(582ds) Porous Cleas of ?-Mannanase Prepared By Macromolecular Cross-Linker

Cross-linked enzyme aggregates (CLEAs),
a carrier-free enzyme immobilization approach, prepared by precipitation and
subsequent cross-linking using glutaraldehyde, have attracted increasing
attentions in recent years because of its advantages of high stability and
enzyme activity due to the exclusion of carrier and the excellent property of
repeated utilization.^{1, 2} However,
mass transfer limitation was considered as the
main shortcoming for the application of CLEAs because of its conglomeration structure formed
by glutaraldehyde.

Therefore,
in this paper, the multi-aldehyde
macromolecule of linear
dextran polyaldehyde (2000 kD) as well as branched
dialdehyde starch (350-750 kD), Fig. A, were introduced as
cross-linker to construct the porous structure and enhance the mass transfer property of beta-mannanase CLEAs.

Scanning electron microscope revealed the inner structure
of CLEAs prepared by different cross-linker. For the CLEAs cross-linked by
glutaraldehyde (Fig. B-a), the SEM
image exhibited a smooth surface with few pores. It was estimated that this
structure had the seriously steric hindrance effect on macro-substrates, which
limited the substrates to enter into the inner CLEAs. Thus, beta-mannanase CLEAs
showed the low activity (4.68%). When using the linearly macromolecular dextran
polyaldehyde (2000 kD) as a cross-linker, beta-mannanases were bonded along its
long chain, which not only prevented the lump of enzyme molecules but also
formed a porous structure of CLEAs for macro-substrates to get into the inner
CLEAs (Fig. B-b). These pores
decreased the steric hindrance and obviously increased the activity of CLEAs (32.01%).
Unlike the linear macromolecule, dialdehyde starch (350-750 kD) is dendritic.
It bonded enzyme molecules along its interlaced branch, which also led to the
congestion of enzyme and the steric hindrance of CLEAs (Fig. B-c). Even then, the activity of CLEAs
prepared with dialdehyde starch (20.98%) was still higher than that
cross-linked by glutaraldehyde due to the flexibility of enzyme.

This
research was supported by the NSF of China (20976125, 31071509,
51173128, 21206113) and Tianjin (10JCYBJC05100), the Ministry of Science and Technology of
China (Nos. 2012YQ090194, 2013AA102204, 2012BAD29B05), the Program for New
Century Excellent Talents in Chinese University (NCET-08-0386), and Beiyang
Young Scholar Program (2012).

References

[1] U.
Roessl, J. Nahalka and B. Nidetzky, Biotechnol. Lett., 2010, 32,
341-350.

[2] L.
Cao, F. van Rantwijk and R. A. Sheldon, Organic Letters, 2000, 2,
1361-1364.

Figure (A)
The molecular structure of dialdehyde starch and dextran polyaldehyde; (B) The
morphology of beta-mannanase CLEAs cross-linked by (a) glutaraldehyde, (b)
dextran (2000 kDa) and (c) dialdehyde starch (magnified 5 k times).