Kai Lin,Ao Xia,Yun Huang,Xianqing Zhu,Kaiyong Cai,Zidong Wei,Qiang Liao
Enzymatic hydrolysis is considered to be a restricted step for lignocellulosic bioethanol production at an industrial scale for its high production costs and low efficiency. In this study, a microreactor loaded with cellulase based on co-deposition of dopamine (DA) and polyethyleneimine (PEI) was proposed, for the first time, to efficiently hydrolyze carboxymethyl cellulose (CMC) and produce sugar. When the concentration of PEI increased from 0 to 0.5 mg mL −1 , the enzyme loading was increased by 27.4%, and glucose production was increased by 33.2%. Besides, the cellulase-loaded microreactor showed better temperature adaptability than free cellulase at elevated temperatures ranging from 55 °C to 65 °C. After storage for 20 days, the cellulase-loaded microreactor retained 84.4% of its initial activity, which is 69.5% higher than the free cellulase. The yield reached 169.7 mg glucose g −1 CMC in the cellulase-loaded microreactor in 16 h. With the same amount of enzyme input, the glucose production per unit time in the cellulase-loaded microreactor is 97.2% higher than that in other reaction systems. This study demonstrated that continuous production of glucose can be achieved in the microreactor at a relatively low enzyme dosage.
Frank Peprah Addai,Jiacong Wu,Feng Lin,Xinnan Ma,Juan Han,Yuelin Liu,Yang Zhou,Yun Wang
In this work, a trimetallic (Ni/Co/Zn) organic framework (tMOF), synthesized by a solvothermal method, was calcinated at 400 and 600 °C and the final products were used as a support for lipase immobilization. The material annealed at 400 °C (Ni-Co-Zn@400) had an improved surface area (66.01 m2/g) and pore volume (0.194 cm3/g), which showed the highest enzyme loading capacity (301 mg/g) with a specific activity of 0.196 U/mg, and could protect the enzyme against thermal denaturation at 65 °C. The optimal pH and temperature for the lipase were 8.0 and 45 °C but could tolerate pH levels 7.0–8.0 and temperatures 40–60 °C. Moreover, the immobilized enzyme (Ni-Co-Zn@Lipase, Ni-Co-Zn@400@Lipase, or Ni-Co-Zn@600@Lipase) could be recovered and reused for over seven cycles maintaining 80, 90, and 11% of its original activity and maintained a residual activity >90% after 40 storage days. The remarkable thermostability and storage stability of the immobilized lipase suggest that the rigid structure of the support acted as a protective shield against denaturation, while the improved pH tolerance toward the alkaline range indicates a shift in the ionization state attributed to unequal partitioning of hydroxyl and hydrogen ions within the microenvironment of the active site, suggesting that acidic residues may have been involved in forming an enzyme–support bond. The high enzyme loading capacity, specific activity, encouraging stability, and high recoverability of the tMOF@Lipase indicate that a multimetallic MOF could be a better platform for efficient enzyme immobilization.
The efficiency of multi-enzyme immobilization is often influenced by the carrier and immobilization techniques employed. In this work, we proposed a ternary enzyme cascade reaction system composed of β-glucosidase (β-G), glucose oxidase (GOD) and horseradish peroxidase (HRP). CaCO 3 microspheres served as a template for in situ immobilization, while poly (allylamine hydrochloride) (PAH) and sodium poly (4-styrene sulfonate) (PSS) were utilized to create PAH-CaCO 3 and PSS-CaCO 3 templates for layer-by-layer multi-enzyme immobilization. The effects of template structure, glutaraldehyde (GA) addition, ethylenediaminetetraacetic acid (EDTA) concentration, and the structure of layer-by-layer assembly were systematically investigated. Our results indicated that an assembly of PSS-CaCO 3 --PAH--β-G--HRP--GOD presented simultaneous high enzyme loading and relative activity. The loadings of β-G, GOD and HRP were 75.2 ± 2.0%, 87.7 ± 4.4% and 85.5 ± 3.2%, respectively, while the relative activities were 81.8 ± 2.7%, 93.6 ± 4.7% and 90.6 ± 3.1%, respectively. Kinetic parameters confirmed that the reaction rate of the immobilized ternary enzymes in this assembly was close to that of the free enzymes. This work provides insights for constructing other multi-enzyme cascade reaction systems using immobilization techniques.
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