*2.3. Thermo-Tolerance and Heat Recovery of Aly08*

When we sought to determine the thermostability of Aly08, we found an interesting phenomenon. After ice-bath, the residual activity of heat-treatment enzymes was always higher than that of enzymes without an ice-bath (Figure 5A,B). After incubation at 30 ◦C and 40 ◦C for 1 h, Aly08 retained only 17.9% and 9.9% of its initial activity when directly assayed its activities. However, when the enzyme incubation at 0 ◦C for 30 min, the residual activity could recover to 43.8% and 39.4% in the same heat treatment condition (Figure 5A). Moreover, even the enzyme was boiled for 5 min, Aly08 was able to recover 78.3% of its initial activity after 30 min incubating in the ice-bath (Figure 5B).

To determine the optimal incubation temperature that contributed to the recovery of activity after boiling for 5 min, the enzyme was incubated for 30 min at various temperatures (0–80 ◦C). The recovered activity of the enzyme reached levels of 76.3%, 63.9%, and 30.1% after incubation at 0 ◦C, 10 ◦C, and 20 ◦C for 30 min, respectively. When the incubation conditions were above 30 ◦C, the recovery of activity was measured at less than 10% (Figure 5C).

To further determine the optimal incubation time that contributed to the recovery of activity, the enzyme was incubated at 0 ◦C for different times. The activity of Aly08 gradually increased with prolonged culture time at 0 ◦C. Aly08 was rapidly re-activated approximately 56.7% and 71.3% of its activities after incubation at 0 ◦C for 5 min and 10 min, respectively. After incubation for 20 min, the activity was restored to 77.9%, after which the activity recovery rate began to decrease (Figure 5D).

The thermo-stability experiment indicated that low temperature may contribute to the recovery of Aly08. The thermo-tolerance of Aly08 could promote effective storage and transportation as the inactivated enzyme with heat treatment is able to successfully restore most of its activity after incubation at 0 ◦C.

**Figure 5.** Thermo-tolerance and heat recovery of Aly08. (**A**) The difference of thermostability of enzymes incubation at ice-bath for 0 min (black columnar) and 30 min (white columnar). (**B**) Effects of boiling times on enzyme Aly08. Black and white columns indicate the activity of the heat-inactivated enzyme following ice-bath for 0 min and 30 min, respectively. (**C**) Effects of different incubation temperatures on the activity recovery of Aly08 under 5 min heat-inactivated conditions. (**D**) Effects of incubation time at 0 ◦C on the activity recovery of heat-inactivated Aly08. The enzyme activity without any treatment was 100%.

#### *2.4. Action Pattern and Final Product Analysis*

The action mode of Aly08 was determined by size-exclusion chromatography with a SuperdexTM peptide 10/300 column (General Electric Company, Boston, MA, USA) using high-performance liquid chromatography (HPLC) platform (Figure 6). The hydrolysis pattern of Aly08 works as an endo-type because of the rapid depolymerization of substrates, the rise in polydispersity, and the production of intermediate oligosaccharides. Meanwhile, the action mode of Aly08 was further monitored by viscosity analysis (Figure S1). The viscosity of the alginate solution decreased rapidly during the first 5 min following the addition of Aly08 but changed little during subsequent time periods. During the whole observation period, the oligosaccharide content which was tested by A235 increased steadily. It can be further suggested that Aly08 is an endo-type enzyme in accordance with this finding.

The hydrolytic degradations were analyzed by thin-layer chromatography (TLC) method after the alginate was completely degraded (Figure 7A). In the hydrolysis proceeds, there was a gradual decrease of alginate polysaccharide and an accumulation of oligosaccharides with various DPs. And two clear spots of end product (2 h) appeared on the TLC plate, indicating that the migration rate was in good agreement with the alginate disaccharide (DP2) and trisaccharide (DP3) marker. The final degradation product was also determined by negative-ion electrospray ionization mass spectrometry (ESI-MS) (Figure 7B). Two main spectra were 351.1 *m*/*z* [ΔDP2-H]− and 527.2 *m*/*z* [ΔDP3-H]−, corresponding to the molecular mass of the unsaturated alginate disaccharides and trisaccharides, respectively [38].

**Figure 6.** Degradation patterns of Aly08 toward sodium alginate. The elution positions of the unsaturated oligosaccharide product fractions with different degrees of polymerization are shown with arrows: DP1 represents unsaturated monosaccharide, DP2 represents unsaturated disaccharide, DP3 represents unsaturated trisaccharide.

**Figure 7.** The hydrolytic products of Aly08. (**A**) TLC analysis of the hydrolytic products of Aly08. *Lane M*, standard alginate oligosaccharides (DP2-3); Line 0, alginate; Lane 1–9, hydrolytic products of Aly08 for different times (1, 2, 5, 10, 15, 30, 60, 90, and 120 min) toward 0.3% (*w*/*v*) high viscosity sodium alginate. DP2 and DP3 indicate alginate disaccharide and trisaccharide, respectively. (**B**) ESI-MS analysis of the end products of Aly08.

Through HPLC, viscosity, TLC and ESI-MS analysis, Aly08 was shown to degrade alginate polymer as an endo-type manner, eventually degrading alginate into disaccharides and trisaccharides. Previous studies have reported that enzymatic oligosaccharide products have a variety of specific biological activities and possess broad potential application prospects in many fields, such as antioxidant activities, regulation of plant root growth, anti-inflammatory activities, and reguation of lipoprotein metabolism [39–42]. The single homogeneous products in the progress of enzymatic production of oligosaccharides is conducive to the oligosaccharide purification and application. Thus far, most of the reported products of alginate lyase are mixtures of DP2–DP5, such as AlyA-OU02 from

*V. spiendidus* OU02, appear to take disaccharides, trisaccharides, and tetrasaccharides as the main hydrolytic products [28]. Additionally, the final degradation products of the alginate lyase FlAlyA from *Flavobacterium* sp. UMI-01 are DP2-DP5 [34]. Compared with those alginate lyase, the end products of Aly08 are a disaccharide and trisaccharide, which are advantageous for further separation and industrial high-efficiency production. Aly08 may have potential as a tool for the preparation of single homogeneous products of monosaccharides which have wide pharmaceutical applications.
