Mesenchymal stem cell (MSC) implantation has emerged as a potential therapy for myocardial infarction (MI). However, the poor survival of MSCs implanted to treat MI has significantly limited the therapeutic efficacy of this approach. This poor survival is primarily due to reactive oxygen species (ROS) generated in the ischemic myocardium after the restoration of blood flow. ROS primarily causes the death of implanted MSCs by inhibiting the adhesion of the MSCs to extracellular matrices at the lesion site (i.e., anoikis). In this study, we proposed the use of graphene oxide (GO) flakes to protect the implanted MSCs from ROS-mediated death and thereby improve the therapeutic efficacy of the MSCs. GO can adsorb extracellular matrix (ECM) proteins. The survival of MSCs, which had adhered to ECM protein-adsorbed GO flakes and were subsequently exposed to ROS in vitro or implanted into the ischemia-damaged and reperfused myocardium, significantly exceeded that of unmodified MSCs. Furthermore, the MSC engraftment improved by the adhesion of MSCs to GO flakes prior to implantation enhanced the paracrine secretion from the MSCs following MSC implantation, which in turn promoted cardiac tissue repair and cardiac function restoration. This study demonstrates that GO can effectively improve the engraftment and therapeutic efficacy of MSCs used to repair the injury of ROS-abundant ischemia and reperfusion by protecting implanted cells from anoikis.

Gayadomonas joobiniege G7 is an agar-degrading marine bacterium belonging to a novel genus. Genomic sequencing of G. joobiniege revealed that AgaJ9 (formerly YjdB) belonging to the glycoside hydrolase (GH) 39 family. It showed the highest similarity (47% identity) to a putative β-agarase from Catenovulum agarivorans DS-2, an agar-degrading marine bacterium sharing the highest similarity in the nucleotide sequence of 16s rRNA gene with G. joobiniege G7. The agaJ9 gene encodes a protein (134 kDa) of 1205 amino acids, including a 23-amino acid signal peptide. The agarase activity of purified AgaJ9 was confirmed by zymogram analysis. The optimum pH and temperature for AgaJ9 activity were determined as 5 and 25 °C, respectively. Notably, AgaJ9 is a cold-adapted β-agarase retaining more than 80% of its activity even at a temperature of 5 °C. In addition, gel filtration chromatography revealed that AgaJ9 exists as two forms, dimer and monomer. Although the two forms had similar enzymatic properties, their kinetic parameters were different. The K and V of dimeric AgaJ9 for agarose was 0.68 mg/ml (5.7 × 10 M) and 17.2 U/mg, respectively, whereas the monomeric form had a K of 1.43 mg/ml (1.2 × 10 M) and V of 10.7 U/mg. Thin-layer chromatography and agarose-liquefying analyses revealed that AgaJ9 is an endo-type β-agarase that hydrolyzes agarose into neoagarotetraose and neoagarobiose. This study is the first report of a GH39 β-agarase with a cold-adapted enzymatic feature, a unique attribute, which may be useful for industrial applications.

A novel β-agarase AgaJ11 belonging to the glycoside hydrolase (GH) 16 family was identified from an agar-degrading bacterium Gayadomonas joobiniege G7. AgaJ11 was composed of 317 amino acids (35 kDa), including a 26-amino acid signal peptide, and had the highest similarity (44 % identity) to a putative β-agarase from an agarolytic marine bacterium Agarivorans albus MKT 106. The agarase activity of purified AgaJ11 was confirmed by zymogram analysis. The optimum pH and temperature for AgaJ11 activity were determined to be 4.5 and 40 °C, respectively. Notably, AgaJ11 is an acidic β-agarase that was active only at a narrow pH range from 4 to 5, and less than 30 % of its enzymatic activity was retained at other pH conditions. The K and V of AgaJ11 for agarose were 21.42 mg/ml and 25 U/mg, respectively. AgaJ11 did not require metal ions for its activity, but severe inhibition by several metal ions was observed. Thin layer chromatography and agarose-liquefying analyses revealed that AgaJ11 is an endo-type β-agarase that hydrolyzes agarose into neoagarohexaose, neoagarotetraose, and neoagarobiose. Therefore, this study shows that AgaJ11 from G. joobiniege G7 is a novel GH16 β-agarase with an acidic enzymatic feature that may be useful for industrial applications.

A novel β-agarase, AgaJ5, was identified from an agar-degrading marine bacterium, G7. It belongs to the glycoside hydrolase family 86 and is composed of 805 amino acids with a 30-amino-acid signal peptide. Zymogram analysis showed that purified AgaJ5 has agarase activity. The optimum temperature and pH for AgaJ5 activity were determined to be 30°C and 4.5, respectively. AgaJ5 was an acidic β-agarase that had strong activity at a narrow pH range of 4.5-5.5, and was a cold-adapted enzyme, retaining 40% of enzymatic activity at 10°C. AgaJ5 required monovalent ions such as Na and K for its maximum activity, but its activity was severely inhibited by several metal ions. The and of AgaJ5 for agarose were 8.9 mg/ml and 188.6 U/mg, respectively. Notably, thin-layer chromatography, mass spectrometry, and agarose-liquefication analyses revealed that AgaJ5 was an endo-type β-agarase producing neoagarohexaose as the final main product of agarose hydrolysis. Therefore, these results suggest that AgaJ5 from G7 is a novel endo-type neoagarohexaose-producing β-agarase having specific biochemical features that may be useful for industrial applications.

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