Engineering highly thermostable xylanase variants using an enhanced combinatorial library method

Abstract

A new directed evolution method was used to enhance the thermostability of the wild-type GH11 xylanase 2 (known as BD-11) from Hypocrea jecorina (Trichoderma reesei). Both Look-Through Mutagenesis (LTM™), which is a method for rapidly screening selected positions in the protein sequence for amino acids that introduce favorable properties, and Combinatorial Beneficial Mutagenesis (CBM™), which is a method for identifying the best ensemble of individual mutations, were employed to enhance the stability of an enzyme that has been thoroughly engineered by various means during the past 20 years. A diverse set of novel mutations was discovered, including N71D, Y73G, T95G and Y96Q. When these mutations were combined into a single construct (Hjx-81), the purified protein was active even after heating at 100°C for 20 min. This time-effective method should be generally applicable for quickly improving the physico-chemical properties of other industrial and therapeutic enzymes in only several months time.

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

“…1062 As discussed above, thermostability may also sometimes (but not always 171–173 ) correlate with evolvability, 175 and is the result of multiple mutations each contributing a small amount. 1064–1069 …”

Microbial xylanases and their biomedical applications: a review

“…30 In some strains, optimal xylanase activity was observed at pH 6.4 and temperature 55°C and xylanase is active up to pH 9 (40.33 IU/ml) and temperature 85°C (48.81 IU/ml) (Agnihotri et al, 2010) 1 and A 27-fold higher production was achieved by cloning and expression (Verma et al, 2011). 89 Recently thermostable xylanase retaining activity at 100 o C for 20 minutes have been found (Hokanson et al, 2011). 35 Bacillus haloduras is a wild (non-genetically manipulated) organism yielding thermo-alkali stable xylanase that acts in submerged fermentation at pH 10.0 (Kumar et al, 2012).…”

“…89 Recently thermostable xylanase retaining activity at 100 o C for 20 minutes have been found (Hokanson et al, 2011). 35 Bacillus haloduras is a wild (non-genetically manipulated) organism yielding thermo-alkali stable xylanase that acts in submerged fermentation at pH 10.0 (Kumar et al, 2012). 48 To the contrary, thermo-acid stable variants act at pH 2.0 and temperature 80°C (Apel et al, 2008…”

Directed Evolution of Penicillium janczewskii zalesk α-Galactosidase Toward Enhanced Activity and Expression in Pichia pastoris

“…This facilitates the transformation in P. pastoris and subsequent screening and sequencing [21]. It is reported that the combinatorial library method focusing on the amino acids that represent side-chain properties is effective for the directed evolution of smaller-sized enzymes [22]. Since our target protein is relatively large, we selected both error-prone PCR and DNA shuffling in this study.…”