周玉玲, 蒋思婧, 贺妮莎, 张桂敏
湖北大学生命科学学院, 湖北省生物资源绿色转化协同创新中心, 省部共建生物催化与酶工程国家重点实验室, 湖北 武汉 430062
收稿日期：2020-08-23；修回日期：2020-10-23；网络出版日期：2020-11-10
基金项目：国家重点研发计划(2018YFA0901100);国家自然科学基金(31970059);2016武汉黄鹤英才人才项目
*通信作者：张桂敏, Tel: +86-27-88663882;E-mail: zhangguimin@hubu.edu.cn, zhangguimin6@hotmail.com.
摘要：几丁质是自然界含量丰富的多糖，难溶于水，常被作为废弃物丢弃，造成资源浪费和环境污染。然而，其水解产物N-乙酰氨基葡萄糖（GlcNAc）是一种重要的功能氨糖类化合物，广泛应用于医药、保健及护肤品等领域，市场需求量大。因此，将几丁质转换为高附加值的GlcNAc具有重要意义。几丁质酶可专一性水解几丁质产生GlcNAc，用于GlcNAc的酶法制备，从而替代化学加工方法，降低环境污染，提高产品质量。本文介绍了微生物来源几丁质酶的特点与分类，重点阐述了微生物来源的几丁质内切酶、几丁二糖外切酶及β-N-乙酰氨基葡萄糖苷酶在几丁质降解生产GlcNAc过程中的作用、方式和产率，这将为酶法生产GlcNAc提供一定的借鉴。
关键词：微生物几丁质酶酶法制备N-乙酰氨基葡萄糖生产
Research progress of microbial chitinase and its application in the preparation of N-acetylglucosamine
Yuling Zhou, Sijing Jiang, Nisha He, Guimin Zhang
State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, School of Life Sciences, Hubei University, Wuhan 430062, Hubei Province, China
Received: 23 August 2020; Revised: 23 October 2020; Published online: 10 November 2020
*Corresponding author: Guimin Zhang, Tel: +86-27-88663882; E-mail: zhangguimin@hubu.edu.cn, zhangguimin6@hotmail.com.
Foundation item: Supported by the National Key R&D Plan of China (2018YFA0901100), by the National Natural Science Foundation of Chian (31970059) and by the 2016 Wuhan Yellow Crane Talents (Science) Program
Abstract: Chitin is the second most abundant carbohydrate polymer in nature, but often discarded as wastes. N-acetylglucosamine (GlcNAc), the final hydrolysate of chitin, is an important functional amino sugar compound that can be used in medicine, healthcare, and skin care products with a great demand. Therefore, it is of great significance to convert chitin to GlcNAc with high value added. Chitinase can specifically hydrolyze chitin to produce high value-added N-acetylglucosamine, to replace chemical processing strategy, thus reducing environmental pollution and improving product quality. This review briefly introduces specific features and classification of microbial chitinases. Then, the roles, mode and yield of endochitinase, chitobiosidase, and β-N-acetylglucosidase in the production of GlcNAc from chitin in recent years are elaborated, to provide references for enzymatic production of GlcNAc.
Keywords: microbial chitinaseenzymatic preparationproduction of N-acetylglucosamine
几丁质又名甲壳素，是由2-乙酰氨基-2-脱氧-D-葡萄糖(GlcNAc)通过β-1，4糖苷键连接形成的高分子碳水化合物，这些化合物和一些蛋白质、酚类、脂类或其他碳水化合物，如β-葡聚糖等交联形成高度有序的高分子支架，构成虾壳、蟹壳、昆虫外壳及真菌细胞壁，在自然界含量非常丰富，仅次于纤维素和半纤维素[1]。几丁质分子间和分子内可形成大量的氢键，致使结构紧密，难溶于水。根据分子链的排列方式，几丁质可分为α、β和γ三种类型：α型几丁质在自然界中含量最丰富，由反向平行排列的GlcNAc聚合长链通过氢键堆积而成，结构致密，难溶于水，普遍存在于虾、蟹和昆虫等节肢动物的甲壳中；β型几丁质由同向平行排列的链形成单斜晶性结构，稳定性比α型几丁质差，遇水会膨胀，主要存在鱿鱼、硅藻与蠕虫等生物体中；γ型几丁质为a型几丁质和β型几丁质的混合体，主要存在于鱿鱼的胃及蚕等昆虫体内。几丁质通过强碱或脱乙酰酶处理，脱去50%以上乙酰基团后成为壳聚糖。几丁质和壳聚糖作为一类可再生资源，在食品、化工、医药和农业等领域有广泛的应用前景，如几丁质或壳聚糖可用于手术缝合线、药物递呈载体和染料吸附剂和重金属吸附剂等[1]。几丁质和壳聚糖进一步的降解产物几丁寡糖、壳寡糖不仅具有几丁质和壳聚糖的某些生物学功能，如植物生长发育调节、吸附螯合重金属等，而且具有自身特殊的性质，如水溶性、抗菌抑菌作用、减肥与降低胆固醇及抗肿瘤活性等功能[1-3]。
几丁质水解的最终产物GlcNAc和GlcN是生物体内合成很多功能大分子物质，如透明质酸、硫酸角质素和硫酸软骨素的前体，在促进关节滑液合成并减缓关节疼痛，增加皮肤透明质酸含量，改善糖尿病与肝炎症状，增进免疫能力和抗癌等方面起到重要作用[4]。全世界已有70多个国家和地区将GlcNAc作为运动营养品、膳食补充剂或药品等用于骨关节病的预防和治疗，成为国际医学界公认的治疗骨关节疾病的首选药物。随着老龄人口的增加及人们保健意识的增强，GlcNAc的市场需求量逐年增加。2004年至2009年，GlcNAc年需求量以7%递增，2014年全球需求量达到2.9万t，2017年进一步增加至4.66万t[5]。从2020到2023年，全球氨基葡萄糖的需求量预计以8.6%的年增长率继续递增[6]。因此，建立绿色环保的生产工艺并提高氨基葡萄糖的产量对满足日益增大的市场需求有着重要意义。
GlcNAc及GlcN的制备方法有化学法和生物法。生物法可细分为微生物发酵法、微生物水解法和酶水解法。微生物发酵法主要通过原始菌株或构建基因工程菌，以粮食或葡萄糖等为底物发酵生产GlcNAc[7]。近年来利用基因工程、代谢工程等方法重构代谢途径，微生物发酵法生产的GlcNAc可达到103.1 g/L[8]。然而，微生物发酵法高耗能耗水，同时耗费粮食等。现今全球粮食资源有限，用微生物发酵法可能加剧粮食消耗。因此，利用非粮食资源或加工后的副产品进行深加工是可持续性发展的重要方向。富含几丁质的虾、蟹壳及真菌菌丝体是加工后的废弃物，每年高达600万–800万t[9]，将其转化为具有功能的寡糖或GlcNAc，具有很大的发展潜力。目前，水解几丁质生产GlcNAc的主要方法有化学法、微生物水解法及酶水解法。化学法简单、高效，但在高温长时间用强酸强碱处理几丁质，会导致产品品质不均、副产物多、设备腐蚀严重及环境污染等问题。微生物水解法由于所用菌株有些是致病微生物或代谢产生有毒副产物，酶的成分不清楚，难以定向改造提高活力等缺点，在应用上受到一定的限制。因此，酶解法是绿色生物法制备GlcNAc及GlcN的主要研究方向和热点。此前，有不少关于几丁质酶的综述，如Oyeleye等从几丁质酶的多样性，糖苷酶18和19家族的催化机理，蛋白质工程对几丁质酶的改造等方面进行了详述；Le等综合阐述了细菌和真菌等微生物几丁质酶的类型、催化机理及几丁质酶在几丁寡糖生产、单细胞生产和真菌病原菌的生物防治等方面的应用[10]。这些文章虽较全面介绍了几丁质酶，但微生物几丁质酶在GlcNAc制备上的应用效果及组合方式提及比较少，因此，本文主要就近年来微生物几丁质酶的发展及几丁质酶在GlcNAc生产制备上展开详述。
