Biochemical characterization and gene expression of two endo-arabinanases from Penicillium chrysogenum 31B

Abstract

We previously described five arabinanolytic enzymes secreted by Penicillium chrysogenum 31B into the culture medium. Here, we describe a sixth such enzyme, termed AbnS1. Analysis of the reaction products of debranched arabinan revealed that AbnS1 cleaved the substrate in an endo manner. The optimum temperature of AbnS1 was 60°C, which was much higher than that of a cold-adapted endo-arabinanase (Abnc) produced by this strain. The abns1 cDNA gene encoding AbnS1 was isolated by in vitro cloning. The deduced amino acid sequence of AbnS1 had 70% identity with that of Abnc. Pfam analysis revealed a Glyco_hydro_43 domain at positions 28 to 318 of AbnS1. Semi-quantitative reverse transcription-polymerase chain reaction analysis indicated that the abns1 gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was only slightly induced with arabinose and arabinan. In contrast, expression of the abnc gene encoding Abnc was strongly induced by arabinose, arabinitol, and arabinan. Using debranched arabinan as substrate, recombinant AbnS1 (rAbnS1) accumulated arabinobiose and arabinotriose as the major products. Recombinant Abnc (rAbnc) released mainly arabinotriose and lesser amounts of arabinose and arabinobiose than did rAbnS1. Branched arabinan was completely degraded to arabinose by the action of rAbnS1 or rAbnc in combination with α-L: -arabinofuranosidase.

Identification and characterization of three Penicillium chrysogenum α-l-arabinofuranosidases (PcABF43B, PcABF51C, and AFQ1) with different specificities toward arabino-oligosaccharides

“…NcoI and EcoRI sites (underlined) were included in the 43B-N-NcoI and 43B-C-EcoRI primers, respectively. Total RNA and singlestranded cDNA used as the template were prepared as described previously [10]. The digested PCR product was ligated into the digested pET-32a vector, resulting in the plasmid pET-Pcabf43B, which was sequenced for confirmation of plasmid identity.…”

Biochemical Characterization and Overexpression of an Endo-rhamnogalacturonan Lyase from Penicillium chrysogenum

“…BamHI and HindIII sites (underlined) were added to the primers, respectively. Total RNA and single-stranded cDNA used as the templates were prepared as previously described [27]. The digested PCR product was ligated with the digested pET-32a vector, resulting in plasmid pET-rgl4A, and then sequenced.…”

“…Previously, we described a series of arabinanolytic enzymes produced by Penicillium chrysogenum 31B [23][24][25][26][27][28][29] isolated from rotten sugar beets. This strain also exhibits considerable ability to degrade sugar beet fibers, which have high pectin content.…”

Characterization of abnZ2 (yxiA1) and abnZ3 (yxiA3) in Paenibacillus polymyxa, encoding two novel endo-1,5-α-l-arabinanases

“…The optimal pH of reported endo-arabinanases is mostly at 6.0 or greater, such as 6.0 to 7.0 for endo-arabinanase AbnS1 from P. chrysogenum [12], 6.0 for an endo-arabinanase from Bacillus licheniformis [24], 6.5 for that from Caldicellulorsiruptor saccharolyticus [25], and 7.0 for Abn2 from B. subtilis [22]. Both AbnZ2 and AbnZ3 reached the highest activity when reaction pH was at 6.0 in 100 mM Naacetate buffer (Figure 7).…”

“…Endo-arabinanases from several microorganisms have been characterized. Their optimal temperatures are 50°C for Aspergillus niger [10], 60°C for Bacillus subtilis [11] and Penicillium chrysogenum [12], 70°C for Bacillus thermodentrificans [13], and 73°C for Thermotoga petrophila [14]. Compared with the mesophilic and thermophilic endoarabinanases, cold-active endo-arabinanases should be more attractive in juice industry as colder conditions hamper spoilage and avoid changes in nutritional properties [15].…”