Secretory production of enzymatically active endo-β-1,4-mannanase from Bacillus subtilis by ABC exporter in Escherichia coli

Eom, G.T.,Oh, J.Y.,Park, J.H.,Jegal, J.,Song, J.K. Elsevier Applied Science 2016 PROCESS BIOCHEMISTRY Vol.51 No.8

To secrete the endo-β-1,4-mannanase of Bacillus subtilis WL7 in Escherichia coli, the ABC exporter (TliDEF) and the C-terminal signal sequences of lipase (TliA) and metalloprotease (PrtA) were used. Six C-terminal fragments of TliA (TliAC1, TliAC2 and TliAC3) and PrtA (PrtAC1, PrtAC2 and PrtAC3) were amplified by PCR, respectively, and were fused to the mature mannanase gene lacking its original N-terminal signal sequence. When coexpressed with TliDEF exporter gene in E. coli, all the fusion mannanases were successfully secreted to the culture supernatant as an enzymatically active form by TliDEF exporter in E. coli. However, the unspecific cell leakage occurred in the fusion mannanases containing TliA-derived signal sequences. Among the fusion mannanases containing the signal sequences of PrtA, the mannanase with the shortest signal sequence fragment (Man-PrtAC3) exhibited the highest secretion level (4.65mg/L). Moreover, the specific activity, the effect of temperature and pH on activity and stability were almost identical to the wild-type mannanase, indicating that the fused signal sequence of PrtA did not affect the enzymatic properties of mannanase. It was concluded that the efficient secretion of enzymatically active mannanase from Bacillus subtilis WL-7 was achieved by using PrtAC3 as the most adequate signal sequence fragment.

Sporolactobacillus sp. M201 균주에 의한 β-Mannanase와 β-Mannosidase의 생산

박원식,김화영,최용진 한국산업미생물학회 1998 한국미생물·생명공학회지 Vol.26 No.3

β-Mannan polymer 분해 효소를 다량 생산하는 균주를 토양으로부터 분리하고 분리균의 형태적 내지는 생화학적 특성을 조사 분석하여 Sporolactobacillus sp. M201로 동정하였다. Sporolactobacillus sp. M2Ol 분리균은 locust bean gum을 탄소원으로 사용, 배양했을 때 세포외 β-mannanase 그리고 세포내 β-mannosidase와 α-galactosidase을 다량 생산하는 것으로 확인되었다. 효소생산 최적 배지와 최적 배양 조건은 β-mannanase 생산에는 2.0% locust bean gum, 0.5% peptone, 0.2% KH_2 PO_4, 80 ㎎/l MgSO_4 및 8 ㎎/l ZnSO_4 배지 (pH 6.0)와 37℃이었고 그리고 β-mannosidase 생산은 질소원으로 peptone 대신 0.5% yeast extract를 첨가한 상기 배지(pH 5.0)와 30℃에서 배양했을 때 가장 높았다. 이상의 각 효소의 최적 생산 조건에서 각각 30시간과 24시간 진탕배양 했을때 β-mannanase(10.6 units/㎖)와 β-mannosidase(1.35 units/㎖)의 최고 생산량을 나타내었다. A bacterial strain producing high levels of an extracellular β-mannanase and intracellular β-mannosidase and α-galactosidase was isolated from soil. The strain isolated was identified as a strain of Sporolactobacillus sp. and designated as Sporolactobacilus sp. M2O1. Synthesis of β-mannanase by Sporolactobacillus sp. M2Ol was induced by sucrose, maltose, or locust bean gum. The highest induction rate was obtained with 2% locust bean gum added to the culture medium as a sole carbon source. On the other hand, induction of β-mannosidase was observed only with locust bean gum. The optimal media for the enzyme production were established as follows: for β-mannanase; 2% locust bean gum, 0.5% peptone, 0.2% KH_2PO_4, 80 ㎎/l MgSO_4, and 8 ㎎/l ZnSO_4 (pH 6.0), and for β-mannosidase; 2% locust bean gum, 0.5% yeast extract, 0.2% KH_2PO_4, 80 ㎎/l MgSO_4, and 8 mg/l ZnSO_4 (pH 5.0). The optimal culture temperatures for production of β-mannanase and β-mannosidase were found to be 37℃ and 30℃, respectively Under the optimal culture conditions, the production of β-mannanase and β-mannosidase reached the highest levels of 10.6 units/㎖ and 1.35 units/㎖ after 30 h and 24 h cultivation, respectively.

