Team:NEAU-China/Project

Overview
Construction
Applications
Results
References

Overview

We utilize enzyme assembling to construct the muti-enzyme complexes and we call it "Enzyme- Brick".

In nature, the macromolecular organization of multi-enzyme complexes have important implications for the specificity, regulation and overall efficiency of biochemical reactions. Artificial multi-enzyme complexes mimicking natural multi-enzyme organization would represent promising biocatalysts.

Immobilized enzyme

Immobilized enzyme technology is an efficient process which the enzyme is attached to an inert, insoluble material and held in place throughout the reaction, following which they are easily separated from the products and may be used again, easy to transport, store and automatize production,so is widely used in industry for enzyme catalyzed reactions.It is a new application technique of enzyme in recent years. However, there are still some aspects needed to improve,such as low stability, high cost of purification.

Cellulosome

This is the natural cellulosome structure. Cellulosome is muti-enzyme complex presents in certain prokaryotes composing of a variety of cellulases, hemicellulases. It is a spatial organization of enzymes into complexes, also it is an important strategy for increasing the efficiency of biochemical pathways and can be a powerful biotechnological tools for improvement of enzymatic syste ms.. The multienzyme cellulosome complexes ,are considered to be the most efficient systems for degradation.Enzymes are flexible during operation to accommodate and process the substrates.In this review, we proposed a development method in the field of multienzyme complex formation using assembling protein scaffolds.So we have combined immobilized enzyme with cellulosome structure , constructed the scadffold protein,cellulose as substrate.

Scaffold proteins:

In nature, scaffold proteins play a key role in multienzyme assembly. Scaffoldin contains a noncatalytic cellulose binding domain(CBM) and multiple copies of cohesin domain. The cohesin binds to the dockerin of cellulase and hemicellulase. The strong interaction between the cohesin and do ckerin domains allows for the integration of the enzymes on a scaffoldin. This integration promotes catalytical efficiency through the synergy effects of enzyme proximity and substrate targeting . Protein scaffolds can readily generate multienzyme complexes by just mixing components together.

CBM：

However, the structure of cellulosic substrates is complex,rigid and variable in nature,the enzymes get access and gegrade the substrates inefficiently in relative .In order to overcome these problems. many of multienzymes that utilize insoluble substrates are modular,comprising catalytic modules appended to one or more non-catalytic cellulose—binding modules(CBMs).CBMs promote the association of the enzyme with the substrate and play the central role in the enzymatic hydrolysis of plant cell wall.

Cohesin-dockerin interactions:

In the traditional model of cellulosome assembly, type-I dockerin of the enzymes bind to type-I cohesin of a primary scaffoldin. Each type-I dockerin contains two highly conserved cohesin-binding sites, which confer quaternary flexibility to the multienzyme complex. The scaffoldin also bears a type-II dockerin that anchors the entire complex to the cell surface by binding type-II cohesins of anchoring scaffoldins. There are three types of Dockerin domains: I, II and III which bind to Cohesin Type I, Cohesin Type II and Cohesin Type III respectively.So the specificility can be utilized.

Construction

Applications

The enzyme which we used in our project, it called Laccases, Laccases are blue multicopper oxidases, which catalyze the monoelectronic oxidation of a broad spectrum of substrates, for example, ortho- and para-diphenols, polyphenols, aminophenols, and aromatic or aliphatic amines, coupled with a full, four-electron reduction of O2 to H2O. Hence, they are capable of degrading lignin and are present abundantly in many white-rot fungi. Laccases decolorize and detoxify the industrial effluents and help in wastewater treatment. They act on both phenolic and nonphenolic lignin-related compounds as well as highly recalcitrant environmental pollutants, and they can be effectively used in paper and pulp industries, textile industries, xenobiotic degradation, and bioremediation and act as biosensors. In addition, Recently, laccase has been applied to nanobiotechnology, which is an increasing research field, and catalyzes electron transfer reactions without additional cofactors.

