We build parts for decolor-assistant proteins (manA1, arfB, xynB^Attention!) to assemble decolor-assistant machines. Compared to existing decolor-assistant proteins, our proteins can work at high temperature and have synergistic effects, which may be useful in paper industry.

manA1, arfB, xynB are three genes which respectively encoding hemicellulase enzyme. With help of these proteins, the paper pulp can be bleached by much less chemical reagent. Since less chemical reagent (especially the chlorinated chemicals) in bleaching can benefit the environment protection, nowadays enzyme-assisted bleaching method is getting popular in paper making industry.

In our project, we build parts for three decolor-assistant proteins (manA1, arfB, xynB) to assemble three decolor-assistant machines. The experiment results confirmed the function of these decolor-assistant proteins, especially their heat-resistant property and synergistic effect.

To better understand the bio-assistant trend in paper industry, we asked for a visit to local paper factory but they all refused us because of some commercial secrets. At the same time, we tightly communicate with several iGEM teams to improve our idea and appreciate their work. This is our first time to join the iGEM competition, and we hope we can have fun and get inspired in the following months.

Pulp And Paper Industry

Introduction

Conventionally, the bleaching of digested pulp by chlorine compounds implies the generation of much adsorbable organic halogen (AOX) which is problematic due to its high toxicity and low biodegradability. Improper treatment and release of these hazardous waste could raise serious environmental and health problems. Finding solutions to reducing or replacing usage of those harmful compounds have aroused much concern.

Since 1986^Ref, several types of hemicellulase have been employed as biotechnological alternative solutions in the bleaching of pulp to help lower the chlorine containing bleaching reagent consumption. Xylanase is a prominent representative. Lab scaled experiments have testified that bleaching process involving Xylanase treatment was rather promising (Shown as table below). Moreover, bleaching process involving xylanase treatment can also improve product performance and save chemical expense^Ref.

The process of biobleach is simple, and does not require major alteration of the conventional flow path. After xylanase is mixed into the pulp and kept in a holding tank for 1-3 hours, the pulp is bleached in the normal way.

Flow Chart Of Paper Bleaching R.C. Kuhad, Ajay Singh, Lignocellulose Biotechnology Future Prospects, 2007

More than two decades have passed since the attempt of reducing pulp bleaching associated AOX emission has been made, yet this field of research still have many questions to ask and problems to solve. Researchers are still making efforts to optimize aspects of the biobleach method ^Ref.

One of the most important aspects of the biobleach method is stability and efficiency of xylanase. It is easy to understand that xylanases from natural hosts are evolved for adaption to the environment, thus not all of them can be in good service for industrial conditions. Many researchers have been seeking a xylanase which can work under conventional pulp treatment conditions, such as high temperature and alkaline.

Availability of the enzyme that is cellulase-free, working at temperature above 80°C and pH >8.0 would be very beneficial. In this respect, the thermostable and alkalistable xylanases have high potential in pulp bleaching process in paper industry.

R.C. Kuhad, Ajay Singh, Lignocellulose Biotechnology Future Prospects, 2007 Sierra et al, 1994

Our Contribution

Three genes of interest, which designated as xynB, arfB and manA1, were derived from a thermophilic bacteria Clostridium stercorarium DSM8532.

xynB gene encodes a endo-1,4-beta-xylanase. We identified this enzyme as an impressive thermostable xylanase because it presented a high activity at high temperature even up to 94.7°C! Since time is limited, other properties (such as synergistic effect and alkalistability) of this enzyme still awaits further validation.

arfB gene encodes an alpha-L-arabinofuranosidase, which has been reported as a thermoactive enzyme with arabinoxylan debranching activity, and is potential for biobleaching^Ref.

manA1 gene, encoding a putative mannan endo-1,4-beta-mannosidase, was identified by complete genome sequencing of Clostridium stercorarium Strain DSM 8532. We construced this gene expression vector to make it expressed in E.coli. The mannosidase activity of this gene was confirmed by our experimental evidences. Given that other mannosidases have been reported to be useful in biobleaching, this enzyme is likely to be a candidate for industrial biobleach agent. Especially considering the thermophilic property of this gene, it could potentially be applied in paper pulp biobleeching.

Future Energy Resource

Besides being useful in paper industry, these hemicellulases might be able to help with solving energy problem.

Lignocellulosic biomass is regarded as a very promising resource of fuel. The conversion is based on pretreatment of raw material to remove unfermantable components such as lignin, and this is exactly what the hemicellulases are capable of.

In fact, someone had already used xylanase to assemble a pretreatment machine^Ref.

Therefore, to the best of our knowledge, our project may be beneficial to industrial pulp bleaching and future energy resource development.