1 微生物几丁质酶 几丁质酶(chitinase，EC3.2.1.14)是一种能专一性地催化GlcNAc-GlcNAc或GlcNAc-GlcNC糖苷键断裂，从而使几丁质降解成为几丁寡糖或单糖的一类酶的总称，在自然界的碳、氮素循环，微生物侵染植物组织、机体免疫、生物防御等方面发挥了重要作用，广泛分布于微生物、植物、脊椎动物及昆虫中。与其他来源的几丁质酶相比，微生物几丁质酶具有种类多、活力高和性质多样等优点，因此成为研究的热点。
1.1 微生物几丁质酶资源丰富 微生物种类繁多，分布在生物圈的各个角落，包括各种极端环境，因而，微生物是最大的酶资源库。几丁质是微生物重要的碳素和氮素来源，几丁质酶在几丁质的循环利用中发挥了巨大的作用。粘质沙雷氏菌[11]、芽孢杆菌[12]、拟杆菌[13]和木霉[14]等微生物甚至可产生多种几丁质酶，如最先被研究的粘质沙雷氏菌体内有3种几丁质酶：ChiA、ChiB、ChiC及一种多糖单加氧酶CBP21[11]。在UniProt数据库(https://www.uniprot.org/)中积累的4378条几丁质酶相关序列[10]，酶学表征的有932条。这些几丁质酶大部分来自于微生物：2779条来自细菌，236条来自真菌，5条来自病毒，占整个几丁质酶序列的70%以上[10]。
1.2 微生物几丁质酶活力高 几丁质是微生物的主要营养来源，为了快速吸收利用几丁质，高活性的微生物几丁质酶发挥了重要作用，很多产几丁质酶的菌种被分离鉴定，并直接用于微生物水解几丁质。Aounallah等分离的Bacillus licheniformis AT6可产高活性的几丁质酶，通过培养基优化和高密度发酵，发酵液中几丁质酶的活力可提高到505.26±22.223 mU/mL，24 h内将几丁质完全水解为GlcNAc[15]。2017年Cardozo等从海洋中分离了10株可产几丁质酶的Aeromonas caviae，用0.5 U/mL的几丁质粗酶液在37 ℃、pH 5.0条件下，可将2% (W/V)胶体几丁质水解为GlcNAc，24 h后GlcNAc的产率可达19%–93%[16]。因此，有些产几丁质酶的微生物被直接用于水解几丁质，该方法简单方便，不需要复杂的基因克隆和操作，但很多微生物是致病微生物或代谢产生有毒副产物，如Aeromonas caviae就属于条件致病菌，用于工业生产就需要对菌株进行鉴定和严格分析。
1.3 微生物几丁质酶性质多样 微生物生活在生物圈的各个角落，为适应环境变化，进化出各种性质多样的酶包括几丁质酶。细菌和放线菌的几丁质酶在pH 4–7时活性最高，pH 3–10还有活力；链霉菌的几丁质酶在pH 8–14仍能保持50%以上活力[10]。大部分微生物来源的几丁质酶较耐热，拟杆菌的几丁质酶在50–60 ℃保温6 h，活力保持80%以上[13]，枯草杆菌的几丁质酶在100 ℃处理20 min，其活力几乎不变[17]。还有部分微生物的几丁质酶在低温也可以发挥作用，如Pseudoalteromonas sp. DL-6来源的几丁质酶ChiA和ChiC的最适温度为20 ℃和30 ℃[18]。另外，海洋中很多生物以贝类为食，富集和进化了许多活力高、性质特殊的几丁质酶，如Das等从海洋中分离的真菌Aspergillus terreus[19]，Yang等从海洋中筛选的Paenicibacillus barengoltzii[20]。它们产的几丁质酶降解能力强、耐高盐高温，可满足几丁质降解的一些特殊条件，如含盐量高的海产品及餐饮垃圾。
2 微生物几丁质酶的分类 同其他几丁质酶一样，微生物几丁质酶根据作用方式不同，可分为内切几丁质酶、外切几丁质酶。外切几丁质酶按照作用部位又分为几丁二糖外切酶和β-N-乙酰氨基葡萄糖苷酶[21-22]，图 1显示了各种几丁质酶的作用位点和产物。
 |
| 图 1 各种几丁质酶的作用位点和产物 Figure 1 The cutting sites and products of chitinases. |
| 图选项 |
2.1 内切几丁质酶 内切几丁质酶(EC 3.2.1.14)从几丁质多糖链的内部随机水解产生几丁寡糖((GlcNAc)n，n≥2)[23] (图 1)，在几丁质的降解中发挥重要功能。微生物来源的内切几丁质酶绝大部分属于糖苷酶18家族，采用(β/α)8-TIM桶状结构，含有保守的“DXXDXDXE”催化模块和“SXGG”底物结合模块。催化模块中的谷氨酸(E)和第二个天冬氨酸(D)为活性位点，其催化过程及机制在很多文献和综述已经详细阐述[11, 24]。利用内切几丁质酶水解几丁质时，通过薄层层析(TLC)和高效液相(HPLC)分析水解产物，研究人员发现几丁质先是被降解成分子量不一的寡糖，随着时间的延长，几丁质最终被水解成几丁二糖(GlcNAc)2及少量的GlcNAc。Yang等报道Paenibacillus barengoltzii的内切几丁质酶PbChi70水解胶体几丁质，最初0–0.5 h内产物以几丁寡糖(GlcNAc)4、(GlcNAc)3和(GlcNAc)2为主，随着时间延长，几丁二糖的比例越来越高，最终产物为(GlcNAc)2和少量的GlcNAc[20]。不同的内切几丁质酶来源不同的环境，其最适温度、最适pH及催化效率都不相同。表 1列出了近几年发掘的微生物内切几丁质酶，这些内切几丁质酶分子量大小不一，最适温度差别比较大。最适温度最高的是来自海洋微生物Paenibacillus barengoltzii的几丁质酶PbChi74，可在65 ℃发挥最大活力；活力最高的是Streptomyces albolongus ATCC 27414的几丁质酶SaChiA4，比活力为66.2 U/mg。
表 1. 微生物几丁质内切酶的酶学性质及比活力 Table 1. Enzymatic properties and specific activity of endochitinases from microorganism
| Endochitinases | Organisms | GH family | Molecular weight/kDa | Temperature optimum/℃ | pH optimum | Specific activity/(U/mg) | References |
| FjChiB | F. johnsoniae UW101 | GH18 | 35.5 | 40 | 6.0 | 26.2a | [25] |
| ChiA | Pseudoalteromonas sp. DL-6 | GH18 | 113.5 | 20 | 8.0 | 42.17a | [26] |
| PbChi74 | Paenibacillus barengoltzii | GH18 | 74.6 | 65 | 4.5 | 19.9a | [27] |
| PbChi70 | Paenibacillus barengoltzii | GH18 | 70.1 | 55 | 6.0 | 30.1a | [20] |
| Chi18H8 | metagenome | GH18 | 45 | 35 | 5.0 | 63.9a | [28] |
| SaChiA4 | Streptomyces albolongus ATCC 27414 | GH18 | 47 | 55 | 5.0 | 66.2a | [29] |
| LinChi35 | Listeria innocua | GH18 | 35 | 50 | 5.0 | – | [30] |
| PbChi67 | Paenicibacillus barengoltzii | GH18 | 67.0 | 60 | 3.5 | 14.1a | [31] |
| –: not mentioned; a: collidal chitin as substrate. | |||||||
表选项
2.2 几丁二糖外切酶 几丁二糖外切酶是外切酶中的一类，主要从几丁质的还原端或非还原端以2个糖为单位水解几丁质，生成终成物为几丁二糖(GlcNAc)2。和几丁质内切酶一样，微生物几丁二糖外切酶属于糖苷酶18和19家族，大部分来自于18家族，同样具有(β/α)8-TIM桶状结构，含有保守的“DXXDXDXE”催化模块和“SXGG”底物结合模块。与几丁质内切酶不同的是，几丁二糖外切酶与底物结合区域的空间构造“深且窄”，可紧密结合底物，并持续外切底物[11]。几丁二糖外切酶在几丁质的降解中也起重要的作用，不同的几丁二糖外切酶来源不同的环境，其最适温度、最适pH及催化效率都不相同。表 2列出了近几年发掘的几丁二糖外切酶，其中，rChit46的活力最高，水解胶体几丁质的比活力为34.5 U/mg，水解几丁质粉末的比活力可达到9.5 U/mg，是目前报道活力最高的几丁二糖外切酶；来自海洋生物Paenicibacillus barengoltzii的PbChi474在高温和酸性环境中发挥最大活性，这些特性可满足几丁质水解的一些酸性及高温条件。
