Thermo-Stable Pullulanase Variants for High-Temperature Liquefaction
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Solution Overview
Problem
Existing pullulanases derived from Bacillus sp. are not sufficiently thermally stable for effective use in conventional starch conversion processes, limiting their efficacy in liquefaction.
Innovation Solution
Development of thermo-stable pullulanase variants with specific amino acid substitutions and deletions at key positions, enhancing their thermal stability and activity, allowing their use in liquefaction processes at temperatures above the initial gelatinization temperature.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Temperature
If conventional pullulanases derived from Bacillus sp. are used in starch conversion processes, then they can hydrolyze alpha-1,6-glycosidic bonds in pullulan and amylopectin, but they lack sufficient thermal stability for effective use in liquefaction at temperatures above the initial gelatinization temperature
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of pullulanase through specific substitutions at defined positions. These sequence changes alter the protein's physical-chemical properties, specifically enhancing its thermal stability parameter to enable operation at higher liquefaction temperatures while maintaining catalytic activity
Solution Approach 2:
The invention creates a composite enzyme system by fusing the N-terminal fragment of pullulanase from Bacillus acidopullulyticus with the C-terminal fragment of pullulanase from Bacillus deramificans. This composite structure combines beneficial properties from both parent enzymes, resulting in improved thermal stability for liquefaction applications
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The thermo-stable pullulanase variants exhibit increased stability and activity, enabling efficient liquefaction of starch-containing materials and subsequent saccharification, thereby improving the production of fermentation products.
Implementation Method 1
Pullulanase is a starch debranching enzyme having pullulan 6-glucano-hydrolase activity (EC3.2.1.41) that catalyzes the hydrolyses the α-1,6-glycosidic bonds in pullulan
Implementation Method 2
the variant has increased thermo-stability and/or increased thermo-activity compared to a parent pullulanase
Data Source
AI summary
The present invention relates to a variant pullulanase, having increased thermo-stability and/or increased thermo-activity compared to a parent pullulanase, comprising a substitution at least a one position selected from a position corresponding to positions 432, 486, 370, 17, 77, 103, 106, 107, 190, 196, 197, 262, 279, 283, 321, 367, 375, 382, 399, 401, 402, 411, 412, 434, 435, 443, 446, 459, 460, 479, 490, 498, 514, 529, 531, 533, 541, 545, 581, 583, 595, 649, 665, 688, 700, 709, 804, 811 of SEQ ID NO: 1, and optionally a deletion of one or more, e.g., all amino acids at positions 821, 822, 823, 824, 825, 826, 827, and 828, wherein the variant has pullulanase activity, and wherein the variant has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to a parent alpha amylase selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5. Further aspect the present invention relates to a process for liquefying starch-containing material at a temperature above the initial gelatinization temperature using an alpha-amylase and a thermo-stable pullulanase of the invention.