Two Highly Similar Chitinases from Marine Vibrio Species have Different Enzymatic Properties.

Xinxin He,Lingman Ran,Xiao-Hua Zhang,Yanhong Wu,Min Yu,Weizhi Liu

doi:10.3390/md18030139

Abstract

Chitinase, as one of the most important extracellular enzymes in the marine environment, has great ecological and applied values. In this study, two chitinases (Chi1557 and Chi4668) with 97.33% amino acid sequences identity were individually found in Vibrio rotiferianus and Vibrio harveyi. They both were encoding by 561 amino acids, but differed in 15 amino acids and showed different enzymatic properties. The optimal temperature and pH ranges were 45–50 °C and pH 5.0–7.0 for Chi1557, while ~50 °C and pH 3.0–6.0 for Chi4668. K+, Mg2+, and EDTA increased the enzymatic activity of Chi4668 significantly, yet these factors were inhibitory to Chi1557. Moreover, Chi1557 degraded colloidal chitin to produce (GlcNAc)2 and minor GlcNAc, whereas Chi4668 produce (GlcNAc)2 with minor (GlcNAc)3 and (GlcNAc)4. The Kcat/Km of Chi4668 was ~4.7 times higher than that of Chi1557, indicating that Chi4668 had stronger catalytic activity than Chi1557. Furthermore, site-directed mutagenesis was performed on Chi1557 focusing on seven conserved amino acid residues of family GH18 chitinases. Chi1557 was almost completely inactive after Glu154, Gln219, Tyr221, or Trp312 was individually mutated, retained ~50% activity after Tyr37 was mutated, and increased two times activity after Asp152 was mutated, indicating that these six amino acids were key sites for Chi1557.

Highlights

Chitin, consisting of β-(1,4)-linked N-acetyl-d-glucosamine (GlcNAc) units, is the most abundant renewable macromolecule organic matter in marine environments [1]
Similar to MmChi60, proteins Chi1557 and Chi4668 are both four-domain structure chitinases annotated by SMART, almost completely overlapping with a TIM β/α-barrel without α+β insertion at N-terminal as catalytic domain, two immunoglobulin-like (Ig-like) domains and a chitin-binding domain (CBM5/12)
By multiple sequence alignment with CLUSTAL-X, two recombinant chitinases both contain the characteristic motif DxDxDxE in their catalytic domain (Figure S5), which is the signature of family GH18 chitinases [11]

Summary

Introduction

Chitin, consisting of β-(1,4)-linked N-acetyl-d-glucosamine (GlcNAc) units, is the most abundant renewable macromolecule organic matter in marine environments [1]. Chitin is abundant in the marine seawater, but almost absent in marine sediments, indicating that chitin is rapidly utilized in the upper layers of seawater, where efficient microbial degradation was taking place [4]. Many marine bacteria could utilize chitin as nutrient source by secreting chitinases, which are the key proteins for chitin degradation [5]. Chitinases (EC 3.2.1.14) degrade chitin by cleaving β-(1,4)-glyosidic bonds and releasing monoand oligomers (i.e., GlcNAc and chitobiose) [6]. According to their hydrolysis properties, chitinases have been classified as exochitinases and endochitinases. Chitin may be cleaved randomly into shorter fragments by endochitinases, whereas exochitinases or chitobiosidase (EC 3.2.1.29) hydrolyze chitin

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Conclusion