-pretreated red alga (Gelidium amansii) as a cheap, abundant carbon source to produce PHA via batch and fed-batch cultivation of Bacillus megaterium KCTC 2194. After acid treatment of 10% (w/v) G. amansii, 25.5 g/L galactose, 3.6 g/L glucose, 6 g/L 5-HMF, and 1.05 g/L levulinic acid were formed. In batch culture at pH 7, the dry cell weight (DCW) and PHA content increased to 5.5 g/L and 51.4%, respectively. The cell concentration was enhanced by fed-batch cultivation using two feeding strategies: pH-stat and intermittent feeding. When the pH-stat feeding strategy was employed to add concentrated hydrolysate to the fermentor, DCW increased to 8.2 g/L, with 53.2% PHA content. When concentrated hydrolysate was fed using the intermittent feeding strategy, higher DCW (10.1 g/L) was obtained, along with a slight increase of PHA content to 54.5%. This study demonstrates that red algae could be used after simple acid treatment, to produce PHA without steps for enzymatic hydrolysis and inhibitor removal.