*Article* **Identification of a Thermostable Levansucrase from** *Pseudomonas orientalis* **That Allows Unique Product Specificity at Different Temperatures**

**Cuie Guang 1, Xiaoqi Zhang 1, Dawei Ni 1, Wenli Zhang 1, Wei Xu 1,\* and Wanmeng Mu 1,2**


**Abstract:** The biological production of levan by levansucrase (LS, EC 2.4.1.10) has aroused great interest in the past few years. Previously, we identified a thermostable levansucrase from *Celerinatantimonas diazotrophica* (Cedi-LS). A novel thermostable LS from *Pseudomonas orientalis* (Psor-LS) was successfully screened using the Cedi-LS template. The Psor-LS showed maximum activity at 65 ◦C, much higher than the other LSs. However, these two thermostable LSs showed significantly different product specificity. When the temperature was decreased from 65 to 35 ◦C, Cedi-LS tended to produce high-molecular-weight (HMW) levan. By contrast, Psor-LS prefers to generate fructooligosaccharides (FOSs, DP ≤ 16) rather than HMW levan under the same conditions. Notably, at 65 ◦C, Psor-LS would produce HMW levan with an average *<sup>M</sup>*<sup>w</sup> of 1.4 <sup>×</sup> <sup>10</sup><sup>6</sup> Da, indicating that a high temperature might favor the accumulation of HMW levan. In summary, this study allows a thermostable LS suitable for HMW levan and levan-type FOSs production simultaneously.

**Keywords:** levansucrase; thermostability; levan; product specificity; application