表 2. 微生物几丁二糖外切酶的酶学性质与比活力 Table 2. Enzymatic properties and specific activity of chitobiosidase from microorganism
| Exochitinases | Organisms | GH family | Molecular weight/kDa | Temperature optimum/℃ | pH optimum | Specific activity/(U/mg) | References |
| PbChi74 | Paenicibacillus barengoltzii | GH18 | 74.6 | 65 | 4.5 | 19.9a | [27] |
| Chi1 | Myceliophthora thermophila C1 | GH18 | 43.8 | 55 | 6.0 | 3.5 a | [32] |
| rChit46 | Trichoderma harzianum GIM3.442 | GH18 | 45.3 | 45 | 6.0 | 34.5 a | [33] |
| Chit42 | Trichoderma harzianum | GH18 | 42.0 | 35 | 6.0 | 5.2 a | [34] |
| RmChi44 | Rhizomucor miehei | GH18 | 44.6 | 50 | 4.5 | 11.3 a | [35] |
| ChiC | Pseudoalteromonas sp. DL-6 | GH18 | 91 | 30 | 9.0 | 159.45b | [22] |
| Echi47 | Pig fecal environment DNA | GH18 | 47.6 | 40 | 5.0 | 6.84 a | [36] |
| ChiT-7 | Metagenome of themangrovetidal flat soil | GH18 | 43.0 | 45 | 6.0 | 0.63 a | [37] |
| NbchiA | Nosema bombycis | GH19 | 21.5 | 40 | 7.0 | 58.6c | [38] |
| a: collidal chitin; b: 4MU-(GlcNAc)2; c: glycolchitin. | |||||||
表选项
2.3 β-N-乙酰氨基葡萄糖苷酶 β-N-乙酰氨基葡萄糖苷酶也属于几丁质外切酶，大部分不能直接降解几丁质或肽聚糖大分子，但可水解分子量低的几丁寡糖。因此，β-N-乙酰氨基葡萄糖苷酶可协同几丁质内切酶或几丁二糖外切酶水解几丁质，从而生成单体GlcNAc。β-N-乙酰氨基葡萄糖苷酶来源广泛，自上世纪70年代枯草芽孢杆菌来源的β-N-乙酰氨基葡萄糖苷酶首次被分离纯化以来[39]，一系列细菌(如E.coli、Bacillus pseudofirmus等)、真菌(如Rhizomucor miehei、Talaromyces emersonii等)、植物及昆虫来源的β-N-乙酰氨基葡萄糖苷酶陆续被分离纯化，这些不同来源β-N-乙酰氨基葡萄糖苷酶的酶学性质、底物特异性和催化机制都被研究，相应的编码基因也被克隆并进行异源表达。2018年，Zhang基于β-N-乙酰氨基葡萄糖苷酶的重要的生物学功能和广泛的工业应用，重点介绍了β-N-乙酰氨基葡萄糖苷酶的酶学性质，包括底物特异性、催化活性、最适pH值、最适温度、热稳定性、各种金属离子和有机试剂的影响以及转糖基作用[40]。表 3仅列出了近3年来β-N-乙酰氨基葡萄糖苷酶的酶学性质与比活力，可得出来源不同，β-N-乙酰氨基葡萄糖苷酶的酶学性质与比活力差别比较大。糖苷酶20家族的比活力普遍比糖苷酶3家族的高，这与Zhang等得出的结论比较一致[40]，活性的差别有可能与糖苷酶20和3家族不同的结构和催化位点有关。
表 3. β-N-乙酰氨基葡萄糖苷酶的酶学性质与比活力 Table 3. Enzymatic properties and specific activity of β - N-acetylglucosidase
| β-N- acetylglucosaminidases | Organisms | GH family | Molecular weight/kDa | Temperature optimum/℃ | pH optimum | Specific activity/(U/mg) | References |
| BpNagZ | Bacillus pumilus | GH3 | 70.3 | 70 | 6.0 | 5.91a | [41] |
| BsNagZ | Bacillus subtilis 168 | GH3 | 70.0 | 60 | 6.0 | – | [39] |
| NagZ703 | Bacillus pseudofirmus 703 | GH3 | 73 | 60 | 6.5 | 10.7a | [42] |
| BaNagase | Bacillus amyloliquefaciens YX-01 | GH3 | 67.5 | 65 | 6.0 | 16.42a | [43] |
| PsNagA | Paenibacillus sp. str. FPU-7 | GH3 | 57 | 47 | 6.5 | 1.40a | [44] |
| SnHex | Stackebrandtia nassauensis | GH20 | 57.8 | 50 | 6.0 | 0.62a | [45] |
| SaHEX | Strep-tomyces spp. | GH20 | 55.7 | 60 | 5.5 | 1149.7a | [46] |
| MthNAG | Myceliophthora thermophila C1 | GH20 | 71.0 | 50 | 4.5 | 432a | [47] |
| rJB10Nag | Shinella sp. JB10 | GH20 | 70.9 | 50 | 6.0 | 538.8a | [21] |
| –: not mentioned; a: pNP-GlcNAc as substrate. | |||||||
表选项
3 微生物几丁质酶在GlcNAc生产上的应用 3.1 单一几丁质酶水解制备GlcNAc 陆续有微生物几丁质酶被表征，发现具有广谱的底物性质，既具有内切酶或外切二糖酶活性，又具有β-N-乙酰氨基葡萄糖苷酶的活力，单独可将几丁质水解为GlcNAc。Zhou等总结有8个几丁质酶具有广谱的底物性质[41]，来源于Rhizomucor miehei的RmChi44[48]，Thermococcus kodakaraensis KOD1的TkChiA[49]，Chitinolyticbacter meiyuanensis SYBC-H1的CmChi1等[48]。这些酶的催化结构域有些只有1个，有些是多个，单独可将几丁质水解为GlcNAc，因此，有些酶被用于水解生产GlcNAc。2015年，Pradeep等纯化Streptomyces sp. CS147的几丁质酶CS147，将0.12 mg/mL的几丁质酶加入2.5% (W/V)的胶体几丁质，在50 ℃，pH 11.0的条件下，反应24 h，可得到1.058 mg/mL的GlcNAc[50]。2018年Zhang等在E. coli中表达Chitinolyticbacter meiyuanensis SYBC-H1的几丁质酶CmChi1，比活力高达15.3 U/mg。该酶既有外切酶和内切酶活力，又有β-N-乙酰葡萄糖苷酶的活力，在pH 5.2，50 ℃，24 h内单独可将1%胶体几丁质水解，水解率可达到100%[48]。2019年Tran等从Streptomyces speibonae TKU048分离纯化的几丁质酶TKU048，既具有外切酶活力又具有β-N-乙酰氨基葡萄糖苷酶活力，在pH 5.0，50 ℃，96 h内可将0.5% β-几丁质粉末水解，得到0.335 mg/mL的GlcNAc，产率73.64%[51]。这些单一几丁质酶不需要其他酶的辅助，单独可将几丁质水解为GlcNAc，工艺简单，因此引起人们的很大兴趣。
