**1. Introduction**

Levansucrase (LS, EC 2.4.1.10), inulosucrase (IS, EC 2.1.4.9), and β-fructofuranosidase (Ffase, EC 3.2.1.26) are three fructansucrases (FSs) that could use sucrose as the substrate to produce fructooligosaccharides (FOSs) and fructans (homopolymers of fructose) [1]. LS and IS belong to the glycoside hydrolase 68 (GH 68) family of enzymes, while Ffase may be categorized as GH68 or GH32 family enzymes. These enzymes can all hydrolyze sucrose and subsequently synthesize fructan, which are defined as hydrolysis reaction (H) and transfructosylation (T), respectively [2]. Both reactions start from sucrose splitting into glucose and a fructosyl moiety. The "T" reaction occurs when the fructosyl moiety is transferred to an acceptor such as the sucrose or the elongating fructan chain. The "H" reaction will happen when the acceptor is water, releasing glucose and fructose [3].

LS, IS, and Ffase have distinguished product specificities. Ffase exclusively synthesizes FOS as its main product, whereas LS and IS could produce FOS and fructan. In addition, the product generated by IS, inulin, primarily consists of β-(2,1) linkages on the polymer backbone, while the fructan generated by LS, levan, harbors β-(2,6) linkages on the main chain [4]. Meanwhile, the identified LSs from more than 40 kinds of microorganisms all alternatively produce β-(2,6) type FOSs, β-(2,6)-type low-molecularweight (LMW, FOS < *<sup>M</sup>*<sup>W</sup> < 5 × <sup>10</sup><sup>4</sup> Da) levan, and <sup>β</sup>-(2,6)-type high-molecular-weight (HMW, *<sup>M</sup>*<sup>W</sup> > 5 × <sup>10</sup><sup>4</sup> Da) levan in the reaction mixture. For instance, the LSs from *Erwinia amylovora* [5] and *Zymomonas* species [6] produced FOSs with a degree of polymerization (DP) below 10 as the main product. However, they only produced a small amount of HMW or LMW levan. The LS from *Bacillus methylotrophicus* SK 21.002 is the only one that produces LMW levan with an *<sup>M</sup>*<sup>W</sup> of 4–5 × 103 Da [7]. By contrast, the LSs from *Acetobacter nitrogenifigens* RG1 [8] and *Lactobacillus reuteri* LTH5448 [9] could synthesize HMW levan as

**Citation:** Guang, C.; Zhang, X.; Ni, D.; Zhang, W.; Xu, W.; Mu, W. Identification of a Thermostable Levansucrase from *Pseudomonas orientalis* That Allows Unique Product Specificity at Different Temperatures. *Polymers* **2023**, *15*, 1435. https://doi.org/10.3390/ polym15061435

Academic Editor: Shashi Kant Bhatia

Received: 18 January 2023 Revised: 9 March 2023 Accepted: 9 March 2023 Published: 14 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the main product, with an *<sup>M</sup>*<sup>W</sup> of 7.1 × <sup>10</sup><sup>6</sup> and 3.9 × 107 Da, respectively. In particular, the LS from *Clostridium acetobutylicum* could exclusively synthesize levan rather than FOSs in the reaction [10].

The properties of levan were varied with *M*W. For instance, the LMW levan was reported to have a potential role in peptic ulcer curing [11] and carcinogenesis initiation stage inhibitory [12], while HMW levan could act as an antivirus agent [13] and pancreatic anticancer agent [14]. Since the practical application of levan dramatically depends on its *M*W, many attempts have been made to explore the potential reason for the product specificity of LSs. Enzyme concentration was regarded as a critical factor. For instance, the LS from *Bacillus subtilis* produced LMW levan (*M*<sup>W</sup> = 7.2 × 103 Da) at a high enzyme concentration (10 U/mL), while synthesizing HMW levan (*M*<sup>W</sup> = 2.3 × <sup>10</sup><sup>6</sup> Da) at a low enzyme concentration (0.1 U/mL) [15]. Additionally, sucrose concentration could also affect product specificity of LS. Relatively high initial sucrose concentrations usually result in the synthesis of FOSs or LMW levan, while lower initial sucrose concentrations favor HMW levan production [16]. On the contrary, the *E*. *amylovora* LS [5] generated FOSs (DP 2–6) at a low sucrose concentration (200 mM), while it synthesized HMW levan at a high sucrose concentration (>500 mM). In addition to enzyme and substrate concentrations, temperature could also affect the product specificity. Lowering temperature was found to favor the T reaction of LS. For instance, the production of HMW levan from *Z*. *mobilis* LS was increased when the temperature was decreased from 40 to 4 ◦C [17].

In this work, a novel LS from the mesophilic bacteria *Pseudomonas orientalis* (Psor-LS) was screened on a *C*. *diazotrophica* LS (Cedi-LS) template. As a result, the Psor-LS showed maximum activity at 65 ◦C, much higher than the other LSs. The Psor-LS retained 46% of its initial activity at 55 ◦C for 9 h and 50% at 45 ◦C for 60 h, exhibiting excellent thermostability. Notably, two thermostable LSs showed a great difference in their product specificity. The Cedi-LS could produce FOSs, LMW (*M*<sup>W</sup> = 4.1 × <sup>10</sup><sup>4</sup> Da), and HMW (*M*<sup>W</sup> = 1.8 × <sup>10</sup><sup>6</sup> Da) levan in the reaction mixture, while the Psor-LS would specifically produce FOS and HMW (*M*<sup>W</sup> = 1.4 × <sup>10</sup><sup>6</sup> Da) levan rather than LMW levan. In particular, temperature was proposed to be significant to the product distribution of Cedi-LS and Psor-LS. When the temperature was changed from 65 to 35 ◦C, Cedi-LS tended to produce HMW with an increased *<sup>M</sup>*<sup>W</sup> of 8.4 × <sup>10</sup><sup>6</sup> Da. By contrast, at 35 ◦C, Psor-LS would produce more FOSs and significantly decrease the HMW levan. This study examines the effect of temperature on the LS product specificity and proposes a thermostable LS suitable for the HMW levan polymer and levan-type FOSs production.