Nonruminant Nutrition : Effect of Dietary β-Mannanase Supplementation and Palm Kernel Meal Inclusion on Laying Performance and Egg Quality in 73 Weeks Old Hens

( Jun Yeob Lee ),( Sang Yun Kim ),( Jae Hwan Lee ),( Jeong Heon Lee ),( Sang Jip Ohh ) 한국동물자원과학회(구 한국축산학회) 2013 한국축산학회지 Vol.55 No.2

This study was conducted to evaluate the effect of dietary β-mannanase supplementation and palm kernel meal (PKM) inclusion (5%) on laying performance, egg quality and nutrient utilizability of laying hens with 73 weeks of age. A total of 240 Lohmann brown laying hens with average 77.5% egg production were randomly allocated with 60 hens per treatment, 4 replicates per treatment and 15 hens per replicate. Experimental design was a completely randomized design with 2 x 2 factorial arrangement, with the factors being (1) two levels of PKM (0 vs. 5%) and (2) with or without dietary β-mannanase (480 IU/㎏ of diet CTCzyme) supplementation. All hens were housed in cages (35 ㎝W ×35 ㎝H) with 2 hens per cage for six weeks feeding trial. Laying performance was recorded daily during feeding trial. Egg quality, nutrients utilizability and blood assays were done at the end of feeding trial. Egg production was improved (P<0.05) by both dietary PKM inclusion and β-mannanase combined supplementation. Either β-mannanase or PKM did not affect feed intakes and feed conversion ratio of all diets. Egg weight of hens fed diet containing 5% of PKM had heavier (P<0.05) eggs compared with hens fed without PKM. Albumen height was improved (P<0.05) by dietary mannanase supplementation. Crude fat utilization of 5% PKM diet was higher than that of no PKM diet regardless of β-mannanase supplementation. Both DM and total carbohydrate utilization were decreased (P<0.05) in hens fed 5% PKM diet. Serum IgG and yolk IgY contents of PKM groups were lower (P<0.05) than those of no PKM groups. This result showed that 5% PKM diet, independent of dietary β-mannanase supplementation, was able to improve egg production. In addition, dietary β-mannanasc supplementation could be used for improving the albumen height of eggs.

Effects of supplemental β-mannanase on in vitro disappearance of dry matter in feed ingredients for swine

( Chan Sol Park ),( Jeonghyeon Son ),( Beob Gyun Kim ) 한국축산학회 2022 축산기술과 산업 Vol.9 No.1

The present study was aimed to investigate the effects of supplemental β-mannanase on in vitro ileal disappearance (IVID) and in vitro total tract disappearance (IVTTD) of dry matter (DM) in barley, canola meal, copra meal, corn, corn distillers dried grains with solubles, cottonseed meal, palm kernel meal, soybean meal, and wheat for pigs. Feed ingredient samples were finely ground and divided into the control group and the treatment group. The samples of the control group were prepared to contain 990 g/kg test ingredient and 10 g/kg cornstarch, whereas the samples of the treatment group were prepared to contain 990 g/kg test ingredient and 10 g/kg β-mannanase product (8,000 units/kg in the mixed sample). A 2-step in vitro ileal digestion technique, which simulated the digestion and absorption processes in the stomach and small intestine, was used to determine the IVID of DM in test ingredients, whereas a 3-step in vitro ileal digestion technique, which additionally simulated the digestion process of the large intestine, was used to determine the IVTTD of DM in test ingredients. The in vitro digestion procedures were performed in triplicate for each sample. The addition of β-mannanase increased (p = 0.003) the IVID of DM in wheat and tended to increase (p = 0.063) the IVID of DM in soybean meal. The IVTTD of DM in barley, cottonseed meal, and palm kernel meal was improved (p < 0.05) by the addition of β-mannanase. In conclusion, the digestibility of nutrients for pigs may be improved when β-mannanase is added into diets containing barley, cottonseed meal, palm kernel meal, soybean meal, or wheat.

