**2. Results and Discussion**

#### *2.1. Analysis of the AlgM4 Sequence*

The alginate lyase gene *algM4* is 1563 bp in length and encodes a 520-amino-acid protein. A signal peptide analysis of AlgM4 using SignalP 4.0 (http://www.cbs.dtu.dk/services/SignalP/) predicted that the N-terminus of AlgM4 contains a 24-amino-acid signal peptide (Figure 1). A sequence alignment using the National Center for Biotechnology Information (NCBI) BLAST (https://blast.ncbi.nlm.nih. gov/) engine revealed that AlgM4 is a dual-domain protease, containing an N-terminal F5/8 type C domain and a C-terminal alginate-lyase 2 domain. The alginate lyases of the PL7 family contain three highly conserved domains: SA3 (RXEXR), SA4 (YXKAGXYXQ), and SA5 (QXH). The BLAST sequence analysis showed that AlgM4 contains the conserved amino acid sequences of the SA3 domain (RTEMR), the SA4 domain (YFKAGVYNQ), and the SA5 catalytic domain (QIH) (Figure 1). Therefore, AlgM4 belongs to the PL7 family of alginate lyases. The amino acid sequence of AlgM4 was compared with the sequences of other alginate lyases of the PL7 family selected in the CAZy database (http://www.cazy.org/), and a phylogenetic tree was constructed using the neighbor joining method (Figure 2). AlgM4 was most closely related to an alginate lyase from *Vibrio litoralis* BZM-2 (ALP75562.1), with an amino acid sequence similarity of 74% observed between AlgM4 and ALP75562.1, without annotation by genome analysis. These results suggest that AlgM4 is a new alginate lyase of the PL7 family.

**Figure 1.** The deduced amino acid sequences of AlgM4. The signal peptide is underlined; SA3, SA5, and SA4 are indicated with blue symbols.

#### *2.2. Purification and Enzymatic Activity of AlgM4*

Using *V. weizhoudaoensis* M0101 genomic DNA as a template, the *algM4* gene was PCR amplified without the N-terminal signal peptide sequence or the stop codon and ligated into the pET30a(+) plasmid. The plasmid was then transformed into *Escherichia coli* BL21 (DE3) cells for AlgM4 expression. Purified AlgM4 protein with a C-terminal 6×histidine (His) tag was obtained by Ni2+ affinity chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a single protein band with a molecular weight of approximately 55 kDa (Figure 3). The specific activity of the purified AlgM4 was 4638 U/mg (Table 1), exhibiting a 36.7% increase in specific activity compared with the crude enzyme extract.

**Figure 3.** SDS-PAGE analysis of purified AlgM4. Lane M, molecular weight markers; Lane 1, purified AlgM4.

**Table 1.** Summary of AlgM4 purification.

