*2.2. Error-Prone Polymerase Chain Reaction (EpPCR) Condition Optimization with Suitable Mutation E*ffi*ciency*

EpPCR randomly introduces mutant sites, and the mismatch rate is related to the magnesium and manganese ion contents [32,33]. In order to build a more efficient mutant library, 1% were selected as the optimal amino acid mutation rate. To determine the appropriate mismatch rate, Mg2<sup>+</sup> concentration gradient ranging from 1 to 8 mM and Mn2<sup>+</sup> gradient ranging from 0 to 0.6 mM were detected respectively. As shown in Figure S1A,B, specific DNA bands were observed following PCR in different Mg2<sup>+</sup> or Mn2<sup>+</sup> concentration gradient. Next, orthogonal test of the two factors (Mg2<sup>+</sup> and Mn2+) was conducted based on the results of the single factor experiment. Appropriate DNA bands were obtained under the 10 orthogonal test conditions (Figure S1C). We randomly selected 100 single colonies of each condition for sequencing. The results demonstrated that under 1 mM Mg2<sup>+</sup> and 0.2 mM Mn2<sup>+</sup> conditions, 70% of single colonies were suffered in 1–3 mutant sites while no mutation was presented in the other 23% of colonies. Fifty percent of the mutant proteins contained no more than 2 mutant sites under the 2 mM Mg2<sup>+</sup> and 0.1 mM Mn2<sup>+</sup> conditions. The average mutation frequency distribution was under 2 mM Mg2<sup>+</sup> and 0.15 mM Mn2<sup>+</sup>, including 2-3 mutant sites in 50% of mutant proteins and 2-5 mutant sites in 83% of mutant proteins. Besides, under this condition, 100 detected mutant libraries existed at least one mutant site. Under 2 mM Mg2<sup>+</sup> and 0.2 mM Mn2<sup>+</sup>, premature translational termination occurred in 30% of mutant proteins, and there were 7–11 mutant sites per protein. Ultimately, based on these results, 2 mM Mg2<sup>+</sup> and 0.15 mM Mn2<sup>+</sup> conditions were selected to maintain the amino acid mutation rate at approximately 1% (Table S2).

#### *2.3. Screening of a Mutation Library Based on a Seamless and EpPCR Strategy*

A random mutagenesis library was built using the selected epPCR conditions. Next, the traditional cloning method (TA cloning) and seamless cloning were separately employed to ligate random mutant fragments and vectors. There were obvious differences in mutation library abundance between the two methods. The number of mutant proteins obtained via seamless cloning was 15–20 times higher than that obtained using TA cloning (Figure 2). Besides, randomly sequencing results showed that 92% of single colonies contained the *momL* gene while only 8% were false positive colonies with self-ligation plasmids.

**Figure 2.** Efficiency comparison between seamless cloning and traditional cloning (TA). All data are presented as mean ± standard deviation (SD, *n* = 3).

We obtained more than 5000 mutant strains for random mutagenesis library. Subsequently, IPTG in situ photocopying technology was utilized to efficiently screen mutant proteins. In the prescreening step, QQ ability of MomL was estimated by whether the visual white halo showed in screening plate. In this step, approximately 3000 strains were screened; 10% of strains that produced larger halo diameters were chosen for second-round screening. In second round screening step, mutants were screened using crude enzyme supernatant in CV026-loaded screening plate. Single colonies M1–M8 were selected from the area with large white halos while M9–M10 were identified from the region lacking white halos (Figure 3 and Figure S2). Two high-activity mutant proteins, M2 and M3, and the mutant proteins M9 and M10, which lacked activity, were selected for sequencing. The results indicated that Ile144 in M2 was mutated to Val (I144V), and Val149 in M3 was mutated to Ala (V149A). In addition, four amino acids in M9 were mutated, namely, E238G, N179S, N51Y and K82R, and four amino acids in M10 were mutated, namely, M228V, T84A, K205E and L254R.

**Figure 3.** Screening target proteins by isopropyl-β-d-thiogalactoside (IPTG) *in situ* photocopying. M1–M8 are single colonies with highly activity; M9–M10 indicate inactive proteins.

#### *2.4. Analysis of Amino Acids in Mutant Proteins*

To further analyze the functions of single amino acids, we mutated the above amino acids loci and constructed 10 single amino acid mutants: MomLI144V, MomLV149A, MomLN51Y, MomLN179S, MomLM228V, MomLK205E, MomLE238G, MomLL254R, MomLT84A, and MomLK82R (Figure 4). Biochemical test indicated that the activities of MomLI144V and MomLV149A were 1.3 and 1.8 times higher, respectively, than that of wild-type MomL (Figure 5A). Furthermore, MomLE238G was inactive, and the activities of MomLK205E and MomLL254R were reduced by 80%–90% compared to MomL. The activities of MomLN179S/MomLN51Y/MomLK82R/MomLM228V/MomLT84A also decreased, ranging from 40–80% of wild-type MomL activity (Figure 5B). The results indicated that Glu238, Lys205, Leu254, Thr84 and Asn179 are related to hydrolysis reaction of C6-HSL. Changes in every single site can reduce the enzyme activity to 50% or more.

**Figure 4.** Multiple sequence alignment of amino acid sequences of MomL, putative homologues, and other representative *N*-acyl homoserine lactone (AHL) lactonases. Sequence alignment was performed by the MUSCLE program in the MEGA software package and enhanced by ESPript 3.0. MomL homologue from *Eudoraea adriatica* (WP\_019670967) showed the highest score when BLASTP searching nonredundant (NR) databases. Other sequences of AHL lactonase are AiiA from *Bacillus* sp. strain 240B1 (AAF62398), AidC from *Chryseobacterium* sp. strain StRB126 (BAM28988), QlcA from unculturable soil bacteria, and AttM (AAD43990), AiiB (NP 396590) from *Agrobacterium fabrum* C58 and YtnP from *Bacillus*. Filled triangles show amino acids which are essential for MomL activity. Filled rhombuses show amino acids, the mutation of which increased MomL activity.

**Figure 5.** (**A**) Enzyme kinetics experiments of MomL and mutant proteins on different substrates C6-HSL and 3OC10-HSL. (**B**) Protein activity test of mutant proteins. All data are presented as mean ± standard deviation (SD, *n* = 3). An unpaired t-test was performed for testing significant differences between groups (\*\*\* *P* < 0.001, \*\* *P* < 0.01, \* *P* < 0.05). (**C**) Multiple-sequence alignment of the amino acid sequences of MomL, putative homologues, and other representative AHL lactonases. The multiple-sequence alignment procedure is the same as described in Figure 4. (**D**) The structure and active site of AiiA, the homologous protein of MomL. A114 and A119 in AiiA are located near the C-loop.

By screening mutant proteins, we rapidly obtained two live mutant proteins and identified seven amino acids that are involved in QQ ability of MomL. By multiple sequence alignment of MomL and other AHL lactonases belonging to the metallo-β-lactonase superfamily, we found that Ile144, Val149, Asn179, Lys205 were variable amino acids in the conserved domain "HXHXDH ~ 60aa ~ H", and may be directly related to the catalytic reaction; while Thr84, Glu238 and Leu254 were amino acids outside the conserved domain, and may be related to maintaining protein stability. In addition, by analyzing the structure of AiiA, the homologous protein of MomL, we found that I144 and V149 in MomL (A114 and A119 in AiiA) are located near the catalytic ring of the active center (C-loop in Figure 5C,D). We speculated that the mutation of I144V and V149A may affect the enzyme activity by affecting the conformation of the C-loop.