Effects of Supplemental Beta-mannanase on Digestible Energy and Metabolizable Energy Contents of Copra Expellers and Palm Kernel Expellers Fed to Pigs

W.B. Kwon,김법균 아세아·태평양축산학회 2015 Animal Bioscience Vol.28 No.7

The purpose of this study was to determine the effect of β-mannanase supplementation on digestible energy (DE) and metabolizable energy (ME) contents of copra expellers (CE) and palm kernel expellers (PKE) fed to pigs. Six barrows with an initial body weight of 38.0 kg (standard deviation = 1.5) were randomly allotted to a 6×6 Latin square design with 6 dietary treatments and 6 periods. Six experimental diets were prepared in a 3×2 factorial treatment arrangement with 3 diets of a corn-soybean meal-based diet, a CE 30% diet, and a PKE 30% diet and with 2 concentrations of supplemental β-mannanase at 0 or 2,400 U/kg. All diets had the same proportion of corn:soybean meal ratio at 2.88:1. The marker-to-marker procedure was used for fecal and urine collection with 4-d adaptation and 5-d collection periods. No interactive effects were observed between diet and β-mannanase on energy digestibility and DE and ME contents of experimental diets. However, diets containing CE or PKE had less (p<0.05) DE and ME contents compared with the corn-soybean meal-based diet. The DE and ME contents in CE and PKE were not affected by supplemental β-mannanase. Taken together, we failed to find the effect of β-mannanase supplementation on energy utilization in CE and PKE fed to pigs.

Trichoderma harzianum 유래 β-Mannanase의 정제 및 Picea abies Galactosyl Glucomannomannan 가수분해 올리고당의 중합도별 Bifidobacterium spp.에 대한 생육활성

이명석 ( Myung Seok Lee ),박영서 ( Young Seo Park ),박귀근 ( Gwi Gun Park ) 한국산업식품공학회 2013 산업 식품공학 Vol.17 No.1

Sephadex G-100 column chromatography에 의해 Trichoderma harzianum 유래 β-mannanase의 정제를 수행하여 비활성 8.44 units/mL 정제배율 56.27배를 나타내었다. SDS-PAGE에 의한 단일밴드를 확인하였고, 분자량은 52.5 kDa으로 결정되었다. 정제효소에 의해 Picea abies Galactosyl glucomannan을 가수분해하여 activated carbon column chromatography에 의해 당가수분해물을 분리 회수하여 TLC 및 Timell`s method에 의해 중합도 6, 8, 9로 결정되었으며, Penicillum purpurogenum유래 정제 β-mannanase와 α- galactosidase를 이용한 enzymatic sequential action에 의해 3 가지 가수분해산물 모두 hetero type galactosyl glucomannooligosaccharides로 확인되었다. B. longum, B. bifidum, B. infantis, B. animalis, B. breve, B. adolessentis, B. auglutum의 생육활성에 대한 중합도 6, 8, 9의 영향을 검토하기 위하여 modified-MRS 배지상에 탄소원으로 중합도 6, 8, 9를 대체하여 생육활성을 비교한 결과 B. longum에서는 D.P. 6 galactosyl glucomannooligosaccharide를 탄소원으로 대체한 경우 표준 MRS배지와 비교하여 14.3배, D.P. 8에서 9.42배, D.P. 9에서 8.0배의 상대활성을 나타내어 가장 우수한 생육활성을 나타내었으며, B. breve의 경우에서도 D.P. 6에서 7.52배, D.P. 8에서 8.19배, D.P. 9에서 6.9배의 높은 활성을 보인 반면, B. bifidum에 대해서는 D.P. 6에서 2.33배, D.P. 8에서 1.33배, D.P. 9에서 2.33배의 낮은 생육활성을 나타내었다. Beta-mannanase from Trichoderma harzianum was purified by Sephadex G-100 column chromatography. The specific activity of the purified enzyme was 8.44 units/mL protein, representing an 56.27-fold purification of the original crude extract. The final preparation thus obtained showed a single band on SDS-polyacrylamide gel electrophoresis. Its molecular weight was determined to be 52.5 kDa. Picea abies galactosyl glucomannan was hydrolyzed by the purified β-mannanase, and then the hydrolysates were separated by activated carbon column chromatography. The main hydrolysates were composed of D.P. (degree of polymerization) 6, 8, and 9 galactosyl glucomannooligosaccharides. To investigate the effects of Picea abies galactosyl glucomannanooligosaccharides on in the vitro growth of Bifidobacterium longum, B. bifidum, B. animalis, B. breve, B. infantis, B. adolescentis, and B. auglutum, Bifidobacterium spp. were cultivated individually on a modified-MRS medium containing carbon sources such as D.P. 6, 8, and 9 galactosyl glucomannooligosaccharides. B. longum grew up 14.3-fold and 9.4-fold more effectively after treatment with D.P. 6 and D.P. 8 galactosyl glucomannooligosaccharides compared to those with standard MRS medium. Especially, D.P. 6 was more effective than D.P 9 galactosyl glumannooligosaccharides with regard to the growth of Bifidobacterium spp.

