Chitin, poly-β-1,4-N-acetylglucosamine (GlcNAc), is presumed to be the most abundant biopolymer in the aquatic biosphere, with an annual production of 1011 tons (Yu et al. 1993). Chitin is widely distributed in nature, e.g., as a constituent of insect exoskeletons, crustacean shells, fungal cell walls, and nematode eggshells and pharynx (Synowiecki and Al-Khateeb 2003). Chitinases (EC 22.214.171.124) are enzymes that catalyze the hydrolysis of chitin. These enzymes are found in both chitin-containing and non-chitin-containing organisms, such as bacteria, fungi, insects, plants, and animals. All most chitinases are classified into two different families of glycoside hydrolases (GH), families 18 and 19, based on the amino acid sequences in their catalytic domains (Henrissat 1991; Henrissat and Bairoch 1993). The catalytic domains of GH18 chitinases have (β/α)8 barrel folds (Perrakis et al. 1994; van Aalten et al. 2000), whereas those of GH19 chitinases have high α-helical content (Hart et al. 1995; Hahn et al. 2000). Bacterial family 18 chitinases can be further classified into three subfamilies A, B, and C. Subfamily A chitinases have an extra domain with a small α + β domain inserted into the seventh and eighth (α/β)8 barrel at the catalytic domain, while subfamilies B and C have no such domain (Suzuki et al. 1999). In addition to GH18 and GH19 chitinases, chitinases belonging to GH23 and GH48 are rarely found in some organisms. For instance, a family 23 chitinase was found in the moderately thermophilic bacterium, Ralstonia sp. A-471 (Ueda et al. 2009; Arimori et al. 2013), whereas a family 48 chitinase was found in the leaf beetle, Gastrophysa atrocyanea (Fujita et al. 2006).
Numerous studies have demonstrated that bacterial chitinases display an important role in inhibiting hyphal growth of phytopathogenic fungi (Ohno et al. 1996; Watanabe et al. 1999; Tsujibo et al. 2000; Kawase et al. 2006; Huang et al. 2012a,b; Pentekhina et al. 2020). In addition, it was reported that chitinolytic bacteria normally produce plant growth-promoting traits, extracellular enzymes, antifungal compounds other than chitinases to control phytopathogenic fungi and promote plant growth (Gu et al. 2017; Tran et al. 2018; Trinh et al. 2019). On the other hand, chitin is the main component of nematode eggshells and the pharynx. Therefore, any disturbance in the synthesis or hydrolysis of chitin could lead to nematode embryonic lethal, laying defective eggs, and/or moulting failure. Hence, the critical components in the chitin metabolic process are targets for the development of anti-nematode agents (Chen and Peng 2019). Bacterial chitinases have been shown to have activity against egg hatching of nematodes (Jung et al. 2002; Lee et al. 2014, 2015). Consequently, chitinase-producing bacteria could be used for crop production as biological agents to reduce the use of chemical agents in controlling fungal phytopathogens (Kurze et al. 2001; Kobayashi et al. 2002; Bhattacharya et al. 2007) and plant-parasitic nematodes (Lee et al. 2015; Chen and Peng 2019).
Chitinolytic bacteria usually produce chitinases and/or auxiliary activities family 10 (AA10) proteins to degrade insoluble chitin efficiently for their carbon and nitrogen sources. AA10 proteins are enzymes that were previously classified into carbohydrate-binding modules in family 33 and have been reclassified into the auxiliary activities family 10 of lytic polysaccharide monooxygenases, according to the CAZy database (Levasseur et al. 2013). Bacterial AA10 proteins were reported to boost the activity of chitinases (Vaaje-Kolstad et al. 2005a, 2010). In addition, combinations of chitinases and AA10 proteins have been demonstrated to have enhanced hydrolytic chitin-degrading activity in comparison with individual chitinases (Gutiérrez-Román et al. 2014; Pentekhina et al. 2020).
The species Bacillus velezensis is widely distributed in the soil environment. It has been reported that B. velezensis formed biofilms to promote biocontrol ability (Krober et al. 2016) and produced secondary metabolites against the growth of plant pathogenic fungi, including fengycin (Koumoutsi et al. 2004), bacillomycin D (Gu et al. 2017), bacillibactin C (Chen et al. 2007), diଃcidin (Wu et al. 2015), bacilysin (Wu et al. 2014), and amylocyclicin (Scholz et al. 2014). In addition, this species produced plant-growth-promoting traits, such as cytokinin (Arkhipova et al. 2007). Based on the evidence, B. velezensis has been widely applied for agricultural cultivation as biocontrol and plant-growth-promoting agents (Cai et al. 2017; Lim et al. 2017). On the other hand, B. velezensis could be a potent chitin degrader due to the presence of family 18 chitinases and AA10 protein according to the CAZy database (http://www.cazy.org/bB.html). However, such chitinases and protein are revealed by genome sequence analysis only, and no experimental studies thus far have described this chitinase system and its utilization in the hydrolysis of chitin. Moreover, it is well known that degradation and utilization of insoluble chitin by chitinolytic bacteria typically consists of two principal steps: (1) bacteria secrete families 18 and 19 chitinases to cleave chitin polymers into oligomers, and dimers (GlcNAc)2 are the major products, and then (2) the dimers are cleaved into monomers (GlcNAc) by the action of family 20 glycoside hydrolase (Beier and Bertilsson 2013). Genome sequences in the CAZy database show that B. velezensis possesses GH18 chitinases and AA10 protein only, posing the question of how this species converts chitin into GlcNAc for its carbon source utilization. In addition, the primary structures of two chitinases of this species contain carbohydrate-binding module family 50 (CBM50) domains. It was reported that the CBM50 domain in plant chitinases and fungal proteins are involved in substrate binding and antifungal activity (Seidl-Seiboth et al. 2013; Inamine et al. 2015), but the role of such domains in bacterial chitinases have not been revealed. Hence, the chitinase system from this species appears unique among those that have already been characterized and may possess antifungal activity.
We previously isolated a chitinolytic strain, RB.IBE29 (formerly DS29), from the rhizosphere of black pepper cultivated in the Central Highlands of Vietnam and identified it as B. velezensis. Strain RB.IBE29 showed high chitinase and antifungal activities against Phytophthora, the main cause of black pepper wilt disease in the region, as examined in vitro and the greenhouse condition (Trinh et al. 2019). Our field study showed that a combination of strain RB.IBE29 and other chitinolytic bacteria exhibited the most significant effect against Phytophthora and Fusarium in the soil and roots of black pepper. This combination also increased the chlorophyll a and b contents of black pepper, indicating that using indigenous bacteria, including B. velezensis, is a good solution for sustainable and green cultivation of black pepper in the Central Highlands of Vietnam (Nguyen et al. 2021). Considering the information on chitinases and AA10 protein genes revealed by genome sequencing and experimental studies on biocontrol and the plant-growth-promoting ability of B. velezensis, our next step was to identify and characterize chitinase molecules from B. velezensis RB.IBE29 by applying gene cloning and expression, then clarifying their bio-properties and synergistic effects for sustainable and green crop production.
In order to analyze the chitinase system of this bacterium, in this initial work, we present (1) isolation, cloning, and sequencing analysis of two chitinase and one AA10 protein genes of strain RB.IBE29, (2) analysis of primary structures of the deduced enzymes, (3) expression of chiB in E. coli BL21-CodonPlus (DE3)-RIPL cells, and examination of purified recombinant enzyme concerning chitinase, antifungal, and anti-nematode activities.