Journal
PROCESS BIOCHEMISTRY
Volume 48, Issue 8, Pages 1181-1187Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.procbio.2013.06.010
Keywords
Candida antarctica lipase B; Silaffin; R1 peptide; Biosilicification; Immobilization; Thermostability
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Funding
- R&D program of MKE/KEIT [10031717]
- Converging Research Center Program [2011K000660]
- National Research Foundation of Korea [NRF20110029249]
- Kwangwoon University
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A large improvement in the thermostability of Candida antarctica lipase B (CALB) was achieved through double immobilization, i.e., physical adsorption and R1 silaffin-mediated biosilicification. The C-terminus of CALB was fused with the R1 silaffin peptide for biosilicification. The CALB-R1 fusion protein was adsorbed onto a macroporous polyacrylate carrier and then subsequently biosilicified with tetramethyl orthosilicate (TMOS). After R1 silaffin-mediated biosilicification, the double-immobilized CALB-R1 exhibited remarkable thermostability. The T-50(60) of the double-immobilized CALB-R1 increased dramatically from 45 to 72 degrees C and that was 27, 13.8, 9.8 and 9.9 degrees C higher than the T-50(60) values of free CALB-R1, CALB-R1 adsorbed onto a resin, commercial Novozym 435, and Novozym 435 treated with TMOS, respectively. In addition, the time required for the residual activity to be reduced to half (t(1/2)) of the double immobilized CALB-R1 elevated from 12.2 to 385 min, which is over 30 times longer life time compared free CALB-R1. The optimum pH for biosilicification was determined to be 5.0, and the double-immobilized enzyme showed much better reusability than the physically adsorbed enzyme even after 6 repeated reuses. This R1-mediated biosilicification approach for CALB thermostabilization is a good basis for the thermostabilization of industrial enzymes that are only minimally stabilized by protein engineering. (C) 2013 Elsevier Ltd. All rights reserved
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