Xylogone sphaerospora 유래 정제 β-Mannanase에 의한 Picea abies Galactosyl Glucomannan 가수분해물의 중합도별 Bifidobacterium spp. 생육활성 비교

이명석 ( Myung Seok Lee ),박영서 ( Young Seo Park ),박귀근 ( Gwi Gun Park ) 한국산업식품공학회 2013 산업 식품공학 Vol.17 No.2

Sephadex G-100 column chromatography에 의해 Xylogone sphaerospora 유래 β-mannanase의 정제를 수행하여 비활성 8.24 units/mL 정제배율 58.86배를 나타내었다. SDS-PAGE에 의한 단일밴드를 확인하였고, 분자량은 42 kDa으로 결정되었다. 정제효소에 의해 Picea abies Galactosyl glucomannan을 가수분해하여 activated carbon column chromatography에 의해 당가수분해물을 분리 회수하여 TLC와 FACE법 및 Timell`s method에 의해 중합도 7, 8, 12 and 13으로 결정되었으며, Penicillum purpurogenum 유래 정제 β-mannanase와 α-galactosidase를 이용한 enzymatic sequential action에 의해 4가지 가수분해산물 모두 hetero type galactosyl glucomannooligosaccharides로 확인되었다. B. longum, B. bifidum, B. infantis, B. animalis, B. breve, B. adolessentis, B. auglutum의 생육활성에 대한 중합도 7, 8, 12 and 13의 영향을 검토하기 위하여 modified-MRS 배지상에 탄소원으로 중합도 7, 8, 12 and 13을 대체하여 생육활성을 비교한 결과 B. longum에서는 D.P. 7 galactosyl glucomanno-oligosaccharide를 탄소원으로 대체한 경우 표준 MRS배지와 비교하여 10.8배, D.P. 8에서 12.5배, D.P. 12에서 10.2배 D.P. 13에서 9.2배의 상대활성을 나타내어 가장 우수한 생육활성을 나타내었으며, B. bifidum의 경우에서도 D.P. 7에서 3.0배, D.P. 8에서 3.3배, D.P. 12에서 3.7배 D.P. 13에서 5.7배의 활성을 보였다. β-Mannanase from Xylogone sphaerospora was purified by Sephadex G-100 column chromatography. The specific activity of the purified enzyme was 8.24 units/mL protein, representing an 58.86-fold purification of the original crude extract. The final preparation thus obtained showed a single band on SDS-polyacrylamide gel electrophoresis. The molecular weight was determined to be 42 kDa. Picea abies galactosyl glucomannan was hydrolyzed by the purified β-mannanase, and then the hydrolysates were separated by activated carbon column chromatography. The main hydrolysates were composed of D.P. (degree of polymerization) 7, 8, 12, and 13 galactosyl glucomanno-oligosaccharides. To investigate the effects of Picea abies galactosyl glucomanno-oligosaccharides on the in vitro growth of Bifidobacterium longum, B. bifidum, B. animalis, B. breve, B. infantis, B. adolescentis, and B. auglutum, Bifidobacterium spp. were cultivated individually on a modified-MRS medium containing a carbon source such as D.P. 7, 8, 12, and 13 galactosyl glucomanno-oligosaccharides. B. longum propagated 10.83-fold, 12.50-fold, 10.25-fold, and 9.25-fold more effectively by the treatment of D.P. 7, 8, 12, and 13 galactosyl glucomanno-oligosaccharides, respectively, compared to those of standard MRS medium. Especially, all four sorts of galactosyl glucomannooligosaccharides were more effective in promoting the growth of B. longum than B. animalis, B. bifidum, B. breve, and B. infantis.

