Hidrolisis karbohidrat manan (bungkil inti kelapa sawit) dengan enzim mananase dari Streptomyses cyaenus untuk menghasilkan oligosakarida

Abstract

A number of oligosaccharides have been produced from carbohydrates starch using various kinds of enzymes such as amylase and cylodextrin glucosyltranferase. Non-starch carbohydrates such as cellulose and hemicelluloses which are abundant in Indonesia, are very potential to be used for oligosaccharides production. Palm kernel cake (PKC) is one of abundant biomass containing non-starch carbohydrates mannan. Mannan is a potential material source to develop a new type of oligosaccharides. Mannanase is an important enzyme that hydrolizes mannan into oligosaccharides. We screened 500 isolates of pure microbe from Indonesia Culture Collection (InaCC) LIPI and found four potential mannanase-producing isolates (Yopi et al., 2010). In this study, these four potential isolates (10, 17, 20, and 78) were then evaluated for mannanase production and production stability. The highest enzyme activity (0,189 U/mL) was obtained from isolate no 17, Streptomyces cyaenus. This isolate was also stable in the enzyme production. The Streptomyces cyaenus 17 was then used for the production of mannanase enzyme in 100 mL and 2 L scale media. In 100 ml fermentation media (pH 7, agitation 150 rpm), this isolate produced enzyme mannanase with the highest activity (0,702 U/mL) at day 2. In 2L fermentation media (pH 7), the Streptomyces cyaenus 17 isolate was incubated for 4 days (30oC) at three different agitation speed, 150, 200, 250 rpm. The mannanase enzyme activity was increased from day-0 to-2 suggesting the formation of monosaccharides and oligosaccharides and then decreased on day-3 and -4 presumbaly because of some monosaccharides or oligosaccharides had been consumed as a carbon source. The results showed that the highest mannase enzyme activity were obtain at day-2, 1619, 1706, and 1637 U/mL at 150, 200, and 250 rpm respectively. Thin layer chromatography (TLC) analysis of the PKC, locust bean gum (LBG), and porang substrat (each contained 6 ml of 0.5% substrate and 6 mL of rough mannanase enzyme with 1706 U/mL activity and incubated for 24h) showed that manan was hydrolyzed into manotriose, manotetrose, manopentose, manoheksose, mannose,and mannobiose. This results also showed that mannanase enzyme from Streptomyces cyaenus could hydrolyze carbohydrate mannan from PKC, LBG, and porang tuber. However, there was variation in the oligosaccharide types produced as indicated by the value of polymerization degree. Reaction of mannan substrates from PKC and LBG with enzyme mannanase from Streptomyces cyaenus 17 produced mannose, mannobiose, or mannotriose, but not porang tuber. HPLC analysis of the PKC substrat reacted with mannase enzim derived from Streptomyces cyaenus produced oligosaccharide with size from disccharide up to heptasaccharide. The oligosachharide produced was observed at 2, 4, 6, 8, 12 and 24 hours after reaction. Observation on the hydrolized sample of PKC substrate two hours after reaction showed the presence of mannobiose, mannotriose, and mannoheksose, with concentration 530, 290, and 1320 ppm respectively. The concentration of oligosaccharide was increased up to 8 hours after reaction. The highest concentration of oligosaccharide was obtained at 8 hour after reaction which produced mannobiose, mannotriose, and mannoheksose with concentration of 880, 190, and 1850 ppm, respectively. In the last stage of this study the oligosaccharide was produced in a reaction of 1 L of 0.5% PKC with 1 L of mannanase enzyme (activity 1.7 U/mL) and incubated for 4 hours (30oC, 150 rpm). The product concentration of 2 L reaction scale using freeze drier and spray drier generated oligosaccharide powder mixture with fawn and white in colour.