3.2 几丁质酶的联合作用 虽然有报道几丁质酶单独可将几丁质水解为GlcNAc，但这样的几丁质酶类型比较少，绝大部分的几丁质酶只有一种活性，要将几丁质完全降解为GlcNAc，需要2种以上的几丁质酶联合作用。因此，很多研究将几丁质内切酶或外切酶与β-N-乙酰氨基葡萄糖苷酶联合使用，利用内切酶或外切酶将几丁质水解为小分子的几丁寡糖，尔后β-N-乙酰葡萄糖苷酶将几丁寡糖水解为GlcNAc。联合水解时，有些是将2种以上的酶一次性添加进去，如Fu等将几丁质酶PbChi74和β-N-乙酰葡萄糖苷酶RmNAG一起加入水解3% (W/V)的胶体几丁质[27]；有些采用两步法：先用几丁质内切酶或二糖外切酶水解一定时间后，再加入β-N-乙酰氨基葡萄糖苷酶。本课题组采用这种两步法做了很多工作，如Song等先用地衣芽孢杆菌ChiA在pH 6.0，50 ℃水解30%的胶体几丁质溶液，12 h后HPLC检测几丁质完全水解为(GlcNAc)2，再加入β-N-乙酰氨基葡萄糖苷酶BsNagZ，在pH 6.0，60 ℃保温0.5 h后，GlcNAc的产率可达到88%[52]。Du等也先用几丁质外切酶AMCase水解2%胶体几丁质，2 h后HPLC检测几丁质完全水解为(GlcNAc)2，再加入β-N-乙酰氨基葡萄糖苷酶BpNagZ[41]，最终在2.25 h内胶体几丁质水解为GlcNAc，产率达到87%。表 4列出了几丁质酶联合水解几丁质的条件和产率，从表中可看出大部分几丁质酶在中温(35–55 ℃)、偏酸性的温和条件下可将胶体几丁质水解。其中，Du等[41]、Jiang等[42]、Zhou等[21]、李等[53]报道在2 h左右胶体几丁质水解基本完成；Song等报道的水解底物浓度可达到30%，是目前最高水平[52]。水解率与几丁质酶的活性和浓度、底物浓度及几丁质来源有很大关系，很难比较得出哪个几丁质酶的组合最好。然而，在相同的底物浓度、酶活性及时间内，Liu等报道的PbChi70和PbNag39组合水解效率[54]比Fu等报道的PbChi74和RmNAG组合效率稍高[27]。另外，PbChi70和PbNag39组合还可水解3%的球磨甲壳素粉，产率达到75.3%，说明这2种酶组合的水解效率比较高，有望用于工业生产。相比单一几丁质酶，2种以上几丁质酶联合使用会缩短几丁质水解时间，提高GlcNAc的产率。
表 4. 几丁质酶联合水解几丁质的条件及产率 Table 4. The conditions and yields of chitin combinatory hydrolysis by chitinases
| Concentration (W/V) and type of chitin | Concentration of endo- or exo-chitinase | Concentration of β-N-acetylglucosaminidases | Condition of hydrolysis | Yield of GlcNAc/% | Hydrolysis time/h | References |
| 30% collidal chitin | 6 U/mL ChiA | 1.25 U/mL BsNagZ | pH 6.0, 50 ℃ and 60 ℃ | 88 | 12.5 | [52] |
| 2% collidal chitin | 0.252 U/mL AMCase | 100 U/mL BpNagZ | pH 6.0, 55 ℃ | 87 | 2.25 | [41] |
| 4% collidal chitin | 0504 U/ mL AMCase | 1.5 U/mL BcNagZ | pH 2.0 and pH 5.5, 55 ℃ | 86.9 | 2.30 | [53] |
| 3% collidal chitin | 5.0 U/mL PbChi74 | 1 U/mL RmNAG | pH 6.0, 45 ℃ | 92.6 | 24 | [27] |
| 0.5% collidal chitin | 0.5 U/mL CtnSg | 0.01 U/mL rJB10Nag | pH 6.0, 37 ℃ | 2.35 times higher than that of chitinase alone | 2 | [21] |
| 0.5% collidal chitin | 0.01 U/mL-1 CtnSg | 0.1 U/mL rNag3HWLB1 | pH 6.0, 25 ℃ | 3.74 times higher than that of chitinase alone | 2 | [55] |
| 1% collidal chitin | 2 mg/mL SgCtn | 50 μg/mL SaHEX | pH 5.5, 45 ℃ | 93.7 | 6 | [46] |
| 1% α-chitin | 1 mg/mL ScChiC | 1 mg/mL ScHEX | pH 5.0, 55 ℃ | 90 | 8 | [56] |
| 1% pretreated crab shells | 39.4 μmol/L BsChi | 0.6 μmol/L OfHex1 | pH 6.0, 40 ℃ | 60 | 24 | [57] |
| 0.5% swollen chitin | 1.2 μmol/L Chi1 | 0.8 μmol/L MthNAG | pH 5.0, 50 ℃ | 37.8 | 1.9 | [47] |
| 3% ball-milled powdery chitin | 5.0 U mL-1 PbChi70 | 1.0 U/mL PbNag39 | pH 5.5, 55 ℃ | 75.3 | 24 | [54] |
| 3% collidal chitin | 5.0 U/mL-1 PbChi70 | 1.0 U/mL PbNag39 | pH 5.5, 55 ℃ | 97 | 24 | [54] |
| 1% mycelial powder | 35 μmol/L mixture of three enzymes (SmChiA, ChiB, ChiC) | 5 μM Of Hex1 | pH 6.0, 37 ℃ | 93 | 6 | [58] |
| 2% collidal chitin | ChiA3 | 3.0 U/mL NagZ703 | pH 6.0, 37° and pH 6.0, 50 ℃ | 100 | 2 | [42] |
表选项
4 总结和展望 微生物来源的几丁质酶，具有来源广泛、催化效率高和性质多样等特点，因此，越来越多的微生物几丁质酶被挖掘并被用于几丁质水解。本文综合比较了单一几丁质酶及2种以上几丁质酶联合水解几丁质的水解条件、时间和水解率，发现2种以上几丁质酶联合水解，时间短、效率高，更适用于工业生产。
利用几丁质酶水解几丁质制备GlcNAc，不但可废物利用，产生可观的经济效益，而且整个生产工艺绿色环保。然而，相对化学法水解生产GlcNAc，酶法生产的成本相对较高。因此，几丁质酶要用于工业化水解几丁质制备GlcNAc，还需要在以下4个方面不断改进。(1) 需要继续从自然界筛选高活性的几丁质酶。海洋微生物资源丰富，是尚待深入挖掘的宝库，国际微生物资源中心(MIRCEN)、中国普通微生物菌种保藏管理中心(CGMCC)和中国典型培养物保藏中心(CCTCC)等可提供大量的菌种资源。另外，借助飞速发展的基因组测序技术，通过宏基因组学和宏转录组学测序也是获得新的几丁质酶资源有效方法；(2) 很多几丁质酶的产量和活性不高，可通过高效表达、理性设计或分子定向进化提高表达量和活性；(3) 协调几种几丁质酶的类型和比例。将文献中表征活力高的几丁质内切酶或外切酶与活力高的β-N-乙酰氨基葡萄糖酶按一定比例复配，提高水解效率；(4) 发展有效的几丁质的预处理方法。几丁质难溶于水，水解反应不是在均相中发生，这极大的降低了酶反应的效率。已有研究人员提出了蒸汽爆破法[59]、高压均质机[60]和球磨等物理方法降低几丁质的晶体结构，提高几丁质的水溶性，这些措施取得了良好的效果。因此，随着微生物几丁质酶基因资源的挖掘、分子结构、机理及其基因水平调控机理研究的深入，未来能够充分利用微生物，生产更高效的几丁质酶，提高GlcNAc生产的经济性。
References
| [1] | Khan FI, Rahman S, Queen A, Ahamad S, Ali S, Kim J, Hassan MI. Implications of molecular diversity of chitin and its derivatives. Applied Microbiology and Biotechnology, 2017, 101(9): 3513-3536. DOI:10.1007/s00253-017-8229-1 |
| [2] | Komi DEA, Sharma L, Cruz CSD. Chitin and its effects on inflammatory and immune responses. Clinical Reviews in Allergy & Immunology, 2018, 54(2): 213-223. |
| [3] | Nagaoka I, Tsuruta A, Yoshimura M. Chondroprotective action of glucosamine, a chitosan monomer, on the joint health of athletes. International Journal of Biological Macromolecules, 2019, 132: 795-800. DOI:10.1016/j.ijbiomac.2019.03.234 |
| [4] | Chen JK, Shen CR, Liu CL. N-acetylglucosamine: production and applications. Marine Drugs, 2010, 8(9): 2493-2516. DOI:10.3390/md8092493 |