Bacillus sp. 유래 β-Mannanase의 정제 및 Picea abies Galactosyl Glucomannan 가수분해 올리고당의 중합도별 Bifidobacterium spp.에 대한 생육활성

이명석 ( Myung Seok Lee ),박영서 ( Young Seo Park ),박귀근 ( Gwi Gun Park ) 한국산업식품공학회 2014 산업 식품공학 Vol.18 No.4

DEAE Sephadex column chromatography에 의해 Bacillus sp. 유래 β-mannanase의 정제를 수행하여 비활성 21.57 units/mL 정제배율 95.33배를 나타내었다. SDS-PAGE에 의한 단일밴드를 확인하였고, 분자량은 38.9 kDa으로 결정되었다. 정제효소에 의해 Picea abies galactosyl glucomannan을 가수분해하여 activated carbon column chromatography에 의해 당가수분해물을 분리 회수하여 TLC, FACE 및 Timell’s method에 의해 중합도 8, 10으로 결정되었으며, Penicillum purpurogenum유래 정제 β-mannanase와 α-galactosidase를 이용한 enzymatic sequential action에 의해 2가지 가수분해산물 모두 hetero type galactosyl glucomannooligosaccharides로 확인되었다. B. longum, B. bifidum, B. infantis, B. animalis, B. breve, B. adolessentis, B. auglutum의 생육활성에 대한 중합도 8, 10의 영향을 검토하기 위하여 modified-MRS 배지상에 탄소원으로 중합도 8, 10를 대체하여 생육활성을 비교한 결과 B. animalis에서는 중합도 8 galactosyl glucomannooligosaccharide를 탄소원으로 대체한 경우 표준 MRS 배지와 비교하여 19.5배, 중합도 10에서 18.7배의 가장 우수한 생육활성을 나타내었으며, B. bifidum에 대해서는 중합도 8의 경우 15.3배와 중합도 10에서 14.3배 그리고 B. longum에서는 중합도 8 galactosyl glucomannooligosaccharide를 탄소원으로 대체한 경우 표준 MRS배지와 비교하여 중합도 8에서 15.2배, 중합도 10에서 13.9배의 상대활성을 우선적으로 나타내었다. β-Mannanase from Bacillus sp. was purified by DEAE sephadex column chromatography. The specific activity of the purified enzyme was 21.57 units/mg protein, representing an 95.33-fold purification of the original crude extract. The final enzyme preparation obtained showed a single band on the SDS-PAGE and its molecular weight was estimated to be 38.9 kDa. Galactosyl glucomannan from Picea abies was hydrolyzed using the purified β-mannanase, and then the hydrolysates were separated by activated carbon column chromatography. The main hydrolysates were composed of galactosyl glucomannooligosaccharides with a degree of polymerization (DP) of 8 and 10. To investigate the effects of Picea abies galactosyl glucomannanooligosaccharides on the growth of the following: Bifidobacterium longum, B. bifidum, B. animalis, B. breve, B. infantis, B. adolescentis, and B. auglutum, each Bifidobacterium spp. was cultivated into the modified-MRS medium containing galactosyl glucomannooligosaccharides with DP 8 or 10 as a carbon source. The growth of B. animalis increased 19.5-fold and 18.7-fold by the treatment of galactosyl glucomannooligosaccharides with DP 8 and 10, respectively, compared to that of the standard MRS medium. B. bifidum grew 15.3 and 14.3-fold and B. longum grew 15.2 and 13.9-fold by the treatment of galactosyl glucomannooligosaccharides with DP 8 and 10, respectively. Especially, galactosyl glucomannooligosaccharides with DP 8 was more effective than that with DP 10 on the growth of B. animalis, B. bifidum and B. longum.

