Preparation of N-Acetyl-D-Glucosamine and chitooligosaccharide by enzymatic hydrolysis of chitin and chitosan with serum from para rubber

Abstract

Serum fraction of para rubber (Hevea brasiliensis) obtained from the process of concentrated latex preparation is known to contain an endo-chitinolytic enzyme, Hevamine. This work presents an investigation of a potential utilization of the serum for the production of N-acetyl-D-glucosamine (GlcNAc) and N,N’-diacetylchitobiose ((GlcNAc)2) from β-chitin. The rubber serum contained 6 mg/mL of protein with chitinolytic activity of 18 mU per milligram of protein. The optimum ratio of enzyme to chitin was found to be 0.22 mU/mg with optimum substrate concentration at 60 mg/mL. The optimum pH range was 2-4 and the optimum temperature was 45℃ where the reaction produced both (GlcNAc)2and GlcNAc with the product mole ratio ((GlcNAc)2/GlcNAc) approximately 2:1. The hydrolysis of 300 mg of chitin yielded 39 mg of GlcNAc and 108 mg of (GlcNAc)2 corresponding to HPLC yield of 11.6% GlcNAc and 35.8% (GlcNAc)2within 8 days when 64 mU of the enzyme was used at the optimum condition. Dialysis technique offered convenient separation of the GlcNAc and (GlcNAc)2 products from the starting chitin and enzymes but reduced the enzyme efficiency. Partial purification of (GlcNAc)2was achieved by gel filtration chromatography. (GlcNAc)2 was recovered in 92% with 36% (w/w) purity. A technique of enzyme combination was used for production of GlcNAc from chitin. This hydrolysis showed an HPLC yield of 52% GlcNAc in 4 days. The serum Hb gave low molecular weight chitosan with Mw 5.4x104-1.5x105 rather than the expected chitooligosaccharide (GlcNAc)2 - (GlcNAc)7) when hydrolyzed with chitosan. The serum thus has potential use for low cost production of GlcNAc and (GlcNAc)2 and low molecular weight chitosan.