Enzymes screening and process development for agarose hydrolysis

Abstract

The enzymatic hydrolysis system have been developed for agarose degradation and production of D-galactose and 3,6-anhydro-L-galactose. First, the $\beta$-agarase AgaG1, which was isolated from Alteromonas sp. GNUM1, was cloned to Escherichia coli BL21 and overexpressed into culture medium by controlling induction condition. A new agarose hydrolysis process using AgaG1 was developed, in which the reaction temperature was adjusted stepwise to avoid gelation problem with no chemical pretreatment step. The enzyme AgaG1 was found to be very effective and highly selective. When 10.0 g/L agarose was hydrolyzed, 98% of the agarose added was converted to 3.8 and 6.4 g/L of neoagarobiose and neoagarotetraose, respectively. Next, to produce neoagarobiose from neoagarotetraose, the $\beta$-agarase DagB, which was isolated from Streptomyces coelicolor A3(2), was cloned to Streptomyces lividans TK24 and overexpressed into culture medium. The DagB could hydrolyze neoagarotetraose to neoagarobiose, whereas agarose degradation rate is relatively low. Because the optimum conditions of AgaG1 and DagB was same, the one-step process which mixing AgaG1 and DagB to produce neoagarobiose from agarose hydrolysis was developed and minimum usage of enzymes was investigated for efficient production of neoagarobiose. Using 0.875 U/ml of AgaG1 and 0.133 U/ml of DagB, 94% of agarose added was converted to neoagarobiose. Next, to produce D-galactose and 3,6-anhydro-L-galactose, new $\alpha$-neoagarobiose hydrolase was screened from Alcanivorax sp. A28-3. The $\alpha$-neoagarobiose hydrolase was characterized and applied to neoagarobiose degradation, and confirmed production of D-galactose and 3,6-anhydro-L-galactose. When 5.0 g/L neoagarobiose was converted to 2.75 g/L of galactose, showed 98% of yield. Finally, agarose was completely hydrolyzed two-stage process of enzymatic hydrolysis without any chemical pretreatment. At the first stage, agarose was hydrolyzed to neoagarobiose using AgaG1, DagB, and produced neoagarobiose was converted to D-galactose and 3,6-anhydro-L-galactose by using α-neoagarobiose hydrolase. As a result, 88% of agarose added was converted to galactose. Produced crude galactose solution was metabolited by Saccharomyces cerevisiae KL17 which was galactose-utilizing yeast. 8.2 g/L of crude galactose was converted to 3.37 g/L of bioethanol without cell growth inhibition effect, compared to chemical hydrolysate solution.