Process Characteristics of Hydrolysis of Chitosan in a Continuous Enzymatic Membrane Reactor

Abstract

Crude enzyme from Bacillus cereus NTU-FC-4 was used to hydrolyze chitosan of 66% deacetylation in a membrane reactor, operated at 45 °C and pH 5, to continuously produce chitooligosaccharides. Major oligomers in the product from the reactor were chitobiose, chitotriose, chitotetraose, chitopentaose, and chitohexaose. When the membrane reactor was operated at an enzyme/substrate ratio of 0.2 (unit/mg) and residence time of 100 min, it reached steady state in 2.5 h. The system could be operated for 15 h and still maintained a stable product composition. When the volume replacement exceeded 2.5, the productivity of the membrane reactor became higher than that of the batch reactor, and the difference between them became even greater when the volume replacement was further increased. The apparent Michaelis constant (K m ) for the enzyme in the membrane reactor was 18.8 mg/mL, but the apparent K m was 5.4 mg/mL for the batch reactor, suggesting that the affinity of the enzyme for chitosan was lower in the membrane reactor compared with the enzyme in the batch reactor. The estimated values of apparent V max were 0.18 and 0.20 mg reducing sugar/mL/min for the enzyme in the membrane reactor and in the batch reactor, respectively, indicating that the enzyme activity was not greatly altered when used in the membrane reactor.

Production of high degree polymerized chitooligosaccharides in a membrane reactor using purified chitosanase from Bacillus cereus

“…The permeate was diverted to a collection vessel and the retentate was returned to the reaction tank for further reaction. The substrate solution (chitosan solution) was continuously fed to the reaction tank from a substrate tank at a rate equals to the permeation rate to maintain a constant volume and substrate concentration of the mixture in the reactor (Kuo et al, 2004).…”

“…After incubation at 30°C for 2 days in a shake incubator, the culture was centrifuged at 4°C and 6200 rpm for 30 min to remove bacteria and colloidal chitin. The crude enzyme was precipitated by 70% acetone and dried by lyophilization (FLEXI-DRY TM , FTS system Co., New York, USA) (Kuo et al, 2004).…”

“…By removing the immobilized enzyme from the reaction mixture when the target pentamers and hexamers reached a maximum, the yield of high DP oligomers was increased in the batch reaction (Ming, Kuroiwa, Ichikawa, Sato, & Mukataka, 2006). Kuo, Chen, and Chiang (2004) demonstrated that chitooligomers could be produced by a continuous membrane reactor (Kuo et al, 2004). In the continuous membrane bioreactor, enzyme is recycled and reused while the product of enzymatic hydrolysis is continuously withdrawn as permeate.…”

Chitosanase production by Paenibacillus ehimensis and its application for chitosan hydrolysis

“…Therefore, this method produces low yields of pentamers and hexamers. Enzymatic hydrolysis has some advantages in producing COS. Chitosanases can catalyze the hydrolysis under mild conditions (Kuo et al 2004;Ming et al 2006) Chitosanases have been recognized as enzymes that attack chitosan but not chitin and defined as the enzyme performing endohydrolysis of β-1,4-linkages between D-glucosamine residues in a partly acetylated chitosan (Su et al 2006). Chitosanase has been found in a variety of microorganisms, including various bacterial species (Kurakake et al 2000;Choi et al 2004;Kim et al 2004;Su et al 2006;Sun et al 2007;Zhu et al 2007;Gao et al 2008;Wang and Yeh, 2008;Wang et al 2008a;Wang et al 2008b) and fungi Ike et al 2007).…”

Microbial Glycosidases for Nondigestible Oligosaccharides Production

“…yielded 60% of COs with 95% of purity [152], whereas Bacillus pumilus BN-262 chitosanase yielded above 80% in a UF membrane reactor [148]. Through the action of a B. pumilus BN-262 chitosanase, a COs productivity of 20 and 15 g/L was obtained in a batch and membrane reactor, respectively [153]. The hydrolysis with B. pumilus chitosanase yielded 52% of COs, producing mainly pentameric and hexameric chitosan oligosaccharides was steadily produced at 2.3 g/L (46% yield) for a month [154].…”