Exogenous fibrolytic enzymes and recombinant bacterial expansins synergistically improve hydrolysis and in vitro digestibility of bermudagrass haylage

摘要

Four experiments were conducted to examine the effectsof a recombinant bacterial expansin-like protein(BsEXLX1) from Bacillus subtilis and a commercialexogenous fibrolytic enzyme (EFE) preparation forruminants on hydrolysis of pure substrates (celluloseand xylan) and in vitro digestibility of bermudagrasshaylage (BMH). Recombinant Escherichia coli BL21strain was used to express BsEXLX1; the proteinwas purified using an affinity column. In experiment1, carboxymethylcellulose, Whatman #1 filter paper(General Electric, Boston, MA) and oat-spelt xylansubstrates were subjected to 4 treatments (1) sodiumcitrate buffer (control), (2) BsEXLX1 (162 μg/g ofsubstrate), (3) EFE (2.3 mg/g of substrate), and (4)EFE + BsELX1 in 3 independent runs. Samples wereincubated at optimal conditions for both additives (pH5 and 50°C) or at ruminal (pH 6 and 39°C) or ambient(pH 6 and 25°C) conditions for 24 h and sugar releasewas measured. In experiment 2, digestibility in vitro ofBMH was examined after treatment with the following:(1) control (buffer only), (2) BsEXLX1 (162 μg/g ofdry matter), (3) EFE (2.2 mg/g of dry matter), and (4)EFE + BsEXLX1 in 3 independent runs at 39°C for 24h. Experiment 3 examined effects of EFE and BsEXLX1on simulated preingestive hydrolysis and profile of releasedsugars from BMH after samples were suspendedin deionized water with sodium azide at 25°C for 24 hin 2 independent runs. In experiment 4, the sequence ofthe BsEXLX1 purified protein was compared with 447ruminal bacterial genomes to identify similar proteinsfrom the rumen. In experiment 1, compared with EFEalone, EFE and BsEXLX1 synergistically increased sugarrelease from carboxymethylcellulose and Whatman#1 filter paper under all simulated conditions; however,hydrolysis of xylan was not improved. In experiment 2,compared with EFE alone, treatment with EFE andBsEXLX1 increased neutral detergent fiber and aciddetergent fiber digestibility of bermudagrass haylage(by 5.5 and 15%, respectively) and total volatile fattyacid concentrations, and decreased acetate-propionateratio. In experiment 3, compared with EFE alone. TheEFE and BsEXLX1 synergistically reduced concentrationsof neutral detergent fiber and acid detergent fiberand increased release of sugars by 9.3%, particularlycellobiose (72.5%). In experiment 4, a similar sequenceto that of BsEXLX1 was identified in Bacillus licheniformis,and similar hypothetical protein sequences wereidentified in Ruminococcus flavefaciens strains alongwith different protein structures in E. xylanophilum andLachnospiraceae. This study showed that an expansinlikeprotein synergistically increased the hydrolysis ofpure cellulose substrates and the hydrolysis and digestibilityin vitro of BMH.