| [5] | Liu L, Liu YF, Shin HD, Chen R, Li JH, Du GC, Chen J. Microbial production of glucosamine and N-acetylglucosamine: advances and perspectives. Applied Microbiology and Biotechnology, 2013, 97(14): 6149-6158. DOI:10.1007/s00253-013-4995-6 |
| [6] | Jose S. Global glucosamine market to reach 46.6 thousand metric tons by 2017, according to a new report by global industry analysts, Inc. 2011. http://www.prweb.com/releases/glucosamine/glucosamine_supplements/prweb8561248.htm. |
| [7] | Gu Y, Lv XQ, Liu YF, Li JH, Du GC, Chen J, Rodrigo LA, Liu L. Synthetic redesign of central carbon and redox metabolism for high yield production of N-acetylglucosamine in Bacillus subtilis. Metabolic Engineering, 2019, 51: 59-69. DOI:10.1016/j.ymben.2018.10.002 |
| [8] | Wu YK, Chen TC, Liu YF, Lv XQ, Li JH, Du GC, Ledesma-Amaro R, Liu L. CRISPRi allows optimal temporal control of N-acetylglucosamine bioproduction by a dynamic coordination of glucose and xylose metabolism in Bacillus subtilis. Metabolic Engineering, 2018, 49: 232-241. DOI:10.1016/j.ymben.2018.08.012 |
| [9] | Kaczmarek MB, Struszczyk-Swita K, Li XK, Szcz?sna-Antczak M, Daroch M. Enzymatic modifications of chitin, chitosan, and chitooligosaccharides. Frontiers in Bioengineering and Biotechnology, 2019, 7: 243. DOI:10.3389/fbioe.2019.00243 |
| [10] | Le B, Yang SH. Microbial chitinases: properties, current state and biotechnological applications. World Journal of Microbiology and Biotechnology, 2019, 35(9): 144. DOI:10.1007/s11274-019-2721-y |
| [11] | Vaaje-Kolstad G, Horn SJ, S?rlie M, Eijsink VGH. The chitinolytic machinery of Serratia marcescens-a model system for enzymatic degradation of recalcitrant polysaccharides. FEBS Journal, 2013, 280(13): 3028-3049. DOI:10.1111/febs.12181 |
| [12] | Itoh T, Kimoto H. Bacterial chitinase system as a model of chitin biodegradation//Yang Q, Fukamizo T. Targeting Chitin-containing Organisms. Singapore: Springer, 2019: 131-151. |
| [13] | Kusaoke H, Shinya S, Fukamizo T, Kimoto H. Biochemical and biotechnological trends in chitin, chitosan, and related enzymes produced by Paenibacillus IK-5 strain. International Journal of Biological Macromolecules, 2017, 104: 1633-1640. DOI:10.1016/j.ijbiomac.2017.04.118 |
| [14] | Yu P, Yan Y, Gu Q, Wang XY. Codon optimisation improves the expression of Trichoderma viride sp. endochitinase in Pichia pastoris. Scientific Reports, 2013, 3: 3043. DOI:10.1038/srep03043 |
| [15] | Aounallah MA, Slimene-Debez IB, Djebali K, Gharbi D, Hammami M, Azaiez S, Limam F, Tabbene O. Enhancement of exochitinase production by Bacillus licheniformis AT6 strain and improvement of N-acetylglucosamine production. Applied Biochemistry and Biotechnology, 2017, 181(2): 650-666. DOI:10.1007/s12010-016-2239-9 |
| [16] | Cardozo FA, Gonzalez JM, Feitosa VA, Pessoa A, Rivera ING. Bioconversion of α-chitin into N-acetyl-glucosamine using chitinases produced by marine-derived Aeromonas caviae isolates. World Journal of Microbiology and Biotechnology, 2017, 33(11): 201. DOI:10.1007/s11274-017-2373-8 |
| [17] | Pan MY, Li JH, Lv XQ, Du GC, Liu L. Molecular engineering of chitinase from Bacillus sp. DAU101 for enzymatic production of chitooligosaccharides. Enzyme and Microbial Technology, 2019, 124: 54-62. DOI:10.1016/j.enzmictec.2019.01.012 |
| [18] | Chen LG, Zhang QF, Chi NY, Wang XH. Recombinant expression and characterization of cold-adapted chitinase genes in Kluyveromyces lactis. Acta Microbiologica Sinica, 2020, 60(1): 172-182. (in Chinese) 陈立功, 张庆芳, 迟乃玉, 王晓辉. 低温几丁质酶基因在乳酸克鲁维酵母中的重组表达及酶学性质表征. 微生物学报, 2020, 60(1): 172-182. |
| [19] | Das S, Dey P, Roy D, Maiti MK, Sen R. N-acetyl-D-glucosamine production by a chitinase of marine fungal origin: a case study of potential industrial significance for valorization of waste chitins. Applied Biochemistry and Biotechnology, 2019, 187(1): 407-423. DOI:10.1007/s12010-018-2822-3 |
