Process Biochemistry 39 (2004) 665–671 Production of -N-acetylhexosaminidase of Verticillium lecanii by solid state and submerged fermentations utilizing shrimp waste silage as substrate and inducer Yoyi Matsumoto, Gerardo Saucedo-Castañeda, Sergio Revah, Keiko Shirai ∗ Biotechnology Department, Universidad Autónoma Metropolitana, Biopolymers Lab. Av. San Rafael Atlixco No. 186. Col. Vicentina C.P. 09340 Mexico city, Mexico Received 13 January 2003; received in revised form 24 February 2003; accepted 6 April 2003 Abstract The aim of this study was to utilise shrimp waste silage both as substrate and inducer of -N-acetylhexosaminidase of Verticillium lecanii in submerged (SF) and solid state fermentations (SSF), taking advantage of the abundance and composition of crustacean wastes (main commercial source of chitin). -N-Acetylhexosaminidase was produced in SF (initial pH 6) inoculated with spores or mycelia, 540 and 965.5 U/g of initial dry substrate (U/g IDS), respectively. SSF were carried out using a mixture of shrimp waste silage and sugar cane pith bagasse at initial pH of 6. The increment of moisture content and mycelia as inoculum in SSF improved the enzyme yield significantly and reduced the lag phase, i.e. 75% of moisture content inoculated with spores or mycelia produced 1016 and 1673 U/g IDS, respectively. SSF produced a higher -N-acetylhexosaminidase yield than SF, but required a longer time (24 h). Specific activity in SSF was only 40% higher than SF, due to impurities from shrimp waste silage and sugar cane bagasse. Shrimp waste silage was an efficient inducer of the extracellular enzyme, compared with media supplemented with sucrose where enzymic activity was not detected. © 2003 Elsevier Ltd. All rights reserved. Keywords: Verticillium lecanii; Submerged fermentation; Solid state fermentation; Chitin; Chitinases; N-Acetylglucosamine; Shrimp wastes; Entomopathogenic fungi 1. Introduction Chitin is a polysaccharide composed of -1,4 N-acetyl- d-glucosamine. It is highly distributed in nature, as a con- stituent of insect exoskeletons, shells of crustaceans and fungal cell walls. The enzymic hydrolysis of chitin is carried out by a chitinolytic system that is classified as: endo-chitinases (EC 3.2.1.1.4), exo-chitinases (EC 3.2.1.14), chitobiase (EC 3.2.1.30) and -N-acetylhexosaminidases (EC 3.2.1.52). Endo-chitinases cleave randomly along the internal chain of chitin, producing low molecular oligomers of N-acetyl glucosamine, such as chitotetraose and chi- totriose, eventually giving diacetylchitobiose as predominant product. On the other hand exo-chitinases release diacetyl- chitobiose without production of N-acetyl glucosamine or oligomers. -N-Acetylhexosaminidases split diacetylchito- ∗ Corresponding author. Tel.: +52-5-804-4921; fax: +52-5-804-4712. E-mail address: smk@xanum.uam.mx (K. Shirai). biose as well as chitotriose and chitotetraose into N-acetyl glucosamine monomers. Since -N-acetylhexosaminidases also acts on di-acetylchitobiose it is also called chitobiase [1]. Chitinases are present in chitin-containing microorgan- isms, bacteria and plants with a diversity of roles, such as chitin metabolism in growing hyphae, defence mechanisms in response to pathogens and abiotic stress, in nutrition and parasitism [2,3]. In addition to the applications as inhibitors and biopesticides, chitinases have been used for the pro- duction of single cell protein for animal and aquaculture feed, for the isolation of fungal protoplasts, preparation of bioactive chito-oligosaccharides and for phytopathogen inhibition [2,3]. The activity and stability of the enzyme preparation to- gether with its cost are the most important factors to be con- sidered for chitinases applications, hence the necessity to study potential producers and inexpensive inducers. Verticillium lecanii has been recognized as a potential biocontrol agent of arthropod pests and also for controlling rust disease, as it produces insect cuticle degrading enzymes 0032-9592/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0032-9592(03)00140-7