Study On Novel Low-temperature-active And Salt-tolerant Mannanases From Bacteria

Hemicellulose is the second renewable resource in the world.Up to now,most of inadequate exploitable hemicellulose are agricultural/industrial wastes.For example,palm kernel cake?PKC?,is a major agricultural by-product of the palm oil industry,which is abundant in Southeast Asia.The annual quantity of PKC is about 9.92 million tonnes.Mannan is one of the main components in the PKC,accounting for about 40%of the dry weight.Mannanase is the key enzyme in the process of mannan degradation.Mannanase is one of the key products released by the National Development and Reform Commission?NDRC?in the<the guidelines for key products and Services of Strategic emerging Industries>?2016?.Therefore,our study explored the novel low-temperature-active and salt-tolerant mannanases,modification of novel low-temperature-active mannanase and potential application of the novel low-temperature-active and salt-tolerant mannanases.we hope to achieve win-win results in terms of economic efficiency,energy saving,emission reduction and environmental protection.1.Exploitation and modification of novel low-temperature-active mannanaseA glycoside hydrolase family 26 mannanase-coding gene?manAGN25?was cloned from Sphingobacterium sp.GN25 isolated from the feces of Grus nigricollis.Before experiment's performance,ManAGN25 was predicted to be a low-temperature active mannanase based on the molecular characterization,including?1?ManAGN25shared the highest identity with the experimentally verified low-temperature active mannanase?ManAJB13?from Sphingomonas sp.JB13;?2?compared with their mesophilic and thermophilic counterparts,ManAGN25 and ManAJB13 had increased number of amino acid residues around their catalytic sites;?3?these increased number of amino acid residues built longer loops,larger total accessible surface area and packing volume.Coding sequence was successfully expressed in Escherichia coli BL21?DE3?.Then the experiments of biochemical characterization verified that the purified recombinant ManAGN25 is a low-temperature active mannanase:the enzyme showed apparently optimal activity at 35–40°C and retained 78.2,44.8,and 15.0%of its maximum activity when assayed at 30,20,and 10°C,respectively;the half-life of the enzyme was approximately 60 min at 37°C;the enzyme presented a K_m of 4.2mg/mL,a V_max of 0.6?mol/min/mg and a k_cat of 0.4/s in McIlvaine buffer?pH 7.0?at35°C using locust bean gum as the substrate;and the activation energy for hydrolysis of locust bean gum by the enzyme was 36.0 kJ/mol.A glycoside hydrolase family 26 mannanase-coding gene?manAJB13?was cloned from Sphingomonas sp.JB13 isolated from slag of a more than 20-year-old phosphate rock-stacking site.Our preliminary study showed that ManAJB3 is a low-temperature-active mannanase.Compared with ManAGN25 and Man5HJ4,ManAJB3 had the highest low-temperature activity.In order to enhance its potential application and reveal the low-temperature-active molecular characteristics,thermostability modification of ManAJB3 was carried out in the study.The intrinsically disordered protein region prediction?IUPred?is based on estimating the capacity of polypeptides to form stabilizing contacts.According to the IUPred,the mutants DeP41 and DeP41P42 were designed to delete the N-terminal redundant sequences for the sake of reducing the disorder trend.In addition,advanced structures of the wild-type ManAJB13N and mutants were constructed by the method of Iterative threading assembly refinement?I-TASSER?.The results of structural comparison showed that the structural rigidity of both mutants was stronger than that of wild enzymes.Combined with the results of energy and advanced structure analysis,the thermal activity and/or stability of mutants should be increased.In order to avoid the effect of redundant N-terminal on the experimental results,the N-terminal sequence introduced by the expression vector was removed by HRV 3C protease.The experimental results show that the thermal activity and stability of the mutants rDeP41N and rDeP41P42N have been improved,which is consistent with the expected results.The mutants rDeP41N and rDeP41P42N showed optimal activity at 40?and 45?,which is 5 and 10?higher than that of the wild-type rManAJB13N?35??,respectively.The half-life of the mutants rDeP41N and rDeP41P42N were 2-and 4-higher than that of the wild-type rManAJB13N at 50°C.2.Exploitation of novel salt-tolerant enzyme and potential application in liquid detergent industryA glycoside hydrolase family 5?-mannanase encoding gene?man5HJ14?was cloned from Bacillus sp.HJ14 isolated from saline soil in Heijing town.Coding sequence was successfully expressed in E.coli BL21?DE3?.Purified recombinant mannanase?rMan5HJ14?exhibited optimal activity at pH 6.5 and 65°C.The enzyme showed good salt tolerance:retaining more than 56%?-mannanase activity at 3.0–30.0%?w/v?NaCl and more than 94%of the initial activity after incubation with 3.0–30.0%?w/v?NaCl at 37°C for 60 min;Surfactants and chelating agents,namely SDS,CTAB,EDTA,and sodium tripolyphosphate,showed little or no effect?retaining>82.4%activity?on enzymatic activity.Liquid detergents,namely Tupperware,Walch,Bluemoon,Tide,and OMO,also showed little or no effect?retaining>72.4%activity?on enzymatic activity at 0.5–2.0%?v/v?.The activity and stability of rMan5HJ14 in various liquid laundry detergents is much better compared with commercial?-mannanase Nanjian.Together,the mannanase may be an alternative for potential use in liquid detergent industry.The enzyme further presents a high Proportion?11.97%?of acidic amino acid residues?D and E?,which may affect the SDS and NaCl tolerance of the enzyme.3.Preparation and prebiotic potential of manno-oligosaccharide by enzymatic hydrolysisHydrolysates of locust bean gum?LBG?and palm kernel cake?PKC?by GH26rManAJB13 and GH5 rMan5HJ14 were analyzed by thin-layer chromatography?TLC?and electrospray ionization mass spectrometry?ESI?,respectively.The results showed that there was no significant difference between hydrolysates of PKC by both mannanases,including mannose to mannotetrose.mannoheptose was got from the hdrolysates of LBG by rMan5HJ14.However,hydrolysates of LBG by ManAJB13 had mannohexaose and higher concentration of mannotriose and mannotetrose than that of rMan5HJ14.The probiotic Lactobacillus plantarum CICC 24202 showed remarkable growth in the media containing the hydrolysates by mannanases in our study.To conclude,three mannanase from the special habitats in Yunnan were studied.?1?GH26 ManAGN25 was a novel low-temperature-active mannanase and the low-temperature-active relevant molecular characteristics were revealed.Based on the above characteristics,the thermal modification of GH26 mannanase ManAJB13 had been rational designed,and it was successfully obtained that the thermal activity and stability of the mutants from the low-temperature-active mannanase ManAJB13 had been improved.?2?GH5 Man5HJ14 was a novel salt-tolerance mannanase.?3?It was revealed that ManAJB13 and Man5HJ14 had potential application in preparation of the manno-oligosaccharides by enzymatic hydrolysis and liquid detergent industry.This study not only enriches the resource and application of novel low-temperature-active and salt-tolerant mannanases,but also provided great information for the molecular modification of low-temperature-active mannanase.