| [20] | Yang SQ, Fu X, Yan QJ, Guo Y, Liu ZQ, Jiang ZQ. Cloning, expression, purification and application of a novel chitinase from a thermophilic marine bacterium Paenibacillus barengoltzii. Food Chemistry, 2016, 192: 1041-1048. DOI:10.1016/j.foodchem.2015.07.092 |
| [21] | Zhou JP, Song ZF, Zhang R, Chen CH, Wu Q, Li JJ, Tang XH, Xu B, Ding JM, Han NY, Huang ZX. A Shinella β-N-acetylglucosaminidase of glycoside hydrolase family 20 displays novel biochemical and molecular characteristics. Extremophiles, 2017, 21(4): 699-709. DOI:10.1007/s00792-017-0935-1 |
| [22] | Wang XH, Chi NY, Bai FW, Du YG, Zhao Y, Yin H. Characterization of a cold-adapted and salt-tolerant exo-chitinase (ChiC) from Pseudoalteromonas sp. DL-6. Extremophiles, 2016, 20(2): 167-176. DOI:10.1007/s00792-016-0810-5 |
| [23] | Synstad B, Vaaje-Kolstad G, Cederkvist FH, Saua SF, Horn SJ, Eijsink VGH, S?rlie M. Expression and characterization of endochitinase C from Serratia marcescens BJL200 and its purification by a one-step general chitinase purification method. Bioscience, Biotechnology, and Biochemistry, 2008, 72(3): 715-723. DOI:10.1271/bbb.70594 |
| [24] | Sugimoto H, Nakamura K, Nishino Y, Idezawa Y, Fujinuma A, Suzuki K, Watanabe T. Differences in the roles of the two surface-exposed tyrosine residues, Y240 and Y481, of Serratia marcescens chitinase B during processive degradation of crystalline chitin. The Journal of General and Applied Microbiology, 2015, 61(6): 255-261. DOI:10.2323/jgam.61.255 |
| [25] | Vaikuntapu PR, Mallakuntla MK, Das SN, Bhuvanachandra B, Ramakrishna B, Nadendla SR, Podile AR. Applicability of endochitinase of Flavobacterium johnsoniae with transglycosylation activity in generating long-chain chitooligosaccharides. International Journal of Biological Macromolecules, 2018, 117: 62-71. DOI:10.1016/j.ijbiomac.2018.05.129 |
| [26] | Wang XH, Zhao Y, Tan HD, Chi NY, Zhang QF, Du YG, Yin H. Characterisation of a chitinase from Pseudoalteromonas sp. DL-6, a marine psychrophilic bacterium. International Journal of Biological Macromolecules, 2014, 70: 455-462. DOI:10.1016/j.ijbiomac.2014.07.033 |
| [27] | Fu X, Yan QJ, Yang SQ, Yang XB, Guo Y, Jiang ZQ. An acidic, thermostable exochitinase with β-N-acetylglucosaminidase activity from Paenibacillus barengoltzii converting chitin to N-acetyl glucosamine. Biotechnology for Biofuels, 2014, 7(1): 174. DOI:10.1186/s13068-014-0174-y |
| [28] | Berini F, Presti I, Beltrametti F, Pedroli M, V?rum KM, Pollegioni L, Sj?ling S, Marinelli F. Production and characterization of a novel antifungal chitinase identified by functional screening of a suppressive-soil metagenome. Microbial Cell Factories, 2017, 16(1): 16. DOI:10.1186/s12934-017-0634-8 |
| [29] | Gao L, Sun JA, Secundo F, Gao X, Xue CH, Mao XZ. Cloning, characterization and substrate degradation mode of a novel chitinase from Streptomyces albolongus ATCC 27414. Food Chemistry, 2018, 261: 329-336. DOI:10.1016/j.foodchem.2018.04.068 |
| [30] | Honda S, Wakita S, Sugahara Y, Kawakita M, Oyama F, Sakaguchi M. Characterization of two Listeria innocua chitinases of different sizes that were expressed in Escherichia coli. Applied Microbiology and Biotechnology, 2016, 100(18): 8031-8041. DOI:10.1007/s00253-016-7546-0 |
| [31] | Fu X, Yan QJ, Wang J, Yang SQ, Jiang ZQ. Purification and biochemical characterization of novel acidic chitinase from Paenicibacillus barengoltzii. International Journal of Biological Macromolecules, 2016, 91: 973-979. DOI:10.1016/j.ijbiomac.2016.06.050 |
| [32] | Krolicka M, Hinz SWA, Koetsier MJ, Joosten R, Eggink G, van den Broek LAM, Boeriu CG. Chitinase chi1 from Myceliophthora thermophila C1, a thermostable enzyme for chitin and chitosan depolymerization. Journal of Agricultural and Food Chemistry, 2018, 66(7): 1658-1669. DOI:10.1021/acs.jafc.7b04032 |
