*Article* **Cloning, Expression and Characterization of a Novel Cold-Adapted** β**-galactosidase from the Deep-sea Bacterium** *Alteromonas* **sp. ML52**

**Jingjing Sun 1,2,\*, Congyu Yao 1,3, Wei Wang 1,2, Zhiwei Zhuang 4, Junzhong Liu 1,2, Fangqun Dai 1,2 and Jianhua Hao 1,2,5,\***


Received: 11 October 2018; Accepted: 23 November 2018; Published: 6 August 2020

**Abstract:** The bacterium *Alteromonas* sp. ML52, isolated from deep-sea water, was found to synthesize an intracellular cold-adapted β-galactosidase. A novel β-galactosidase gene from strain ML52, encoding 1058 amino acids residues, was cloned and expressed in *Escherichia coli*. The enzyme belongs to glycoside hydrolase family 2 and is active as a homotetrameric protein. The recombinant enzyme had maximum activity at 35 ◦C and pH 8 with a low thermal stability over 30 ◦C. The enzyme also exhibited a *K*<sup>m</sup> of 0.14 mM, a *V*max of 464.7 U/mg and a *k*cat of 3688.1 S−<sup>1</sup> at 35 ◦C with 2-nitrophenyl-β-d-galactopyranoside as a substrate. Hydrolysis of lactose assay, performed using milk, indicated that over 90% lactose in milk was hydrolyzed after incubation for 5 h at 25 ◦C or 24 h at 4 ◦C and 10 ◦C, respectively. These properties suggest that recombinant *Alteromonas* sp. ML52 β-galactosidase is a potential biocatalyst for the lactose-reduced dairy industry.

**Keywords:** *Alteromonas*; deep sea; cold-adapted enzyme; β-galactosidase; lactose-free milk

#### **1. Introduction**

Beta-galactosidase (EC 3.2.1.23), a glycoside hydrolase enzyme, catalyzes the hydrolysis of terminal non-reducing β-d-galactose residues into β-d-galactosides and also catalyzes transgalactosylation reactions [1–3]. Beta-galactosidases exist naturally in many organisms, including microorganisms, plants and animals [4,5]. Most industrial β-galactosidases are obtained from microorganisms. For example, the enzymes isolated from bacteria [6] and yeast [7], with neutral optimum pH, were used in milk products, and fungal [8] enzymes with an acid optimum pH were used in acid whey products. The main application of β-galactosidase is to hydrolyze lactose in milk in the dairy industry to provide lactose-free milk for lactose-intolerant consumers [9]. Another application of β-galactosidase is to transfer lactose and monosaccharide to a series of galacto-oligosaccharides (GOS) which are functional galactosylated products [10–12]. However, β-galactosidase catalyzed at moderate temperatures may cause some issues, e.g., increased production costs, wasted energy and producing undesirable microbial contamination [13]. Cold-adapted β-galactosidases, with low optimum temperatures, could catalyze hydrolysis or transgalactosylation reactions at refrigerating temperatures (4–10 ◦C), thus potentially

overcoming these shortcomings. It may be especially beneficial to the dairy industry which could improve the hydrolysis of lactose at low temperatures.

While a minority of β-galactosidases from fungus are secreted to the extracellular medium, e.g., an acid β-galactosidase from *Aspergillus* spp. [14], β-galactosidases are generally intracellular enzymes in yeast and bacteria. Most reported β-galactosidases are recombinant enzymes derived from heterologous expression than from a natural source. In recent years, the number of cold-adapted β-galactosidases were isolated from psychrophilic and psychrotrophic microorganisms obtained from isothermal cold environments such as polar [15–17], deep-sea [18] and high mountainous regions [19]. The main source of enzymes has been obtained from bacterial strains such as *Arthrobacter psychrolactophilus* strain F2 [20], *Arthrobacter* sp. 32c [21], *Halomonas* sp. S62 [22], *Paracoccus* sp. 32d [23], *Pseudoalteromonas haloplanktis* [24] and *Rahnella* sp. R3 [19]. Only a few cold-adapted β-galactosidases have been discovered from other sources, including psychrophilic-basidiomycetous yeast *Guehomyces pullulan* [25] and Antarctic haloarchaeon *Halorubrum lacusprofundi* [26]. The β-galactosidase from *Arthrobacter psychrolactophilus* strain F2 showed the lowest optimum temperature at 10 ◦C with an optimum pH of 8.

Based on the specific features of sequence, structure, substrate specificity and reaction mechanism, β-galactosidases have been classified into GH1, GH2, GH35 and GH42 families [27]. Most reported microorganism β-galactosidases belong to the GH2 [15,20,23,24,28] and GH42 [19,21,29] families. A typical GH2 β-galactosidase from *E. coli* is made up of five sequential domains and forms a functional tetramer [30]. Most of the characterized cold-adapted β-galactosidases from the GH2 family are tetrameric enzymes, except for a dimeric enzyme from *Paracoccus* sp. 32d and a hexameric enzyme from *Arthrobacter* sp. C2-2 [31]. Hitherto, three crystal structures of cold-adapted β-galactosidases have been obtained: GH42 β-galactosidase from *Planococcus* sp. L4 [32] and two GH2 β-galactosidases from Antarctic bacteria *Arthrobacter* sp. C2-2 [31] and *Paracoccus* sp. 32d [33].

In this study, we report on a gene of β-galactosidase from the marine bacterium *Alteromonas* sp. ML52, isolated from a deep-sea sample. This novel cold-adapted β-galactosidase belongs to the GH2 family and was overexpressed and characterized.

#### **2. Results**

#### *2.1. Characterization and Identification of Strain ML52*

Strain ML52 was isolated from deep-sea water in the Mariana Trench at a depth of 4000 m and found to produce intracellular β-galactosidase at 4 ◦C. Database searches showed that strain ML52 is related to the genus *Alteromonas*. As shown in the neighbor-joining tree (Figure 1) [34,35], strain ML52 formed a monophyletic cluster with *Alteromonas addita* R10SW13<sup>T</sup> (99.9% identity), *Alteromonas stellipolaris* LMG 21861<sup>T</sup> (99.8% identity) and *Alteromonas naphthalenivorans* SN2T (99.3% identity). Strain ML52 was able to produce β-galactosidase on 2216E X-Gal agar in the presence of either lactose or glucose (Figure 2), while the expression of β-galactosidase in *E. coli* containing a lac operon was repressed by glucose [36].

**Figure 1.** Neighbor-joining tree based on 16S rRNA gene sequences showing the phylogenetic position of strain ML52 and closely related *Alteromonas* and *Pseudoalteromonas* species. Bootstrap values (>50%) were calculated for 1000 replicates. The reference 16S rDNA sequences were collected from EzTaxon-e server (www.bacterio.net/) and the National Center for Biotechnology Information (NCBI) Database and aligned using the ClustalX 2.1 program (Conway Institute UCD Dublin, Dublin, Ireland). The phylogenetic tree was obtained using MEGA 7.0 software (Institute for Genomics and Evolutionary Medicine, Temple University, Tempe, AZ, USA).

**Figure 2.** Effects of glucose and lactose on the expression of β-galactosidase of strain ML52. *E. coli* strain BL21(DE3) was used as a control. A, 2216E X-Gal agar with 2% glucose; B, 2216E X-Gal agar with 2% lactose; C, 2216E X-Gal agar with 2% glucose and lactose.
