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In tropical and subtropical areas, peanuts are a very important legume crop that is widely cultivated for food and cooking oil. They are, however, extremely susceptible to a wide range of phytopathogens, particularly soil-borne fungi, which result in low yields and poor seed quality. This study aimed to express three Trichoderma asperellum SH16 42 kDa chitinase-encoding genes in peanut to improve their resistance to some soil-borne fungi. Chi42 is a synthetic, intronless, wild-type gene, whereas syncodChi42-1 and syncodChi42-2 are peanut codon-optimized synthetic genes. These genes were introduced into a local peanut strain through Agrobacterium-mediated transformation. Analysis of the transgenic peanut lines showed that chitinase-specific activities from the syncodChi42-1 and syncodChi42-2 genes were approximately 1.2 and 1.4 times higher than those of the wild-type gene, respectively. The engineered peanut plants also exhibited antifungal activity against the soil-borne pathogenic fungus Sclerotium rolfsii. The transgenic peanut lines transformed with the two synthetic genes have stronger antifungal activities than those transformed with the wild-type version, suggesting that they could be used as novel peanut lines to combat phytopathogenic fungi. [ABSTRACT FROM AUTHOR]

Chitinases are enzymes that catalyze the degradation of chitin, a major component of the cell walls of pathogenic fungi and cuticles of insects, gaining increasing attention for the control of fungal pathogens and insect pests. Production of recombinant chitinase in a suitable host can result in a more pure product with less processing time and a significantly larger yield than that produced by native microorganisms. The present study aimed to express the synthetic chi42 gene (syncodChi42), which was optimized from the chi42 gene of Trichoderma asperellum SH16, in Escherichia coli to produce 42 kDa chitinase (Ta-CHI42); then determined the activity of this enzyme, characterizations and in vitro antifungal activity as well as its immunogenicity in mice. The results showed that Ta-CHI42 was overexpressed in E. coli. Analysis of the colloidal chitin hydrolytic activity of purified Ta-CHI42 on an agar plate revealed that this enzyme was in a highly active form. This is a neutral chitinase with pH stability in a range of 6–8 and has an optimum temperature of 45°C with thermal stability in a range of 25–35°C. The chitinolytic activity of Ta-CHI42 was almost completely abolished by 5 mM Zn2+ or 1% SDS, whereas it remained about haft under the effect of 1 M urea, 1% Triton X-100 or 5 mM Cu2+. Except for ions such as Mn2+ and Ca2+ at 5 mM that have enhanced chitinolytic activity; 5 mM of Na+, Fe2+ or Mg2+ ions or 1 mM EDTA negatively impacted the enzyme. Ta-CHI42 at 60 U/mL concentration strongly inhibited the growth of the pathogenic fungus Aspergillus niger. Analysis of western blot indicated that the polyclonal antibody against Ta-CHI42 was greatly produced in mice. It can be used to analyze the expression of the syncodChi42 gene in transgenic plants, through immunoblotting assays, for resistance to pathogenic fungi. [ABSTRACT FROM AUTHOR]

• This is the first study that successfully expressed genes encoding chitinase 42 kDa from Trichoderma asperellum SH16 in peanuts (Arachis hypogaea) cultivar L14 through Agrobacterium tumefaciens -mediated transformation. • Two synthetic genes (syncodChi42–1 and syncodChi42–2) optimized for codon usage for plant expression proved to be more efficient than their original gene (Chi42) from T. asperellum SH16 in transgenic peanuts. • The high expression level of two synthetic genes in peanut roots as well as their strong resistance to Sclerotium rolfsii promise potential applications in the field of transgenic crops against phytopathogenic fungi. This study aims to extracellularly express the 42 kDa chitinase from Trichoderma asperellum SH16 in peanut (Arachis hypogaea) roots under the regulation of a tissue-specific Asy promoter and the driving of the signal peptide of the rice amylase 3D gene. The 42 kDa chitinase genes were employed in this study, which included one wild-type sequence (Chi42) and two synthetic sequences (syncodChi42–1 and syncodChi42–2) optimized for codon usage for plant expression. From 102 42 kDa chitinase transgenic peanut lines, seven elite lines were discovered, among them two for syncodChi42–1, three for syncodChi42–2, and two for Chi42. The study also revealed chitinase from two synthetic genes had higher expression levels than chitinase from the wild-type gene, with the highest specific activities of approximately 901 U/mg and 1124 U/mg, respectively, compared to 739 U/mg. All seven elite transgenic peanut lines exhibited strong extracellular chitinase activity, and some of them were able to fight the soil-borne pathogenic fungus Sclerotium rolfsii in both in vitro and in vivo assays. The significant chitinase activity of seven elite transgenic peanut lines, particularly five lines carrying one of two synthetic genes, suggests that transgenic crops might be used to prevent phytopathogenic fungi. [ABSTRACT FROM AUTHOR]

The objectives of this study were to purify 42 kDa chitinase derived from Trichoderma asperellum SH16 produced in Nicotiana benthamiana by a polyethylene glycol (PEG)/salt aqueous two-phase system (ATPS). The specific activities of the crude chitinase and the partially purified chitinase from N. benthamiana were about 251 unit/mg and 386 unit/mg, respectively. The study found the 300 g/L PEG 6000 + 200 g/L potassium phosphate (PP) and 300 g/L PEG 6000 + 150 g/L sodium phosphate (SP) systems had the highest partitioning efficiency for each salt in primary extraction. However, among the two types of salt, PP displayed higher efficiency than SP, with a partitioning coefficient K of 4.85 vs. 3.89, a volume ratio V of 2.94 vs. 2.68, and a partitioning yield Y of approximately 95 % vs. 83 %. After back extraction, the enzymatic activity of purified chitinase was up to 834 unit/mg (PP) and 492 unit/mg (SP). The purification factors reached 3.32 (PP) and 1.96 (SP), with recovery yields of about 59 % and 61 %, respectively. SDS-PAGE and zymogram analysis showed that the recombinant chitinase was significantly purified by using ATPS. The purified enzyme exhibited high chitinolytic activity, with the hydrolysis zone's diameter being around 2.5 cm–3 cm. It also dramatically reduced the growth of Sclerotium rolfsii ; the colony diameter after treatment with 60 unit of enzyme for 104 spores was only about 1 cm, compared to 3.5 cm in the control. The antifungal effect of chitinase suggests that this enzyme has great potential for applications in agricultural production as well as postharvest fruit and vegetable preservation. • N. benthamiana expressed the Chi42 gene from T. asperellum to make 42 kDa chitinase. • 42 kDa chitinase was then successfully purified by the aqueous two phase system. • The activity of pure chitinase (834 unit/mg) is 3.32 times higher than the control. • This enzyme shows notable efficacy against the hazardous fungus Sclerotium rolfsii. • High antifungal activity points to possible uses for chitinase in plant protection. [ABSTRACT FROM AUTHOR]