Production in Pichia pastoris, antifungal activity and crystal structure of a class I chitinase from cowpea (Vigna unguiculata): Insights into sugar binding mode and hydrolytic action

Abstract

A cowpea class I chitinase (VuChiI) was expressed in the methylotrophic yeast P. pastoris. The recombinant protein was secreted into the culture medium and purified by affinity chromatography on a chitin matrix. The purified chitinase migrated on SDS-polyacrylamide gel electrophoresis as two closely-related bands with apparent molecular masses of 34 and 37 kDa. The identity of these bands as VuChiI was demonstrated by mass spectrometry analysis of tryptic peptides and N-terminal amino acid sequencing. The recombinant chitinase was able to hydrolyze colloidal chitin but did not exhibit enzymatic activity toward synthetic substrates. The highest hydrolytic activity of the cowpea chitinase toward colloidal chitin was observed at pH 5.0. Furthermore, most VuChiI activity (approximately 92%) was retained after heating to 50 °C for 30 min, whereas treatment with 5 mM Cu caused a reduction of 67% in the enzyme's chitinolytic activity. The recombinant protein had antifungal activity as revealed by its ability to inhibit the spore germination and mycelial growth of Penicillium herquei. The three-dimensional structure of VuChiI was resolved at a resolution of 1.55 Å by molecular replacement. The refined model had 245 amino acid residues and 381 water molecules, and the final R-factor and R values were 14.78 and 17.22%, respectively. The catalytic domain of VuChiI adopts an α-helix-rich fold, stabilized by 3 disulfide bridges and possessing a wide catalytic cleft. Analysis of the crystallographic model and molecular docking calculations using chito-oligosaccharides provided evidences about the VuChiI residues involved in sugar binding and catalysis, and a possible mechanism of antifungal action is suggested.

Antifungal Peptides as Therapeutic Agents

“…The strains differ in their genotypes, which affects the selection of selectable markers, typically antibiotic resistance genes or auxotrophic markers. Histags are commonly used here also to facilitate the purification of recombinant proteinsaffinity metal-chelating chromatography (Niu et al, 2015;Landim et al, 2017). Plant oleosin fusion technology (Ling, 2007;Bhatla et al, 2010) has also been used for the production of iturin A (Popa et al, 2019).…”

Soybean seed coat chitinase as a defense protein against the stored product pest Callosobruchus maculatus

“…Antifungal chitinase with a strong activity toward Fusarium oxysporum, Fusarium solani, Rhizoctonia solani and Trichoderma viride was also purified from the Baozhu pear ( Pyrus ussuriensis ) . A cowpea class I chitinase (VuChiI) recombinant protein inhibited the spore germination and mycelial growth of Penicillium herquei . The expression of a Brassica juncea class IV chitinase gene increased in response to Alternaria infection, jasmonic acid (JA) treatment and wounding .…”

“…27 A cowpea class I chitinase (VuChiI) recombinant protein inhibited the spore germination and mycelial growth of Penicillium herquei. 28 The expression of a Brassica juncea class IV chitinase gene increased in response to Alternaria infection, jasmonic acid (JA) treatment and wounding. 29 A study of the constitutive proteome of tomato genotypes resistant and susceptible to late blight, an important disease of the tomato, showed that a 26-kDa chitinase was more abundant in the resistant genotype and could be associated with the resistance.…”

Chitinases As The Key To The Interaction Between Plants And Microorganisms

“…Jednym z najważniejszych utrudnień jest złożona optymalizacja aktywności chitynaz, która wynika ze zróżnicowania ich cech, począwszy od obecności i lokalizacji wielu domen [45,48], czy różnice w mechanizmach hydrolitycznych, w stosunku do natury wytworzonych produktów [37,91]. Skutkuje to złożonością procesu optymalizacji produkcji chityny w tym doboru warunków jak pH czy temperatura, zastosowanie różnych źródeł węgla, dopasowanie specyficznego substratu i jego stężenia a także obecność induktorów i inhibitorów [25,36,58,65].…”

“…W ten sposób pozyskiwano także rekombinowane chitynazy pochodzenia roślinnego o aktywności antygrzybiczej. Wykorzystując jako system ekspresyjny drożdże Pichia pastoris pozyskano chitynazy klasy I z wspięgi wężowatej (Vigna ungui culata) o aktywności przeciwko Penicillium her quei [48], z kukurydzy o aktywności przeciwko Fusa rium graminearum [16]. Wykorzystując bakteryjny system ekspresyjny jakim jest Escherichia coli uzyskano chitynazę z klasy I pochodzącą z pietruszki (Hordeum vulgare L.) o potwierdzonej aktywności przeciwko Alter naria solani, Fusarium spp., Rhizoctonia solani i Verticillum dahliae [93].…”