Isolation and Molecular Characterization of Chitinase-Deficient Bacillus licheniformis Strains Capable of Deproteinization of Shrimp Shell Waste To Obtain Highly Viscous Chitin

Abstract

Proteolytic but chitinase-deficient microbial cultures were isolated from shrimp shell waste and characterized. The most efficient isolate was found to be a mixed culture consisting of two Bacillus licheniformis strains, which were first determined microscopically and physiologically. Molecular characterization was carried out by sequencing the 16S rRNA gene of both strains. According to the residual protein and ash content, the chitin obtained by fermentation of such a mixed culture was found to be comparable to a commercially available, chemically processed product. However, the strikingly high viscosity (80 versus 10 mPa of the commercially available sample) indicates its superior quality. The two strains differed in colony morphology and in their secretion capabilities for degradative extracellular enzymes. Sequencing of the loci encoding amylase, cellulase, chitinases, and proteases, as well as the degS/degU operon, which is instrumental in the regulation of degradative enzymes, and the pga operon, which is responsible for polyglutamic acid production, revealed no differences. However, a frameshift mutation in chiA, encoding a chitinase, was validated for both strains, providing an explanation for the ascertained absence of chitinolytic activities and the concomitant possibility of producing highly viscous chitin in a fermentational deproteinization process.Chitin (a polysaccharide consisting of ␤-1,4-linked N-acetyl-D-glucosamine moieties) is the second most abundant biopolymer on earth; thus, it represents a nearly constant source of renewable raw material. Along with its deacetylated derivative chitosan, chitin recently has gained biotechnological significance, not only because of favorable pharmaceutical features, such as antimicrobial, anticholesterol, and antitumor activities, but also because of its potential for wastewater treatment, drug delivery, and wound healing and as a dietary fiber (26).Approximately 50 to 60% of the total weight of shellfish, such as shrimp, crab, and krill, consists of nonedible material, i.e., "heads" and exoskeletons rich in chitin but also protein, which form, on the one hand, major environmental pollutants as a result of uncontrolled dumping (12). On the other hand, however, due to their chemical composition (20 to 30% chitin, 20 to 40% protein, 30 to 60% minerals, and 0 to 14% lipids) and their actual availability from seafood industries, shrimp waste also constitutes the major source for chitin and chitosan production (21). Currently applied methods to purify and modify chitin from such material and to transform it to useful carbohydrate products involve harsh chemical treatments accompanied by uncontrollable hydrolysis and chemical modifications that eventually result in the formation of undesired by-products such as irregularly deacetylated polymers (33).Exploitation limits are mainly set by the purification costs, which mainly arise from removal of proteins and calcium carbonate by alternating acid and alkali treatment, ultimately resulting in large amounts of aque...

Chitinolytic Bacteria Isolated from Chili Rhizosphere: Chitinase Characterization and Its Application as Biocontrol for Whitefly (<i>Bemisia tabaci</i> Genn.)

“…Shanmugaiah et al (2008) reported that among the 39 chitinolytic bacteria which were isolated from rice rhizosphere, only 11 isolates produced zone of clearance over 0.5 cm on chitin colloidal agar. Chitinase production was reported in different species of Bacillus such as B. amyloliquefaciens (Wang et al, 2002), B. cereus (Huang et al, 2005), B. circulans (Chen et al, 2004), B. licheniformis (Waldeck et al, 2006), B. megaterium (Donderski and Brzezinska, 2005), B. stearothermophilus (Sakai et al, 1994), B. subtilis (Wang et al, 2006), B. thuringiensis sub sp. aizawai (De la Vega et al, 2006), B. thuringiensis sub sp.…”

Genetic Improvement of Bacillus licheniformis Strains for Efficient Deproteinization of Shrimp Shells and Production of High-Molecular-Mass Chitin and Chitosan

“…For a comparison of the mutants generated with a strain carrying the reconstituted chiA gene (the formerly published frameshift mutation in chiA of F11 [43], yielding a premature translational stop), the defective chiA sequence from B. licheniformis F11 was exchanged for that of the intact chiA gene of B. licheniformis DSM13 (see Materials and Methods). The relevant genotypes of the respective strains and the deviations from wild-type strain F11 are depicted in Fig.…”

“…B. licheniformis DSM13 possesses an operon with two chitinase genes, chiB and chiA, which possibly hampers the recovery of high-molecular-mass chitin due to degradation. B. lichenifor-mis strain F11 has a frameshift mutation in the chitinase A gene (chiA); it was efficiently used in shrimp shell protein hydrolysis, at the same time leaving the chitin largely unaffected (43). However, the other chitinase-encoding gene (chiB) localized immediately upstream of chiA as part of the operon may still have a negative impact on chitin recovery.…”

“…Subsequently, the remaining proteins can be hydrolyzed by proteolytic enzymes, which are secreted by a variety of microorganisms. Deproteinization of shrimp shells by applying different microorganisms has been repeatedly attempted (5,7,8,43,46,49,51). Representatives of Bacillus licheniformis are major bacterial workhorses in the industry (37); their genome sequence was published already a couple of years ago (34, 42), clarifying not only the close relationship to Bacillus subtilis but also facilitating efficient genetic strain improvement.…”

Overexpression and characterization of thermostable chitinase from Bacillus atrophaeus SC081 in Escherichia coli

“…The modified agar diffusion method was used to visualize SCChi-1 chitinase activity (18,19). First, the colloidal chitin, glycol chitin, glycol chitosan, and soluble chitosan plates were prepared.…”