On 2017 May 21, Misha Koksharov commented:

This paper provides interesting insights on the role of the 471–481 and 487–495 flexible regions in P. pyralis luciferase and on their utility in improving its thermostability.

For those interested to investigate this topic further, I suggest to look also at the substitution D475P. As can be seen in the alignments below, this substitution occurs naturally in Luciola and in many other beetle luciferases:

D476: http://forumbgz.ru/user/upload/file33577.gif

D489: http://forumbgz.ru/user/upload/file33578.gif

Moreover, in the available structures of L. cruciata luciferase the region 471–481 is not flexible - in contrast to P. pyralis enzyme - so I expect that this mutation should work (where the mutations D476P (Yu H, 2015) and D476N (Amini-Bayat Z, 2012) were inefficient). Interestingly, the successful substitution H489P also naturally occurs in many pH-insensitive luciferases (as shown in the alignments above).

On 2017 May 21, Misha Koksharov commented:

This paper provides interesting insights on the role of the 471–481 and 487–495 flexible regions in P. pyralis luciferase and on their utility in improving its thermostability.

For those interested to investigate this topic further, I suggest to look also at the substitution D475P. As can be seen in the alignments below, this substitution occurs naturally in Luciola and in many other beetle luciferases:

D476: http://forumbgz.ru/user/upload/file33577.gif

D489: http://forumbgz.ru/user/upload/file33578.gif

Moreover, in the available structures of L. cruciata luciferase the region 471–481 is not flexible - in contrast to P. pyralis enzyme - so I expect that this mutation should work (where the mutations D476P (Yu H, 2015) and D476N (Amini-Bayat Z, 2012) were inefficient). Interestingly, the successful substitution H489P also naturally occurs in many pH-insensitive luciferases (as shown in the alignments above).