Ferulic acid (FA) groups esterified to the arabinan side chains of pectic polysaccharides can be oxidatively cross-linked in vitro by horseradish peroxidase (HRP) catalysis in the presence of hydrogen peroxide (H(2)O(2)) to form ferulic acid dehydrodimers (diFAs). The present work investigated whether the kinetics of HRP catalyzed cross-linking of FA esterified to α-(1,5)-linked arabinans are affected by the length of the arabinan chains carrying the feruloyl substitutions. The kinetics of the HRP-catalyzed cross-linking of four sets of arabinan samples from sugar beet pulp, having different molecular weights and hence different degrees of polymerization, were monitored by the disappearance of FA absorbance at 316 nm. MALDI-TOF/TOF-MS analysis confirmed that the sugar beet arabinans were feruloyl-substituted, and HPLC analysis verified that the amounts of diFAs increased when FA levels decreased as a result of the enzymatic oxidation treatment with HRP and H(2)O(2). At equimolar levels of FA (0.0025-0.05 mM) in the arabinan samples, the initial rates of the HRP-catalyzed cross-linking of the longer chain arabinans were slower than those of the shorter chain arabinans. The lower initial rates may be the result of the slower movement of larger molecules coupled with steric phenomena, making the required initial reaction of two FAs on longer chain arabinans slower than on shorter arabinans.
A thin alginate layer induced on the surface of a commercial polysulfone membrane was used as a matrix for noncovalent immobilization of enzymes. Despite the expected decrease of flux across the membrane resulting from the coating, the initial hypothesis was that such a system should allow high immobilized enzyme loadings, which would benefit from the decreased flux in terms of increased enzyme/substrate contact time. The study was performed in a sequential fashion: first, the most suitable types of alginate able to induce a very thin, sustainable gel layer by pressure-driven membrane filtration were selected and evaluated. Then, an efficient method to make the gel layer adhere to the surface of the membrane was developed. Finally, and after confirming that the enzyme loading could remarkably be enhanced by using this method, several strategies to increase the permeate flux were evaluated. Alcohol dehydrogenase (EC 1.1.1.1), able to catalyze the conversion of formaldehyde into methanol, was selected as the model enzyme. An enzyme loading of 71.4% (44.8 μg/cm(2)) was attained under the optimal immobilization conditions, which resulted in a 40% conversion to methanol as compared to the control setup (without alginate) where only 10.8% (6.9 μg/cm(2)) enzyme was loaded, with less than 5% conversion. Such conversion increased to 60% when polyethylene glycol (PEG) was added during the construction of the gel layer, as a strategy to increase flux. No enzyme leakage was observed for both cases (with/without PEG addition). Modeling results showed that the dominant fouling mechanism during gel layer induction (involving enzyme entrapment) was cake layer formation in the initial and intermediate phases, while pore blocking was the dominant mechanism in the final phase. Such mechanisms had a direct consequence on the type of immobilization promoted in each phase. The results suggested that the strategy proposed could be efficiently used to enhance the enzyme loading on polymer membranes.
Enzymatic reduction of carbon dioxide (CO2 ) to methanol (CH3 OH) can be accomplished using a designed set-up of three oxidoreductases utilizing reduced pyridine nucleotide (NADH) as cofactor for the reducing equivalents electron supply. For this enzyme system to function efficiently a balanced regeneration of the reducing equivalents during reaction is required. Herein, we report the optimization of the enzymatic conversion of formaldehyde (CHOH) to CH3 OH by alcohol dehydrogenase, the final step of the enzymatic redox reaction of CO2 to CH3 OH, with kinetically synchronous enzymatic cofactor regeneration using either glucose dehydrogenase (System I) or xylose dehydrogenase (System II). A mathematical model of the enzyme kinetics was employed to identify the best reaction set-up for attaining optimal cofactor recycling rate and enzyme utilization efficiency. Targeted process optimization experiments were conducted to verify the kinetically modeled results. Repetitive reaction cycles were shown to enhance the yield of CH3 OH, increase the total turnover number (TTN) and the biocatalytic productivity rate (BPR) value for both system I and II whilst minimizing the exposure of the enzymes to high concentrations of CHOH. System II was found to be superior to System I with a yield of 8 mM CH3 OH, a TTN of 160 and BPR of 24 μmol CH3 OH/U · h during 6 hr of reaction. The study demonstrates that an optimal reaction set-up could be designed from rational kinetics modeling to maximize the yield of CH3 OH, whilst simultaneously optimizing cofactor recycling and enzyme utilization efficiency.
Type A chitinases (EC 3.2.1.14), GH family 18, attack chitin ((1 → 4)-2-acetamido-2-deoxy-β-D-glucan) and chito-oligosaccharides from the reducing end to catalyze release of chitobiose (N,N'-diacetylchitobiose) via hydrolytic cleavage of N-acetyl-β-D-glucosaminide (1 → 4)-β-linkages and are thus "exo-chitobiose hydrolases." In this study, the chitinase type A from Serratia marcescens (SmaChiA) was used as a template for identifying two novel exo-chitobiose hydrolase type A enzymes, FbalChi18A and MvarChi18A, originating from the marine organisms Ferrimonas balearica and Microbulbifer variabilis, respectively. Both FbalChi18A and MvarChi18A were recombinantly expressed in Escherichia coli and were confirmed to exert exo-chitobiose hydrolase activity on chito-oligosaccharides, but differed in temperature and pH activity response profiles. Amino acid sequence comparison of the catalytic β/α barrel domain of each of the new enzymes showed individual differences, but ~69% identity of each to that of SmaChiA and highly conserved active site residues. Superposition of a model substrate on 3D structural models of the catalytic domain of the enzymes corroborated exo-chitobiose hydrolase type A activity for FbalChi18A and MvarChi18A, i.e., substrate attack from the reducing end. A main feature of both of the new enzymes was the presence of C-terminal 5/12 type carbohydrate-binding modules (SmaChiA has no C-terminal carbohydrate binding module). These new enzymes may be useful tools for utilization of chitin as an N-acetylglucosamine donor substrate via chitobiose.
Certain enzymes of the glycoside hydrolase family 20 (GH20) exert transglycosylation activity and catalyze the transfer of β-N-acetylglucosamine (GlcNAc) from a chitobiose donor to lactose to produce lacto-N-triose II (LNT2), a key human milk oligosaccharide backbone moiety. The present work is aimed at increasing the transglycosylation activity of two selected hexosaminidases, HEX1 and HEX2, to synthesize LNT2 from lactose and chitobiose. Peptide pattern recognition analysis was used to categorize all GH20 proteins in subgroups. On this basis, we identified a series of proteins related to HEX1 and HEX2. By sequence alignment, four additional loop sequences were identified that were not present in HEX1 and HEX2. Insertion of these loop sequences into the wild-type sequences induced increased transglycosylation activity for three out of eight mutants. The best mutant, HEX1GTEPG , had a transglycosylation yield of LNT2 on the donor that was nine times higher than that of the wild-type enzyme. Homology modeling of the enzymes revealed that the loop insertion produced a more shielded substrate-binding pocket. This shielding is suggested to explain the reduced hydrolytic activity, which in turn resulted in the increased transglycosylation activity of HEX1GTEPG .