We build parts for decolor-assistant proteins(ManA1, ArfB, XynB) 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 each encoding an enzyme (three types of xylanase). With help of these proteins, the pulp(which then become paper) can be bleached by only little chemical reagent. Since using less chemical reagent(especially the chlorinated) can help protect the environment, 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. Through experiments, we confirm their decolor-assistant function, and we observe their heat-resistant property and synergistic effect.

To better understand the bio-assistant trend in paper industry, we also talk to local paper factory (we asked for a visit, but they all refused us. Some said this is the secret, and other suggested they are under emission standard). 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

Our parts contains 3 genes from a thermophilic bacteria Clostridium stercorarium DSM8532, designated xynB, arfB and manA1.

xynB gene encodes a endo-1,4-beta-xylanase. Our experiment identified this enzyme as an impressive thermostable xylanase which presents efficiency at high temperature up to 94.7°C! Due to time limit, 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 use in biobleach^Ref.

manA1 gene was identified by complete genome sequencing of Clostridium stercorarium Strain DSM 8532 and encodes a putative mannan endo-1,4-beta-mannosidase. We expressed this protein in E.coli, and confirmed its mannosidase activity. Given that other mannosidases have been reported to be useful in biobleaching, and this one is found in a thermophilic natural host, this enzyme is likely to be a candidate for industrial biobleach agent.

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.

Recently, Morais et al. have integrated six dockerin-bearing cellulases and xylanases from the highly cellulolytic bacterium, Thermobifida fusca, into a chimeric scaffolding, engineered to bear a cellulose-binding module and the appropriate matching cohesin modules. The resultant hexavalent designer cellulosome was fully functional and achieved enhanced levels (up to 1.6-fold) of untreated wheat straw degradation compared to those of the wild-type free enzymes.

Lignocellulose processing: a current challenge in ROYAL SOCIETY OF CHEMISTRY S. Morais, E. Morag and E. A. Bayer, mBio, 2012, 3(6), e00508–00512.

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