**1. Introduction**

Quorum sensing (QS) is a communication system that many bacteria aggregates used to regulate their aggregate size via small molecules called autoinducers [1,2]. The process of interfering with QS through degradation of signals is termed as quorum quenching (QQ). *N*-acyl homoserine lactones (AHLs) are QS signals used by a wide range of Gram-negative bacteria. AHL lactonase is one major type of AHL-degrading enzymes, which hydrolyses the lactone ring of AHL molecule to produce corresponding *N*-acyl-homoserine. Since some bacteria use QS to mediate virulence factors and antimicrobial resistance, QQ is considered to be a promising alternative for bacterial disease control, which can attenuate QS-regulated virulence factors production in many bacterial pathogens without any lethal effect and impart less-selective pressures for resistant mutants than conventional antibiotics [3–5]. QQ enzyme is one of the most well-studied methods of QQ. AiiA, the earliest identified QQ enzyme, can

decrease extracellular pectolytic enzyme activity and attenuate pathogenicity of *Erwinia carotovora* [6]. A recent study shows that QQ enzyme (AiiA) and QS inhibitor (G1) demonstrated enhanced QS inhibiting effects on reducing AHL concentration when applied together [7].

MomL, a novel AHL lactonase, was isolated from*Muricauda olearia* Th120 [8,9]. This protein consists of 294 amino acids and has a molecular weight of 32.8 kDa. MomL belongs to the metallo-β-lactamase superfamily, and shows the highest identity of 56.8% with protein Aii20J, which belongs to *Tenacibaculum* sp. 20J [10]. Moreover, MomL shares 54.4% and 24.5% identity with FiaL from *Flaviramulus ichthyoenteri* T78T and AiiA from *Bacillus* sp. 240B1 [10–14]. The wide-ranging substrate properties of MomL confer great advantages in disease prevention because different pathogenic bacteria produce AHL molecules with different chain lengths. For example, AHL produced by *Burkholderia* is C8-HSL [15–17], while that of *Vibrio harveyi* is 3OC4-HSL [18]. Moreover, the ability of MomL to degrade C6-HSL is approximately 10 times higher than that of AiiA [14]. MomL exhibited degradative activity on both short and long-chain AHLs and inhibited the pathogenicity of different pathogenic bacteria [9,19]. In order to investigate its application value, MomL was heterologously expressed by *Bacillus brevis*, and the recombinant strain showed a broad antibacterial spectrum than original strain [20]. Although MomL shares the "HXHXDH~H~D" motif with other AHL lactonases in the metallo-β-lactamase superfamily, this motif of MomL performs different functions from AiiA [14,21,22]. Furthermore, little is known about its catalytic mechanism and other amino acids that are involved in the active site remain unclear. Therefore, elucidating the action mechanism helps to expand the application of MomL and paves a way for marine-derived QQ enzyme research.

*Pectobacterium carotovorum* subsp. *carotovorum (Pcc)* is a bacterial pathogen that can cause severe soft rot of cabbage [23–25]. Extracellular enzymes such as pectate lyases, pectinases, cellulases and proteases produced by *Pcc* are main causes for tissue maceration [26]. Disease factors produced by *Pcc* can be induced by the AHL-based QS system [27]. Thus, as an environmentally friendly biocontrol strategy, QQ can be used to prevent or alleviate symptoms caused by such infections.

Protein engineering is a multi-faceted field that can create desired protein properties via various approaches including protein structure prediction to protein selection from random mutagenesis library [28]. As an early example, the *ebgA* gene of *E. coli K12*, was deleted to lead to the synthesis of ebg enzyme and show enhanced activity toward lactose [29]. The catalytic function of cytochrome c from *Rhodothermus marinus* was enhanced more than 15-fold than industrial catalysts in forming carbon-silicon bonds [30,31]. Building high-quality mutant libraries and high efficiency screening system are crucial steps for selecting functional proteins. Site-directed mutagenesis is a valuable tool for understanding the relationship between enzyme activity and amino acids.

In this study, we improved the efficiency of mutant library establishment using a combination method of error-prone polymerase chain reaction (epPCR) and seamless cloning. In addition, an IPTG in situ photocopying technology was used to perform high-throughput screening of random mutagenesis library. We rapidly obtained two high-activity mutant proteins and identified seven amino acids which are vital for QQ ability of MomL. Furthermore, we investigated the ability of MomL and its mutants to inhibit the agricultural pathogenic bacterium *Pcc* virulence factors and the formation of soft rot on Chinese cabbage.

#### **2. Results**

#### *2.1. Overview of the High-E*ffi*ciency Strategy of Constructing and Screening a Random Mutagenesis Library*

In this study, we built a highly efficient and rapid method to obtain the required variants. This method mainly combined three types of technology, specifically epPCR, seamless cloning and isopropyl-β-d-thiogalactoside (IPTG) in situ photocopying. We selected an appropriate amino acid mutation rate and generated PCR products containing randomly mutated amino acids by performing optimized epPCR of three rounds. The PCR products were cloned into pET-24a(+) vectors via seamless cloning, and the recombinant plasmids were transformed into *E. coli* BL21(DE3). *Chromobacterium*

*violaceum* CV026 can produce violacein in the presence of AHLs with *N*-acyl side chains from C4 to C8 in length. When QQ substances were added, the production of violacein was inhibited. Therefore, in the screening plate containing exogenous C6-HSL and the indicator CV026, C6-HSL can be degraded and the plate will not turn violet when the imprinted *E. coli* BL21 colonies of the random mutagenesis library produced active MomL enzyme. Single colonies were imprinted on the screening plates containing IPTG and indicator CV026. The QQ ability of MomL was estimated by either the white halo or the halo diameter produced in the screening plate and positive mutants were selected. The method used in this study was highly efficient and faster than the traditional method (Figure 1). The analyzation for the efficiency and feasibility of this method were performed using MomL protein as an example.

**Figure 1.** The schematic diagram of high efficiency strategy of constructing and screening random mutagenesis library (**A**) and the process of error-prone polymerase chain reaction (epPCR) and seamless cloning (**B**).