| [33] | Deng JJ, Shi D, Mao HH, Li ZW, Liang S, Ke Y, Luo XC. Heterologous expression and characterization of an antifungal chitinase (Chit46) from Trichoderma harzianum GIM 3.442 and its application in colloidal chitin conversion. International Journal of Biological Macromolecules, 2019, 134: 113-121. DOI:10.1016/j.ijbiomac.2019.04.177 |
| [34] | Kidibule PE, Santos-Moriano P, Jiménez-Ortega E, Ramírez-Escudero M, Limon MC, Remacha M, Plou FJ, Sanz-Aparicio J, Fernández-Lobato M. Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42:enzymatic activity and structural basis of protein specificity. Microbial Cell Factories, 2018, 17(1): 47. DOI:10.1186/s12934-018-0895-x |
| [35] | Yang SQ, Fu X, Yan QJ, Jiang ZQ, Wang J. Biochemical characterization of a novel acidic exochitinase from Rhizomucor miehei with antifungal activity. Journal of Agricultural and Food Chemistry, 2016, 64(2): 461-469. DOI:10.1021/acs.jafc.5b05127 |
| [36] | Liu YC, Yan QJ, Yang SQ, Jiang ZQ. Novel protease-resistant exochitinase (Echi47) from pig fecal environment DNA with application potentials in the food and feed industries. Journal of Agricultural and Food Chemistry, 2015, 63(27): 6262-6270. DOI:10.1021/acs.jafc.5b01457 |
| [37] | Li RK, Hu YJ, Ng TB, Guo BQ, Zhou ZH, Zhao J, Ye XY. Expression and biochemical characterization of a novel chitinase ChiT-7 from the metagenome in the soil of a mangrove tidal flat in China. International Journal of Biological Macromolecules, 2020, 158: 1125-1134. DOI:10.1016/j.ijbiomac.2020.04.242 |
| [38] | Han B, Zhou K, Li ZH, Sun B, Ni Q, Meng XZ, Pan GQ, Li CF, Long MX, Li T, Zhou CZ, Li WF, Zhou ZY. Characterization of the first fungal glycosyl hydrolase family 19 chitinase (NbchiA) from Nosema bombycis (Nb). The Journal of Eukaryotic Microbiology, 2016, 63(1): 37-45. DOI:10.1111/jeu.12246 |
| [39] | Jiang S, Jiang HY, Zhou YL, Jiang SJ, Zhang GM. High-level expression of β-N-acetylglucosaminidase BsNagZ in Pichia pastoris to obtain GlcNAc. Bioprocess and Biosystems Engineering, 2019, 42(4): 611-619. DOI:10.1007/s00449-018-02067-5 |
| [40] | Zhang R, Zhou JP, Song ZF, Huang ZX. Enzymatic properties of β-N-acetylglucosaminidases. Applied Microbiology and Biotechnology, 2018, 102(1): 93-103. DOI:10.1007/s00253-017-8624-7 |
| [41] | Du C, Jiang S, Jiang SJ, Zhou YL, Zhang GM. A Bacillus pumilus originated β-N-acetylglucosaminidase for chitin combinatory hydrolysis and exploration of its thermostable mechanism. International Journal of Biological Macromolecules, 2019, 132: 1282-1289. DOI:10.1016/j.ijbiomac.2019.04.054 |
| [42] | Jiang S, Hao SH, Xiang L, Song L, Zhou YL, Jiang SJ, Zhang GM. A new N-acetylglucosaminidase from facultative alkaliphilic Bacillus pseudofirmus 703. Acta Microbiologica Sinica, 2020, 60(1): 69-80. (in Chinese) 姜顺, 郝少华, 向腊, 宋俐, 周玉玲, 蒋思婧, 张桂敏. 一种来源于兼性嗜碱菌Bacillus pseudofirmus 703的β-N-乙酰氨基葡萄糖苷酶. 微生物学报, 2020, 60(1): 69-80. |
| [43] | Chen BL, Qin Z, Zhao LM. Gene cloning, expression and characterization of β-N-acetylglucosaminidase from Bacillus amyloliquefaciens. Food Science, 2020, 41(8): 123-129. (in Chinese) 陈宝莉, 秦臻, 赵黎明. 解淀粉芽孢杆菌β-N-乙酰氨基葡萄糖苷酶基因的克隆表达及其酶学表征. 食品科学, 2020, 41(8): 123-129. |
| [44] | Itoh T, Araki T, Nishiyama T, Hibi T, Kimoto H. Structural and functional characterization of a glycoside hydrolase family 3β-N-acetylglucosaminidase from Paenibacillus sp. str. FPU-7. Biochemistry, 2019, 166(6): 503-515. |
| [45] | Wang M, Zheng F, Wang T, Lyu YM, Alteen MG, Cai ZP, Cui ZL, Liu L, Voglmeir J. Characterization of Stackebrandtia nassauensis GH 20 beta-hexosaminidase, a versatile biocatalyst for chitobiose degradation. International Journal of Molecular Sciences, 2019, 20(5): 1243. DOI:10.3390/ijms20051243 |
| [46] | Lv CY, Gu TY, Xu KY, Gu JG, Li LC, Liu XN, Zhang AD, Gao SX, Li WJ, Zhao GG. Biochemical characterization of a β-N-acetylhexosaminidase from Streptomyces alfalfae and its application in the production of N-acetyl-d-glucosamine. Journal of Bioscience and Bioengineering, 2019, 128(2): 135-141. DOI:10.1016/j.jbiosc.2019.01.017 |
