Biochemical Characterization of the First Fungal Glycoside Hydrolyase Family 3 β-N-Acetylglucosaminidase from Rhizomucor miehei

Abstract

A novel β-N-acetylglucosaminidase gene (RmNag) from Rhizomucor miehei was cloned and expressed in Escherichia coli. RmNag shares the highest identity of 37% with a putative β-N-acetylglucosaminidase from Aspergillus clavatus. The recombinant enzyme was purified to homogeneity. The optimal pH and temperature of RmNag were pH 6.5 and 50 °C, respectively. It was stable in the pH range 6.0-8.0 and at temperatures below 45 °C. RmNag exhibited strict substrate specificity for p-nitrophenyl β-N-acetylglucosaminide (pNP-GlcNAc) and N-acetyl chitooligosaccharides. The apparent Km of RmNag toward pNP-GlcNAc was 0.13 mM. The purified enzyme displayed an exo-type manner as it released the only end product of GlcNAc from all the tested N-acetyl chitooligosaccharides. Besides, RmNag exhibited relatively high N-acetyl-β-D-glucosaminide tolerance with an inhibition constant Ki value of 9.68 mM. The excellent properties may give the enzyme great potential in industries. This is the first report on a glycoside hydrolyase family 3 β-N-acetylglucosaminidase from a fungus.

Characterization of a NaCl-tolerant β-N-acetylglucosaminidase from Sphingobacterium sp. HWLB1

“…Although the activity of purified rNag3HWLB1 was not obviously affected by most metal ions and chemical reagents tested in this study, the enzyme was completely inhibited by HgCl 2 and AgNO 3 . The drastic inhibition in the presence of Ag + and Hg 2+ is commonly observed for most GH 3 GlcNAcases, such as the GH 3 GlcNAcases from Rhizomucor miehei (Yang et al 2014) and Streptomyces cerradoensis (da Silva Junior Sobrinho et al 2005). Ag + and Hg 2+ can react irreversibly with sulfhydryl (or thiol) groups and Hg 2+ can oxidize the indole ring.…”

“…Chitin is largely produced from marine sources, but more than 80,000 tons of chitin per year is considered as waste (Patil et al 2000). Endochitinases (EC 3.2.1.14) randomly catalyze the hydrolysis of β-1,4-glycosidic bonds in chitin to release N-acetyl chitooligosaccharides, which can inhibit the activities of most endochitinases (Yang et al 2014). β-N-Acetylglucosaminidase (GlcNAcase, EC 3.2.1.52) further hydrolyzes N-acetyl chitooligosaccharides to yield N-acetylglucosamine (GlcNAc), thus allowing the endochitinases to act efficiently (Yang et al 2014).…”

“…Endochitinases (EC 3.2.1.14) randomly catalyze the hydrolysis of β-1,4-glycosidic bonds in chitin to release N-acetyl chitooligosaccharides, which can inhibit the activities of most endochitinases (Yang et al 2014). β-N-Acetylglucosaminidase (GlcNAcase, EC 3.2.1.52) further hydrolyzes N-acetyl chitooligosaccharides to yield N-acetylglucosamine (GlcNAc), thus allowing the endochitinases to act efficiently (Yang et al 2014). The synergistic action of the two types of chitinolytic enzymes degrades chitin completely to produce GlcNAc.…”

“…The inhibition effects of GlcNAc to GlcNAcases are different. The GlcNAcase from R. miehei is inhibited by 49.9 % in the presence of 9-mM GlcNAc (Yang et al 2014), which also inhibits the activity of rNag3HWLB1 by approximately 50 %, whereas the GlcNAcase from Trichoderma harzianum is inhibited by approximately 50 % in the presence of only 0.21-mM GlcNAc (Nieder et al 2004). Furthermore, many GH 3 GlcNAcases with two domains have a molecular mass of approximately 60 kDa, such as the GH 3 GlcNAcases from Streptomyces thermoviolaceus (66 kDa; Tsujibo et al 1998), Cellulomonas fimi (60 kDa; Mayer et al 2006), Symbiobacterium thermophilum (56 kDa; Ogawa et al 2006), and Thermotoga neapolitana (53 kDa; Choi et al 2009).…”

“…The optimal pH of rNag3HWLB1 is the same or close to that of some GH 3 GlcNAcases, but the optimal temperature of rNag3HWLB1 is lower than that of most GH 3 GlcNAcases. For example, R. miehei GlcNAcase is optimal at pH 6.5 and 50 °C (Yang et al 2014), Clostridium paraputrificum GlcNAcase is optimal at pH 7.0 and 50 °C (Li et al 2002), and Thermotoga maritima GlcNAcase is optimal at pH 7.0 and 65 °C (Choi et al 2009). The inhibition effects of GlcNAc to GlcNAcases are different.…”