Team:Tongji/project-zh

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iGEM Tongji 2014

项目介绍

我们将几个助脱色(助漂)蛋白(manA1, arfB, xynB注意!)组装成了助漂生物机器。比起现有的助漂蛋白,我们的蛋白能在极高温下工作,而且具有协同效应,在造纸工业中具有潜在价值。

manA1, arfB, xynB 是同类的三种蛋白,各自编码一种酶。在这些酶的帮助下,纸浆脱色时需要的化学试剂量大大减少,尤其是含氯试剂的减少。这意味保护环境方面的重大意义。也正因此,如今酶助漂工艺在造纸工业中越来越受到重视。

在我们的项目里,我们将几个助漂蛋白(manA1, arfB, xynB)组装成了助漂生物机器。通过实验,我们验证了它们的助漂活性,而且我们还发现了它们具有令人惊讶的耐高温能力以及相互之间的协同效应。

为了看看业界的生物助漂现状,我们业跟不少造纸厂取得联系(我们请求参观一下工厂,但是他们都拒绝了我们。有一些说这是商业机密,另一些暗示我们别来参观,他们排放不达标)。同时,我们紧密地和几支上海地区地iGEM队伍进行交流,改进我们的想法,也欣赏他们的成果。这是我们第一次参加iGEM大赛,希望我们能在加下来的几个月能享受它!

工业制浆及造纸

前言

传统的纸浆漂白因为使用氯化物带来的AOX(可吸附有机卤素)污染而受人诟病。如果不采取改进措施,人们的健康和生活环境都会受到极大的威胁。所以这引起了人们对于环保纸浆漂白的思考。

1986年Ref几种半纤维素酶被尝试用来在纸浆漂白中降低含氯试剂使用量。其中木聚糖酶表现得很有潜力(见下表)。而且用了木聚糖,造出来的纸质感也更好。Ref.

作者及年份 漂白步骤 纸浆类型 ClO2 使用减少量
Viikari, 1986
X(DC)E
SWKP
25%
Shan, 2000
XD0ED1ED2
HWKP-O
25%
Paice, 2004
XDE0D
HWKP
13.4%

这种生物助漂技术使用流程其实挺简单,而且只是稍微改变一下传统的流程。

纸浆漂白流程对比图 R.C. Kuhad, Ajay Singh, Lignocellulose Biotechnology Future Prospects, 2007

尽管已经过去二十年,但是纸浆的环保漂白这个领域里仍然有许多待解决的问题,而且学者们也仍然在不断优化其中的技术。Ref.

其中一个问题就是,要如何才能提高木聚糖酶的稳定性和效率。而来源于不同天然宿主的木聚糖酶都有不一样的工作属性,于是人们希望找到一种能在高温和碱性条件下工作的木聚糖酶。这样就能更好地和传统的工艺相结合。

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

我们的工作

我们本次制作的三个零件xynB, arfB and manA1来自一种嗜热菌Clostridium stercorarium DSM8532.

xynB 编码木聚糖酶,在我们的实验里它极其耐高温,能在高达94.7度的环境中工作。由于时间原因,它的其他性质(比如和另外两个酶的协同作用以及嗜碱性仍在验证中)

arfB 编码阿拉伯呋喃糖酶, which has been reported as a thermoactive enzyme with arabinoxylan debranching activity, and is potential for use in biobleachRef.

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 machineRef.

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

同济大学, 2014