| [47] | Krolicka M, Hinz SWA, Koetsier MJ, Eggink G, van den Broek LAM, Boeriu CG. β-N-acetylglucosaminidase MthNAG from Myceliophthora thermophila C1, a thermostable enzyme for production of N-acetylglucosamine from chitin. Applied Microbiology and Biotechnology, 2018, 102(17): 7441-7454. DOI:10.1007/s00253-018-9166-3 |
| [48] | Zhang AE, He YM, Wei GG, Zhou J, Dong WL, Chen KQ, Ouyang PK. Molecular characterization of a novel chitinase CmChi1 from Chitinolyticbacter meiyuanensis SYBC-H1 and its use in N-acetyl-D-glucosamine production. Biotechnology for Biofuels, 2018, 11: 179. DOI:10.1186/s13068-018-1169-x |
| [49] | Tanaka T, Fukui T, Imanaka T. Different cleavage specificities of the dual catalytic domains in chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. The Journal of Biological Chemistry, 2001, 276(38): 35629-35635. DOI:10.1074/jbc.M105919200 |
| [50] | Pradeep GC, Yoo HY, Cho SS, Choi YH, Yoo JC. An extracellular chitinase from Streptomyces sp. CS147 releases N-acetyl-D-glucosamine (GlcNAc) as principal product. Applied Biochemistry and Biotechnology, 2015, 175(1): 372-386. DOI:10.1007/s12010-014-1267-6 |
| [51] | Tran TN, Doan CT, Nguyen MT, Nguyen VB, Vo TPK, Nguyen AD, Wang SL. An exochitinase with N-acetyl-β-glucosaminidase-like activity from shrimp head conversion by Streptomyces speibonae and its application in hydrolyzing β-chitin powder to produce N-acetyl-D-glucosamine. Polymers, 2019, 11(10): 1600. DOI:10.3390/polym11101600 |
| [52] | Song W, Zhang N, Yang M, Zhou YL, He NS, Zhang GM. Multiple strategies to improve the yield of chitinase a from Bacillus licheniformis in Pichia pastoris to obtain plant growth enhancer and GlcNAc. Microbial Cell Factories, 2020, 19(1): 181. DOI:10.1186/s12934-020-01440-y |
| [53] | Li CN, Jiang S, Du C, Zhou YL, Jiang SJ, Zhang GM. The properties and its application in preparating GlcNAc of β-N-acetylglucosaminidases from Bacillus coagulans DSM1. Chinese Journal of Biotechnology, 2021, 37(1): 1-10. (in Chinese) 李丛娜, 姜顺, 杜超, 周玉玲, 蒋思婧, 张桂敏. 凝结芽孢杆菌耐热β-N-乙酰氨基葡萄糖苷酶在大肠杆菌的分泌表达及其在制备GlcNAc中的应用. 生物工程学报, 2021, 37(1): 1-10. |
| [54] | Liu YH, Jiang ZQ, Ma JW, Ma S, Yan QJ, Yang SQ. Biochemical characterization and structural analysis of a β-N-acetylglucosaminidase from Paenibacillus barengoltzii for efficient production of N-acetyl-D-glucosamine. Journal of Agricultural and Food Chemistry, 2020, 68(20): 5648-5657. DOI:10.1021/acs.jafc.9b08085 |
| [55] | Zhou JP, Song ZF, Zhang R, Ding LM, Wu Q, Li JJ, Tang XH, Xu B, Ding JM, Han NY, Huang ZX. Characterization of a NaCl-tolerant β-N-acetylglucosaminidase from Sphingobacterium sp. HWLB1. Extremophiles, 2016, 20(4): 547-557. |
| [56] | Nguyen-Thi N, Doucet N. Combining chitinase C and N-acetylhexosaminidase from Streptomyces coelicolor A3(2) provides an efficient way to synthesize N-acetylglucosamine from crystalline chitin. Journal of Biotechnology, 2016, 220: 25-32. DOI:10.1016/j.jbiotec.2015.12.038 |
| [57] | Wang D, Li AJ, Han HY, Liu T, Yang Q. A potent chitinase from Bacillus subtilis for the efficient bioconversion of chitin-containing wastes. International Journal of Biological Macromolecules, 2018, 116: 863-868. DOI:10.1016/j.ijbiomac.2018.05.122 |
| [58] | Zhu WX, Wang D, Liu T, Yang Q. Production of N-acetyl-D-glucosamine from mycelial waste by a combination of bacterial chitinases and an insect N-acetyl-D-glucosaminidase. Journal of Agricultural and Food Chemistry, 2016, 64(35): 6738-6744. DOI:10.1021/acs.jafc.6b03713 |
| [59] | Tian ZQ, Wang SK, Hu XF, Zhang ZM, Liang L. Crystalline reduction, surface area enlargement and pore generation of chitin by instant catapult steam explosion. Carbohydrate Polymers, 2018, 200: 255-261. DOI:10.1016/j.carbpol.2018.07.075 |
| [60] | Wei GG, Zhang AL, Chen KQ, Ouyang PK. Enzymatic production of N-acetyl-D-glucosamine from crayfish shell wastes pretreated via high pressure homogenization. Carbohydrate Polymers, 2017, 171: 236-241. DOI:10.1016/j.carbpol.2017.05.028 |