In the future, we may explore the more solid and special immobilized substrate. Some studies used the zirconium oxide as the substrate,where there are many specific perssads which could make ligands with the corresponding binding domain,thus the specificity could be promoted. Some studies take the inorganic-hybrid silica gel as the subatrate, the tiny ducts could expand the immobilized area greatly. In addition, the gelatin nano-particles could realize absorption and release triggerd by the temperature, which make the purification process more rapidly. After all, t is promosing to try other substates in addition to the cellulose binding domain and starch binding domain.

In our project,the same kind of cohesion structure domain is connected to the same level of scaffold. We can imagine that the different structural domain is linked to the same level of scaffold so that different enzymes can be adsorded on the scaffolds. If these enzymes have metabolisn causalities ,then we achieve to fix a metabolic pathway on immobilization mechanism ,so as to improve the value of enzyme caralysis.

Except connecting cohesion structure domain on the same level of scaffold,we can also explore another way--connecting matrix association regions to cohesion structure domain,and connect the exagenous enzymes to docking domain to form a kind of protein organization form without scaffold.This kind of structure is more flexible ,and can increase the amount of immobilized enzyme.

Results

1 original vector; 2 original vector after absorption; 3 original vector resuspended by the cellulose; 4 original CtCBM-Coh; 5 original CtCBM-Coh after absorption; 6 original CtCBM-Coh resuspended by the cellulose; 7 original CtCBM-2Coh; 8 original CtCBM-2Coh after absorption; 9 original CtCBM-2Coh resuspended by the cellulose; 10 original CtCBM-3Coh; 11 original CtCBM-3Coh after absorption; 12 original CtCBM-3Coh resuspended by the cellulose; 13 original CtCBM-4Coh; 14 original CtCBM-4Coh after absorption; 15 original CtCBM-4Coh resuspended by the cellulose; 16 original CtCBM-5Coh; 17 original CtCBM-5Coh after absorption; 18 original CtCBM-5Coh resuspended by the cellulose.

Figure1.Sample 1 is the control, 2~6 are five first-stents,Sample 7 is the second-stent enzyme activity of laccase mcoI. All the data is based on the same experimental condition and determined by ABTS which purified by Ni-Agarose His-Tagged protein purification kit.

Sample	10 times diluent	20 times diluent	30 times diluent
1	0	0	0
2	8.43	4.3	2.65
3	8.82	4.21	2.22
4	8.92	4.34	2.01
5	8.32	4.08	1.89
6	7.98	3.83	1.98
7	7.31	3.91	1.69

Reference

【1】Hidehiko Hirakawa , Tomoaki Haga ,Teruyuki Nagamune:Artificial Protein Complexes for Biocatalysis .Top Catal (2012) 55:1124–1137
【2】Quentin M. Dudley, Ashty S. Karim, and Michael C. Jewett:Cell-free metabolic engineering: Biomanufacturing beyond the cell .Biotechnol. J. 2015, 10, 69–82
【3】C. Eric Hodgman and Michael C. Jewett:Cell-Free Synthetic Biology: Thinking Outside the Cell .Metab Eng. Author manuscript; available in PMC 2013 May 01.
【4】Chun You and Y.-H. Percival Zhang:Self-Assembly of Synthetic Metabolons through Synthetic Protein Scffolds: One-Step Puriﬁcation, Co-immobilization, and Substrate Channeling .dx.doi.org/10.1021/sb300068g | ACS Synth. Biol. 2013, 2, 102−110
【5】Wei Hong, Jie Zhang, Yingang Feng, Georg Mohr, Alan M Lambowitz, Gu-Zhen Cui, Ya-Jun Liu1*and Qiu Cui1:The contribution of cellulosomal scaffoldins to cellulose hydrolysis by Clostridium thermocellum analyzed by using thermotargetrons .Hong et al. Biotechnology for Biofuels 2014