Alpha-amylase variants for baking thermostability
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Solution Overview
Problem
Current alpha-amylases used in baking, particularly those from fungal and bacterial sources, have limitations such as low heat stability, which affects starch modification during baking, leading to suboptimal crumb texture and shelf-life extension, and bacterial amylases can result in an unacceptable crumb structure with small doses.
Innovation Solution
Development of a variant alpha-amylase with improved properties, including increased thermostability, specific activity, sucrose tolerance, and altered substrate specificity, achieved through specific amino acid substitutions, to enhance dough development, crumb structure, and shelf-life of baked products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If fungal or bacterial alpha-amylases are used in baking, then starch modification and gas production are improved, but heat stability is insufficient leading to suboptimal crumb texture and shelf-life
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues in the alpha-amylase protein sequence to alter its thermal stability parameters. The invention identifies specific position-residue pairs (e.g., position 13 Q→E, position 61 L→F, position 68 T→A, position 70 W→Y, position 75 L→F, position 88 G→A, position 128 F→I, position 133 S→T, position 168 F→Y, position 188 F→I, position 200 S→N, position 222 A→V, position 254 V→F, position 261 D→G, position 264 A→S, position 281 V→L, position 282 L→F/M/I/T, position 327 N→S, position 371 N→G, position 388 A→L, position 421 Y→F, position 450 F→Y, position 505 S→N, position 536 T→N, position 548 E→Q, position 554 A→T, position 583 E→Q, position 588 A→T, position 603 T→N, position 637 S→N, position 648 T→N, position 651 A→S, position 652 T→N, position 660 A→S, position 676 T→N, position 677 A→S) that when substituted improve the enzyme's thermostability while maintaining its catalytic activity for starch modification during baking.
2Productivity
If bacterial alpha-amylase is used to improve starch modification, then gas production is enhanced, but crumb structure becomes unacceptable with small doses
Solution Approach 1:
The patent modifies the bacterial alpha-amylase parameters through specific amino acid substitutions that fine-tune the enzyme's activity and stability characteristics. These parameter changes enable the enzyme to maintain optimal gas production while improving crumb structure quality by preventing excessive starch breakdown that occurs with bacterial amylases at small doses.
3Productivity
If alpha-amylase is used to improve dough leavening, then gas production and retention are enhanced, but crust color and shelf-life are adversely affected
Solution Approach 1:
The patent applies parameter changes to the alpha-amylase enzyme by substituting specific amino acid residues that modify the enzyme's thermal stability and activity profile. These changes enable the enzyme to maintain optimal activity during dough leavening for gas production and retention while improving stability during baking to prevent adverse effects on crust color and extend shelf-life by controlling starch modification rates.
4Productivity
If conventional alpha-amylase is used for starch modification, then dough leavening is improved, but thermostability during baking is insufficient
Solution Approach 1:
The patent modifies the conventional alpha-amylase parameters through systematic amino acid substitutions at specific positions in the protein sequence. These parameter changes increase the enzyme's thermostability allowing it to withstand baking temperatures while maintaining sufficient activity for effective dough leavening and starch modification.
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 variant alpha-amylase improves dough strength, elasticity, crumb softness, and shelf-life, while maintaining optimal texture and preventing gumminess, by effectively modifying starch during baking and storage.
Implementation Method 1
The variant alpha-amylase improves dough strength, elasticity, crumb softness, and shelf-life, while maintaining optimal texture and preventing gumminess, by effectively modifying starch during baking and storage
Implementation Method 2
Development of a variant alpha-amylase with improved properties, including increased thermostability, specific activity, sucrose tolerance, and altered substrate specificity
Implementation Method 3
During baking the starch gelatinization process is taking place. Amylose is leaking out of the granule and forms a continuous gel in the baking dough
Implementation Method 4
During storage of the bread over several days, the amylopectin starts to re-crystallize (also called retrogradation). The staling of bread is believed to be a direct reflection of the retrogradation of amylopectin
Data Source
AI summary
A variant polypeptide having alpha-amylase activity, wherein the variant has an amino acid sequence which, when aligned with the alpha-amylase comprising the sequence set out in SEQ ID NO: 2, comprises at least one substitution of an amino acid residue. Such a variant polypeptide may be used in the preparation of a baked product.