Abstract Beauveria bassiana chitinases are involved in degrading the chitin in insects’ exoskeletons and internal structures, and thus are important virulence factors as they participate in initial to final steps of infection. In this work, the B. bassiana (Bv062 isolate) open reading frame (ORF) encoding a chitinase identified as Chit37 (orthologous Bvchit1) was molecularly cloned and expressed in Escherichia coli, and the potential of the recombinant protein (rChit37) to enhance the insecticidal activity of Bv062 conidia against second instar Diatraea saccharalis larvae was studied. rChit37 was produced in both soluble and insoluble fractions of an E. coli culture. Both fractions expressing endo- (90 mU/µL) and exochitinase (170 mU/µL) enzymatic activity, with optimum conditions for enzyme activity of 45 °C and pH 5.0. His-tag affinity chromatography was used to purify the rChit37 from the soluble fraction. Purified rChit37 was then diluted to 200 and 300 µg/mL for use as Bv062 conidia additive (1x106con/mL) in a laboratory bioassay against D. saccharalis larvae. No significant differences were observed between the efficacy of Bv062 conidia applied alone or mixed with 200 µg/mL purified rChit37. However, 300 µg/mL rChit37 increased BV062 conidia insecticidal activity, achieving 96.7% efficacy (14 days post-infection) and 6.2 days median lethal time (LT50), compared to 60% efficacy and 8.9 days for conidia alone. rChit37 addition did not affect conidial viability in terms of germination (96.6% after 24 h) or vigour estimated as germ-tube elongation rate. This work provides proof of concept about soluble recombinant chitinase as an additive to enhance B. bassiana virulence against D. saccharalis.

中文翻译：

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使用具有内切和外切壳多糖酶活性的可溶性重组酶增强白僵菌对糖衣藻的毒力

摘要 球孢白僵菌几丁质酶参与降解昆虫外骨骼和内部结构中的几丁质，因此是重要的毒力因子，因为它们参与了感染的最初到最后步骤。在这项工作中，B. bassiana（Bv062 分离株）开放阅读框（ORF）编码几丁质酶被鉴定为 Chit37（直系同源 Bvchit1）被分子克隆并在大肠杆菌中表达，重组蛋白 (rChit37) 的潜力增强了研究了Bv062分生孢子对二龄Diatraea saccharalis幼虫的杀虫活性。rChit37 在大肠杆菌培养物的可溶性和不溶性部分中产生。表达内切 (90 mU/µL) 和外切壳多糖酶 (170 mU/µL) 酶活性的两个部分，酶活性的最佳条件为 45 °C 和 pH 5.0。His标签亲和层析用于从可溶性部分纯化rChit37。然后将纯化的 rChit37 稀释至 200 和 300 µg/mL，用作 Bv062 分生孢子添加剂 (1x106con/mL) 在实验室生物测定中针对 D. saccharalis 幼虫。在 Bv062 分生孢子单独应用或与 200 µg/mL 纯化 rChit37 混合应用的功效之间未观察到显着差异。然而，300 µg/mL rChit37 增加了 BV062 分生孢子的杀虫活性，达到 96.7% 的功效（感染后 14 天）和 6.2 天的中位致死时间（LT50），相比之下，单独使用分生孢子的功效为 60% 和 8.9 天。添加 rChit37 不影响分生孢子的萌发活力（24 小时后为 96.6%）或以胚管伸长率估计的活力。这项工作提供了关于可溶性重组几丁质酶作为添加剂来增强 B.