Various levels of copra meal supplementation with β-Mannanase on growth performance, blood profile, nutrient digestibility, pork quality and economical analysis in growing-finishing pigs

( H. J. Kim ),( S. O. Nam ),( J. H. Jeong ),( L. H. Fang ),( H. B. Yoo ),( S. H. Yoo ),( J. S. Hong ),( S. W. Son ),( S. H. Ha ),( Y. Y. Kim ) 한국축산학회 2017 한국축산학회지 Vol.59 No.7

Background: To reduce use of main feed ingredient like corn, soy bean meal (SBM) and wheat, alternative ingredients has been studied like copra meal (CM). Production amount of CM which has been high makes CM to be an alternative feed stuff. However, low digestibility on AA and low energy content by high fiber content can be an obstacle for using CM. This experiment was conducted to evaluate the effects of CM supplementation with β-mannanase on growth performance, blood profile, nutrient digestibility, pork quality and economic analysis in growing-finishing pigs. Methods: A total of 100 growing pigs ([Yorkshire × Landrace] × Duroc) averaging 31.22 ± 2.04 kg body weight were allotted to 5 different treatments by weight and sex in a randomized complete block (RCB) design in 5 replicate with 4 pigs per pen. Treatments were 1) Control (corn-SBM based diet + 0.1% of β-mannanase (800 IU)), 2) CM10 (10% copra meal + 0.1% β-mannanase (800 IU)), 3) CM15 (15% copra meal + 0.1% β-mannanase (800 IU)), 4) CM20 (20% copra meal + 0.1% β-mannanase (800 IU)) and 5) CM25 (25% copra meal + 0.1% β-mannanase (800 IU)). Four phase feeding program was used: growing I (week 1-3), growing II (week 4-6), finishing I (week 7-9) and finishing II (week 10-12). Results: In growth performance, there was no significant difference among treatments during whole experimental period. In growingI phase, G:F ratio tended to increase when CM was increased (P = 0.05), but ADG and ADFI tended to decrease in finishingII phase (linear, P = 0.08). Also, increasing CM reduced ADG (linear, P = 0.02) and feed efficiency (linear, P = 0.08) during the whole finishing period. In blood profiles, BUN was linearly increased as CM increased (linear, P = 0.02) at growingII period. In digestibility trial, there was no significant difference in dry matter, crude fat, crude ash and nitrogen digestibility. However, crude protein digestibility was decreased linearly (linear, P = 0.02). In economic analysis, feed cost per weight gain and total feed cost per pig were reduced in overall period when CM was provided by 25% (linear, P = 0.02). Conclusion: CM with 0.1% of β-mannanase (800 IU) could be supplemented instead of corn and SBM up to 25% without detrimental effects on growth performance and pork quality of growing-finishing pigs.

Effects of bacterial β-mannanase on apparent total tract digestibility of nutrients in various feedstuffs fed to growing pigs

Jang Ki Beom,Zhao Yan,Kim Young Ihn,Pasquetti Tiago,Kim Sung Woo 아세아·태평양축산학회 2023 Animal Bioscience Vol.36 No.11

Objective: The objective of this study was to determine the effects of β-mannanase on metabolizable energy (ME) and apparent total tract digestibility (ATTD) of protein in various feedstuffs including barley, copra meal, corn, corn distillers dried grains with solubles (DDGS), palm kernel meal, sorghum, and soybean meal. Methods: A basal diet was formulated with 94.8% corn and 0.77% amino acids, minerals, and vitamins and test diets replacing corn-basal diets with barley, corn DDGS, sorghum, soybean meal, or wheat (50%, respectively) and copra meal or palm kernel meal (30%, respectively). The basal diet and test diets were evaluated by using triplicated or quadruplicated 2×2 Latin square designs consisting of 2 diets and 2 periods with a total of 54 barrows at 20.6±0.6 kg (9 wk of age). Dietary treatments were levels of β-mannanase supplementation (0 or 800 U/kg of feed). Fecal and urine samples were collected for 4 d following a 4-d adaptation period. The ME and ATTD of crude protein (CP) in feedstuffs were calculated by a difference procedure. Data were analyzed using Proc general linear model of SAS. Results: Supplementation of β-mannanase improved (p<0.05) ME of barley (10.4%), palm kernel meal (12.4%), sorghum (6.0%), and soybean meal (2.9%) fed to growing pigs. Supplementation of β-mannanase increased (p<0.05) ATTD of CP in palm kernel meal (8.8%) and tended to increase (p = 0.061) ATTD of CP in copra meal (18.0%) fed to growing pigs. Conclusion: This study indicates that various factors such as the structure and the amount of β-mannans, water binding capacity, and the level of resistant starch vary among feedstuffs and the efficacy of supplemental β-mannanase may be influenced by these factors.