A novel family 9 beta-1,3(4)-glucanase from thermoacidophilic Alicyclobacillus sp. A4 with potential applications in the brewing industry.
An endo-beta-1,3(4)-glucanase gene, Agl9A, was cloned from Alicyclobacillus sp. A4 and expressed in Pichia pastoris. Its deduced amino acid sequence shared the highest identity (48%) with an endo-beta-1,4-glucansae from Alicyclobacillus acidocaldarius that belongs to family 9 of the glycoside hydrolases. The purified recombinant Agl9A exhibited relatively wide substrate specificity, including lichenan (109%), barley beta-glucan (100%), CMC-Na (15.02%), and laminarin (6.19%). The optimal conditions for Agl9A activity were pH 5.8 and 55 degrees C. The enzyme was stable over a broad pH range (>60% activity retained after 1-h incubation at pH 3.8-11.2) and at 60 degrees C (>70% activity retained after 1-h incubation). Agl9A was highly resistant to various neutral proteases (e.g., trypsin, alpha-chymotrypsin, and collagenase) and Neutrase 0.8L (Novozymes), a protease widely added to the mash. Under simulated mashing conditions, addition of Agl9A (20 U/ml) or a commercial xylanase (200 U/ml) reduced the filtration rate (26.71% and 20.21%, respectively) and viscosity (6.12% and 4.78%, respectively); furthermore, combined use of Agl9A (10 U/ml) and the xylanase (100 U/ml) even more effectively reduced the filtration rate (31.73%) and viscosity (8.79%). These characteristics indicate that Agl9A is a good candidate to improve glucan degradation in the malting and brewing industry.
Bai
Yingguo
Y
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, People's Republic of China.
Wang
Jianshe
J
Zhang
Zhifang
Z
Shi
Pengjun
P
Luo
Huiying
H
Huang
Huoqing
H
Luo
Chunliang
C
Yao
Bin
B
D056450:Alicyclobacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D044644:Endo-1,3(4)-beta-Glucanase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D005285:Fermentation; D005936:Glucans / Q000378:metabolism; D006358:Hot Temperature; D007218:Industrial Microbiology; D007700:Kinetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D016415:Sequence Alignment; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005936:Glucans; D044644:Endo-1,3(4)-beta-Glucanase