*Article* **Activity Improvement and Vital Amino Acid Identification on the Marine-Derived Quorum Quenching Enzyme MomL by Protein Engineering**

**Jiayi Wang 1,**†**, Jing Lin 1,**†**, Yunhui Zhang 1, Jingjing Zhang 1, Tao Feng 1, Hui Li 1, Xianghong Wang 1, Qingyang Sun 1, Xiaohua Zhang 1,2,3 and Yan Wang 1,2,3,\***


Received: 4 April 2019; Accepted: 17 May 2019; Published: 21 May 2019

**Abstract:** MomL is a marine-derived quorum-quenching (QQ) lactonase which can degrade various *N*-acyl homoserine lactones (AHLs). Intentional modification of MomL may lead to a highly efficient QQ enzyme with broad application potential. In this study, we used a rapid and efficient method combining error-prone polymerase chain reaction (epPCR), high-throughput screening and site-directed mutagenesis to identify highly active MomL mutants. In this way, we obtained two candidate mutants, MomLI144V and MomLV149A. These two mutants exhibited enhanced activities and blocked the production of pathogenic factors of *Pectobacterium carotovorum* subsp. *carotovorum (Pcc)*. Besides, seven amino acids which are vital for MomL enzyme activity were identified. Substitutions of these amino acids (E238G/K205E/L254R) in MomL led to almost complete loss of its QQ activity. We then tested the effect of MomL and its mutants on *Pcc*-infected Chinese cabbage. The results indicated that MomL and its mutants (MomLL254R, MomLI144V, MomLV149A) significantly decreased the pathogenicity of *Pcc*. This study provides an efficient method for QQ enzyme modification and gives us new clues for further investigation on the catalytic mechanism of QQ lactonase.

**Keywords:** quorum quenching enzyme; error prone PCR; high-throughput screening; site-directed mutagenesis; catalytic ability; *Pectobacterium carotovorum* subsp. *carotovorum (Pcc)*
