title: Expression of an extremely acidic beta-1,4-glucanase from thermoacidophilic Alicyclobacillus sp. A4 in Pichia pastoris is improved by truncating the gene sequence.
abstract: BACKGROUND
  Alicyclobacillus sp. A4 is thermoacidophilic and produces many glycoside hydrolases. An extremely acidic beta-1,4-glucanase (CelA4) has been isolated from Alicyclobacillus sp. A4 and purified. This glucanase with a molecular mass of 48.6 kDa decreases the viscosity of barley-soybean feed under simulated gastric conditions. Therefore, it has the potential to improve the nutrient bioavailability of pig feed. For the study reported herein, the full-length gene, CelA4, of this glucanase (CelA4) was identified using the sequences of six peptides and cloned from strain A4. The gene fragment (CelA4F) encoding the mature protein was expressed in Pichia pastoris. Sequence truncation and glycosylation were found for recombinant CelA4F, both of which affected the expression efficiency. The physical properties of various forms of CelA4 as they affected enzymatic activity were characterized.
  
  
  RESULTS
  We located the full-length 2,148-bp gene for CelA4 (CelA4) in the genome of Alicyclobacillus sp. A4. CelA4 encodes a 715-residue polypeptide with a calculated molecular mass of 71.64 kDa, including an N-terminal signal peptide (residues 1-39), a catalytic domain (residues 39-497), and a C-terminal threonine-rich region (residues 498-715). Its deduced amino acid sequence and that of an Alicyclobacillus acidocaldarius endo-beta-1,4-glucanase were identical at 44% of the residue positions. When the experimental molecular mass of CelA4F--a recombinant protein designed to mimic the CelA4 sequence lacking the N-terminal signal peptide that had been expressed in Pichia pastoris--was compared with its hypothetical molecular mass, it was apparent that CelA4F was truncated, possibly at residue 497. An artificially truncated gene fragment (CelA4T) without C-terminal threonine-rich region was expressed in P. pastoris, and the expression efficiency of CelA4T was substantially greater than that of CelA4F. Purified CelA4F and CelA4T had similar molecular masses (~60 kDa) and enzymatic properties (optimum pH, 3.4; optimum temperature, 60 degrees C); they were relatively stable between pH 1.2 and 8.2 at 70 degrees C and resistant to acidic and neutral proteases. However, their molecular masses and thermostabilities differed from those of CelA4 isolated from Alicyclobacillus sp. A4. A deglycosylated form of CelA4 (CelA4D) had properties similar to that of CelA4 except that it was thermoliable at 60 degrees C.
  
  
  CONCLUSIONS
  Truncation during expression of CelA4F or artificial truncation of its gene--both of which produced a form of CelA4 lacking a threonine-rich region that includes a putative linker--increased the level of enzyme produced in comparison with that produced by cultivation of Alicyclobacillus sp. A4. Glycosylation increased the thermostability of CelA4. Of the four forms of CelA4 studied, CelA4T was produced in highest yield and had the most favorable physical properties; therefore, it has potential for use in the feed industry.
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China.
authors.forename: Yingguo
authors.initials: Y
authors.lastname: Bai
authors.forename: Jianshe
authors.initials: J
authors.lastname: Wang
authors.forename: Zhifang
authors.initials: Z
authors.lastname: Zhang
authors.forename: Pengjun
authors.initials: P
authors.lastname: Shi
authors.forename: Huiying
authors.initials: H
authors.lastname: Luo
authors.forename: Huoqing
authors.initials: H
authors.lastname: Huang
authors.forename: Chunliang
authors.initials: C
authors.lastname: Luo
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
mesh_terms: D000383:Agriculture; D056450:Alicyclobacillus / Q000201:enzymology* / Q000235:genetics; D000821:Animal Feed; D003001:Cloning, Molecular / Q000379:methods*; D004795:Enzyme Stability; D015870:Gene Expression; D044643:Glucan 1,4-beta-Glucosidase / Q000096:biosynthesis* / Q000235:genetics; D006031:Glycosylation; D010843:Pichia / Q000235:genetics*; D017384:Sequence Deletion
chemical_list: D044643:Glucan 1,4-beta-Glucosidase
references.citation: Plant Physiol. 1990 Feb;92(2):316-26
references.pmid: 16667276
references.citation: Appl Microbiol Biotechnol. 2006 Sep;72(2):297-305
references.pmid: 16493554
references.citation: Eukaryot Cell. 2006 Oct;5(10):1664-73
references.pmid: 16936142
references.citation: Appl Microbiol Biotechnol. 2002 Dec;60(4):428-36
references.pmid: 12466883
references.citation: Gene. 1997 Apr 29;190(1):55-62
references.pmid: 9185849
references.citation: Huan Jing Ke Xue. 2004 Sep;25(5):127-32
references.pmid: 15623039
references.citation: BMC Biochem. 2006 Dec 05;7:23
references.pmid: 17147821
references.citation: FEMS Microbiol Lett. 2004 Sep 1;238(1):71-8
references.pmid: 15336405
references.citation: Biochem Int. 1991 Oct;25(3):585-92
references.pmid: 1805802
references.citation: Gene. 1995 Nov 7;165(1):25-9
references.pmid: 7489911
references.citation: Eur J Biochem. 2003 Sep;270(17):3593-602
references.pmid: 12919323
references.citation: Methods Enzymol. 1996;266:554-71
references.pmid: 8743706
references.citation: Appl Microbiol Biotechnol. 2010 Jun;87(1):251-9
references.pmid: 20169343
references.citation: Appl Environ Microbiol. 1997 Jun;63(6):2449-53
references.pmid: 9172367
references.citation: J Microbiol. 2009 Jun;47(3):270-6
references.pmid: 19557344
references.citation: Protein Eng Des Sel. 2004 Mar;17(3):245-9
references.pmid: 15051866
references.citation: Appl Environ Microbiol. 1999 Feb;65(2):359-66
references.pmid: 9925554
references.citation: Protein Pept Lett. 2008;15(9):1017-21
references.pmid: 18991780
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Extremophiles. 2004 Oct;8(5):421-9
references.pmid: 15480866
references.citation: Appl Environ Microbiol. 2007 May;73(10):3215-24
references.pmid: 17384308
references.citation: Annu Rev Biophys Biophys Chem. 1991;20:175-203
references.pmid: 1867715
references.citation: J Ind Microbiol Biotechnol. 2010 Feb;37(2):187-94
references.pmid: 19916085
references.citation: J Agric Food Chem. 2010 Feb 10;58(3):1970-5
references.pmid: 20070105
references.citation: Biochem Biophys Res Commun. 1990 Sep 14;171(2):596-602
references.pmid: 2119576
references.citation: Biochemistry. 2005 Jun 28;44(25):9197-205
references.pmid: 15966744
references.citation: J Anim Sci. 1995 Apr;73(4):1074-6
references.pmid: 7628950
references.citation: Appl Microbiol Biotechnol. 2009 May;83(1):99-107
references.pmid: 19107475
references.citation: Nucleic Acids Res. 1994 Nov 11;22(22):4673-80
references.pmid: 7984417
references.citation: Protein Expr Purif. 2006 May;47(1):152-61
references.pmid: 16290006
references.citation: J Biol Chem. 2005 Apr 29;280(17):17549-61
references.pmid: 15737989
references.citation: Microbiol Rev. 1991 Jun;55(2):303-15
references.pmid: 1886523
references.citation: FEBS Lett. 2002 Mar 13;514(2-3):163-7
references.pmid: 11943144